Control Of Reproduction In Domestic Animals
BANAT UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE KING MICHAEL I OF ROMANIA TIMIȘOARA
HABILITATION THESIS
Control of reproduction in domestic animals
Assist.Prof.Călin Mircu, PhD
Medicine Veterinary Faculty
Timișoara, 2015
These 25 years Assistant Professor Călin Mircu, PhD in medical sciences since 1997, has been teaching courses on physiology and pathology of animal reproduction, on clinical reproduction, (assisted) reproduction biotechniques, and medical and molecular genetics, with Veterinary Medicine Faculty, of Banat University of Agricultural Sciences and Veterinary Medicine KING MICHAEL I OF ROMANIA, Timișoara, actually the same that he graduated in 1988.
Assist.Prof. Călin Mircu disseminated his research (run intra and extra muros, as on the field, in clinics, in Horia Cernescu Research Labs…) in various national and world conferences; in publications (some 100 science papers in Romania and abroad, most BDI, also ISI); in joint projects (as Coaching young vets for business, or Romania – Serbia Joint Agrobusiness); last but not least, as students courses, on paper and on the www, in Romanian and in English. At times our faculty team research substantiated into fully designed and implemented projects, such as Development of Research Infrastructure for Education and Servicing in Veterinary Medicine and for Innovative Technologies – West Development Region Romania – POSCCE 2669, 2009 to 2014.
As a staff of the Reproduction, Obstetrics and Gynecology Chair in our quoted university, Assist.Prof. Călin Mircu greatly benefitted from first hand skills and knowledge acquired as working with Prof. D.E. Noakes, in LAMS Department, Royal Veterinary College London, UK, 1993; Prof. G. Husenicza, in Veterinary Medicine Budapest University, 1998, and again in 2001; Prof. Jan Kotwica, in Animal Reproduction and Food Research Institute, Polish Sciences Academy, Olsztyn, Poland; and Dr. Claus Leiding, BVN Neustadt an der Aisch, Germany. Assist.Prof.Călin Mircu has been passing on such precocious human experience as working with, and for, his students, whether guiding them to proficiency (coaching for no less than 50 graduation theses, or assisting Prof. Horia Cernescu doctoral candidati); and seeing that such research became visible, nation or worldwide, as published science articles
As a university professor to-be, Assist.Prof. Călin Mircu is expected to run ever ampler research (happily furthered by the research infrastructure available through quoted POSCCE 2669 project), be it with current courses, or as guiding graduation and doctoral theses, or science articles on topics pertaining to (assisted) reproduction based on genetic markers, obstetrics and gynecology; as he will also be expected to meet further academic tasks pertaining to leveling home curricula to the best in the western world; or, designing partnerships apt to promote inter-doctoral schools mobilities; or raising academic standards to home and world labor markets’ (ever more exacting) specific requirements.
Habilitation thesis is a well rounded synthesis at present time of his academic research.
Prof. Ioan Huțu, Veterinary Medicine Faculty,
Banat University of Agricultural Sciences and Veterinary Medicine
KING MICHAEL I OF ROMANIA, Timișoara
Habilitation thesis being developed based on author’s prior team research (further indicated as quoted below), acknowledgement to colleagues is gratefully extended.
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R1. Mircu C., Cernescu H., Bonca Gh., Ardelean V., Igna Violeta, Șerdean C. Monitorizarea funcției de reproducere la vaci cu ajutorul nivelului progesteronului din lapte. 1998. Revista Română de Medicină Veterinară, 8, no.2: pp.47 to 56
R2. Mircu C., V.Ardelean, H.Cernescu, Gh.Bonca, Violeta Igna, C. Șerdean. Diagnosticul precoce al gestației la vacă cu ajutorul PGF2alfa. 1998. Revista Română de Medicină Veterinară, 8, no.3: pp.61 to 72
R3. Mircu C., H.Cernescu, Gh. Ghișe, V. Ardelean, Gh.Bonca, Violeta Igna. Effects of imunization with porcine zona pellucida and oocytes upon reproductive function in the bitch. 2000. Acta Veterinaria (Faculty of Veterinary Medicine Beograd), vol.50, 4: pp.215 to 224
R4. Mircu C., H.Cernescu, Gh. Ghișe, V. Ardelean, Gh.Bonca, Violeta Igna. The influence of immunization with porcine zona pellucida upon bitch ovaries. 2001. Acta Veterinaria (Faculty of Veterinary Medicine Beograd), vol.51, 4: pp. 235 to 244
R5. Simona Marc Zarcula, Gabriel Godja, Cornelia Milovanov, Camelia Tulcan, Horia Cernescu, Gabriel Otavă, Gheorghe Bonca, Andreea Ciobota, Ioan Huțu, Călin Mircu. Morphological aspects of cumulus –oocyte complexes in different species. 2014. Lucrările Științifice USAMV Iasi, vol. 57, no.3-4, pp. 91 to 97, 2014, First International Congress Life sciences – a challenge for the future, Iași (Romania), October 23-25, 2014
R6. Simona Marc Zarcula, Gabriel Godja, Ioan Huțu, Camelia Tulcan, Gheorghe Bonca, Gabriel Otavă, Adela Markovski, Vasile Ardelean, Călin Mircu. Comparison of morphological aspects and nuclear status of bovine COCs cultured in medium with/without sheep FSH. 2015. The 3rd International Virtual Conference on Advanced Scientific Results – SCIECONF, Scientific Conference. Conference home page, pp. 299-301, May, 25 – 29, 2015
R7. Mircu C., Oana Boldura, Camelia Tulcan, I.Hutu. Evaluation of BCL2 and PTX3 genes expression in swine cumulus cell cultured in different media. 2015. 6th ISAFG Piacenza, Italy. Proceedings, Transcriptomics – Abstract no 67.
the authorCONTENTS
HABILITATION THESIS
CONTROL OF REPRODUCTION IN DOMESTIC ANIMALS
ABSTRACT
Habilitation thesis sums up part of our research activity, ran since our PhD thesis in 1997. Selected aspects are relevant and pioneering, focused on three main areas, as further detailed.
Luteal function and luteolysis
Immunocontraception based on zona pellucida
Factors of fertilization in vitro
A sum of relevant details is quoted in thesis abstract. Our PhD thesis is not a terminus set to our research around PGF2alpha role in cow reproduction. Major aspects of luteal function have been considered, as related to corpum luteum (CL) formation and cell composition thereof, steroidogenesis and luteolysis. PGF2alpha is not the only factor responsible for luteal regression. Hence our further research (as far as early 90’s in Romania allowed) targeted luteolysis, as well as various phenomena inducted by PGF2alpha, somehow or other present in cow reproductive tract. Both hypophiseal oxytocin and corpus luteum oxytocin play a key role in luteolysis; as also do estrogens and progesterone.
The way pregnancy interferes with luteolysis is highly relevant. Presence of an embryo in sheep uterus was found to suppress the PGF2alpha secretion from uterus, starting on day 12 post estrus. On the other hand, gestant sheep corpus luteum was noticed to resist exogenous PGFs and (effluent) venous blood of pregnant uterine horn, exerting (with same animal) luteotrophic action on its contralateral pair. Such statements allow us to conclude that embryo may play a double role in maintaining the corpus luteum functional, i.e. by inhibiting PGF2alpha on one hand, and by causing a luteotroph principle, on the other. The embryo signals basic to pregnancy recognition are PGE2, PGI2 and, especially, trophoblastin (IFNtau), one of the key events being suppression of uterine receptors for oxytocin expression, with subsequent suppression of PGF2alpha secretion.
Research of oxytocin–PGF2alpha relation during luteolysis proved a surprising theory basis for a novel practical application; more specifically, use of a sub-luteolytic PGF2alpha dose for early pregnancy diagnosis (on pregnancy day 20) post artificial insemination (Labussiere – 1990). In Labussiere’s view, a sub-luteolytic PGF2alpha dose injected in the subcutaneous abdominal cranial vein, causes oxytocin release from pregnant CL (and subsequent milk ejection), expressed as CL being fully functional, if accompanying current pregnancy. As applying such method, we concluded that pregnancy diagnosis by TPGF on day 20 post artificial insemination is a reliable method, allowing for identification of 87.7 to 90% of the pregnant females, while top accuracy manifests with non-gestant diagnosis. Progesterone levels in blood and milk closely reflect sexual cycle stage, based on which reason progesterone (P4) level proves a highly accurate indication of pregnancy. The experiments we ran allowed for conclusions as further detailed: low progesterone level (below 3 ng/ml) during artificial insemination (AI) is critical for pregnancy to occur; levels of milk progesterone varying by over 1.5 ng/ml on days 8 to 10 post artificial insemination may indicate luteolysis being triggered and subsequent pregnancy failure; progesterone levels on days 16, 21 and 24 post artificial insemination differ widely for non-gestant cows, as against the pregnant; there is a positive correlation between luteal phase progesterone level and pregnancy rate.
The concepts suggested by our previous studies, plus availability, today, of our high tech equipments, enable us to direct ourselves towards investigating such phenomena through molecular genetics. Early embrionary development is ruled by maternal transcripts and by the proteins stored in oocyte during ovogenesis. During development process, maternal transcripts and proteins will be destroyed concomitantly with embrionary genome activation (EGA). We find identification useful, of genes for oxytocin (OT), PGF2alpha and the estrogens active in different phases of corpus luteum (CL) function; as equally useful proves to be the expression level of a number of genes, related to pregnancy and embryonic mortality, or lack of genome activation, and so on.
Based on results of a project we ran, in the Reproduction, Obstetrics and Gynecology Clinic, focused on the side effects of steroid contraception in pet dogs, we started researching other contraceptive methods, apt to yield similar results, yet no adverse side effects. Main obstacles, when applying immune contraception, manifest as: toxic side effects; lack of proper means of spreading steroids within animal organism; and incomplete reversibility. Zona pellucida (ZP) plays a basic role in fertilization, as well as in embryogenesis preimplantation stage. Our experiments and investigations targeted determination of antiZP antibody titers subsequent to bitch immunization, using both pig pellucida (ZP), and pig oocyte together with zona pellucid (ZPp), i.e. (OV+ZPp), in order to show how presence of anti ZP antibodies interfere with sexual activity of immunized females. Final results of such research allowed for conclusions as further detailed: bitch immunization using pig zona pellucida generated an antibody titer inhibitory for fertilization, which lasted for 7 months with no bearing on sexual cyclicity; using oocyte together with zona pellucida, for immunization, generated higher antibody titers, also inhibitory for fertilization, lasting for a comparatively longer period (10 months), yet accompanied by reproductive disorders; such methods granted complete (100%) contraception.
We tend to think it useful that synthesis pattern of zona pellucida (ZP) proteins should be clearly determined, as equally useful would be to determine the potential structural differences between ZP proteins synthesized by follicular cells, and ZP proteins produced in oocytes, as displayed in either protein sequence or by glycosylation? degree. Spermatozoa penetration during fertilization could be prevented by proteasome inhibitors and by antiproteasomal antibodies. Proteasome generated by acrosome may be an interesting contraception target. Targeting 26S proteasome or proteasome 20S content may favor investigation of the interaction sperm – zona pellucida, during in vitro fertilization (IVF); and investigation or of polysperm management.
Our research labs, developed through Project POSCCE, broke new horizons for our faculty, thus enabling us to direct our interest and efforts towards researching in vitro techniques, mainly such as related to in vitro fertilization (IVF). IVF is a procedure consisting in oocyte harvesting, oocyte maturation and fertilization occurring in artificial conditions, the resulting embryo being transferred to a surrogate mother. There are a number of operational IVF protocols by now, due to each species gametes needing specific conditions. On the other hand, there is a growing tendency manifest, for refining such technique, so as the highest number of transferrable embryos should be obtained. Approaching new research directions implies thorough and accurate knowledge of all physiological aspects; we therefore think fully justified a thorough presentation of folliculogenesis and fertilization, as loosely correlated with cellular cycle. Our first study targeted assessment in vitro maturation of bovine oocytes; when all of the subsequent experiment stages were completed, we concluded that presumptive low reproductive parameters of culled cows is reflected in the biological material harvested, quality of oocytes thus obtained affecting maturation capacity thereof; FSH supplementation of culture medium has no bearing on maturation of bovine cumulus oocyte complex (COC). Maturation degree is determined only based on assessment of nuclear stages of female ova during meiosis, staining by substances with high affinity for nucleic acids following; staining bovine oocytes with 1%, aceto-orcein, in order to assess nuclear maturation, is easy to run; yet, by such procedure, the stained oocytes remain bound, hence invalid for the IVF. Assessment of nuclear maturation is insufficient for thoroughly assessment of bovine oocyte maturation, cytoplasmic maturation assessment being also needed.
Next step we took was choosing the proper IVF technique, fully compatible with our research routine, and lab equipments; with all stages completed and final analyses run, our statement is that ovaries begot from slaughterhouses contain a heterogeneous oocyte population. Such samples, regardless of follicular dynamics, and of cow individual characteristics (such as age, physiological status, and breed) will impact oocytes developmental capacity; IVF protocols success rate relies on the reconstruction of close to perfectly similar conditions of in vivo maturation, fertilization and development; the expertise of the lab staff plays a major role, as such technicians would be able to handle cells, or small quantities of culture medium, fast, in order to cut on the time cultures are outside incubator, as being harvested or moved around. Implementing an IVF protocol requires long time delays, depending on a series of factors, related to both the biologic material used, and the good technical facilities and well- trained staff available.
Our third experiment, rendered in the last chapter, targeted improvement of sow oocyte viability, by adding more cysteine to the culture medium. For the purpose, beside oocytes morphological assessment post maturation, we also studied Bcl2 genes expression in sow cumulus cells, before and after in vitro maturation. Our conclusion was that, by supplementing cysteine to the maturation medium, the number of oocytes reaching maturation does not increase; also, that no morpho-structural changes in oocytes occur during maturation.
Nevertheless, as quantifying Bcl2 apoptotic genes expression, we recorded a high rise in oocyte-cumulus complexes cultured in media supplemented with cysteine, as compared to the non-supplemented medium. A high level of Bcl2 genes expression in cumulus cells was noticed to lead to inhibition of apoptosis thereof on mitochondrial path, leading to marked enhancement of oocytes viability and of fertilization rate.
A high successful fertilization rate is basic to in vitro fertilization, especially for economicity reasons, such as availability of oocytes, and costs of semen, disposables, and handling; also, the higher the fertilization rate, the lower the handling time. Based on such results, further investigation is recommended on the topic. Supplementing cystein in more culture media may be researched for other type studies, targeting increased cells viability: cells viability is highly likely to increase by increasing the expression level of genes, such as, mainly, Bcl2, which can also act molecular marker, as assessing cell viability, implicitly oocytes fertilization rate, too.
Data resulted from the experiments run enabled us to identify a number of directions for our further research. Both the Golgi complex, and the endoplasmic reticulum are major research topics, in terms of how the former contribute to embryo development; and interference of inositol triphosphate (IP3) path on calcium release may express a control mechanism of oocyte activation during fertilization.
Since cytoskeleton filaments dynamics of bovine oocytes is related to completion of nuclear development competence, determination of the magnitude and intensity of cytoskeleton alterations might indicate a potential for nuclear development opportunity. Starting from studies revealing fertilin function (the dimeric glycoprotein bound to oocyte plasma membrane) investigation becomes possible of ways in which fertilin induces a fusion process.
Research could accurately determine the role of cumulus cells in meiotic maturation, especially by identifying the factors acting in meiosis inhibition mechanism. Another topic of interest proves to be embryo oxygen consumption, quantifiable by various methods, due consideration given to the role cumulus cells play in oxygen tension variation, in embryo close vicinity.
Oocyte morphology and quality are established during oogenesis and folliclegenesis, being complete at final maturation time. Early embryo development is closely associated with oocyte-cumulus complex (COC) morphology. Determining a correlation oocyte morphology and protein contents (a study on proteome) would be most welcome, for revealing fertilization capacity.
Morphological features assessment of oocyte-cumulus complexes helps selection of the oocytes apt for IVF and embryo production. Such aspect is worth investigating in terms of the transcripts (transcriptomics); as well as of proteins abundance (proteomics).
As staining with 1% aceto-orcein impairs oocyte for further IVF, a staining dye should be used that would have no bearing on oocyte viability (e.g. Hoechst 33342); also, staining techniques are recommended which reveal cytoplasmic maturation, by organelle staining, or other type enhancement of activity thereof.
Chapter 1. LUTEAL FUNCTION AND LUTEOLYSIS
CONCEPT ANALYSIS AND EVOLUTION
Luteolysis and luteal function
Prostaglandin F2alfa and role thereof in controlling cow reproduction was our doctoral thesis topic, further research on aspects pertaining to luteolysis as well as on progesterone levels variations being left for a later time. Present chapter will state a number of theory aspects on such topics, followed by brief presentations of two research papers on same topics, ending with directions quoted, that we deem worth turning our efforts to.
Major aspects of luteal function are CL formation and cellular composition, i.e. steroidogenesis and luteolysis.
Corpus luteum (CL) formation
Fast cellular transformation occurring early in CL life is also characterized by a series of key proteins vanishing, as well as by other proteins showing up, or, at least, manifesting a highly intensified expression. FSH receptor, a protein expressed only in follicular granulosa and Sertoli cells, becomes undetectable over CL formation time (Gibori-1993). A series of key enzymes expression, as well as of P45017β and P450AROM, implied in synthesis of androgens and estrogens, fall to low levels, even undetectable. Inhibition of such enzymes synthesis may be short-term (e.g. in humans and rat) or is sustained for as long as CL life (e.g. in bovine, ovine and rabbits).
Angiogenesis is a major component of CL development and differentiation process. Angiogenic effect is mediated by paracrine release of angiogenesis inductors, which cause endothelial cells migration, as well as capillaries formation and proliferation in CL. Several proteins manifest such effect, e.g. bFGF (basic fibroblast growth factor); VEGF (vascular endothelial growth factor); and angiotensina 2. Such proteins are responsible for capillary endothelial cells proliferation; for extracellular matrix degradation of local venule post protease release; as are also responsible for the migration of the capillary-like tubules. Whatever the basic mechanism of extensive angiogenic activity manifest in CL, absence of vascular smooth muscles in developing CL seems to correlate with the fast division and migration of the endothelial cells. Such migration results in formation of sinusoidal vessels over a time delay insufficient for ingrowth, or for the smooth muscles in such vessels to differentiate.
The two types of luteal cells population, i.e. small and large, have first differentiated based on size, morphology and tinctorial characteristics. Generally, small luteal cells are under 20 µm diameter; while large luteal cells are over 20 to 22 µm. Ultrastructural differences consist in the large luteal cells manifesting higher contents of smooth and rough endoplasmic reticulum (ER), mitochondria, Golgi complex and secretory granules, as against the small luteal cells. The secretory characteristics of the large luteal cells correlate to intensified secretory activity. To say that large luteal cells are originally granular, while small luteal cells originate in preovulatory follicle sheath, would be an oversimplified statement. It was proven, by means of monoclonal antibodies, that large luteal cells may be of dual origin. Small luteal cells may turn into large luteal cells along CL functional life. There are around 10 to 12 million large luteal cells in an ovine CL, such number staying relatively constant during sexual cycle. By contrast, number of small luteal cells per CL is around 10 million, during early luteal phase, increasing to a maximum 50 to 60 million mid luteal phase.
One more hypothesis (Gibori -1993) says that luteal cells size depends not on follicular origin but, rather, on individual responsivity to growth signals. In rats, only large luteal cells respond to IGF-1 (insulin-like growth factor 1) stimulation; containing most of the receptors for such growth factor, IGF-1 could cause such cells’ protein capacity for synthesis and metabolism to increase, triggering ampler size of such cells. Such stimulation holds for both type luteal cells in ovine mature CL.
Fields and col. (1991) reveal presence of a series of secretory granules clusters in large luteal bovine cells, containing oxytocin and neurophysin. One cluster only is formed at sexual cycle debut. Once the secretory granules are depleted, the cell is unable to generate a new secretory granules population. Our research showed presence of two type large luteal cells populations, i.e. α and β. Type α large luteal cells show higher contents of mitochondria and smooth endoplasmic reticulum, as against smaller type β luteal cells. Moreover, we proved type α luteal large cells to be much more sensitive to PGF2alpha action, than β. Type α cells, sensitive to subluteolytic PGF doses, are the first to regress; followed by type β large luteal cells regression, when PGF2alpha levels have increased, a process made evident by regression of all large luteal cells up to day 19 of sexual cycle, in absence of fertilization.
Various species’ large luteal cells would normally secrete 10 to 30 times more progesterone (P4) than small luteal cells. Large luteal cells produce over 80% of progesterone secreted by CL, mid luteal phase (Niswender and col., 1994). Cell types also differ in the way lipoprotein derivatives are incorporated and metabolized.
Luteal cells populations are specifically regulated, an aspect proven firstly by LH acutely stimulating P4 production by the small luteal cells, yet having no detectable effect on (or else needing much higher concentrations in order to minimally increase) large luteal cells production. Lack of functional receptors for LH in large cells might account for such aspect (Niswender and col., 1994).
Steroidogenesis.
Mechanisms implied in P4synthesis and secretion are complex, even though such hormone is the first active biological compound resulted in steroids biosynthesis. In most cases, cholesterol (COL) used as a sublayer is obtained in low density lipoproteins (LDL) or high density lipoproteins (HDL), and less as synthesized de novo in acetate. LDL incorporation occurs by receptor-mediated classical endocytosis, while HDL incorporation implies attachment thereof to specific sites on membranes. Cholesterol resulted from various sources can later be used for steroids synthesis, or can be incorporated into cholesteryl esters and stored in lipid droplets. As P4 secretion increases over sexual cycle luteal phase, the number of lipoprotein binding sites on luteal cells surface also increases.
During early luteal phase (i.e. days 3 to 5), both small and large luteal cells respond to stimulation exerted by HCG, PGE2 and AMPc, as increased progesterone production. Mid luteal phase (i.e. days 6 to 12), only large luteal cells still respond to action of quoted three agonists; while over late luteal phase (i.e. post day 15), only HCG and AMPc still exert a slight stimulation of progesterone production, at large luteal cells level. It seems that a lower CL steroidogenic capacity, over mid to late luteal phase, is (partly) due to luteal cells population mutation (i.e. selective loss of the large, highly steroidogenic, luteal cells), rather than to loss of steroidogenic capacity. Adenosine intensifies luteal cells functional response (to luteotropic HCG, PGE1 or PGE2) and reduces luteolitic response to action of PGF2alpha. Thus, adenosine could act as second messenger in LH-regulated stereoidogenesis.
COL esters release depends on a neutral cholesterol esterase, also known as hormone-sensitive lipase. Activity of such enzyme is regulated by phosphorylation of serine residues. AMPc-dependant protein kinase (PKA) causes phosphorylation of the former residual serine and enzyme activation, while Ca2+ /calmodulin-dependent protein kinase phosphorylates the latter residual serine and prevents enzyme activation.
COL side-chain cleavage indicates P4 biosynthesis limit threshold. Such process implies transport of COL to the cytoplasm inside the mitochondrial membranes, where it finds side-chain cytochrome P450 (P450scc) cleavage enzyme, which transfer seems to involve the cytoskeleton (Niswender and col. -1994). Transport can be mediated by a number of factors, including peptide activating steroidogenesis, sterol carrier proteins 2 (SCP2), endozepine/ benzodiazepine as well as lipoxygenase metabolites.
Three proteins are implied in COL conversion to pregnenolone (PREG), i.e.: adrenodoxin (Adx), adrenodoxyn reductase (ADXR) and cytochrom P450scc. The ARNm for such proteins are similarly regulated. P4Secretion is differently regulated in the two types ovine luteal cells (Niswender and col. -1994). LH activates protein kinase (PKA) and stimulates P4secretion in small luteal cells, yet not in the large. PKC activation inhibits P4secretion in LH stimulated small luteal cells, as well as in large cells luteal. PGF2alpha activates PKC in large luteal cells, yet it is unclear what activates PKC in small luteal cells. PGF2alpha also stimulates Ca2+ flow within large luteal cells, which seems to induce changes associated with cellular degeneration.
Development of corpus luteum of pregnancy is the norm, which implies an inner capacity for sustaining progesterone synthesis; whereas not-inseminated, as well as the inseminated non gestant, cows, having manifested lower progesterone rates post day 10, are taken to be deviant from such norm. Results grant circumstantial evidence for indicating antiluteolytic effect of bovine trophoblast/ interferon tau to be late on day 12 post insemination. Day 8 may mark the end of a critical period in progesterone activity, yet necessary before PGF2alpha can be released; whereas progesterone level fluctuations in non gestant inseminated cows on days 9 and 10 may indicate luteolysis debut, which may later cause embryonic death (Miqueu, 1988). Such remark is also supported by Thibodeaux’s and col. (1994) study conclusions, which states that there is evident marked growth of progesterone level in gestant cows plasma and milk, as against in non gestant, starting on day 8 post estrus.
TOPIC DEFINITION AND CONTENTS
Luteolysis
Luteolysis is an extremely complex process, with major connotations at enzymatic level. PGF2alpha is not the only factor implied in luteal regression. Both neurohypophiseal oxytocin and the corpus luteum play an important role in luteolysis process; as also do estrogens and progesterone.
Actually oxytocin plays a basic role in quoted prostaglandin release episodes leading to luteolysis, oxytocin stimulating prostaglandin synthesis in myometrial and endometrial cells. Estrogens impact uterine prostaglandins secretion, by both directly intervening in synthesis thereof, and by stimulating formation of uterine receptors for oxytocin.
Neurohypophiseal oxytocin is synthesized by pulses and can induce each uterine PGF2alpha release episode. Uterine PGF2alpha stimulate release of oxytocin from corpus luteum.
Luteal oxytocin then stimulates PGF2alpha secretion by uterus and may induce short-term oxytocin-stimulated uterine refractoriness, at a later time. Refractoriness will decrease in around 6 hours. A similar desensitization phenomenon occurs in response to PGF2alpha action at CL level. Uterine and luteal refractoriness may be solidarily responsible for the PGF2alpha interpulse noticed during luteolysis. Uterine secretory responsiveness to oxytocin increases at luteolysis time, when pulsatile endogenous PGF2alpha secretion normally starts. Hence, presence of uterine responsiveness to oxytocin could be determined when PGF2alpha endogenous secretion may occur over all of the sexual cycle time. Uterine secretory responsiveness to oxytocin develops slowly, in presence of progesterone.
Episodic nature of PGF2alpha release seems to result from a positive feed-back loop, where oxytocin secreted by CL acts upon the uterus in order to stimulate PGF2alpha release, which acts back upon the CL, causing more oxytocin release. Positive feed-back is even said to mature (Silvia and Raw, 1993); uterus PGF2alpha secretion capacity in response to oxytocin action does not develop before day 13 or 14 post estrum. Silvia and Raw (1993) deem neurohypophiseal oxytocin to be a triggering signal for a number of events to snowball. Each PGF2alpha secretion episode is accompanied by an oxytocin release episode, and secretion stop post each episode is thought to be due to one of the factors as further detailed: luteal oxytocin depletion; depletion of PGF2alpha precursor in uterus; CL refractoriness to PGF2alpha, or uterus refractoriness to oxytocin. Evidence for oxytocin being involved in luteal process regression covers facts such as immunization against oxytocin delaying luteolysis, which also does administration of an antagonist to oxytocin receptors.
Uterine circulatory sensitivity to oxytocin is determined by level of oxytocin receptors in the endometrial cells. Concentration of receptors for oxytocin increases by 500 times in endometrium, at the time of luteal regression; moreover, inducing oxytocin receptors prematurely, by administering estrogens, causes premature luteolysis; while inhibition of receptor expression by continuous oxytocin administration (which causes receptors to malfunction) generates delayed luteolysis. As uterus responsiveness to oxytocin is determined by expression of receptors, generally shortening the cycle by oxytocin is not possible; as soon as expression of receptors occurs, luteolysis will result consecutive to action of oxytocin present in circulation. Thus, a positive correlation was proven to exist between plasma estradiol concentrations and uterine receptors for oxytocin level; also, a negative correlation between serum progesterone level and receptors for oxytocin level (Beard and col., 1994).
Consequently, it will be noticed that luteal regression moment is determined mainly by the time when oxytocin receptors are formed within uterus. During uterus exposition to P4, receptors for oxytocin levels may vary: over the first 10 days of exposure to P4, receptors number is low, while post such period it will increase.
Bovine endometrium responsiveness to oxytocin increases over days 13 to 19 post estrus, oxytocin potentially activating phospholipase C, catalyzing release of inositol phosphate and of dyacilglycerol (secondary messengers) thus stimulating PGF2 alpha luteolytic secretion over days 13 to 19. It will be remembered that oxytocin-induced PGF2alpha stimulation will be by protein kinase C effector. Estrogens regulate concentration of uterine receptors for oxytocin, while myometrial receptors for oxytocin buffer PGF2 alpha release.
Progesterone exerts two type effects which contribute to regulation of PGF2 alpha secretion. Firstly, acute exposure to progesterone seems to promote storage of arachidonic acid (a prostaglandins precursor) within uterus, of peroxidic prostaglandin synthetase and of other substances needed in prostaglandins synthesis. Secondly, progesterone exerts an inhibitor effect upon such secretion, effect which vanishes post acute exposure. When such effects of progesterone occur simultaneously, PGF2 alpha is caused to be secreted only close to time when luteolysis is induced. Also, progesterone suppresses basal release of prostaglandins in the endometrium (Mitchell and Stevenson, 1995).
Over the luteal phase, progesterone inhibits action of estrogens, by blocking estrogens storage in receptor nuclei, hence inhibits estrogens capacity for synthesizing receptors for oxytocin. Thus, endometrial PGF2 alpha production leads to progesterone-shutdown. Yet, after 10 progesterone administration days, progesterone action decreases, due to the loss of own (progesterone dependent) receptors. Consequently, estradiol will again synthesize receptors for oxytocin, allowing resumption of oxytocin induced PGF2alpha secretion. This time, in response to oxytocin, PGF2alpha secretion will be 100 times higher, as against secretion amount before progesterone intervention, possibly because progesterone (while inhibitory) causes storage of fatty acids precursors in endometrium. Precisely such intensified synthesis is what generates massive discharge of uterine prostaglandins at end of luteal phase cycle, which, after passing counter-current, reach target cell, starting and later finalizing regression thereof.
Estradiol promotes regulation of PGF2alpha uterine secretion, manifesting direct effects upon the uterus, which maximizes responsiveness to oxytocin and is likely to modulate hypophyseal pulsatile oxytocin secretion. Such endocrine interactions generate a mechanism specific for uterine PGF2alpha secretion. The first PGF2alpha pulses are relatively narrow, and likely to start luteal regression. Once luteolysis is triggered, pulses become wider, as impacted by the changes occurred in progesterone and estradiol concentration, associated with luteal regression. Such wide pulses could complete the luteolytic process. Over 13 to 17 cycle days time, numerous peak PGF2alpha levels are manifest, which does not happen with gestant females.
Progesterone and estrogens intervene in regulation of endometrial PGF2alpha production, by affecting both phospholipase C, and analogs thereof. Progesterone promotes estrogen, increasing oxytocin-induced PGF2alpha secretion, pointing to possible synergic action of such hormones in inducing luteolysis. Estrogens act at CL level, promoting luteolytic response to PGF2alpha.
In order to explain PGF2alpha luteolytic effect, several mechanisms are accepted, as further detailed: a.) fast dramatic fall of blood inflow to CL; b.) fall of the number of receptors for LH; c.) impossible coupling of receptors for LH to adenylate cyclase; d.) cytotoxic effects; e.) immune phenomenon.
Wiltbank and Niswender (1992), as well as Wiepz and col. (1993) describe PGF2alpha action patterns as further detailed: (1) coupling with a specific receptor on plasma membranes of LLC; (2) activating phosphoinositide phospholipase C pathway , thus causing production of inositol triphosphate (IP3) and dyacilglycerol; (3) increased intracellular free Ca2+ concentrations, due to either IP3 higher concentration, or to receptor activated Ca pathway; (4) activating protein kinase c, by increased free Ca concentrations and higher dyacilglycerol level; (5) decreasing progesterone production by protein kinase C effector system, apparent through inhibition of cholesterol transport intracellularily (6) causes cell degeneration and LLC cells death, due to protein kinase C activation, as well as to the sustained concentrations of intracellular free Ca (see. Fig.1.1.).
Hansel and col. (1991) deem that the remarkable capacity of arachidonic acid, and of a number of metabolites thereof, to mobilize intracellular Ca, suggests the sequence of events below, as a work hypothesis for initiating luteolysis.
(1) Protein kinase C Activation in SLC causes, at first, increased production of progesterone, of arachidonic acid and metabolites thereof (PGF2alpha, PGE2 and PGI2), accompanied by luteotropic action in the SLC. At any rate, chronic activation of protein kinase C in the small luteal cells causes desensitisation of the LH-induced progesterone production, while prostanoids production continues uninhibited.
(2) Protein kinase C induces high levels of arachidonic acid, and/or metabolites thereof, generate critical levels of intracellular Ca, in the LLC. Arachidonic acid and other long-chain unsaturated fatty acids (i.e.eicosapentaenoic, docosatetraenoic and docosapentaenoic) may produce direct luteolitic effects, as a result of capacity thereof of inducing intracellular Ca level rise.
(3) Intracellular Ca high levels induce luteolysis in the LLC derived from granulosa cells, probably due to causing exocytosis in oxytocin secretory granules; and, as likely, by direct effects upon the cell, including Ca-activated DNA sequencing, which generates apoptosis.
(4) Finally, inhibant substances released in luteal large cells act directly, by reducing steroidogenesis in the small luteal cells. Oxytocin released in the LLC may also be transported to uterus, at a later time causing release of arachidonic acid and/or of PGF2alpha.
PGF2alpha luteolytic action in ruminants implies interaction among the large and the small luteal cells. As the LLC have PGF2alpha receptors, such prostaglandins could represent production start impact site, of substances apt to act upon the LLC for the suppression of (LH mediated) progesterone production, as well as for triggering LLC destruction.
The most striking morphological feature of luteal cells is the storage of large lipid droplets during regression thereof; cells in functional LC accumulate low amounts of cytoplasmic lipids. During luteolysis, the number of electron-dense granules in the steroidogenic large cells decreases, which suggests that PGF2alpha causes release of such granules. Autophagy and intense lysosomal enzymes activity occur with loss of cytoplasmic granules (Braden and col., 1994).
Involvement of oxygen-reactive species (ORS) in luteolysis is important and possibile (Behrman and col., 1993). PGF2alpha induces fast depletion of ascorbic acid stock (a substance potentially highly antioxidant) in LC (Lucy and Zhao, 1993).
Minutes post PGF2alpha administration, and before detection of changes occurred in progesterone production, increasing amounts of O2 manifest within luteal cells membranes. 1 to 2 hours after administration of PGF2alpha, H2O2 production in rat luteal tissue increases (Behrmann and col. 1993). One among the possible H2O2 action mechanisms could be the fall of luteal plasmalemma fluidity, associated with luteal regression, which is partly determined by Ca-dependent phospholipase activity. H2O2 inhibits progesterone synthesis, by interfering COL transport towards mitochondrial inner membranes. H2O2 is a strong inhibitor of protein synthesis in luteal cells.
PGF2alpha produces highly intensified lipid peroxidation of luteal tissue, which occurs at the same time with luteal ascorbic acid depletion and progesterone production stop.
H2O2 low levels block AMPc-dependent LH and P4production over several minutes, concomitantly with fall in ATP cellular levels. Even though the decline caused by H2O2 in LH-sensitive to AMPc storage is a process partly reversible, inhibition of AMPc-dependent steroidogenesis is irreversible. As peroxide promote synthesis of eicosanoids, H2O2 could cause increased local production of PGF2alpha, thereby generating positive feedback on ROS and PGF2alpha.
PGF2alpha-induced regression of bovine corpus luteum was proven possible starting sexual cycle day 5, several synthetic PGF2alpha analogs manifesting 30 times more active than natural PGF. PGF2alpha, administered in case of CL physiological regression, speeds up autolysis and structural changes specific to such process, causing marked level changes of various sexual hormones. Thus, after a short time (under 2 hours) after growth start, serum progesterone level falls continuously in cows administered PGF2alpha, with concomitant increase of 17β-estradiol concentration becoming manifest.
Pregnancy
Pregnancy impacts luteolytic process in important specific ways. Presence of an embryo in ovine uterine cavity was noticed to suppress PGF2alpha release in uterus, starting day 12 post estrum. On the other hand, it was noticed that pregnant ovine corpus luteum acts refractory to exogenous prostaglandins action; also that venous (affluent) blood of same animal’s pregnant uterine horn, manifests a luteotropic effect on ipsilateral corpus luteum. Such conclusions entitle us to conclude that the embryo can play a double role in sustaining corpus luteum integrity: on the one hand, by inhibiting PGF2alpha; on the other hand, by developing a luteotropic principle. Embryonic signals allowing detection of sheep pregnancy are PGE2, PGI2 and, to a larger extent, trophoblastin (proteins 1 of ovine trophoblast-oTP-1, in interferons class – Păunescu and col., 1996). One of the key events is suppression of expression of uterine receptors for oxytocin, which will finally generate suppression of PGF2alpha release (Beard and col., 1994).
Prostaglandins synthesized by the conception products are directly involved in: intrauterine migration of embryo; release of blastocyst in zona pellucida; ion transport across the trophoblast; storage of blastocoel fluid; increased permeability of endometrial capillaries; and carbohydrates metabolism of blastocyst.
Inhibition of PGF synthesis is higher in early pregnancy, than over the cow sexual cycle. As extra exogenous arachidonic acid does not suppress inhibitory activity, it appears that bovine endometrial cytosol inhibits cyclooxigenase, rather than the early stages of arachidonate metabolism. Placental capacity for inactivating prostaglandins correlates with the vasoactive effects thereof, exerted both upon the maternal, and upon the fetal, component. Prostaglandins are deemed to play a specific regulatory role of placental blood irrigation. Degeneration of the decidual cells and frailty of decidual lysosomes, manifest over the last pregnancy weeks, cause increased prostaglandin synthesis, and thereby trigger labour.
Prostaglandins have been detected in fetal placental blood circulation; quite interestingly, prostaglandins concentrations in the fetal placental blood are higher than respective levels in plasma and in the maternal blood serum; also, in preterm spontaneously delivered new born animals, concentrations of prostaglandins F, of plasma and of blood serum, are higher than same type concentrations in new born animals by c-section, post slack, or even absent, labour. Functionally, such prostaglandins can be suspected to play a controlling role, of fetal and placental irrigation. Experiments run on sheep indicate that exogenous PGF2alpha promotes both fetal arterial system blood pressure, and the umbilical blood flow. Quite differently, PGE2 decreases fetal blood irrigation, consecutively to strong vasoconstriction of the fetal placental vascular bed. PGE2 present in ovine fetus plasma, and concentrations of fetal blood thereof, increase over the intrauterine life last phase, accounting for the higher concentration of fetal plasma corticosteroids at such time, at least in sheep fetus. PGF2alpha has no impact on ovine fetal plasma corticosteroids level.
P4 mediates own gene functions by two types nuclear receptors (Okumu and col., 2010), i.e.: P4R isoform A (P4R-A) which is N-terminal (164 amino acids lacking) and P4R isoform B (P4R-B). Both isoforms are regulated by the same genes, yet by distinct promoters. P4R-A mediates progesterone actions at uterus and ovary levels, while P4R-B is more important for mammary gland development.
P4 blocks expression of alpha receptors for estrogens (ESR1) and of receptors for oxytocin (OTR) for around 10 days; post which interval, P4 suppresses expression of receptors for progesterone (P4R) in uterine epithelium, which will permit fast increase of ESR1 and OTR genes expression (see Fig. 1.2).
Subsequently, pulsatile release of OT, from posterior hypophysis and CL, will induce uterine epithelium pulsatile release of PGF2alpha, on days 15 and 16, which will cause functional and structural CL regression, followed by estrus.
The maternal organism acknowledges pregnancy, expecting: (1) IFNT to attenuate ESR1 gene transcription, hence also expression of estradiol-induced OTR in uterine glandular epithelium, in order to suppress development of the endometrial luteolytic mechanism which implies OT-induced PGF2alpha luteolitic pulse; (2) basal PGF and PGE2 production to appear higher in pregnant sheep, as against cyclic females, due to continuous prostaglandin-synthase 2 (PTGS2) expression in uterine epithelium; (3) attenuation exerted by IFNT upon ESR1 expression to block estradiol induced P4R in endometrial epithelium; and (4) P4R in uterine epithelium to vanish, as necessary for the expression of progesterone-induced and interferon-stimulated genes, such genes enhancing embryonic development.
Bovine IFNT secreted between pregnancy days 12 and 38 prevents uterine epithelia pulsatile release of PGF2alpha, counteracting E2 and OT release of PGF2alpha stimulating effects (see Fig. 1.3). Either messenger ARN expression for ESR1 and OTR decreases, or receptors appear non-receptive to E2 and OT, with both pregnant cows and cyclic cows subjected to intrauterine inoculated natural IFNT, or recombinant bovine IFNT.
Fig. 1.2 In ruminants, IFNtau is the pregnancy detection hormone which acts for an attenuated expression of alpha receptor for estrogens (ESR1), and of receptor for oxytocin (OTR), preventing luteolytic mechanism; such mechanism needs the oxytocin in CL and in posterior hypophysis for inducing the PGF2alpha luteolytic pulses. Thus, IFNT abrogates uterus capacity for triggering luteolytic mechanism, yet does not inhibit either prostaglandin synthase 2 (PTGS2), or PGF basal production, over pregnancy time. (Bazer and col., 2008).
IFNT attenuates expression of ESR1, to grant for the organism that estradiol will not stimulat expression of ESR1 in uterine epithelium during pregnancy. Consequently, uterine glandular epithelium will not express FSR1, P4R, IRF9 or STAT1, as IFNT induces expression of IRF2, a strong inhibitor of transcription in such epithelial areas as are in direct contact with embryo trophectoderm (see Fig. 1.4). Consequently, uterine epithelium in direct contact with embryo expresses a unique IFN-stimulated genes type, i.e. the genes for transport of nutrients within uterus, promoting growth and development of the conception product. Uterine glandular epithelium is affected by progesterone, whose action is mediated by P4R-positive uterine stromal cells, which secrete one or several progestamedines.
Effects of IFNT upon the glandular uterine epithelium are mediated by a cellular signaling mechanism, JAK/STAT-independent. Consequently, on the one hand IFNT abrogates the uterine luteolytic mechanism, in order to prevent pulsatile release of PGF, on the other hand amplifying expression of a number of genes basic to uterine receptiveness for embryo implantation and developement (see Fig. 1.3).
Such genes cover MMTV integration site family members, i.e. IFNT-induced 7A (WNT7A), LGALS15 (Galectin 15), CTSL (cathepsin L), CST3 (cystatin C), SLC2A1 (solute carrier family 2 facilitated glucose transporter), SLC7A2 (cationic amino acid transporter), HIF2A (hypoxia-inducible factor A2) and GRP (gastrin releasing peptide) which are progesterone-induced, and later stimulated by IFNT and/or prostaglandins.
Fig. 1.3. Hypothetical role of progesterone (P4), of progestamedines (FGF7, FGF10 and HGF) and of tau interferon (IFNT), in gene expression and secretory functions of ovine uterine lumen and of the superficial glandular epithelium (LE/sGE), which lack both progesterone receptor (PGR), and signal transducer factor, activator of transcription-1 (STAT1). Ovine uterine LE and sGE are PGR- and STAT1- undetectable, which indicates P4 and IFNT to use unusual signaling pathways in order to regulate expression of P4-induced and IFNT-stimulated genes. (Bazer and col., 2008).
Fig. 1.4. P4R expression is attenuated in the uterine epithelium, which is a prerequisite for implantation. Progesterone (P4) acts through the P4R-positive uterine stromal cells, to intensify expression of progestamedines (e.g. FGF7 – fibroblast growth factor7 or FGF10 and HGF – hepatocyte growth factor) in ovine uterus. Similarly to IFNT, progestamedines exert paracrine effects upon the uterine epithelium and the embryonic trophectoderm which express receptors for FGF7 and FGF10 and HGF for stimulating cellular signaling pathways, including phosphatidylinositol kinase 3 kinase (PI3K) and mitogen activated protein kinase (MAPK), in order to stimulate expression of genes and secretory response by trophectoderm and glandular epithelium which not express signal transducer and activator of transcription (STAT1 / STAT2). Thus, IFNT activates indefinite cell signaling pathways which may include PI3K and MAPK, in order to influence expression of genes on glandular epithelium (Bazer, 2013).
Study of Jerome and Sristava (2012) reveals role of gene mutations, as well as fibroblast growth factor 2 (FGF2), signal transducer and activator of transcription 5A (STAT5A), growth hormone (GH), prolactin (PRL), prolactin receptor (PRLR), osteopontin (OPN), uterine milk protein (UTMP) in fertilization and early embryo survival.
Bovine IFN is produced over pregnancy days 16 and 26, having antiluteolytic, antiproliferative, antiviral and immunomodulatory effects. Genome and DNAc screening revealed 12 variants of bovine IFN tau.
Cyclooxygenases 1 and 2 (COCS-1 and COX-2) have a bearing on PGF2alpha synthesis, as also do prostaglandin-synthase (PGFS); whereas low levels IFN tau decreases COX-2. ARNm for COCS-2 is expressed at low levels over cycle days 1 to 12, and lat higher levels over days 13 to 21, peaking on days 16 to 18.
Progesterone exerts two type effects which contribute to PGF2alpha secretion regulation. First effect, acute exposure to progesterone seems to promote uterine storage of arachidonic acid – a precursor of prostaglandins, of peroxidic prostaglandinic synthase and of other substances required for prostaglandins synthesis. Second effect, progesterone exerts an inhibitor effect upon secretion, which effect vanishes post acute exposure. Such two progesterone effects acting concomitantly cause PGF2alpha to be secreted only around luteolysis occurrence time. Also, progesterone suppresses basal prostaglandins secretion in the endometrium (Mitchell and Stevenson, 1995). IFNT is only expressed short-term during embryonic development, e.g. expression in the extra embryonic bovine trophectoderm (Demers and col., 2001). Bovine mRNA detected in embryo in low levels over pregnancy days 10 to 12, increases over days 13 to 15, to peak level on pregnancy day 17. Drastic growth of INFtau gene expression on bovine pregnancy day 15 occurs coincidently with blastocyst morphological change, from globules to filaments, only loosely correlated with pregnancy day.
Debut of IFNtau expression is genetically programmed, independently of uterine maternal medium, as IFNtau is expressed in vitro, post in vitro fertilization and maturation; however, maternal circulating progesterone – which controls uterine glands secretion – correlates with level of IFNtau produced by the bovine embryo. ETS2 growth factor is involved in debut of IFNtau gene expression.
Occurrence – over luteolysis – of the a series of phenomenons related to apoptotic phenotype (Păunescu and col., 1996), respectively DNA oligonucleosomal fragmentation, as well as expression of Ca/magnesium-dependent endonuclease becoming manifest, entitles us to see apoptosis as a possible catalyst of luteolysis. High Ca levels in cell is deemed to be associated with the production of a series of programmed cell death types, probable mechanism being activation of endonuclease, which degrades chromatin, generating multiple nucleosome oligomers.. Among the genes responsible for debut and run of such process stages, we considered genes Bcl2 (Mircu and col., 2015). Bcl2: Bcl-2 (B-cell lymphoma 2), encoded in humans by the Bcl2 gene, is founder member of the Bcl-2 family of regulator proteins involved in cell death (apoptosis), either by inducing (pro-apoptotic) or inhibiting (anti-apoptotic) apoptosis. Bcl-2 protein is stated to be a specifically major anti-apoptotic, thus classified as oncogene. Bcl-2 reads B-cell lymphoma 2, as being the second member of a gamut of proteins initially described in chromosomal translocations involving chromosomes 14 and 18, in follicular lymphoma.
Much research has been run on growth factors in various embryonic developmental stages, as well as on acknowledgement of pregnancy by the maternal organism. The two transcription factors, i.e. CDX2 (caudal related homeobox 2 transcription factor) and OCT4 (octamer-binding transcription factor 4) coexist in initial stages of embryonic development (Kim Min-Su and col., 2013). CDX2 acts as a transcription factor, required for the IFNT gene transcription. IFNT transcription is only possible post formation of CREBBP (CREB binding protein) complex, JUN (transcription factor A1) and ETS (transcription factor de which encoding an oncogene). As blastocyst is repelled, CDX2 expression, as well as formation of complex JUN-CREBBP-ETS2 transcription, causes increased IFNT expression, leading to pregnancy set in. As signaling is distinct based on messages original site, the question arises which, among the embryo cells assemblies, is the first to exert such role, the most efficiently possible. Thus, as comparing cell type effect (embryonic switch – ICM and trophectoderm) to gene expression, ICM was noticed to intensely expresses eight (KDM28, NANOG, SOX2, SPIC, STAT3, ZX3HAV1, OTX2 and IL6R) while in TE, six (DAB2, DSP, GM2A, SCD, SSFA2și VAV3) embryo cell assemblies; while genes specific to ruminants, i.e. IFNT1, PAG2 and TKDP1, appear to be intensely expressed in trophectoderm. Such genes cause embryo presence to be signaled by the maternal organism (Ozawa and col., 2012).
H2O2 inhibits progesterone synthesis by interfering COL transport towards inner mitochondrial membranes. H2O2 is strongly inhibitory of protein synthesis in luteal cells. All type oxidative stress can drastically intervene in growth processes and in embryonic development, impeding pregnancy acknowledgement by maternal organism. Chromosome X inactivation is of paramount importance in the early stages of embryonic development. The major factor responsible for chromosome X inactivation is XIST (X-inactive specific transcript). Genes basic for metabolism are found on chromosome X, such as glucose-6–phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyltranseferase (HPRT). Such genes will also be inactivated, expression thereof being involved in control of free radicals; embryo survival is directly dependent on capacity thereof for sustaining cell homeostasis. G6PD plays a role in ROS detoxification, hence is responsible for cell equilibrium sustainment. For such reason, embryos and fetuses manifesting such transcript deficiencies will be much more vulnerable to oxidative stress. Expression of G6PD, by the embryo, controls production of IFNtau, being associated with intensified transfer of pentose-phosphate cycle (Merighe and col., 2009).
There is a functional relation between expression of cathepsin in cumulus cells and oocytes competency in cows, suggesting that expression thereof may foretell developmental potential of embryo originating in respective oocyte. Around 12% of the proteins are shared by oocyte and cumulus cells. Proteins have been found that represent 338 transcription factors and 241 receptor-ligand mechanisms present both in cumulus cells and in oocyte, among the number 18 growth factors patterns. In such terms, ovarian and cumulus cells may represent oocyte development competency indices (Schilling and Smith, 2011).
Several genes have been found that are associated with the time of first cell division and development competency (Peelman, 2011). Comparisons between mRNA contents of bovine embryos which have undergone first cell division more or less slowly or fast, have also revealed differences between expression of a series of genes, such as: histone 3 (H3A), pre-implantation embryo development (Ped), HPRT, G6PD, IGF-I and IGF-IR, GLUT-5, sarcosine (SOX), Mn- superoxide dismutase (MnSOD), Cx43, IFNtau, IGF-II, BAX, histone 2 (H2A), isocitrate dehydrogenase (IDH), YY1 and E4TF1 (YEAF1) – associated factor. E.g. Ped allele, originated in the faster developing embryo, may reach three times higher transcription speed in two-cell embryo phase, as against embryos developing at a slower pace. The widest variance has been noticed starting 16-cell stage. H3A proved more abundant in embryos which had divided earlier. Such aspects indicate that embryos need to store enough histone mRNA for normal development, while abundence could act as development competency marker.
Oxygen radicals are necessary for normal embryo development. Regulation of free radicals production amount is also interfered, among others, by two genes on chromosome Y, glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine phosphoribosyl transferase (HPRT), such genes also playing a major role in energy metabolism. There are differences in expression of both genes, between female and male blastocysts, a correlation with manifest division rates. For HPRT, it was proven that one polyadenylate tail (poly-A) correlates with a low development capacity.
Genes (such as SOX, MnSOD, BAX, IFNtau and G6PD) whose activation is supposed to be stress-induced, are found expressed at higher rates in slow development and in vitro generated embryos, as against fast developing embryos. Contrariwise, genes (such as GLUT-5, Cx43, IGF-II and IGF-IR) involved in metabolism, growth and differentiation, manifest at higher mRNA levels, in fast development embryos. Both patterns may express embryo health state.
P4 level variation over days 8 to 10 post IA, by much more than 1.5 ng/ml, may indicate luteolysis being triggered and pregnancy loss (Mircu and col., 1998).
Coordinated actions of P4, prostaglandins and IFNT regulate uterus receptiveness for embryo implantation, by controlling endometrial genes expression, while actions thereof are basic to embryo elongation (Dorniak and Spencer, 2013). Blastocyst growth to elongated embryo stage does not occur in vitro, as depending on ovarian progesterone, as well as on endometrial secretions. Acting as a pregnancy detection signal, IFNT causes continuous P4 production by the CL. Moreover, IFNT stimulates transcription of a number of genes, and of a few enzymes involved in determining uterine receptiveness, implantation and embryonic development. Administration of extra P4 cannot save inherently genetically marred embryos, or embryos of cows producing very much milk. P4 induces expression of many genes involved in elongation and in embryo implantation.
IFNT stimulates expression of many genes involved in elongation and embryo implantation, expression initially induced by P4 (CST3, CST6, CTSL, GRP, HSD11B1, IGFBP1, LGALS15, SLC2A1, SLC2A5, SLC5A11, SLC7A2) specifically at endometrium level.
PTGS2 is cyclooxygenase predominantly expressed in both endometrium and the embryonic trophectoderm. IFNT acts as a molecular switch which stimulates type E2 prostaglandin production in endometrium. Determination of PTGS2 expression in biopsies run on day 7 blastocysts is an index for blastocyst development and for pregnancy development, safe and on schedule (Dorniak and Spencer, 2013).
IGF1 and IGF2 impact IFNtau production by embryo, as soon as blastocyst expands and little time before zona pellucida breaks and eclosion? enclosion? occurs.
Interferon detectable at early (7 or 8 days) stage indicates embryonic development state, yet interferon amount produced has no bearing on embryo viability (Neira and col., 2005). Close to the sensitive time when embryo fate is decided, there occurs a joint action of intrinsic embryonic factors, as well as of the factors pertaining to the maternal organism. Among the growth factors, Oct-4 is strongly expressed in embryonic switch cells, such expression being detectible in trophectoderm on pregnancy day 10, some three days post blastocyst formation. IFNtau is first detectible as early as blastocyst formation time, while peak production thereof will only occur several days later, i.e. when Oct-4 is completely repressed (Toshihiko Ezashi and col., 2001).
Oct-4 is a strong inhibitor of IFNtau promoters, a phenomenon manifest by transcriptional coupling of promoters and by transcriptional interference. Oct-4 contributes to cells sustainment in an undifferentiated, pluripotent or totipotent stage, in two ways, i.e. by the activation of a number of specific genes, and by inhibition of others.
RESEARCH THEORY AND EXPERIMENT
Oxytocin is a major CL secretory product. Besides the role played in luteolysis, oxytocin is also involved in muscular activity regulation of the genital tractus during estrus, as well as in modulation of ovarian steroidogenesis. Both synthesis and secretion of oxytocin occur by a cyclic mechanism, detectable at low levels in granulosa cells in preovulatory follicles. Consecutively to ovulation, oxytocin levels and levels of correspondent mRNA, increase inside CL, with mRNA levels peaking on day 3, whereas oxytocin contents peaks on day 6. Further on, oxytocin and mRNA levels fall, to relatively low, before luteolysis debut. In absence of fertilization, luteal concentrations of such hormone decrease, long before progesterone level does. Such production pattern stands in contrast to hypothalamic pattern, where no cyclic variation occurs. As per Wathes and col. (1992) ovarian capacity to synthesize oxytocin varies drastically with each sexual cycle phase, rising and falling directly with the changes in progesterone concentrations, and touching at a minimum during estrus.
Oxytocin capacity for positive feed-back to PGF2alpha release, causing down-regulation of own uterine receptors, prompts the concept that oxytocin may fine-tune the PGF2alpha pulsations, so as luteolysis may be efficiently triggered (Jenkin, 1993). Oxytocin and PGF2alpha act interconditional, and mutually controlling of one another.
PGF2alpha level in uterine vein on day 18 post estrus is markedly lower with pregnant cows, as against the non-gestant. Oxytocin-stimulated PGF2alpha secretion is low in pregnant females, starting day 18 post estrus (Parkinson and col., 1990). In pregnant females, CL resistance to PGF2alpha luteolytic action increases over days 10 to 13 post-estrus, to gradually vanish on days 16 and 26 post-estrus.
Endometrial oxytocin receptors play a major role in selecting between luteolysis and pregnancy, in ruminants (Flint and col.- 1992, Wathes and col. -1992). Receptors expression of determines time at which luteolysis occurs, in non-gestant females, thereby determining sexual cycle duration. Trophoblastic interferon (IFNtau)-induced inhibition of receptors expression blocks luteal regression, causing progesterone secretion, and conception product development, to continue. bTP-1 may be required as early as normal luteolysis time, and all along the cycle, until luteal oxytocin levels rise up to switching stimulator mechanism to episodic PGF2alpha release, required by luteolysis.
Antisteroidogenic effect of PGF2alpha may be mediated by the PKC second messenger system. PGF2alpha activates phospholipase C, which causes hydrolysis of membrane phosphatidylinositol (PIP2) into inositol-1,4,5-trisphosphate (IP3) and 1,2-dyacilglycerol (DAG). PKC activity depends on membrane phospholipids, as regulated by free Ca, as well as by DAG, concentration.
DAG enhances PKC affinity for Ca, while IP3 releases Ca in intracellular deposits; higher intracellular free Ca concentrations result, as well as activation of PKC. PKC acute antisteroidogenic effect may be exerted by inhibiting transport COL to cytochrome P450scc, rather than by mRNA low level for cytochrome P450scc. PKC activation may have long term effects upon steroidogenic enzymes. PGF2alpha was noted to decrease by 80% the mRNA basal levels for 3β HSD. During bovine CL regression, decline of plasma progesterone precedes changes in mRNA levels for steroidogenic (side-chain cleavage of cholesterol and/or 3β-HSD) enzymes. Second messenger system involved in mediating PGF2alpha luteolytic effect is intracellular free Ca. Rise of PGF2alpha-induced intracellular free Ca levels can no longer be balanced by the large luteal cells, even though P4 blocks, by P4 receptors, redistribution and rise of intracellular Ca.
Luteal cells exposure to PGF may induce homologous desensitization of the PGF-stimulated inositol phospholipid-phospholipase C signaling system in luteal cells; which points to existence of a series of multiple mechanisms modulating PGF2alpha and LH effects on the inositol phospholipid-phospholipase C. Other arachidonic acid metabolism products (e.g. 5-hydroxyeicosatetraenoic acid -HETE- in lipoxygenase pathway) also impact steroidogenesis at luteal cell level. The gamut of arachidonic acid metabolites may well exert a basic role in regulation of progesterone secretion.
Study of Poehland and col. (1996) reveals that apoptosis may play a role in ovarian cyclic phenomenons; thus, the number of apoptotic cells increases, early phase to middle, to further drop towards luteal end phase (58.1% vs. 77.1% respectively 40%). Murdoch (1995) reveals marked rise of heat shock protein -70 (HSP -70) level, post PGF2alpha administration, suggesting the possibility that HSP-70 induction by PGF2alpha may precede steroidic depletion, and activate apoptotic death of luteal cells.
Early diagnosis of pregnancy in the cow, by PGF2alpha
Researching oxytocin vs. PGF2 interrelation in luteolysis process produced a surprising theory to a novel practical application, i.e. administration of a PGF2alpha sub-luteolytic dose for early diagnosis of pregnancy, on day 20 post artificial insemination (Labussiere – 1990).
As per quoted author, a PGF2alpha sub-luteolytic dose administered in abdominal subcutaneous cranial vein causes release of oxytocin in corpus Iuteus (consequently, milk ejection), in presence of pregnancy at such time, revealed by a functional corpus luteum (CL).
Theory and practice premises of the method rest on functional processes, as further detailed:
bovine large luteal cells containing secretory granules with oxytocin;
luteal oxytocin releasable by PGF2alpha;
beta large luteal cells sensitive to PGF2alpha sub-luteolytic doses, regressing the first;
alpha luteal cells regressing when PGF2alpha levels are high, an aspect manifested by all large luteal cells regressed before day 19;
post day 19, all of such cells having regressed, no more secretory granules exist to release oxytocin.
As per study of Wathes and colab. (1992), in absence of pregnancy, oxytocin level falls before drop of progesterone level, hence major fall of oxytocin level precedes luteolysis debut. Later oxytocin release, over luteal regression, decreases the luteal oxytocin reserves.
Flint and col., 1992, admit that luteal oxytocin stimulates PGF2alpha uterine secretion, responsible for luteolysis. PGF2alpha also stimulates oxytocin secretion in corpus luteum. As PGF2alpha secretion pulsation occurs at the same time as oxytocin pulsations, during luteolysis, the hypothesis was advanced that a positive feed-back loop may tie luteal oxytocin and uterine PGF2alpha (Flint and col. 1992, Labussiere -1990).
Acting at the level of the newly formed specific endometrial receptors, presence of luteal oxytocin is a prerequisite for luteolitic effect of PGF2alpha pulsatile release. Time when such receptors development is final depends on the ovarian steroids involved in regulation of the binding sites, as well as on the very binding process. Presence of an embryo suppresses uterine expression of receptors for oxytocin, which implicitly inhibits PGF2alpha release.
Pregnancy set-in depends, with cows, on a fine equilibrium of start of maternal luteolytic mechanism, and trophoblastic antiluteolytic interferon production by the conception product (Mann and Lamming, 1995). Failure of such equilibrium will generate embryonic death, which continues to be a major reproductive disorder, manifested by 28% dairy cows (Lamming and Mann, 1995).
Progesterone level, in plasma or milk, accurately indicating the sexual cycle phase, allows for reading such steroid concentration as a marker of pregnancy presence, or absence.
Experiment I. 180 cows (lot 1) were subjected to a protocol as follows: pregnancy diagnosis by administration of one PGF2alpha subluteolytic dose, on day 20 post artificial insemination (as per Labussiere – 1990); collection of 10 ml milk samples (per 1.5 mg potassium bichromate, for storage) batch on days 20, respectively 24, post insemination; and transrectal examination on days 60, respectively 90, post insemination.
Experiment II. 90 cows (lots 2, 3 and 4, 30 cows each) were subjected to same protocol (as described for experiment I), where there only differ the pregnancy diagnosis (by PGF2alpha) day, and the day when first milk samples were collected, respectively day 19 post insemination for lot 2, day 20 for lot 3 and day 21 for lot 4. Such procedure is based on taking interval 19 to 21 days post insemination to be proximal to potential luteolysis time, where pregnancy is found absent.
Day 24 was set for collecting the second milk samples batch, as several authors take such day to be statistically the best for pregnancy early diagnosis (Beate Holtschlag-Aple -1989, Miqeu -1988, Simon Edda -1989).
Milk samples were refrigerated (+4°C). Determination of progesterone in milk was run by technique ELISA, and Ovucheck kits (Cambridge Ltd., UK). Transrectal examination, ascertaining presence of pregnancy, was run on days 60, respectively 90, post artificial insemination, checking against results for pregnancy early phase, i.e. days 19 to 24.
The test run implied as further detailed: post the morning milking and thorough antiseptic udder wash, sterile mammary probe was introduced into the rear right teat. Last milk droplets were rubbed off the mammary cistern, by light massage. 250 microgram dose cloprostenol (1 ml Flavoliz) was injected into the subcutaneous cranial vein. For the 3 minutes the intravenous administration of the PGF2alpha subluteolytic dose took, mammary probe was held within papillary duct.
The over 20 second continuous jet milk ejection was taken to ascertain presence of a functional corpus Iuteus, sample reading positive for pregnancy diagnosis. Absence of milk ejection in quoted time delay, or presence of a number of de milk droplets, or of discontinuous jets, were read as negative results.
Experiment I (lot 1) on day 20 post insemination (D20) as testing by Flavoliz (further indicated as TPGF): 158 pregnant females (87.7%) and 22 non-gestant females were diagnosed, out of the 180 cows examined. The same day, D20, progesterone level in milk allowed for diagnosing pregnancy present in 136 females (75.5%); while on D24, 129 pregnancies (71.1%) were diagnosed, still based on progesterone level
Consistency of diagnosis set on day 20, by TPGF, and diagnosis based on progesterone level on D20, was 86.07%; while consistency of TPGF and results yielded by dosage progesterone in milk, on D24, was 81.08%.
For such test, Labussiere (1990) got positive results accuracy to be 72.2%, higher than accuracy resulted by progesterone dosage by RIA technique, i.e. 68.8%. Negative diagnoses accuracy is 93.2%, by such method detection of non-gestant females being on the spot.
Experiment I yielded 100% accuracy for the negative diagnoses, i.e. all of the diagnoses indicated absence of pregnancy, as subsequently ascertained by all of the other methods.
In veterinary practice, routine pregnancy diagnosis is determined by transrectal examination, run 60 days post artificial insemination. In lot 1, at 60 days post artificial insemination time, 142 pregnant females were diagnosed, while at 90 days post insemination, 132 of the females were, i.e. 73.3%, which ascertains conclusion in study of Laing (1976), i.e. positive results (yielded based on level of milk progesterone) on day 21, or day 24, post artificial insemination, grant only 75 to 80% diagnosis accuracy, due to early embryonic death set-in.
Experiment I records consistency of positive results determined on D20 by TPGF, and results by ETR, as being 89.8% for day 60, and 88.54% for day 90.
Experiment II run on lots 2, 3, and 4, of 30 females each, watched for chance differences which might show with TPGF run on days 19, 20 or 21 post insemination.
Thus, based on TPGF run on day 19 post artificial insemination (D19), 22 (73.3%) pregnant females were diagnosed; while by level analysis of milk progesterone, on same day, 18 (60%) pregnant females were detected. Consistency of such methods (for day 19) was 81.8%, while consistency of TPGF run on DI9 and progesterone level on D24, was found to be 95.65%.
Consistency of results yielded by TPGF and results of transrectal examination, run 60 days post artificial insemination, was 81.84%; while, with transrectal examination run 90 days post insemination, results indicated 84.61% consistency.
Comparing results of progesterone level determination on day 24, and results of transrectal examination on day 60, consistency was found to be 85.18%.
Simon Edda (1988) determined consistency to be 78.1%, of results yielded by monitoring progesterone on day 19, and results of transrectal examination run 45 days post insemination, case in which 5 ng/ml milk is the down limit admitted for progesterone (in order to ascertain pregnancy), consistency being 84%, if progesterone threshold was set to be 15 ng/ml.
For lot 3, with TPGF 20 days post insemination, 27 positive diagnoses (90%) were set, 25 (83.3%) pregnant females being detected by dosage of progesterone in milk, on same day. Rate of females diagnosed pregnant by dosage of progesterone level in milk, on day 24, is the same (i.e. 83.3%)
Consistency of results yielded by TPGF on day 20, and results revealed by dosage of progesterone on days 20 or 24, was found to be 92.59%.
Transrectal examination run on day 60 post insemination detected 24 pregnant females (80%), consistency with result by TPGF being 88.8%; while results of transrectal examination run on day 90 detected 23 pregnancies (76.6%), consistency thereof being recorded to be 85.18%.
Beate Holtschlag-Apel (1989), indicate consistency to be around 85% (in-between 70 and 97%) for positive diagnoses, and 95% (90 to 100%) for negative diagnoses, as comparing results yielded by pregnancy diagnosis based on level of progesterone in milk, on day 20, and results of the transrectal examination run on day 45.
17 pregnancies (56.6%), on day 21, were found by quoted test, and 23 de pregnancies (76.6%), based on level progesterone in milk, consistency of such being 73.91%, clearly inferior to consistency figures for lots 2 and 3, in same context (81.81%, respectively 92.59%).
Low consistency (65.38%) was found, of results yielded by TPGF and by progesterone level on day 24; as well as of TPGF and diagnoses determined by transrectal examination on day 60, or on day 90 (68%).
Simon Edda’s study (1988), claims 73.3% consistency of results yielded by determining level of progesterone in milk, on day 21, and results of transrectal examination run at 45 days post insemination time, with the down limit below 5 ng/ml milk, while for 15 ng/ml progesterone threshold, consistency was found to be 74%.
Consecutive to running Van der Waerden rank statistic test, marked differences were noted, between results revealed by running PGF2alpha test, 20 days post insemination, and results yielded on both day 19, and day 21. Differences are slight, as comparing results yielded in the three lots, regarding both determination of progesterone level, 24 days post artificial insemination, and transrectal examination results, run 60 days post insemination, respectively 90.
Conclusions
Pregnancy diagnosis by TPGF run on day 20 post artificial insemination is a safe method, allowing determination of 87.7 to 90% of the pregnant females.
Accuracy is maximum (100%) for the negative diagnoses.
Consistency is 89.3%, of such test, and transrectal examination run on day 60.
Test accuracy is conditional on such test being run on day 20, when luteal receptors for PGF2alpha appear to manifest optimal concentration.
Debut of luteal regression is affected by the drastic decline of P4 serum concentration, followed by CL weight loss. Morphological changes over luteal regression time cover accumulations of lipid droplets in cytoplasm of luteal cells; degeneration of capillaries; and increased primary lysosomes. During CL regression, number of steroidogenic luteal cells may fall; also, number of receptors for PGF2alpha may increase (Lamming and Mann, 1995). Number of small luteal cells and of endothelial cells is noted to fall, during induced luteolysis, over first 24 to 36 hours interval, before any change in large luteal cells number, or in fibroblasts. Effects of prostaglandins upon the CL are mediated by the large luteal cells. The binding sites with high affinity for PGE2 and PGF2alpha are initially located on the large luteal cells, as PGE2 stimulates progesterone production via an APMc independent mechanism, at large luteal cells level, yet not of the small luteal cells.
Wiltbank and col. (1991) noted that PGF2alpha decreases progesterone production amount in large luteal cells in presence of high density lipoprotein (HDL) in medium. Authors advance the hypothesis that PGF2alpha luteolytic effect might be due (at least partly) to inhibition of lipoproteins-stimulated steroidogenesis. CL progesterone secretion post PGF2alpha administration, falls drastically (in 7.5 hours) as against the drop in the number of receptors for LH, occupied or not (in 22.5 hours).
Monitoring reproductive function in cows by milk progesterone level
HEAP and col.(1989) focus on specific pregnancy characteristics of corpus Iuteum (C.L.); asserting that, over early pregnancy phase, Iuteal progesterone synthesis increases by stages, continuing beyond day 25 post insemination, thereby leading to elevated progesterone over days 10 to 16 interval, as against during similar sexual cycle phase. Day 8 may mark end of a critical P4 activity time delay, which is a prerequisite for PGF2 alpha to be released. Days 9 and 10 of P4 level fluctuations, in inseminated non-gestant cows, may indicate luteolysis debut, later causing embryonic death, a phenomenon endorsed by study of Thibodeaux and col. (1994) who state that a marked rise is obvious, of plasma and milk P4 levels, in pregnant, as against non-gestant, cows, starting day 8 post estrus.
Blood and milk progesterone levels accurately reflect sexual cycle phase, based on which P4 level acts as an accurate index for diagnosing presence of pregnancy. In bovine, over the first pre-ovulation days and the three days post ovulation, P4 level drops below 1ng / ml in plasma, respectively 2ng/ml in milk (Boitor – 1979, Miqueu -1988). Starting day 4, P4 level rises, peaking on day 5, when concentrations touch at 6 to 7 ng/ml in plasma, and 15 ng/ml in milk, or even more. Checked against constant rise uring cycle debut, P4 level fall starting some 5 days before ovulation appears much faster. As per research data in Bulman’s study (1978), P4 levels post artificial insemination (AI), whether successful or failed, were found to be similar to data recorded 21 days before and 13 days post insemination, i.e. falling in pregnant cows, and rising up to day 22, in non-gestant cows.
Results published by Lamming and Bulman (1976), on correlation between progesterone level around end of luteal phase time, and pregnancy, indicate that a low plasma P4 level will trigger a strong luteolytic mechanism, thereby possibly predisposing cows for early embryonic loss. Inseminated cows show past luteolysis lower blood and milk P4 levels, than cows sustaining pregnancy.
Such experiment considered 25 BNR cows, milk samples being collected on insemination day, as well as on days 8, 9, 10, 16, 20, 21 and 24 post AI. The morning milking samples (potassium bichromate added), were refrigerated (+2 to 8 C) and stored up to processing time.
Determination of milk P4 level was run by ELISA technique, and kits OVUCHECK (Cambridge Ltd., UK). Statistic processing was based on statistics rank Van der Waerden test. Pregnancy diagnosis by transrectal examination (TRE) was run 60 days post AI.
The kit test ascertained presence of estrus, at milk P4 level below 3 ng/ml. Pregnancy is present when milk P4 goes beyond 10 ng/ml, as per same kit standards. Out of the 25 cows considered: post first AI, 17 (68%) were found to be pregnant, as diagnosed by ETR 60 days later. Based on P4 level on day 21, 16 cows (64%) would have been found pregnant, i.e. 94.11% consistency of the two pregnancy diagnosis methods.
In the non-gestant cows lot, 8 of the females (32%) were diagnosed on day 60, by ETR; based on P4 level on day 21, the females found to be non-gestant were 9 (36%), hence an 88.8% consistency of the two data.
Results based on progesterone level on day 24 post AI were proven identical with results yielded by the ETR method, run on day 60.
Variation analysis of P4 level at AI time indicates optimal run time, except for cow no.8832, progesterone level thereof being below 3 ng/ml milk. Adequate fertilization basically requires a P4 low level at AI time (Foulkes and col., 1982), a condition of female genital apparatus strongly impacted by estrogens, which satisfactory boost transport of spermatozoa. Average P4 at AI time is higher than value indicated by McCaughey and Cooper (1980), who record average value to be 0.8 ng /ml defatted milk; yet close to value indicated by Abdel Rahim and col. (1980) i.e. 1.4 ± 0.28 ng/ml milk.
P4 levels, on days 8 to 10, certify presence of Iuteal tissue. The differences recorded between P4 levels on days 9 and 10 are slight (i.e.0.23 ng/ml), an aspect subsequently proven consistent in presence of pregnancy. Such phenomenon was noted by Heap and col. (1989), which claim that day 8 marks the end of a critical period in progesterone activity, decisive for pregnancy sustainment, or else triggering luteolysis.
P4 level 21 days post AI led to pregnancy diagnosis in 15 cows, 88.2% consistency resulting, with ETR. P4 level 24 days post AI indicated 16 pregnancies set-in, and 94.11% consistency with ETR run on day 60. In all of the cases considered, progesterone level rose in the interval 10 to 16 days post AI, a phenomenon correlated by Heap and col. (1989) with presence of pregnancy.
In cows diagnosed as non-gestant by ETR run on day 60, data were found as further detailed: as early as debut, the average progesterone value (3.88 ±2,16 ng/ml milk) at AI time, was clearly above limit, which ascertains absence of Iuteal tissue (3 ng/ml), which, besides other factors, contributes to failure of pregnancy set-in.
There are higher than 1.00 ng/ml differences (i.e. 1.96±0.93 ng/ml) between P4 levels on day 9 and day 10, same aspect appearing evident when considering levels on day 8 and on day 10; such are important statistic differences.
Except for 2 (25%) in 8 cases, P4 level down trend is obvious, over day 10 to day 16 interval, post IA.
Both P4 levels on day 21 and the on day 24 indicate absence of pregnancy, in consistency with results yielded by ETR run 60 de days post AI. Cow no. 92110A is special, as, based on P4 level chart, we would expect embryonic death. A number of cows manifested new estrus post day 42, suggesting a pathological corpus Iuteum which subsequently underwent luteolysis. P4 level on day 21 clearly indicated no pregnancy; moreover, no clinic specific manifestations of estrus were there, either.
Data analysis reveals a number of aspects as further detailed: on AI day, marked differences are manifest between average P4 levels in cows expected to enter pregnancy, as against in the non-gestant; such differences continue wide, on days 16, 20, 21 and 24, post AI.
Pregnancy diagnosis based on milk P4 level on days 21 and 24 post AI indicates that there are no marked differences between average values thereof; in such terms, diagnoses on days 21 and 24 post AI are proven equally efficient.
Conclusions
Under 3 ng/ml progesterone level at AI time is basic for pregnancy set- in.
Milk progesterone level variation, over days 8 to 10 post AI, ampler than 1.5 ng/ml may indicate luteolysis being triggered, hence absent pregnancy.
Progesterone levels of pregnant and of non-gestant cows, on days 16, 21 and 24 post AI, differ widely.
There is a positive correlation of progesterone levels over luteal phase, and pregnancy rates.
Consistency of results yielded by transrectal examination run on day 60, and milk progesterone levels on days 21 to 24 post AI, confer the latter of the diagnosis methods practical applicability.
DEVELOPMENT TRENDS ANALYSIS
P4 is a key hormone which regulates embryonic development in ruminants. Elevated progesterone immediately before ovulation markedly alter mechanism for endometrial genes expression, by quickening pace of changes normally occurring in time, as well as and by intensifying blastocyst elongation. Such effect results from preparation of endometrium for embryo binding, which alters histotroph composition, rather than directly affecting the blastocyst.
Pregnancy set-in involves snow-balling of basic phenomenons such as maternal organs secretions, determining uterine receptivity for implantation, and adequate endometrial response to IFNT production. Transcriptomic analysis of the endometrium indicated that modulation of circulatory P4 level alters endometrial expression of genes likely to contribute to composition of histotroph, an aspect beneficial to embryo development when extra P4 is produced, or detrimental if P4 level is low (Forde and Lonergan, 2012). Inhibitory progesterone receptors – required for determining uterine receptivity – are affected in females with P4 altered profile endometrium. IFNT induces expression of a large number of ISGs genes (classical Type I IFN-stimulated genes) starting pregnancy day 15. Comparative analysis of gene expression on day 18, of pregnant females and of non-gestant, revealed 87 distinctly expressed genes, close to half of which ISGs: OASI (2`5`-oligoadenylate synthetase? I), ISG15 (ubiquitin-like modifier ISG15), CXCL5 chemokine (C- X-C motif) ligand 5, and such like.
Transcriptomic changes, developing since estrus time over luteal phase interval, succeed similarly, weather fertilization was successful or failed. Starting day 16, differences in gene expression (in 27 genes) may occur, as part of the early endometrium response to presence of embryos; which indicates that, during the peri-implantation period, induction of an IFN type response is the major effect of embryos on endometrium.
With circulating progesterone normal level, starting day 7 (when embryo is enclosed by zona pellucida) up to day 13 (when enclosed blastocyst starts elongating) numerous genes contributing to histotroph production undergo temporal modulation. Such modulation is correlated with presence (on day 7) or absence (on day 13) of endometrial nuclear progesterone receptors.
Elevated progesterone does not affect embryo capacity for reaching early blastocyst stage in vivo, yet induces subtle changes in embryo transcriptome, possibly an associated elongation phenomenon going on post enclosion by zona pellucida (Carter and col., 2010). Progesterone-induced changes in uterine medium may interfere with transcriptome of blastocysts. Early embryo is somewhat autonomous during first week of life, and, up to a point, contact thereof with maternal reproductive tract is no longer required. Temporal changes in the endometrium are similar in pregnant females and in the non-gestant, up to the time when luteolysis normally occurs.
In both pregnant females and the non-gestant, similar gene expression changes in endometrum occur, up to debut time of blastocyst elongation, suggesting that the organism is programmed to prepare for hosting pregnancy. Only starting day 16, when maternal organism acknowledges presence of pregnancy, are there notable profile transcriptome changes, in both pregnant and the non-gestant females. Low progesterone level will generate a uterine medium below physiological requirements (in terms of amino acids, ions, and energy reserves), which will manifest low capacity for enhancing blastocyst elongation (Forde and col., 2011).
Success of implantation is granted by the several basic stages which cover uterine medium adjustment, so as to boost embryo development; as well as drastic remodelling of the endometrial structure, as required by the apposition, adherence and invasion processes thereof.
Study run by Mansouri-Attia and col. in 2009, targeted determination of factors in the carruncular (C), as well as in the intercarruncular (IC) areas, enhancing bovine embryo implantation. By? 13,257-oligonucleotids microarray, on day 20 post insemination, 446, respectively 1295 differentially expressed genes were found, in areas C and IC. Based on primary cultures of bovine endometrial cells, target genes were determined, for IFNT (PTN, PLAC8, CXCL2) and IFNT-regulated genes (MSX1 and CXCR7). Transcriptomic data provided by such study generated new molecular patterns for biological functions related to areas C and IC, apt to contribute to determining potential biomarkers for normal and abnormal early pregnancy. (see Fig. 1.5).
Early embryonic development is regulated by maternal transcripts and proteins embodied in oocytes, during oogenesis. Developing maternal origin transcripts and proteins will degrade, as embryonic genome activation (EGA) is initiated. Development switch from maternal genes to embryonic production is known as maternal-to-embryonic transition (MET). EGA acts in multiple waves, based on specific species; yet, in bovine, such process occurs in the 8 to16 cell embryo. Graf and col. (2014) considered the RNA sequence, stating that most of the activated genes are found in the 8 cell embryo. Respective genes are involved in chromatin structure, in transcription, RNA processing, proteins biosynthesis, transmission signals, cells attachment and in sustainment of pluripotency.
Fig. 1.5. Cyclic, or pregnant, endometrium is parted into C and IC areas.
Each DEG (differentiatedly expressed gene) is represented by a rounded rectangle, located in the compartment where respective biological action is exerted. Position of each DEG results by compiling data relative to transcriptomic comparative analysis of estrous cycle vs. pregnancy, and C vs. IC. Each EDG was initially located based on gene expression difference between estrous cycle and pregnancy; we later also considered location in C or IC. When no statistic differences between C and IC were found, DEG was located along the interface line of the two endometrial areas.
We deem it useful to determine the genes for OT, PGF2alpha and E2 active in various phases of CL functional life, as well as expression level of specific genes in correlation with presence of pregnancy and embryonic death/ inactivated own genome, and such like.
Also, an investigation would be most welcome, of factors impacting embryonic genome activation, relative to the genes responsible for the production of receptors for OT, PGF2alpha and E2.
Chapter 2. IMMUNOCONTRACEPTION BASED ON ZONA PELLUCIDA
CONCEPT ANALYSIS AND EVOLUTION
Zona pellucida. General.
Several pathways were explored in order to prevent pregnancy set-in; however, reversible interference of pharmacological agents into oocyte-spermatozoon interactions is still a target hard to meet. These 20 years, animal experimental models yielded ever more evidence that immunological agents directed against mammalian gametes may efficiently and irreversible inhibit fertilization. Progress also came with the novel breakthrough in the field of gamete binding specific macromolecules involved in fertilization; such surface cell molecules manifest as promising targets for satisfactory development of immunocontraception (IMMC).
Major problems, such as side effects, lack of adequate difusion systems within organism, as well as incomplete reversibility, manifest when using immunological agents in prevention of fertilization.
Zona pellucida (ZP) plays a basic role in fertilization, as well as in preimplantation embryogenesis. ZP glycoproteins mediate fertilization basic stages, in mammals, as further detailed:
1.) start adhecion, next tight binding of spermatozoon (SPERM CELL) and ZP;
2.) ZP induced acrosome reaction;
3.) ZP block to polyspermy.
ZP manifests as a thick acellular viscous glycoprotein layer, in-between developing oocyte surface and granulosa cells; components thereof are polysaccharides and mucopolysaccharides, as well as proteins and glycoproteins (Dunbar and Raynor 1980, Wassarman –1988, Wassarman -1998).
In a number of species, ZP is made of several cell layers, manifesting various chemical composition. The number of microvilli and of follicular cells in ZP structure increases as oocyte and granulosa cells differentiate, surface expanding as oocyte develops.
Deep ZP structure manifests as a fibrous mesh of numerous pores, canaliculi which, interconnected by fibers, act as ducts along which chemicals in the granulosa cross ZP and interact with surface vitelline.
Spermatozoon binding
Binding of sperm cell head to ZP appears to be regulated by receptors on ZP surface. Treating oocyte with antibodies anti-zone or with proteolytic enzyme-trypsin, blocks sperm cells binding to ZP.
Binding can be also inhibited by prior treating of sperm with antisperm antibodies, or with glycoproteins extracted from ZP antibodies; sperm cells or ZP block or masque receptors on surface of sperm cell, or on ZP surface.
Specific sperm cell receptor to ZP was found to be one among the three major glycoproteins which form ZP, an extracellular matrix. The three glycoproteins, found and accepted by several authors to be ZP1, ZP2 and ZP3, are synthesized by the maturing oocyte; even though there may appear species-based structure variations of such proteins, such glycoproteins appear to be present in all of the mammals.
ZP3 acts a sperm receptor, which only binds to acrosome-intact spermatozoa. Binding will be by O-linked oligosaccharide, to terminus ZP3. Pig ZP1 acts as second receptor for binding to sperm cell, by sulphate sugar chains (Tsubamoto and col., 1996).
Presence of glycosyltransferase, of protease and of the glycosidases on plasma membranes covering the spermatozoon, cause hinge binding to ZP3, similar to enzyme binding to sublayer.
Acrosome reaction
Sperm cell penetrates ZP in 5 to 15 minutes post attachment. Acrosome reaction (ASR) may occur before or post binding of sperm cell head to ZP glycoproteins; yet, in order for the attachment to be possible, presence of an intact acrosome is mandatory. Sperm cell head binding to ZP3 allows for interaction with other zona pellucida components, which stimulates acrosome activation. Acrosome reaction allows for release of lysine, sperm cell thus cutting a path to ZP vitelline membranes.
As acrosome-activated sperm cell triggers ZP penetration, glycoprotein ZP2 acts second receptor, engaging sperm cell adhesion, during binding to ZP.
Polyspermy block
With most of the species (e.g.ovine, swine, and carnivorous), block to polyspermy occurs at ZP level, a number of species (e.g. rabbits) sustaining the process physiologically at vitelline membranes level.
Block to polyspermy is triggered when spermatozoon penetrates the ovum, hence cortical granules are released within perivitelline space. Contents of such granules release will generate extensive reconfiguration, of ZP and/or of vitelline surface, a phenomenon known as cortical reaction. Such reaction involves release of a series of enzymes, which cause ZP surface to harden, as well as inactivation of sperm receptors (ZP3). Enzymatic digestion, consecutive to terminus ZP3O-linked oligosaccharides, removes specific carbohydrates involved in binding. ZP2 proteolysis may cause modifications of ZP physical characters, in order to prevent spermatozoa penetration, at a later time.
DEFINITION OF TOPIC AND CONTENTS
Immunogenicity and antigenicity of ZP proteins
ZP antigens (Ag) are often thought to be an interesting target for imunocontraceptive vaccine (Porter –1965), for several reasons as further detailed.
1. Tissue-specific antibodies (Ab) directed against ZP antigens may prevent fertilization, yet not be abortogenic.
2. ZP is exposed to Ab action in follicular fluid for several months.
3. Normally only one oocyte is ovulated per cycle (with monovular species), hence there is a limited Ag ZP amount, to be neutralized by Ab.
4. ZP matrix molecular constituents do not exit ovary in order to enter systemic circulation; hence no problems should develop, related to immune complexes.
Notwithstanding such advantages, still numerous problems are to be dealt with, before a contraceptive vaccine is developed, that is safe for human use. One of the major statements would be that ZP glycoproteins manifest extreme variability in expression of antigenicity and of immunogenicity.
Species specific variations in ZP proteins molecular structure
In most species, ZP matrix contains 3 major glycoproteins, which express considerable heterogeneity of weight and molecular mass, especially due to massive post-translational modifications.
Most such post-translational modifications involve N-terminal glycolysis and O-terminal glycolysis, causing microheterogeneity; such microheterogeneity is responsible for the differences in molecular weight of various such proteins, as determined by electrophoretic methods. Hence, initial ranking of various mammals ZP, based on electrophoretic mobility thereof, is confuse and shallow.
Due to isolation and sequencing of a number various species ZP clone, today we can classify such proteins based on sequentiality similitude (Dunbar and col- 1994; Harris and col. – 1994, quoted by Prasad -1996).
Unfortunately, there is no unanimously accepted systematic nomenclature for ZP glycoproteins in various species. Harris and col. (1994) quoted by Hedrick (1996) advance a unique nomenclature for identifying ZP as ZPA, ZPB and ZPC, as per Table 2.1:
Table 2.1 Equivalence nomenclature for ZP glycoproteins (Hedrick, 1996)
Such terminology is based on on genes size, with ZPA manifesting the largest DNA sequence, ZPC manifesting the smallest. Nomenclature based on DNA size, rather than glycoprotein size, or electrophoretic mobility, is necessary, as post-translational modifications of glycoproteins expression, as well as electrophoretic mobility thereof, are quite variable, species to species.
Today’s model considered, relating to ZP glycoproteins functions during fertilization is based on Wassarman’s findings (1988) for mouse. Such model was not tested on many other species, being not representative for gametes interaction. E.g. mouse ZPC is admitted to act as ligand for sperm-egg binding, while ZPB does the same for pig.
Mouse ZPC is accepted to induce acrosome reaction (ASR) post sperm cell binding to ZP, a phenomenon little tested on other mammals, and proven invalid for pig (Berger and col. -1988, as quoted by Hedrick –1996).
As per Table 2.1, ZP glycoproteins may be classified in 3 large classes (families) based on similitude deduced to exist between amino acids sequences, even though molecules vary in size. It is to be noted, however, that, within each family, considerable numbers and positions of cystein residues are stable, as also are potential N-terminal glycolysis sites. Moreover, one ZP module, consisting in 260 amino acids, with 8 cystein residues preserved, was found in all of the proteins families. Such statement suggests that three-dimensional ZP proteins structure is preserved, in each protein family. The concept was advanced, that mouse ZP1 may be homologous to the rabbit 55 kDa protein, yet only 51% similar to homologue protein in humans, hence deemed to be the 55 kDa proteins family ortholog in ZP.
Functional characteristics of ZP glycoproteins
Even though, in terms of evolution, the genes encoding ZP proteins are preserved, major differences manifest in terms of such proteins’ immunochemical and functional characteristics. Functional characteristics advanced for various species ZP proteins are indicated in Table 2.2. Major part of such investigation was run in order to determine sperm receptor function in ZP molecules.
Mouse ZP3 was proven to be a primary sperm receptor, with a specific oligosaccharides class involved in sperm cell binding; while in rabbit and pig, 55 kDa protein family was involved in sperm receptor activity. Litscher and col. (1995) quoted by Prasad and col. (1996) indicated that – galactose in structure Gal1-Gal3, manifest on ZP3, is involved in sperm cell binding; however, such type parted oligosaccharide is absent in humans and in apes ( yet developed normally and fertile in mice) where such type oligosaccharide was destroyed. Contrariwise, both proteins and carbohydrates appear to be involved in pig sperm cell binding, and the multiple ZP proteins possibly involved in such interaction, in pig and rabbit. With pigs, sperm cell receptor protein, partly deglycosilated (ZP3) was proven to be a ligand much more efficient for spermatozoon, than for the native protein, suggesting that terminal oligosaccharides may not be basic for binding sperm cell. Pig ZP3 , a mouse ZP3 homologue, does not bind to boar spermatozoon membranes, while ZP3 binds to acrosine.
Such studies proved that there may be multiple ZP proteins involved in sperm cell acknowledgement process, in binding thereof or in both processes; also, that such involvement may be species- specific.
ZP proteins immunogenic and antigenic capacity
Numerous studies proved complexity of ZP proteins immunogenic and antigenic capacity, as well as physiological effect thereof on ovary development.
Immune response depends on both ZP immunogenic source, and on the species immunized. E.g. rabbit immunization by pig ZP causes infertility and complete ovarian dysgenesis, whereas mouse and rat immunization, by same immunogen, has no effect on fertility, even though circulant antibodies may be detected, which acknowledge ZP. Immunogenic capacity of ZP glycoproteins is due, firstly, to foreign epitopes associated to various species ZP; while alloimmunization by same species ZP protein causes no marked immune response, determined by presence of circulant antibodies, for own ZP. Carbohydrates appear to influence ZP immunogenic proteins; ZP deglycosilated proteins are less immunogenic than ZP native glycosilated proteins.
Studies also proved that immunization by a number of ZP proteins induces an immune response which affects normal follicular development. Such response may be desirable with sterilizing vaccines, in a number of animal species; yet, it is not desirable in contraceptive vaccines for humans. Hence, identification is required, of ZP antigens which induce antibodies and inhibit fertility, yet do not affect follicular development. Identification of such antibodies which bind to sperm cell receptor and inhibit fertilization, in various species, could be a solution.
Immunization of apes and rabbits by pig ZP3 diminishes fertility, yet does not alter ovarian functions; whereas immunization by pig ZP3 (a mouse ZP3 homologue) generates ovarian dysgenesis.
Mouse ZP3 and homologues act as in vitro spermatozoa receptors; however, such ZP immunogenes do not appear to be adequate candidati for contraceptive vaccines for humans. Pig ZP3 and 55 kDa rabbit protein appear promising, identification of specific regions producing maximum fertility inhibition being required, that does not alter follicular development and ovarian functions.
1.3. ZP molecular genetics
ZP plays a basic role in fertilization and preimplantation embryogenesis. With mice and humans, ZP is the extracellular matrix, containing three sulphate glycoproteins (ZP1, ZP2 and ZP3). Primary amino acids sequence (AA) of such – in mouse and in humans – 3 proteins is deduced from nucleic acids sequence of related DNA thereof (Castle and Dean –1996).
Of all types, human and mouse ZP1 manifest the lowest stability. Mouse ZP (623 amino acids) is by 83 AA longer than human amino acids (540 AA), due to an elongated exon 3 in mouse genes; when overlapping, such ZP are only 43% identical.
Each ZP protein manifests post-translational modifications, being secreted as a glycoprotein, a portion of extracellular insoluble matrix.
A ZP around 7m thick, containing 3 to 4 ng protein, surrounds mouse mature oocyte. Electron microscopy indicates that the 3 glycoproteins form a relatively homogenous mesh of 2 to 3 m long, interconnected, filaments. Each filament contains a repetitive heterodimer ZP2-ZP3 structure, filaments appearing to cross-link by ZP1, a homodimeric protein.
Table 2.2 Various species ZP characteristics
Molecular weights differ species to species, due to glycolysis variations. In vitro studies data indicate that carbohydrates side-chains play an important role in ZP biological function.
Each of such 3 proteins has an amino-terminal signal mature peptide, cleaved off from a polypeptide chain, pushing such protein through a secretory pathway. Each ZP protein has an around 34 to 47 AA transmembrane domain, close to terminal-carboxyl group, manifesting the potential of a tetra-basic (R-X-R-R) cleavage site, human ZP1 cleavage site being only tri-basic (R-R-R).
Proteolytic transformation of transmembrane domains may be an intermediate state of ZP proteins secretion and incorporation thereof into ZP matrix. Specific protein regions were preserved among ZP classes in mammal, as well as among ZP domain of species unrelated along evolution chain.
Zona pellucida genes
Mouse ZP1, ZP2 and ZP3 are unique copy genes located on chromosomes 19, 7 and 5. Mouse ZP1 is made of 12 exons, 82 to 364 bp long, 6.5 kbp span.
Transcription of the whole ZP1dimension (i.e. 1963 nucleotides – nt) has one only open-reading frame (made of 1869 nt), flanked by 2 untranslated regions, 5’ and 3’ short, 57 and 37 nucleotides long.
Mapping of mouse ZP1 exons, and of ZPB human ortholog thereof, is remarkably well preserved. Even though human gene is much more dispersed (11 kbp), most exons are almost the same size, except for exons 3 and 12, which are larger in mice.
Mouse ZP2 contains 18 exons, varying 45 to 190 bp, interrupted by 17 introns (81 to 1490 kbp). Genes span 12.1 kbp in the DNA. ZP2 is transcribed and processed into a 2201 nt mRNA, with untranslated very short regions, 5’ (30 nt) and 3’ (32 nt). Human genes Zp2 are made of 19 exons (one more than mouse genes), nucleic acids sequence being 70% similar to mouse Zp2 gene sequence.
Mouse Zp3 genes (8.6 kbp) contain 8 exons (92 to 338 kbp) and 7 introns (125 to 2320 kbp). 1317 nt mRNA encoding Zp3 has 5’ (29 nt) and 3’ (16 nt) 2 short untranslated regions, the last of which is shortened to the point that TAA stop codon is included into AATAAA polyadenylation signal. Human Zp3 gene, too, contains 8 exons.
Transcription process of the 3 proteins is only detectable in the ovary, expression thereof being restrained to only oocyte level. Mouse ZP2 transcription is detectable in oocytes before birth, much before ovocitary growth stage, and the earliest on pregnancy day 16. By contrast, ZP1 and ZP3 are detected only after oocytes start developing.
ZP1-, ZP2- and ZP3-encoded mRNAs accumulation appears to be coordinated, markedly increasing during the ovogenesis early stages, and declining during ovocitary maturation. An activation zone protein (ZAP-1) was identified, that is involved in regulation of genes expression for ZP (Castle and Dean, 1996).
RESEARCH. THEORY AND EXPERIMENT.
1.4. Identifying ZP proteins for IMMC
1.4.1. Passive immunization against ZP
Administration of anti-zona antibodies manifests two major advantages:
1. Contraceptive effect appears to be reversible;
2. Regulation is possible of infertility, by adjusting dosage and frequency of antibodies administration.
Injecting AZP-Ab generated infertility in a number of species’ females (Henderson 1988). E.g. parenteral administration of rat monoclonal specific to mouse Ab ZP2 and ZP3 generated reversible infertility over 15 sexual cycles in 100% of the female mice tested. Injected animals contained Ab surrounding developing oocytes. Fertility came back when circulant antibodies levels fell below critical threshold where developing oocytes did no longer accept specific antibodies. Fertilization in vitro indicated that the spermatozoa bound to Ab-lined oocytes, yet did not penetrate ZP. Ovaries of the female mice treated had normal morphology; and embryos appeared unaffected when anti-zona antibodies were administered post fertilization.
Passive immunization manifests several potential drawbacks. Antibodies must be administered at set times, in order for the contraception to be sustained on the long run term, and may determine premature loss of oocytes by triggering complement activation, and inflammatory response.
Repeated administration of heterologous proteins can determine recipient organism to produce own antibodies against administered antigen, by inactivating such antigen; while chronic presence of such immune complex may generated immune complex diseases. New technologies emerged, that may fully counteract a number of such drawbacks, as further detailed.
1. Antibodies may be incorporated in slow intermittent release devices (reviewed by Saltzman- 1993, quoted by Castle and Dean -1996);
2. Determining antibodies species is possible, so that antibodies should not be identified as foreign entities, either by recombinant DNA technique (Hoogenboom and col- 1992; Lewis and Crowe-1993, quoted by Castle and Dean-1996), or by producing human antibodies from transgenic mice (Lonberg and col- 1994, quoted by Castle and Dean -1996).
1.4.2. Active immunization
When targeting ZP, generation is important of a series of peptides vaccines composed of B-cell epitopes, which will create Ab against ZP, yet not inducing an immune response to T-cell epitopes, leading to destructive inflammation. T-cell epitopes (nedeed for triggering helper T-cell response required to activate production of B-cells antibodies) may be provided by binding peptides containing B-cell epitopes to immunogenic transport molecules, or synthesized and polymerized epitopes for defined (non-zone) T-cells. By such strategies, helper T- cells directed to immunogenic foreign T- cell epitopes are stimulated to proliferate, yet preventing generation of a memory T-cell population able to recognize ZPs.
If peptides are identified, that trigger the first, yet not the second, immune response, such peptides are appropriate candidates for contraceptive agents. It is still debatable whether or not Ab against ZP causes loss of intraovarian oocytes.
A mice line was established, which is a null mutation to ZP3 site, based on using embryonic stem cells technology. Even though heretozygous females are fertile, homozygous females do not manifest visible ZP.
1.4.3. Autoimmune ovarian diseases generated by IMMC
Lou and col. (1996) used a mouse model as investigating mechanism of ovarian pathology induced by active mice immunization with 13 polymeric ZP3 peptide (mZP3330-342). Such peptide includes a native B-cell epitopes, and two nested T-cell epitopes. Ovarian pathology was transferred to neutral recipients for CD4+ T-cells; however, transfer was not possible by antibodies to immunized mice, suggesting the importance of T-cell mechanism of in ovarian pathogenesis. Thus induced oophoritis spontaneously remitted some 4 months later, ovaries manifesting a normal aspect, no lymphocytic infiltrations, containing developing follicles, cyclicity and fertility at a later time not affected. Reimmunization of females by same procedure yielded a much lower immune response.
Moreover, immune responses, as well as disease induction, were restricted to H-2a,k,u,s,axb haplotypes. Based on such model, a strategy is described for generating a contraceptive anti-ZP antibodies response, yet not a T-cells pathogen- triggered response. B-cells epitope was modified by substituting a series of amino acids, in order to eliminate epitope for oophoritogenic T-cells, as bound to foreign T-cells epitope, i.e. bovine Rnase(94-104). Resulted chimeric peptide (CP2) induced anti ZP antibodies in 100% of the 8 mouse strains with various haplotypes for H2, yet not inducing marked disease forms, respectively not activating pathogenic ZP3-specific T-cells. A female mouse lot, immunized with CP2, proved that anti-ZP antibodies were associated with fertility decrease. Infertility was found to be reversible.
Active immunization with mZP3330-342 induced autoimmune oophoritis. Such disease is characterized by lymphocyte infiltration of ovary. Low grade 1 and middle grade 2 oophoritis are associated with presence of infiltrates located predominantly in ovarian interstitial tissues, involving atretic follicles. Chronic oophoritis is associated with diffuse involvement of developing follicles, presence of giant cells, as well as formation of granulomas, or presence of atrophy.
In Grootenhuis and co-workers’ view (1996), presence of ZP3 on primordial follicles and in granulosa is what generates ovarian pathology post immunization with ZP3. Thus, feedback drop, of signals from follicles recruited to primordial follicles, could trigger fast recruitment of the whole pool of primordial follicles available. Also, presence of antibodies, and of joint complement thereof, could generate a toxic microenvironment for the sensitive primordial follicles. Separation of B-cells epitopes from T-cells could be the solution.
Yahuan Lou and col. (1995) managed to induce anti-ZP antibodies and afferent reversible infertility; yet not ovarian pathology, by chimeric peptide (CP2) and altered variant for CP1, manifesting basic same 13 peptide-polymer, derived from mouse ZP3 (mZP3330-342).
1.5. Immunogenicity of ZP vaccines
The preoccupation for ovarian contraceptive vaccines dates back to 1965, when immunization with ovarian homogenate proved, for the first time, strong ZP immunogenicity, as well as correlation thereof with infertility (Porter, 1965). Availability of unlimited amounts of isolated zonae pellucidae made possible the study of immunogenicity, of antigenicity and of ZP biochemistry. However, inducing ovarian pathology generated post immunization appears to be an applicability problem, aspect noted in several species (Skinner and col., 1984; Mahi-Brown and col., 1988; Dunbar and col., 1989 – quoted by Ndolo and col. – 1996). Such research employed DNA-encoded ZP molecules to express large amounts of recombined ZP in bacteria; which solved the issue of large-scale production of ZP native proteins (Ndolo and col. –1996). Isolation, at a later time, of the DNA specific to each of all three ZP protein families, in several species, also solved the issue of large-scale production of peptide immunogens.
It is quite important noting that immunization with rabbit ZP recombinant proteins (rec55) appears to efficiently separate the two immune responses generated against ZP own proteins; such responses would involve (1) induction of antibodies which inhibit sperm binding to ZP; and (2) induction of immune responses causing ovary pathology. Consequently, today’s techniques can produce recombinant ZP immunogens inducing antibodies which block spermatozoa binding in vitro, yet manifest no obvious effect upon normal follicular development. The new generation of recombinant vaccines is thus expected to emerge.
As attempting to explain how contraceptive vaccines induce ovary pathology, Mahi-Brown and col. (1992) as well as Paterson and col. (1996) focus on ZP3, starting from premise that active immunization against ZP antigens not only induces infertility by simply blocking interaction sperm cell – oocyte, mediated by antibodies, but also disturbs ovarian function, a number of epitopes ZP3 being suspected to generate pathological effects. A peptide was isolated, made up of seven AA (336 to 342) which encodes a continuous epitope, able to suppress fertility and induce oophoritis. The fact was also shown that active immunization with ZP proteins leads to primordial follicles depletion, as well as depletion of recruited ovarian follicles, by a mechanism still unknown. Among other reasons, small amounts of ZP glycoproteins present on primordial follicles or granulosa cells may lead to occurrence of ovarian pathology. It was also stated that presence of ZP3 in primordial follicles and in granulosa cells plays a role in affecting ovarian function post active immunization.
Immunocontraception with ZP found a unique application in wild animals population (Kirkpatrick and col., 1996). Vaccination with porcine ZP (ZPp) was successfully used in a series of wild horses and donkey population. One unique administration per year provided effective ongoing contraception.
Treatment of wild mares for 7 years running, did not generate side effects, fertility being reversible in females treated for 4 consecutive years. Long time (5 to 7 years) treatment was accompanied by a number of ovulation disorders, as well as of fall of estrogens levels in urine. Social behavior of horses was not affected by treatment. Immunocontraception with ZPp was also successfully applied to white tail stag, yet no alteration of ovarian histology was manifest after 2 treatment years. 74 captive zoo species were treated with ZPp for immunocontraception, results being satisfactory in 27, including members of order Perissodactyla (Equidae), Artiodactyla (Cervidae, Capridae, Giraffidae, Bovidae) as well as Carnivora (Ursidae, Mustelidae, Felidae). Immunocytochemistry studies proved high cross reactivity between anti-ZPp antibodies and zona pellucida in African Elephant. The need was felt, for one unique administration of the vaccine; which led to incorporating ZPp into lactide-glycolide microspheres, which caused slow ZPp release. Immunocontraception with ZPp in wild animals manifests ample potential, as: (1) efficacy goes over 90%; (2) possibility to administer antigen from a safe distance, by shooting; (3) reversibility over the short time run administration; (4) efficacy in many species; (5) lack of side effects, even post long time treatment; (6) minimal effects on social behavior and (7) incapacity, of vaccine or of antibodies resulted, to diffuse into trophic chains. In order to block undesirable effects on ovaries, Kaul and col. (1996) recommend a cocktail of synthetic peptides or recombinant glycoproteins or proteins, as adequate to the ZP wherefrom the epitopes were removed, that are responsible for the inflammatory phenomenons. Mentioned authors set the sequence AA in ape (Macaca radiata) ZP3, in correlation with other species sequences (see Table 2.3):
Table 2.3 Similitude of ZP3 amino acids sequences in Macaca radiate vs. human, Callithrix jacchus, mouse and hamster
Determination of ZPp zona responsible for inducing contraceptive effect was possible owing to studies of Koyama and col. (1996). ZPp was initially separated into four major glycoproteins (ZPp1, ZPp2, ZPp3 and ZPp4) families; while integral cloning of the corresponding DNA proved that peptide ZPp4 (50 to 59) is the one which could inhibit sperm cell binding to oocyte, thereby possibly generating a contraceptive effect.
Cherrie Ann Mahi-Brown (1996) recommends using immunogenic peptides for immunocontraception, advancing a basic theory in point: once the proteins sequence is known, epitopes mapping is possible, as also is testing of specific immunogen. Each antigen contains an array of regions or epitopes, which are recognized in specific ways, by receptors on B- and T-cells. Anti-body response to protein origin antigen requires contribution from both B-cells, for antigen presentation and antibody production, and of T-cells, for helping B-cells, providing factors for activation and differentiation thereof. T-cells are also inflammation mediators, being involved in several autoimmune diseases. Normally, for B-cells epitopes are not the same as T-cell epitopes; hence the possibility that a synthetic antigen is generated, which should contain B-cell epitopes in an autoantigen like ZP, and a T-cell epitope in a foreign antigen, such as the carrier protein. Thus, a response may be generated, where antibodies are directed against ZP, yet T-cells able to recognize ZP are not activated.
Peptide antigens are small size and simple make, hence needing to be conjugated to a larger carrier protein, so as to act immunogenic. In long term immunization, a strong response to carrier proteins may, in fact, interfere with immune response to peptides. Peptides antibodies often bind to a free, or conjugated, peptide; yet do not recognize native proteins epitopes, due to tertiary structure of protein. Such is the reason why testing is required, of all anti-peptides serum variants, in order to check on binding thereof to native proteins, unaltered and undiminished. Response to an only epitope is often under genetic control, so that various individuals in same species respond in distinct ways to same epitopes, or do not respond at all. Hence, in order to be efficient in most of a population, a vaccine should probably contain more than one unique epitope.
Injection of ZPp proteins in bitch and cat may generate own antibodies response to ZP (Lowrey, 1993). Such animals’ target oocytes, whose surface was lined with antibodies, were infertile, as sperm cell was no longer able to bind, implicitly fertilization did not occur. Inoculation of solubilized and partly purified ZPp as antigen in impuber bitches delayed heat start by 17 months. Histological sections showed absence of follicular activity, especially lack of primordial follicles in the ovarian corticala. 8 antigens were cloned and sequenced, out of the 12 of potential interest for immunocontraception vaccine in bitch and cat.
Immunocontraception in bitch
Sperm cell binding to receptors on ZP, for fertilization start, as well as embryo exiting zona pellucida for implantation, are two basic events vulnerable to the blocking exerted by anti ZP antibodies. Anti ZP antibodies in follicular fluid of immunized bitches, bound to immune-complexes on oocytes surface in ovaries of same animal, suggest that antibodies in follicular fluid form a complex with the ZP antigens in oocytes, indicating that infertility could be generated by blocking sperm cell binding. Immune-complexes could masque for sperm cell receptors, or could block mechanic sperm cell binding, as proven by numerous studies, in vivo or in vitro, run in various mammalian species.
Contraceptive effect duration, of active immunization, depends, firstly, on levels of antibodies in serum and follicular fluid; or, with passive immunization, on duration of antibodies lined oocytes vanishing from ovary. Contraceptive effect vanishes post fall of antibodies titers, or when lined oocytes vanish from ovary, either by ovulation, or by atresia.
Zona pellucida is a weak alloantigen, yet a strong xenoantigen; hence an antigenic response is yielded, consistent with ZP of species used to immunize other species (Mahi-Brown and col., 1988). In several cases, such xenogenic anti-zona anti-bodies interact with ZP of such species where antibodies were produced, generating infertility.
1.6. Pig ZP for IMMC in bitch
Experiments and investigations were run in order to determine antiZP antibody titers resulted post immunization in bitch, with ZPp, as well as with oocyte and ZPp (OV+ZPp); determination was also in order how presence of antiZP antibodies impacts sexual activity of the females immunized.
Body weight of the four bitches included in experiment varied 12 to 15 kg. At onset, females were subjected to general clinical examination; abdominal echography included, to rule out presence of pregnancy or estrus. Also, the bitches were deparasitized with Drontal plus.
All of the 4 females were kept in individual cages, water available at all time, fed in the morning, on (based on size) 150 to 200 g ABC (market food) granting 100% the daily share of protein, energy, vitamins and microelements. Twice a week, each bitch underwent estrous detection cytovaginal examination. 7 days after encagement, first blood samples were collected and first antigen was administered.
Antigen preparation. Sow ovaries were collected from one slaughterhouse, only the normal looking (non-cystic) being preserved. Ovaries were deep frozen prior to processing, so antigenic characteristics thereof should not be impaired (Mahi and Yanagimachi – 1978, Shivers and col. –1981). Mahi and Yanagimachi (1976, 1978), Gwatkin and col. (1980) as well as Henderson and col. (1988) recommend a relatively simple and fast method for obtaining sow ovaries oocytes; however, for lack of a series of basics, such as synthetic filters, mesh size 215 to 136 and 100 micrometers (Shivers and col., 1981), we had to aspirated fluid in each follicle visible on ovary surface; we then poured such fluid into a Petri box containing TCM 199. Subsequently, under magnifying glass, each oocyte surrounded by ZP was aspirated. We isolated, average figures, 25 oocytes per sow ovary. Oocytes were denuded by preservation in 3% collagenase, for 60 minutes, at 37C. Doses were prepared, of 300 oocytes with afferent ZP, or only ZP from the 300 oocytes, suspended in 2 ml TCM 199 and preserved in plastic tubes, inside deep freezer. In order to determine antibodies titers, ZP was boiled for 45 to 60 minutes, at 70C (Mahi-Brown and col., 1985), until solubilization was noticed, under microscope, to occurr.
Shivers and col. (1981) applied 3 immunizations in 7 days, by 2000 ZPp per dose, suspended in 1 ml Freund adjuvant, for immunocontraception in bitch.
Serum was prepared from blood samples and inactivated by incubating for 30 minutes, in water bath, at 56° Celsius, later stored at –50° C.
Testing for presence of anti-zone Ab in serum
Presence in serum of Ab which interacted with CZP (canine zona pellucida) was tested by treating bitch oocytes denuded of radiate crown by serum; while assessment of Ab binding was run as further detailed:
1. presence of precipitate on ZP surface, visible on phase contrast microscope;
2. indirect immunofluorescence (IIF);
3. reaction of ZP penetration in vitro.
Mahi and Yanagimachi (1978) devised and applied a specific technique, in order to isolate oocytes in freshly excised bitch ovaries, to remove radiate crown, to prepare capacitated spermatozoa, and to inseminate oocytes in vitro. Procedures described at a later time (Mahi and Yanagimachi –1979) was applied for assessment of ZP surface precipitation and ZP penetration by sperm cell; except that, at such latter time, oocytes were treated with undiluted serum, as against earlier diluted serum. Experiments involved two oocytes groups, i.e. a group treated only with sperm cell capacitating medium, and the control group treated with preimmune serum. Each group contained 10 to 20 oocytes and tests were repeated twice, at the very least. Ab titer in serum in CZP binding was determined by indirect immunofluorescence. Bitch oocytes were treated with antiserum serial dilutions in PBS containing 1% polyvinylpyrrolidone (PBSp); or only with PBSp (control group). Oocytes were immersed into 100 l diluted serum or PBSp, on a watch glass, overlaid with mineral oil, in order to prevent evaporation. After being treated for 1 hour at room temperature (27ºC) in an orbital shaker (75 rpm), oocytes were washed twice, for 10 minutes each time, in fresh PBSp, and later translated to a watch glass, containing 30 l in 0.1 mg/ml protein A conjugated with fluorescein isothiocyanate (FITC) in PBSp. Protein A binds specifically to Fc-region of IgG subclasses 1, 2 and 4 (Kronvall and Williams, 1969). Post treatment for one more hour, as above quoted, oocytes were washed four times for 10 minutes each time, in PBSp; then set on glass plates and examined on fluorescence microscope. Intense fluorescnce on ZP surface indicated Ab surface binding. The highest of the serum dilution at which fluorescence occurred indicated the anti-ZP Ab titer, for respective serum.
Demonstrating specificity
In order to demonstrate that precipitation, and A protein binding to ZP surface, are caused by species-specific Ab binding to ZP surface, procedure applied was as further detailed. An antiserum anti-PZP strongly positive (1:10000) was absorbed into PZP, by mixing 0.75 ml serum with 0.25 ml PBS containing 2000 solubilized PZP. One more anti-PZP strong positive serum sample was absorbed by liver, intestines and plasma, in order to remove non-specific Ab against pig tissues. 4 ml serum was mixed with 200 mg liver acetone powder, or intestines and 50 mg lyophilized plasma. Low titer (1:1000) bitch anti-PZP antiserum was absorbed by homgenizing 4 sow ovaries (priorly sectioned and centrifuged, in order to remove most of the follicular fluid) in 0.5 ml antiserum. In each case, serum was incubated with absorbant, for 45 minutes, at 37° C, and 4° C overnight when later centrifuged, in order to remove precipitates and tissues. In order to prove that material bound to ZP surface was really Ab, IgG was precipitated out of the non-absorbed positive antiserum with saturated ammonium sulphate (40%) final concentration three times redisolved in original PBS volume, dialyzed against PBS in order to remove ammonium sulphate; and preserved deep frozen, 10 mg protein per ml. Serum absorbed and IgG preparations was later tested, as quoted above.
Immunization of females
6 consecutive immunizations were run inside 3 weeks, as further detailed: bitches A and B only with pig ZP (one dose made of ZP from 300 oocytes), while bitches C and D with doses containing 300 sow oocytes, with afferent ZP (OV+ZPp).
Before administration, each dose was mixed with an equal volume (2 ml) sterile Al(OH)3. If not an adjuvant yet, ZP does not generate an immune response (Mahi-Brown and col., 1985) and was let settle for 5 minutes at room temperature. Dose was administered subcutaneously, on side of the neck.
Blood samples were taken from antebrachial vein, as further detailed: before first immunizations and 14 days post each immunization, starting 6 months after first immunization, every 30 days up to circulant antibodies vanished. After expression of serum, blood samples were stored in a deep freezer, up to analysis time thereof.
At debut of vulvovaginal leakage, each 48 hours a color cytovaginal smear May-Grunwald-Giemsa (MGG) was run. Based on such cytovaginal smear, the mounting was run, daily, for so long as female accepted the male. When leakage stopped, cytovaginal smears were further run twice a week.
Antibodies titer was set by fast agglutination reaction.
Results and debate
Results are indicated in Table 2.4 and pictured in charts 2.1 la 2.3. Analysis thereof allows for noting that, in bitch A, the first titers (1:200) are detectable only after third immunization; while, in order to go beyond 1:1000 titer (which Mahi-Brown and col., 1982, deem enough for inhibiting spermatozoa penetration), 5 immunizations were required. AntiZP antibodies titer rose beyond such level for 8 months since first immunization, while after 10 months no more antibodies were detected. Female A manifested heat 5.5 months after first immunization. Vaginal leakage lasted for 17 days, the bitch being mounted 3 days later (on days 12, 13 and 14) yet not getting pregnant (see Table 2.4). Cytovaginal smears, run by MGG technique, indicated normal development at estrus time.
Table 2.4 Sexual activity characteristics of immunized females
Second bitch, B, manifested antiZP antibodies as early as post first immunization, titer thereof rising continuously up to time of 6th immunization, when peaking (1:4000). Antibodies titers rose over threshold 1:1000 only after three immunizations, as against bitch A, that required 5 immunizations. Antibodies titer settled at sperm cell inhibitive level, for 7 months since first immunization, by 30 days less than with female A, as indicated quite obviously, on chart 2.1.
Bitch B entered estrus 6 months after first immunization, vaginal leakage lasting for 14 days; two mountings were run, on cycle days 11 and 12. Based on such characteristics, female B comes closest to average physiological figures admitted for cycle duration and bitch mating time. Cytovaginal smear did not indicate pathological aspects. Post mating, bitch B did not get pregnant.
Mahi-Brown and col. (study run in 1985) determined antiZP antibodies titers to be in-between 1:1000 and 1:4000, through a series of 3 immunizations, run 30 days apart each, with 2000 ZPp doses.
Chart 2.1. AntiZP antibodies titers in bitches immunized with ZPp
In 1982, Mahi-Brown and col. determined a maximal 1:100.000 titer, post 6 consecutive immunizations, one month apart each, with same 2000 ZPp / dose.
Chart 2.3 indicates data for bitches C and D, immunized with OV+ZPp, by protocol above quoted. In female C, fertilization inhibitive antibodies titer was recorded post second immunization, peaking at 1:7000, after fifth immunizations. Let it be highlighted that inhibitive titer persisted for 11 months since first immunization, while antibodies vanished in blood serum only 14 months after protocol debut.
Chart 2.2. AntiZP antibodies titers in female dogs immunized with OV+ZPp
During all of such time, bitches C did not enter estrus, cytovaginal smear providing confirmation. Starting month 6 post first immunization, erythrocytes manifested in relatively large numbers, in the cytovaginal smear, a phenomenon persisting up to month 10 post first immunization; other smear elements permanently indicated metestrus.
Bitch D manifested antibodies post third immunization, touching at inhibitive level only post fourth immunization, as against bitch C, where such level was reached only after two immunizations. Maximum antibodies titer (1:6000) was generated after 5 administrations, 11 months post first immunization no antibodies existing yet. Inhibitive level persisted up to month 9 since protocol debut. Bitch D entered estrus 8 months since first immunization, vulvovaginal leakage lasting for 28 days. 5 mountings were run (in-between days 15 and 20), bitch D not getting pregnant. Cytovaginal smear indicated erythrocytes to be still in large numbers 7 days after leakage stopped, insufficient number of basophil cells being present, which indicates possible ovulation disorder.
Mahi-Brown and col. (1985) obtained antiZp antibodies titers up to 1:20.000, with unpurified ZPp, as well as abnormal sexual cycles in the 3 bitches observed.
Chart 2.3 pictures an overall view of such study data. It will be noted that, for immunization with ZPp, inhibitive antibodies titer (average figures 1:1900) was reached post 5th immunization, sustained at such level for 7 months since protocol debut; whereas for immunization with OV+ZPp, inhibitive titer was reached post second immunizations (average figures 1:1100), sustained at inhibitive level up to month 10 post first immunization.
Chart 2.3. Average antibodies titer figures for the two bitch classes.
With the first antigenic variant (ZPp), estrus was manifested by both females, 5.5 months (average figures) since first immunization; whereas with the second variant (OV+ZPp), estrus was manifested by one female, 8 months post initiating protocol, yet not by the second female, observed all along the study 14 months.
As per Mahi-Brown and col. (1988), even though bitches immunized with ZPp produced antibodies which inhibited fertilization in vitro, inhibition of fertilization did not appear to be the only infertility cause. Other infertility causes were indicated, such as abnormal cycles and lack of ovulation; as well as extensive destruction of the oocytes, which is likely to lead to permanent infertility.
Yurewicz and col. (1983) state that apparent ovarian function disorders in immunized females suggest that resulting antiserum is not necessarily specific to zona pellucida; which would explain titers recorded to be higher for bitches C and D, as against A and B, one more explanation being that antibodies were produced in response to ovarian antigens Ab other than zona pellucid.
Skinner and col. (1983) recorded decreasing number of primary, secondary and tertiary follicles, 6 weeks post last immunization; while post 20 weeks, only few (if any) developing follicles could be noted. Based on such statements, Skinner suggested that antiZP antibodies acted upon cells responsible for zona pellucida synthesis.
Experiments allowed for final conclusions as further detailed.
Immunization of bitches with pig zona pellucida generated antibodies titer inhibitive for fertilization, which persisted for 7 months, not affecting sexual cyclicity.
Immunization with oocytes, concomitantly with afferent zona pellucid, caused higher antibodies titers, equally inhibative for fertilization, which persisted for a comparatively longer time (10 months), yet accompanied by reproductive function disorders.
Such methods cause 100% contraception.
DEVELOPMENT TRENDS ANALYSIS
Contraceptive vaccines which inhibit fertilization, yet do not affect ovarian function (Carino and col., 2001) should meet requirements as further detailed: (a) inhibit fertilization, yet not induce abortion; (b), manifest low enough antibodies titer to inhibit fertilization, in case of multiple ovulation/poly ovulation; and (c) expect no immune complex to develop, ZP antigens being tissue-specific, not resulting in a process involving blood system (circulatory system apparatus). As well known, antibodies developed against ovarian tissue inhibit, or totally block, spermatozoa binding to ZP. ZP glycoprotein immunogenicity is quite complex, immune reaction depending on both antigen source and on species to be immunized. ZP antigenicity is triggered by presence of foreign epitopes correlated with a different species ZP, as alloimmunization causes a quite low immune response.
Antibodies specific to various regions, or to native ZP, or recombinant ZP carbohydrates, will inhibit recognition, as well as oocyte adhesion and/ or fusion with spermatozoon. In bovine, fusion of gametes is inhibited by monoclonal antibodies produced against carbohydrates formed by N-acetylglucosamine and galactose. Such is TEC-2 epitope, where specific antibodies were generated by low binding of the gametes, depending on dose.
Zona pellucida is the site of acrosome reaction. ZP provides specific spermatozoon binding site, and modifiers structure post spermatozoon – oolemma contact, thus blocking polyspermy. Post fertilization, ZP is required for sustaining juxtaposition of precompact blastomeres, as well as for protecting early embryo against physical aggression and environment negative impact. Study of Bogner and col. (2004) indicates that expressed ZPA and ZPC amount differ, based on folliculogenesis stage. Results of such study, which targeted ZPA, ZPB and ZPC genes expression, by RT-PCR, revealed that marmoset ZPA, ZPB and ZPC biosynthesis occurs in both oocytes and follicular cells at different follicular stages. The oocyte is the primary ZP proteins synthesis site, follicular cells being also involved in de novo ZP proteins synthesis.
As for immunocontraception, ZPA antigens in marmoset primordial follicles may induce irreversible infertility, as primordial follicles destruction will exhaust follicles at rest on stock. When temporary (reversible) infertility is the target, ZPC is to be preferred.
As antibodies generated against ZP3 block oocyte-spermatozoon interaction, ZP3 was considered as potential candidate for contraceptive vaccine. A DNAc sequence generated by PCR reaction encoding 342 amino acids (23 to 364) residues in mouse ZP3 (ZP3m) was cloned (Zhang and col., 1997) in a Asd+ vector, i.e. a vector based on aspartate-β-semialdehyde dehydrogenase-asadh, which is stable for an acute expression in vivo, and ideal for developing a vaccine based on Salmonella. A non virulent Salmonella strain, yet expressed ZP3m, colonized mouse inner organs post inoculation per os. Thus, marked levels were induced, of IgG antibodies against own ZP, as well as IgA antibodies in vaginal secretions. Thus induced IgG antibodies bound to ZP, in vivo. 3 of the 6 females injected with recombinant Salmonella were infertile, an aspect proven post mounting. None of the females injected with plasmid Salmonella (as a vector) generated anti ZP antibodies, all were fertile, and none manifested ovary inflammations.
Anti ZP IgG antibodies in the circulatory system may be extremely important in immunocontraception, such Ab being proven apt to penetrate ovarian follicles, and bind to ZP in vivo.
Contraceptive vaccines must be efficient over the long term run, cheap, easy to administer, and side effect proof. One of the major problems is lack of specificity; in such terms, targeting functional regions of antigens would be an interesting approach. Antigenic peptides may enhance species specificity, as also may, concomitantly, lower side effects; however, polyepitope vaccines are to be preferred, as triggering simultaneous immune response against a number of distinct antigens. Hardy and col. (2004) proved mouse fertility to fall by 50% post immunization with peptides and polyepitope antigens targeting a large number of proteins involved in the reproductive processes. Peptides were selected based on a short sequence of amino-acids shared with other species’ homologous proteins, deemed to manifest antigenic contraceptive potential, in mouse. Peptides were produced by two different methods, i.e. by chemical synthesis and binding to a carrier protein, expressed in bacteria as recombinant polyepitopes. The most consistent response, as antibodies titer, was obtained against two peptides derived from ZP1 and ZP3 proteins. Antibodies response was preferentially directed against epitopes pertaining to the comparatively longer peptide sequences. Such longer peptides (SP56 and ZP1) are much more likely to contain T-cell and B-cell specific multiple antigen epitopes, which could explain preferential production of antibodies against such epitopes (in polyepitopes).
Potential drawback becomes thus evident, in choosing peptides for developing contraceptive vaccine, as being, possibly, occurrence of epitope immunodominance post selective and dominant activation of the B-cell specific peptides.
Contraceptive vaccines can be improved in various ways. Firstly, contraceptive response to immunization with polyepitopes can be improved based on a series of repetitive epitopes. Copies number increases directly with the correlated higher antibodies levels. Improvement of subdominant epitopes immunogenicity can be reached by using such immunity enhancers as cytokines. Secondly, polyepitope vaccines which concomitantly target species-specific regions of a number of proteins, structurally and functionally similarly important, as well as the three proteins, ZP – ZP1, 2 and 3 – may induce a much more efficient contraception. Contraceptive efficiency is high when ZP1 and ZP3 are used concomitantly. Thirdly, immune response other than antibodies response, such as cytotoxic response triggered by T lymphocytes (CTLs) may generate immunocontraceptive effects, as proven post immunization with ZP3 (Hardy and col., 2004), when contraception was triggered by both T-cells and antibodies response. Determining T-cell epitopes apt to induce CTLs, as well as incorporation thereof in polyepitopes, enhances immunogenicity. One development method of a fertilization inhibitor vaccine is based on transfection of Escherichia coli and ZP glycoproteins expression, from various species, such as sow, rabbits, bitch and marmoset monkey. Thus, immunocontraception based on using glycoproteins ZP expressed in E.coli acts as a pregnancy prevention method, in various species.
Rabbit zona pellucida is made of three glycoproteins, i.e. ZPA (R75), ZPB (R55) and ZPC (R45), ZPB is preferred for immunization, as manifesting receptor activity for the rabbit spermatozoa. ZPB (R55) is also structurally homologous to sow ZP3alfa, which was nominated for potential candidate to immunocontraception. Study of Foley and col. (2007) focused upon the rZP (two proteins were used in rabbit ZP, i.e. ZP55 and ZP75, based on molecular weight thereof, 55 respectively 75 kDa; while plasmid pRSET was used as an expression vector for the purpose) targeting generation of cow antibodies titers apt to block oocyte – spermatozoon interaction. Even if such approach resulted less than wholly successful, the cows manifesting obvious high ZP proteins titers had cyclicity and ovaries function impaired. High antibodies titers, or anti-ZP immune complex, may affect ovarian function by interference of follicle-oocyte relation, a mechanism involved in follicular growth regulation. If such interference was produced over follicular differentiation time, when proteins ZP were synthesized and secreted, when ovulation may fail, then ovulation and CL normal development may also fail.
ZP constituent glycoproteins are intensely glycolized, and such carbohydrates fragments are required for fertilization.
In terms of mechanics, sequencing or blocking for carbohydrates (receptors) sites, or altering ZP chemistry, will generate immunocontraception.
Immunocontraception can be defined as the possibility of using reproductive processes specific protein for inducing a humoral immune response, which should lead to contraception for a predetermined period de time. Administration of sow ZPp glycoproteins determined immunocontraception in various species. In dogs vaccinated with ZPp, initially IgG level rise is enhanced (Fayrer-Hosken and col., 2000) to a point high enough to prevent fertilization. Heterologous ZP induced ovarian pathology in a number of species; such pathology is deemed to have resulted from (a) initial stimulation, exerted by human immunoglobulins followed by T-cell response; and (b) B-cell mediated response. It was proven that Zp peptides used had epitopes for T-cells and induced oophoritis (CD4- and T-cell induced autoimmune disorder). Altering ZP epitopes for T-cells solved such problem.
Sequencing of the four ZP glycoproteins of various species indicated that a specific ZP protein manifests variable levels of amino acids sequence conservation. Such characteristic of ZP proteins permitted heterologous immunization, as a preferable immunocontraception method, antibodies generated against pig ZP3 manifesting cross-reactivity with human ZP.
ZP-based DNA vaccines may elicit bioactive antibodies. Live genetically modified vectors trigger immune response against zona pellucida proteins. Side effects (oophoritis) were mediated by presence of epitope of mouse ZP3 oophoritogenic T-cell. Immunization with ZP3 B-cell epitopes separated from oophoritogenic T-cell epitopes caused fertility to be blocked, yet not accompanied by ovarian pathology (Kumar and col., 2014).
Bitch ZP3 recombinant glycoprotein associated with diphtheria toxin (DT) was used for inducing infertility. In order to prevent ZP3 conjugating to a carrier protein (which could cause differences among the batches of vaccine yielded) and produce recombinant proteins with no affinity tag peptides sequences, Shrestha and col. (2015) advanced the concept of generating a synthesis protein with no tag containing masked epitope for T-cells expressed on tetanus toxin (TT) as well as of a canine ZP3 ectodomain, in residues 23 to 348. Thus recombinant protein TT-KK-ZP3 resulted, to be used in contraception.
The optimized synthetic gene was designed for encoding T-cell epitopes masked on tetanus toxin (TT) as 830 to 844 amino acids residues, followed by dilysine (KK) binding, and dog ZP3 ectodomain, amino acids residues 23 to 348. Synthetic gene was flanked by restriction sites Nde1 and BamH1 for 5` and 3` respective ends. Stop codon (TAA) was set upstream BamH1, in order to prevent concomitant of His6-tag expression.
Immunization protocols considered both administration procedure (to determine which of the immune responses – mucosal, or systemic – is more efficient) and the number of administrations.
Use of CpG nucleotides sequences, which, when not methylated, act as immunostimulant, activated immune response. Hence, the number of administrations required for generating an efficient immune response was low.
Native, or recombinant, ZP pathogenic effects are induced by presence of T-cell epitopes in peptide-based skeleton of such molecules; hence, determining epitopes for B-cell permitted using a series of formulas where ZP was bound to antigenic helper T cell clones. Such usage neither triggered pathological aspects, nor generated contraceptive effects (McLaughlin and col., 2003). Consequently, a high antibodies titer being required for reaching target advanced, the idea emerged that ZP may not be the ideal candidate for developing a fertility control vaccine.
Hence, investigations focused on the male, several candidates being considered: SP-17, PH-20 and PH-30/fertiline, lactate dehydrogenase (LDH-C4), SP-10, FA-1, SOB-2, and CD-52. All of such candidates involve the spermatozoon (directly or indirectly), spermatozoon membranes or metabolism thereof. SP-56 is the male counterpart of ZP3, while antibodies generated against SP-56 inhibit interaction oocyte-spermatozoon in vitro. SP-56 is an intra-acrosomal protein which cannot express on cell surface, thus pushing even farther demonstration of contraceptive effect in vivo. Integrins figure among the best decoded biomarkers of spermatozoon-oocyte interaction. Integrins are a heterodimeric cell adhesion molecules family, which promote intercellular interactions, as well as interactions between cell and extracellular matrix. The 18 alpha subunits and the 8 beta subunits found up to now, compound and generate 24 different type integrins. Many such integrins were able to recognize tripeptide sequence arginine – glycine – aspartic acid (RGD). Subsequently, integrins bind to specific motifs in extracellular matrix, and later play a role in cell migration.
Integrins are involved in fertilization process (Goncalves and col., 2013). Oocytes and the spermatozoa express a large number of integrins, as well as molecules which contain integrin recognition sites. Integrins are expressed as active or inactive.
Integrin-mediated spermatozoon-oocyte interaction was proven by presence of subunits alpha6, alpha4, alpha2, alphaV and beta3 on plasma membranes of bovine oocytes surface; and reiterated by capacity of RGD-peptide integrins to modify intracellular Ca level. Integrin alpha6 is part of the bovine oocyte.
Samoilova and col. (2010) devised a procedure for determining beech peptides (by selection in peptide library), which were set in contact with intact oocytes surrounded by ZP proteins, for the purpose of determining the spermatozoon antigens binding to ZP and enhancing antisperm antibodies
Foreign oligonucleotides translation inserted into beech DNA yielded peptides sequence which bound to ZP. Peptides synthesized subsequently, which manifested characteristics required for binding to ZP, were administered mediated by a carrier protein. Immunogenic capacity thereof was proven, as well as capacity of generating dog antisperm antibodies.
Anterior head plasma membrane (APM) of spermatozoon is the first contact site with zona pellucida. Binding process intensity peaks post spermatozoa capacitation. Protein profiles generated by mass spectrometry of APM yielded by non-capacitated and capacitated spermatozoa, revealed that proteins binding to ZP were more intensely manifested in capacitated spermatozoa (Kongmanas and col., 2015). Proteomic analysis indicated that a number of the proteins with acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP (acrosome binding protein) were high molecular weight (HMW) protein complexes components of APM, among which zonadhesin was the most amply represented. A fraction of such acrosomal proteins was translated to capacitated acrosome-intact surface. Acrosomal proteins, especially zonadhesin, play a major role in the process of capacitation, enhancing initial spermatozoon binding to oocyte.
In 2012, Samoylova and col. have indicated an assembly of beech peptides that bind to ZP, mediated by epitopes which mimic the proteins of spermatozoon apt for fertilization. When administered to animals, such beech-peptides assemblies cause production of antisperm antibodies, with contraceptive characteristics. As beech antigens stimulate production of antisperm antibodies, the concept emerges that administration thereof could generated contraceptive effects.
Using IVF in pigs, Stovsky and col. (2004) proved the ubiquitin-proteasome signaling pathway to be involved in ZP fertilization and penetration. Ubiquitin induced post translational modification occurs by covalent link of the 76 de amino acids (AA) ubiquitin residue to a residue of lysine (K) in AA sublayer sequence, mediated by ubiquitin residue end, i.e. C-terminal glycine residue (G76). Monoubiquitination is used for a number of purposes, e.g. protein lysosomal degradation, membranes receptors endocytosis, as well as signals translation and transcription control. Polyubiquitination enhances docking of proteins to 26S proteasome. Proteasome specific inhibitors block penetration of mouse and sow ZP.
Inhibitive proteasome, as well as anti-proteasome antibodies, blocked ZP penetration by the boar spermatozoa, yet did not affect mobility thereof, neither affecting spermatozoon-oocyte binding, or acrosomal exocytosis.
A number of proteins on ZP outer surface modify post translation by ubiquitination – a mechanism similar to various ZP proteins glycolization types. While ZP3 and other ZP proteins are secreted mainly by the oocyte, mRNA for ZP3alfa is manifest in high concentrations in pig follicular fluid. Consecutively, ubiquitinated ZP proteins may be stored directly on ZP surface.
In terms of the above statements, we deem that, in the future, several aspects could be targeted, as further detailed.
Further research should accurately explain mechanisms of ZP proteins synthesis; also, determine a series of possible structural differences between ZP proteins synthesized by follicular cells, and proteins produced in oocytes, differences reflected either in protein sequence, or in glycolysis type.
Inhibition of spermatozoon-oocyte binding, as well as recognition of antibodies by integrin 6alpha subunit, proves integrin 6 alpha to be involved in spermatozoon-oocyte interaction, as well as in polyspermy prevention. Mammalian oocytes can block polyspermy, at both zona pellucida level, and at plasma membrane level. The ways in which plasma membranes can prevent polyspermy, are not fully determined.
Determination would be highly interesting, of a series of ZP-based contraceptive vaccines, by immunogens corresponding to B-cell epitopes, devoid of oophoritogenic T-cells epitopes, in order to get at oophoritis proof contraception.
Development and optimization of vaccines based on peptides binding to ZP by generating a series of different administration forms, such as peptides attaching to carrier proteins, as well as peptides using viral vectors, or beech vectors.
Penetration of ZP by spermatozoa during fertilization can be prevented by proteasome inhibitors and anti-proteasome antibodies. Acrosomal proteasome may represent an interesting target for contraception. 26S proteasome was targeted, as well as 20S proteasome contents, which may boost investigation of the stages of spermatozoon- zona pellucida interaction in IVF, and polyspermy management.
Chapter 3. IN VITRO FERTILIZATION AND CONNEX TECHNIQUES
Aspects approached in Chapter 3 focus preponderantly on oocyte and connex structures, as well as transformations occurring during fertilization; presentation is therefore in order, of aspects of folliculogenesis physiology and fertilization, so as a series of in vitro techniques basic details are indicated.
CONCEPT ANALYSIS AND EVOLUTION
Folliculogenesis and ovulation physiology
Folliculogenesis is a process responsible for development of ovulatory follicles and release of one mature oocyte or more, in a predetermined interval in females’ reproductive life. Entire bovine follicular growth process, from primordial follicle stage (50 to 60 µm) up to follicle de Graaf (15 to 20mm), lasts for around 180 days. Folliculogenesis can be basal (part), a process occurring in absence of gonadotropins; and tonic – a process requiring gonadotropins, occurring when cow follicle diameter is over 4 mm.
Follicular selection occurs by interfering (passively or actively) into provision of gonadotropins; and the dominant follicle prevails by preferential gonadotropins release and storage, as well as by comparatively higher sensitivity for gonadotropins.
Tertiary (antral) follicles manifest a cavity known as antrum. Such antrum is filled with follicular fluid. Tertiary follicles grow into a wide gaps junction, which allows transfer of nutrients and regulation signals in-between oocytes and granulosa cells.
Intensity of RNA oocytes synthesis is highly correlated with developmental stage thereof, as well as with the changes occured in nucleoli morphology. RNA synthesis activity gradually falls, as oocytes reach maximum development. Bovine oocytes, harvested from 0.5 to 3 mm follicles, actively synthesize RNA; whereas, in oocytes hosted by over 3 mm diameter follicles, activity decreases.
Hypophysial hormones FSH (folliculostimulant hormone) and LH (luteinizing hormone), whose secretion is regulated by feedback from hypothalamic gonadotropin-release hormone (GnRH), are equally involved in stimulation of follicular surge and dominance sustainment.
LH pulse frequency, going up to one per hour, causes the dominant follicle to produce maximum estradiol level, estradiol subsequently inducing gonadotropin discharge and ovulation.
Oocyte maturation
Oocyte maturation is a long term process, whereat oocytes acquire the intrinsic ability to sustain further development stages, so that, eventually, embryo genome is activated (Ferreira and col., 2009). Such process covers both cytoplasmic and nuclear maturation.
Nuclear maturation mainly covers chromosome segregation, while cytoplasmic maturation covers organelles redistribution, as well as storage of messenger RNA, proteins and transcription factors, all involved in assembly maturation process, and, also, fertilization and embryogenesis (Ferreira and col., 2009).
2.1. Cytoplasmic maturation
2.1.1. Organelles redistribution
Ultrastructural analysis proved that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and Golgi complex figure in different stances during transition from germinal vesicle stage, to metastage II (Ferreira and col., 2009).
Mitochondria
Activation of the predetermined metabolic pathways involved in proteins synthesis and phosphorylation is mandatory for cytoplasmic maturation. In such context, mitochondria plays a basic role, as a major metabolic mechanism component, responsible for providing the energy consumed over the maturation process. Mitochondrial movement in high energy consumption areas is basic for oocytes, and for embryonic blastomere, at critical times of cell cycle.
Ultrastructural analysis of bovine oocytes subjected to in vitro maturation indicated that mitochondria migrate from being somewhat peripheral, to appearing dispersed in all of the cytoplasm, post 12 to 18 culture hours. Such process is similar to in vivo, which implies a comparatively more perypheral distribution, before LH peak, a cortical formation resulting, distributed in bunches during the later stages of nuclear maturation; as well as a dispersed distribution, post polar body release, around 19 hours after LH peak.
As metaphase II is reached, mitochondria in bovine oocytes concomitantly with lipids droplets, will move to cell center. Also, the number of mitochondria present in cytoplasm varies based on cell development stage. During maturation, there are over 100 000 mithocondrial DNA (mtDNA) clones (Shoubridge and Wai, 2007, Tarazona and col., 2006).
Fig. 3.1 Schematic overview of cytoplasmic organelles distribution, during maturation, fertilization and bovine zygote formation. (Ferreira and col., 2009)
Zygotes entering early the first mitotic division stand better chances for reaching blastocyst stage. Analogy also holds with the comparatively more active oocytes. Mitochondrial activity may be considered, as well as amount of ATP released over development early stages, when determining bovine embryos competency. Low ATP level embryos in cytoplasm manifested slower development and less cells.
Ribosomes, Golgi complex and endoplasmic reticulum (ER)
Proteins synthesis is required for both oocytes maturation, and for zygote formation and early embryogenesis. An adequate amount of ribosomes must, eventually, result, for maturation. Ribosomes are synthesized by transcription of ribosomal RNA (rRNA) genes, by processing transcripts and by extra ribosomal proteins added to the two subunits thereof. Nucleolus is the formation site of ribosomal subunits; during oocyte growth and activation of the embryonic genome, nucleolus appears fibrogranular, indicating comparatively higher activity of ribosomal synthesis, and, implicitly, of proteins synthesis.
During meiosis metaphase I, proteins synthesis in oocytes is around three times more intense, than during germinal vesicle breakdown (GVBD) stage. When cell reaches metastage II, oocyte manifests a basal translation level of mRNA (RNA messenger). Possibly, absence of one functional nucleolus may lead to rRNA transcription block, or to ribosome production for mRNA translation. Presence of ribosomes is directly correlated with proteins synthesis during the most important development periods (Ferreira and col., 2009).
Dynamics of Golgi complex membranes, at maturation and fertilization time, requires thorough research. Bovine oocytes in germinal vesicle stage are known to contain fragments of Golgi complex, which are transformed into vesicles at germinal vesicle break-down time.
Endoplasmic reticulum membranes are physiologically active, interact with cytoskeleton and contain domains specialized for various functions. Among the known ER functions, there figure proteins packaging and degradation, lipid metabolism, nucleolus compartmenting, regulation of Ca²+ ions gradient and membranes synthesis. By storing and releasing Ca, such system plays a basic role in intracellular signalling. Ca2+ release by IP3 (inositol triphosphate) and by receptor inositol 1,4,5 trisphosphate (IP3R) was determined to be basic for oocyte activation, at fertilization time (Machaca, 2007).
ER is located in cortical areas and accumulates in 1 to 2 µm wide arrays in cytoplasm (outside vicinity of meiotic apparatus) during transit to metastage II (Ferreira and col., 2009) – (Fig. 3.1) .
System sensitivity against Ca release is amplified post maturation debut. During fertilization, penetration of spermatozoon into oocyte leads to marked Ca release from reticulum, followed by debut of embryonic development. During formation of second polar body, ER arrays start disintegrating 3 to 4 hours post insemination.
Cortical granules (GC)
Cortical granules are derived from Golgi complex. GC exocytosis involves cytoskeleton filaments, as well as protein complexes. With oocytes at germinal vesicle stage (GV-germinal vesicle), cortical granules are distributed in arrays in all of the cytoplasm. At end of maturation time, when such oocytes are in metaphase II (MII), granules are distributed all of over inner surface, close to plasma membranes, in a strategic pattern enhancing spermatozoon penetration of oocyte, and further activation of the latter. (Ferreira and col., 2009). (Fig. 3.1)
Cortical granules are organites present exclusively in oocytes, composition thereof containing a diverse population of proteins, structural molecules, enzymes and glycosaminoglycans.
Cortical granules exocytosis (cortical reaction) is one of the most common polyspermy prevention mechanisms, used by oocyte (Coy and col., 2005).
If fertilization occurs by more than one spermatozoon, resulted zygote will be subjected to abnormal cleavage and will become unviable, degeneration starting at mitotic divisions debut. Blocking mechanism, based on fast modifications in oocyte extracellular matrix, is well preserved in animals. Such modification involves CG discharge on outer surface, post oocyte activation by Ca release from ER, in response to spermatozoon penetration into oocyte oolemma (Ferreira and col., 2009). (Fig. 3.1)
Data available indicate that, with mammalian oocytes, cortical reaction is controlled by intracellular signals, involving activation of inositol phosphate cascade. Spermatozoon-oocyte fusion generates two secondary messengers, i.e. inositol 1, 4, 5 trisphosphate (IP3) and diacylglycerol (DAG).
IP3 is involved in Ca release, from intracellular deposits, when DAG activates C protein kinase, which triggers cortical granule exocytosis (Ferreira and col., 2009).
2.1.2. Cytoskeleton dynamics
Microtubules and microfilaments present in cytoplasm enhance such movement and act upon chromosomal segregation (Ferreira and col., 2009).
Fig. 3.2 Dynamics of cytoskeleton filaments during cytoplasmic and nuclear maturation of bovine oocytes A. Detail of meiotic spindle in metaphase I, and centriole/centrosome structure; B. Detail of meiotic spindle telophase I, where microtubules may be noted, among chromosomes sets (Ferreira and col., 2009)
Cytoskeleton filaments are dynamic and perfectly adaptable structures, involved in chromosomal meiosis and mitosis, in cell division being responsible for transport molecules and for organites inside the cell. The three type filaments in cytoskeleton are formed into subunits characteristic to each. Microtubules consist of globular compact tubulin subunits, whereas actin filaments are formed in globular compact actin subunits. Intermediate filaments are formed into elongated fibrous polypeptide subunits (Fan and Sun, 2004).
Such filaments play a major role in mechanical resistance mainly, in response to stress. The three type cytoskeletal polymers are sustained by weak non covalent interactions, which may associate or dissociate fast, not requiring covalent bonds or breaks. Of such three types, only microtubules are involved in cell organites movement. Microtubules subunits adhere to motile proteins, such as dynein, dynactin and kinesin, which bind to organite molecules and membranes, hence the movement along microtubules.
During oocyte developing stage GV, organites will reconfigure, in direct correlation with cytoskeleton distinct structuring into a mesh, wherein organites, bound to one membrane, move and take specific stances. When entering cellular cycle stage M (i.e.meiosis, with female gamete), microtubules formed as star arrays settle close to condensed chromatin, in bovine oocytes post GVBD. Moreover, during germinal vesicle (GV) transition to anaphase I, microfilaments and actin filaments are distributed into cortical area under oolemma, not binding to microtubules (Ferreira and col., 2009).
During metaphase I, microtubules are nucleated; whereas, by polymerization of tubuline in oocyte cytoplasm with centrosome, meiotic division spindle and metaphase plate are formed, where chromosomes are arranged in ecuatorial pattern (Fig. 3.2. A.). In metaphase I, metaphase plate is proportionally larger than plate formed during MII; during metaphase I, actin filaments are distributed abundently in the cortical area, but are absent along microtubules (Ferreira and col., 2009) (Fig.3.2).
As cell reaches anaphase I, chromosomes start separating, during such segregation a large part of microtubules arranging in-between the two chromosomes sets. Division spindle elongates, and a large amount of actin filaments surround the chromosomes (Fig. 3.2). In telophase I, microtubules in-between the two chromosomes sets form into a taper structure, manifesting one large end, and one narrower end (Fig.3B).
The large microtubule end binds to cell extrusion chromosome set, thus first polar body formation becoming final. The narrower end is associated with the set which will stay in oocyte and enter meiosis II, again forming metaphase plate (Li and col., 2005).
To conclude with, during oocyte meiosis, cytoskeletal structural system plays a basic role, granting that cell cytoplasm keeps dividing within secondary oocyte. Only a small part of cytoplasm will be extruded concomitantly with extrusion of polar globules (Ferreira and col., 2009). Cytoskeleton filaments dynamics correlates with acquisition of nuclear development competency, in bovine oocytes (Ferreira and col., 2009).
2.1.3. Molecular maturation
Molecular maturation covers transcription, storage and processing of maternal mRNA, which is stably stored and inactive up to translational recruiting. Polyadenylation is the main triggering mechanism of proteins translation, consisting in extra adenosine residues added to mRNA terminal part 30. Cell cycle regulators, proteins, cytoplasmic maturation markers and antioxidant enzyme system components will be transcripted during such stage, mainly. (Ferreira and col., 2009).
2.2. Nuclear maturation
Nuclear maturation occurs in stages as further detailed: germinal vesicle breakdown; chromosomes condensation; first meiotic spindle formation; extrusion of first polar body to perivitelline area (in-between oocyte surface and zona pellucida); stop second meiosis in metaphase (metaphase II).
Meiotic maturation stages are indicated in Table 3.1.
During maturation, oocytes develop competency for meiotic resumption, a process triggered by luteinizing hormone (LH). Germinal vesicle breaks down (GVBD – germinal vesicle brake-down stage) while chromosomes separate, as further detailed: one set is extruded to the first polar globule, the second set reconfiguring to form metaphase plate in metaphase II.
Meiosis is blocked at such stage, up to reactivation by spermatozoon. Consequently, the spermatozoon acts as basic factor of meiosis resumption. (Bilodeau-Goeseels and Magyara, 2012).
Table 3.1. Meiotic maturation phases, synopsis apud Karp, (2002)
3. PHYSIOLOGY OF FERTILIZATION
3.1 Fertilization stages
3.1.1 Penetration of cumulus oophorus
Cumulus oophorus is defined as a granular cells association (in granulosa) which surrounds oocyte during antral follicle stage. In response to preovulatory surges of gonadotropins, oocyte re-enters meiosis, while cumulus cells start producing hyaluronic acid, to be stored in intercellular spaces and stabilized by accessory proteins, a phenomenon known as cumulus expansion. Expanded cumulus exerts own functions, at three basic times, as further detailed:
before ovulation, causing oocyte maturation;
during ovulation, leading oocyte along oviduct;
post ovulation, partaking in control of penetration by the spermatozoa.
Abundant information is recorded on role of cumulus oophorus in oocyte maturation and ovulation; however, functions thereof in fertilization are only partly known. Interestingly, in a number of species (human included), cumulus layer denudation before insemination enhances low quality oocytes fertilization; whereas bovine and pig denuded oocytes before in vitro fertilization will impact negatively penetration of zona pellucida by spermatozoa and/or male pronuclei formation.
In order to fecundate the oocyte, capacitated spermatozoa must cut a pathway through cumulus layer. Only the spermatozoa which cross through cumulus layer, and are morpho-physiologically intact, can get in contact with zona pellucida, and, implicitly, act in fertilization.
Spermatozoon – zona pellucida interaction
Zona pellucida (ZP) is the extracellular matrix which incorporates oocyte even post fertilization, preimplantation embryos included. ZP sets a barrier between oolemma and corona radiata.
Structurally, ZP is made of glycoproteins (ZP1, ZP2, ZP3) onto which oligosaccharides (fructose and N- acetylglucosamine) are grafted
Zona pellucida thickness varies based on species, in bovine being 27 μm. By electron microscopy, at ZP outer surface level porous structures were noted, which, are smallest in bovine, than in any other species, e.g. in mouse and cat being large.
First stage of spermatozoon-zona pellucida interaction is spermatozoon binding; only capacitated spermatozoa will bind. ZP3 is the first binding site of capacitated spermatozoa. The spermatozoa bind to terminal sugars in oligosaccharides components of such glycoproteins. ZP3-α appears to contribute to species-specific binding.
Post capacitating, an exocytotic process occurs, known as acrosome reaction, characterized by spermatozoon plasma membranes fusing with acrosome outer membranes. By such fusion a series of membrane vesicles are formed, limited by the two membranes, vesiculation thus occurring.
When the spermatozoon penetrates ZP, membrane vesicles persist on oocyte surface, sperm cell undergoing so-called denuding. Acrosome reaction will grant the spermatozoon enzymatic equipment required in order to cross zona pellucida (Nir and col., 2009). The next stage is ZP penetration, by spermatozoon, along a slanting path.
Gametes fusion
Once past ZP, spermatozoon enters perivitelline space, later adheres to side plasma membranes of oocyte, turns immobile and next fusion of the two gametes occurs. Not much is known about molecular aspect of spermatozoon binding to oocyte, yet; however, some research does focus on function of fertilin, a dymeric glycoprotein, that binds to oocyte plasma membranes, being responsible for inducing fusion.
Oocyte activation
After the spermatozoon is attached, oocyte will undergo a series of metabolic and physiological modifications, known as oocyte activation, wherein Ca plays a major role. Complex of modifications undergone by oocyte during activation would cover as further detailed:
release of intracellular Ca;
exocytosis of cortical granules (acrosome homolog, in spermatozoon);
completion of oocyte 2nd meiotic division; extrusion of 2nd polar body;
modifications in cortical ovule characteristics;
zona pellucida reaction, i.e. ZP hardening, and sperm receptors break.
Pronuclear formation and migration
Penetration of spermatozoon into oocyte cytoplasm causes instant activation thereof. Oocyte undergoes second maturation division, forming mature ovule and also second polar body. Mature ovule nucleus is known as female pronucleus. Spermatozoon tail degenerates, while nucleus will further be known as male pronucleus.
Male pronucleus closes in to the female pronucleus, both subsequently reaching center position, where DNA replication occurs at such time, the two pronuclei undergoing amphimixis, i.e. fusion. Meiotic spindle forms in-between the two centrioli, whereon chromosomes settle. Formation of ovum, or zygote, is final act of fertilization, which, once completed, is followed by embryogenesis.
TOPIC DEFINITION AND CONTENTS
4. In vitro fertilization
In vitro fertilization (IVF) is a procedure covering oocytes harvesting from ovary, maturation and fertilization occurring under artificial conditions, embryo later resulting being inoculated to receptor females.
4.1. In vitro fertilization stages
There are numerous IVF protocols today, as each species gametes require specific differing conditions. However, the trend is continuous for ever better performing techniques, in order to obtain the highest possible number of transferrable embryos.
IVF stages are as further detailed:
harvesting of oocytes;
in vitro maturation of oocytes;
capacitating of the spermatozoa;
in vitro fertilization;
culture of embryos obtained.
4.1.1. Oocytes retrieval (OCR – ovum collection retrieval)
Such stage exerts major influence on quality embryo obtainment. Several approaches are known, as further detailed:
in vivo oocyte retrieval, i.e.:
by ultrasound-guided transvaginal harvesting;
harvesting ovaries from culled cows, by:
follicle aspiration;
slicing of ovaries;
both above techniques.
4.1.1.1. In vivo oocyte retrieval – ultrasound-guided transvaginal harvesting
For bovine, of all in vivo oocytes recovery techniques, ultrasound-guided harvesting is preferred (Ovum Pick-Up/OPU), a non invasive technique for oocyte harvesting from antral follicles. Such technique was initially devised to be used in IVF protocols for humans; yet, research made applicability thereof obvious, to bovine reproduction biotechnologies. In 1987, an OPU protocol was advanced in Denmark; and in 1988, the first bovine oocytes harvesting was run by a research team, in Germany. Statistics indicate techniques OPU+FIV to possibly obtain over 50 embryos per donor cow per year (Meiyu and col., 2013).
Such technique may be run in two ways: by hormonal stimulation, and in absence thereof. With no hormonal stimulation OPU protocol, oocytes recovery is run twice a week, thus yielding oocytes of the quality required for embryos obtainment. OPU Protocol with hormonal stimulation involves FSH administration, and, later, oocytes recovery (Meiyu and col., 2013).
4.1.1.2. Oocytes retrieval from culled cows’ ovaries
When oocytes are harvested from culled cows ovaries, two factors interfere with oocytes maturation rates, and, implicitly, the number of embryos obtained. Firstly, time factor, i.e. time delay inter-cullings, and transport duration from slaughterhouse to lab. Second factor is transport medium average temperature. Ovaries will be transported in sterile vessels, wherein 0.9 % salt solution containing 100 mg/l streptomycin is introduced, at 35ºC room temperature, in thermal insulated systems.
Follicular puncture
Such technique involves 2 to 8 mm follicles content aspiration, by 18 G needle syringes. There are numerous studies regarding correlation of follicle size and oocyte quality. In bovine, oocytes recovered from 2 to 5 mm follicles are better fit for development, than oocytes harvested in smaller follicles.
Oocytes manifest maximum development competency with 8 mm follicles.
Main advantages of such techniques are high operating speed and method simplicity. Contrariwise, oocytes being only 30 to 60% recoverable from punctured follicles is taken to be a major drawback.
Slicing of ovaries
Such technique implies using a device provided with two glass, 1.5 mm interdistanced, parallel plates.
Using both techniques consecutively
Such method involves harvesting oocytes by follicular puncture, later slicing the ovaries. By using such two techniques, a high rate COC is reached, in no way impairing oocytes quality.
4.2.1. Morphological assessment of oocyte quality; postharvest classification
Harvested oocytes differ widely in terms of quality; hence, selection and classification thereof is particularly important in embryos obtainment. Oocytes morphological characters are therefore assessed (such as aspect of zona pellucida, of cumulus cells, of cytoplasm) as well as morphocytometric sizing.
Table 3.2. Description of morphological assessment classes, for bovine complex oocyte – cumulus
Table 3.3. Assessment criterions for bovine oocytes quality
In the labs producing marketable bovine embryos, a very large number of oocytes are processed; hence a fast oocytes quality assessment system is required, so that such oocytes may reach the maturation medium as fast as possible. Hawk and Wall’s 1994 system is used for the purpose.
Table 3.4. Selection criterions for bovine oocytes
Fig. 3.3 COC classified: A- class I; B- class II; C- class III.
4.2.2 In vitro maturation of oocytes
Oocytes maturation is a process whereby oocytes develop intrinsic capacity for the next development stages, up to embryo stage.
Maturation process involved two complex, distinct events, i.e.:
nuclear maturation: germinal vesicle breakdown, chromosomes condensation, first meiotic spindle formation, extrusion of the first polar body in perivitelline space (in-between oocyte surface and zona pellucida), second meiotic division stop, in metaphase (metaphase II);
cytoplasmic maturation: cellular organites reconfiguration, cytoskeleton dynamics, molecular maturation (mRNA material transcription, storage and processing).
Mitochondrial structuring and metabolic continuous activity are required by cytoplasmic maturation and by meiosis resumption, affecting post fertilization development. Bovine oocytes undergo major relocation at maturation time, influenced by hormones and by the energy sublayers in maturation medium.
First visible sign of oocyte maturation is nuclear membranes break (GVBD – germinal vesicle breakdown), first polar globule (PB – polar body) manifesting, and formation of the second meiotic spindle. Even though changes at nucleus level may be easily traced, modifications associated with cytoplasmic maturation are less obvious.
In vivo follicular maturation occurs concomitantly with oocyte maturation. Shortly prior to ovulation, LH action triggers luteinizing process. Such process coincides with the rise of progesterone level and with release of hyaluronic acid secretion by cumulus cells, in response to LH secretion. Hyaluronic acid hydration will cause cumulus cells interspacing to widen, and next expansion thereof. Hence, bovine oocytes maturation level assessment may be based on cumulus cells expansion level (Yan-Guang and col., 2007).
Table 3.5. Bovine oocytes assessment post maturation: classification based on cumulus cells expansion degree
Fig. 3.4 COCs post maturation: A- class I; B- class II; C- class III.
4.2.2.1. Culture technique for in vitro oocytes maturation
In vitro maturation can be run by culture in microdroplets.
Microdroplet culture is the most common maturation technique, involving culture of a small number of oocytes (10 to 15) in 50 μl medium. Oocytes are translated, in microdroplets, on plate, later covered by mineral oil (Miller and col., 1994). Advantages of microdroplet culture are as further detailed: easy monitoring; cutting on toxic medium substances; preventing evaporation and pH control; preventing temperature variations during handling and during assessment outside incubator; adequate for embryos culture as well.
Maturation media main component is a basic medium, to which hormones are added. Basic media must grant optimal pH for the oocytes to develop, so sodium bicarbonate is used to such purpose, targeting a 7.45 pH.
Besides pH, such medium must also grant an energy substrate, which oocytes require during maturation. Cumulus cells use glucose, to metabolize in pyruvate, which oocyte later uses in nuclear maturation. Lactate is one more major compound in oocyte development process. A 2003 study made it obvious that, in bovine, during lactate production, maturation stayed constant, carbohydrates consumption stayed level, while pyruvate consumption rose. Similarly, a 2004 study notes that, for lack of pyruvate, maturation rate results low.
Like any other live cell, an oocyte is sensitive to action of reactive oxygen species (ROS) which may induce noxious chemical reactions (e.g. lipids, or protein, peroxidation). In order to prevent such reactions, medium is used that contains antioxidant molecules (mercaptoethanol, cysteine, proteins, and vitamins A, C, E), chelating agents (EDTA, taurine, hypotaurine, and transferine) or antioxidant enzymes (superoxide dismutase, catalasis).
Basic media are supplemented with surfactant agents (BSA-bovine serum albumin), hormones (FSH, LH, and estradiol), growth factors (FCS – fetal calf serum, ECS – estrus cow serum, EGF – epidermal growth factor). Such supplements enhance obtainment of high rate maturated oocytes. Also, a penicillin-streptomycin mix is used for antimicrobial effect. The most common in vitro maturation medium for bovine oocytes would be as further detailed:
TCM 199 ( Tissue Culture Media – 199)
MEM (Minimum Essential Media)
Waymouth
Ham- F12
Maturation plates are introduced into incubator, CO2 kept at 38.5 to 39ºC, for 22 to 24 hours.
4.3. Role played by cumulus oophorus cells. Bovine oocytes classification post harvest, based on morphological assessment of quality thereof.
Communication between oocyte and surrounding cumulus cells is particularly important, firstly for sustaining oocytes blocked in meiosis prophase I, and secondly, for causing oocytes to resume meiosis at ovulation time. Communication will be by joint gaps (Boni, 2012).
Functionally, oocytes quality may correlate with development competency thereof. Sirard and col., (2006) quoted by Boni (Boni, 2012) describe five levels for oocyte competency development, i.e.:
ability to rerume meiosis;
ability to multiply post fertilization;
ability to develop up to blastocyst stage;
ability to induce and sustain pregnancy, to final term;
ability to develop soundly, up to final term.
Ideal oocytes are identified based on: morphological criterions (cumulus cells layers, cytoplasmic granulation, granular area location); biochemical criterions (adequate lipid contents, stored especially as cytoplasmic lipids droplets); molecular criterions (mRNA, proteins – required for embryonic development); biological criterions (based on ovarian-follicular microclimate, maternal signals mediated by granulosa and cumulus cells); metabolomic criterions (based on follicular fluid and culture media contents – intermediate glycolytic products and amino acids sustaining oocyte development); cellular criterions (intrinsic markers – mitochondrial status, activity of glucose-6-phosphate dehydrogenase 1; extrinsic markers–follicular cell apoptosis, and TGF-beta superfamily level, in follicular fluid or serum).
Most of the oocytes assessment criterions are based on morphological assessment thereof, yet morphological and functional criterions do not always coincide (Bon i, 2012).
Classification of cumulus-oocyte cells complex may consider factors as further detailed:
presence of cumulus oophorus cells, light color, compact layer, ooplasm transparent.
dark color compact cumulus, and dark color ooplasm.
cumulus extended and dark; ooplasm dark color.
One more classification type, of cumulus-oocyte complex, on 40x microscope lens, would be as further detailed:
Degree I groups cumulus-oocyte complex, with cytoplasm even darker (medium brown) and germinal vesicle located eccentrically, and identifiable. Cumulus cells layers five or more.
Degree II groups cumulus-oocyte complex, cytoplasm even darker (medium brown) and germinal vesicle identifiable, located eccentrically. Cumulus cells layers less than five.
Degrees III and IV group cumulus-oocyte complex. In advancing cytoplasmic degradation stages, as indicated by transparence or mosaic sequencing, and part, or total, loss, of cumulus oophorus cells layers.
A fast assessment method of oocytes quality, useful for granting instant distribution of oocytes in maturation medium, is as further indicated (Fig. 3.5).
Good quality oocyte – cumulus compact, intact, manifesting several cumulus cells layers. Cytoplasm aspect dense, even, fine granulation.
Intermediate quality oocyte – cumulus granulosa thick, adherent, cytoplasm unclear, manifesting several cumulus cells layers, totally or partly covering zona pellucida. Cytoplasm aspect varies from even, dense, fine granulation, to moderate size granulation.
Inferior quality oocyte – cumulus expanded partly or totally, tends to disperse. Oocytes manifest no cumulus layers (denuded), very small or very large, cumulus dark or quite light. Granulosa aspect uneven, dark spots alternating with light. Unusually shaped oocytes.
Fig. 3.5. COC classified: A- class I. B- class II. C- class III. (Zarcula Marc and col., 2012)
4.4. Oocyte maturation techniques, post harvest
Extrusion of oocytes completely developed in follicles before gonadotropins surge, as well as and culture in culture medium, allow for resumption of meiosis, and first in vitro meiotic division becoming final. Extra gonadotropins and steroids in maturation medium are yet required, for stimulating extrafollicular oocytes in vitro physiological maturation, generating higher potential for fertilization and embryonic development.
Oocytes released from origin follicle persist in germinal vesicle stage (GV), which is first meiotic division diplotene, at maturation process debut. Cultured bovine oocytes go through germinal vesicle breakdown stage (GVBD) after a 10 hour maturation. First meiotic division comes next, and metaphase II, reached 20 to 24 hours maturation. Stages are seen as per Fig. 3.6.
GV GVBD Metaphase II
Fig. 3.6 Oocyte maturation
One of the basic aspects of oocytes maturation in culture medium is pH, as implicitly affecting oocyte intracellular pH. Useful maturation media are, among others TCM199, alpha MEM, SOF, and HAM F10.
Most commonly, in vitro maturation media for bovine oocytes are Ham F10 and M199, composition thereof being complex. To such maturation media, fetal calf serum (FCS) or estrus cow serum (ECS) may be added, as well as gonadotropins (FSH/LH) and steroids (17ß estradiol). Maturation medium amount used varies 0.6 to 1 ml, respectively 10 to 100 µl. As nutritional support, granulosa cells in small or preovulatory follicles, bovine oviduct epithelial cells, and special cell lines, can be used.
Culture media for oocytes maturation must contain distinct additives. In bovine oocytes, a major role is played by glutamine, mixed with glucose or lactose, as enhancing development competency. Optimal glutamine metabolism requires COC (complex oocyte-cumulus) to be intact, as boosted in presence of LH. Glutamine plays a basic role in oocyte maturation, as also does glucose for development, metabolized by COCs, yet neither by denuded acolytes, nor by cumulus cells as such. Protein supplements are serum and albumin-based preparations. (Gardner and col., 2009).
Cellular modifications generated by oxygen reactive species are highly detrimental for normal cell development. Glutathione (GSH) is a major component of non-protein sulfhydryl in mammalian cells, protecting cells against oxydative stress. GSH synthesis during oocyte maturation was proven to occur in bovine as well. Glutathione is synthesized by gamma-glutamyl cycle, where such synthesis depends on presence of cysteine in medium, which is unanimously accepted to play a major role in cytoplasmic maturation. Measurement performed on bovine GSH, post in vitro maturation, may be an index for cytoplasmic maturation. TCM 199 is a mix of 0.6mmol/l and 83.2 mmol/l cysteine; however, cysteine could not be proven to cause auto oxidation. Cysteine generated by auto oxidation may be converted into cumulus cell cysteine, to be later incorporated into GSH synthesis.
Extra cysteamine, cysteine, and β- mercaptoethanol in maturation medium, boost GSH synthesis, in in vitro maturated bovine oocytes (De Matos and col., 1997, Gardner and col., 2009).
Oocyte culture will be run with higher oxygen levels in vitro, than in vivo; hence larger amount reactive oxygen species (ROS) result, such as superoxide anion, hydrogen peroxide and radical hydroxyl. ROS may lead to cell death, playing a role in oocyte meiotic stagnation, as well as in embryonic development stagnation. Effect thereof can be annihilated by GSH, in bovine oocytes.
Gonadotropins normally added into culture medium are FSH (follicular stimulation hormone) and LH (luteinizing hormone), and frequently estradiol (E2) as well. In vitro maturation of bovine oocytes in presence of FSH inhibits nuclear maturation by AMP-mediated pathway. However, basic role FSH plays is boosting fertilization capacity, as well as bovine oocyte development capacity. Gardner and col. (2009) indicate that 0.5 UI/ml extra LH does not led to major modifications of results yielded in oocytes maturation.
Other oocyte maturation major factors are as further detailed: insulin-like growth factor, epidermal growth factor, and somatotropic hormone (boosting nuclear maturation), to which maturation media may be added (Gardner and col., 2009).
Culture gaseous atmosphere is basic for in vitro maturation of oocytes. Normally, for in vitro maturation, CO2 level in air is 5%. Oxygen level is not controlled, but would be close to atmosphere air, i.e. 20%. Oxygen level in female reproductive tract is 5%. Hashimoto and col. (2000) indicate that 5% oxygen level is beneficial to bovine oocytes, an aspect also mentioned by Leivas and col. (2006).
Culture proper will be in microdroplets, 10 to15 oocytes being introduced in 50 µl culture medium. Plate will be later covered by mineral oil, such culture method blocking oxygen access to surface.
Staining techniques for oocyte maturation efficiency assessment
Assisted reproduction techniques involve protocols for in vitro oocytes maturation, efficiency thereof varying with culture media factors and oocytes quality. Meiotic competence development, and expression thereof, vary directly with oocyte growth change, chromatin structure in germinal vesicle, and transcriptional activity in oocytes.
Assessment of nuclear maturation in oocytes, at various time delays, makes it possible to quantify efficiency of in vitro maturation protocols. Ooplasm contains dark lipid fragments, which prevent visualization of nuclear material (Prentice-Biensch and col., 2012).
During maturation, oocytes undergo changes of nuclear status, by stages, starting with meiosis diplotene prophase I, up to metaphase II, when first polar body is extruded. Viewing oocyte chromosomes can yield highly accurate data, at such stage, as regards definition of in vitro maturation progress (Mehmood and col., 2011).
Two assessment reading variants are accepted, of bovine oocyte nuclear maturation, i.e.: contrast microscopy staining and assessment; as well as UV microscopy staining and assessment; hence, staining dyes must be selected that are consistent with assessment type, considering the effects such dyes induces in oocyte.
4.4.1. Aceo-orcein staining
Aceto-orcein staining is an assessment method of oocytes nuclear maturation, in vitro culture being currently used. Substantial oocytes loss occurs during staining procedure, due to penetration of staining dye in-between glass plate and colored glass plate, as also due to washing solutions; also, a large number de oocytes cannot be classified, on account of ambiguous morphology, or of staining problems (Prentice-Biensch and col., 2012).
Mechanism of acetic orcein action is not known in detail. Stain is actually made of a series of phenazones, which probably interact with acid pH in negatively charged groups, or else in hydrophobic interaction with chromatin.
The fact must be considered that oocytes stained with acetico-orcein lose viability and are no longer apt for fertilization in vitro and embryos obtainment.
Post maturation, bound oocytes are stained with different concentrations orcein. Space in-between glass plate and plate colored with glycerol-acid-acetic, 1:1, will be washed by capillarity. Assessment will be on contrast phase microscope, assessing nuclear maturation phase (Fig. 3.7).
Germinal vesicle stages will be identified (distinct oocyte nuclear membranes, no detectable chromatin), germinal vesicle sequencing, metaphase I (highly stained chromatin, at mitotic spindle level), telophase I or metaphase II (highly stained chromatin, in presence of polar body).
One more drawback of acetic-orcein staining is the limited number de oocytes which may be set on glass plate for one examination (Mehmood and col., 2011, Prentice-Biensch and col., 2012).
Oocytes in telophase I and metaphase II, are taken to be mature.
Fig. 3.7 Bovine oocytes nuclear maturation stages, aceto-orcein stained.
a. GV (germinal vesicle), b. Condensed (Condensation), c. Metaphase I (MI), d. Anaphase I (A), e. Telophase I (TI), f. Metaphase II (MII) and polar body I (Pb) in perivitelline space.
4.4.2. Hoechst 33342 reactive staining
First polar globule (body) being extruded is not enough a criterion, for assessment of oocytes accomplished nuclear maturation. The certitude must exist that oocyte, after a specific time delay in maturation medium, not only extrudes the first polar globule, but also aligns along secondary metaphase plate, at around two hours post extrusion of the first polar globule (Gardner and col., 2009) (Fig. 3.8).
A satisfactory assessment variant, of nuclear maturation and of abnormal modifications, is by determining oocyte meiosis, staining DNA with reactive Hoechst 33342, and UV visualizing nuclear stage (Gardner and col., 2009). Hoechst 33342 is also known as bisbenzimide (Chazotte, 2011). Staining dye mechanism involves binding to DNA areas rich in adenine-thymine, at minor groove point, where the two DNA strands come closest. By such binding, DNA fluorescence rises substantially. (Chazotte, 2011).
Fig. 3.8 Bovine oocytes nuclear maturation, stained with Hoechst 33342.
DNA was visualized in blue fluorescence: a. condensed nucleus; b. metaphase I; c. anaphase I; d. telophase I; e. metaphase II; asterisk for polar globule I; arrow for metaphase II plate (Roth and Hansen, 2005)
Staining dye can bind to all nucleic acids; however, with staining dye binding, fluorescence is enhanced twice as much at areas rich in adenine-thymine, as against areas rich in cytosine and guanine.
Advantage of staining dye Hoechst 33342 is membrane permeability, which makes such dye adequate for staining live cells, as manifesting toxicity acceptable for cells (Chazotte, 2011).
When first polar body is extruded, chromosomal plates yet not physically lined up (pro-metaphase II), or else randomly arranged chromosomes, or a metaphase II normal plate, become visible. If development is abnormal, a number of chromosomes will not be aligned on metaphase plate, or cytoplasmic oocyte chromosomes can even appear dislocated (Gardner and col., 2009).
With Hoechst 33342 staining, oocyte activation, too, can be determined. Thus, chromosomes will be noticed to accumulate in cytoplasm. Hoechst 33342 will detect presence of chromosomes in polar body, attesting chromosomes segregation, concomitant with polar body extrusion (Gardner and col., 2009).
UV microscopy reading will be at 350 to 461 nm wavelengths.
4.4.3. In vitro fertilization
Fertilization is a fusion process, of female and male gametes, ending in embryonic stadial development.
In vitro fertilization occurs in several stages, as further detailed:
seminal material preparation: capacitation and selection of mobile spermatozoa;
oocyte preparation: oocytes are transferred from maturation medium to fertilization medium.
Capacitation is a process whereat spermatozoa develop fecundancy capacity. Under natural conditions, potential fertile spermatozoa are selected at migration time, through female reproductive tract; whereas in assisted reproduction techniques, selection is artificial. Sperm separation procedures, by removal of seminal plasma from cytoprotective agents, diluents, detritus and potential infectious agents, as well as increased progressive motility and morphology selection, improve spermatozoa quality. Capacitation process is supposed to remove decapacitation factor, as glycoproteins present on sperm surface interfere with fertilizing and acrosome reaction. Consecutively, hydrolytic enzymes will be released, hyaluronidase and acrosin playing a role in penetration of oocyte zona pellucida. Such transformations occur in 1 hour, or 1 hour 30 minutes, at 38.50C in controlled 5% CO2 atmosphere air.
In the 1980's, a technique known as Swim-Up was developed, by which capacitated spermatozoa are obtained for IVF protocols. Such technique is supposed to use contents of one sperm sample, onto which a medium nutritious for sperms is added, such sample being later incubated at 38.5 to 39ºC for 60 minutes. Meanwhile, good motility spermatozoa will move towards medium.
Higher spermatozoa level is obtained when tubes slant by 45º. As an alternative to Swim Up technique, Percoll gradient solution can be used (colloidal suspension of silica particles coated by polyvinylpyrrolidone).
Such technique involves two Percoll solutions, concentration levels 45%, respectively 90%, onto which sperm sample is added; when centrifuged for 5 minutes, viable spermatozoa reach tube basis.
Research results indicate the differences between the two methods. Thus, Cesari and col. (2006) noted higher selection capacity of bull viable spermatozoa in Percoll medium, as against Swim-up; while Mehmood and col.(2011) obtained a better index for selected buffalo spermatozoa embryos, by Swim-up method, rather than Percoll.
Fig. 3.9 Swim UP Technique – work stages
For fertilization stage, oocytes are translated from maturation medium to fertilization medium. Fertilization media may be TALP (Tyroide Albumine Lactate Piruvate) or SOF (Synthetic Oviductal Fluid) to which, based on lab-specific protocols, various concentrations hypotaurine, epinephrine and heparin are added, thus enhancing spermatozoa motility.
Fig. 3.10. Percoll gradient selection of spermatozoa – aspects before and post centrifugation
Penicillin can be used as an antimicrobial. Oocytes and spermatozoa are added on a plate, then placed in an incubator, for 18 to 22 hours.
4.4.4. Culture embryos
In vitro growth of embryos is stage running from oocyte fertilization up to blastocyst stage. In vitro embryo growth medium is a factor affecting embryo quality and viability, particularly over the first 5 to 6 days, up to reaching compaction stage, such stage being basic for the selection of high quality embryos apt to survive deep freezing.
Macromolecular embryo culture medium plays a major role for such stage. Serum, or serum albumin, in in vitro culture media, prevents embryos adhering to glass or plastic surfaces, binds to heavy metals ions and sustains pH medium; bovine serum albumin (BSA) plays a major role in acrosome reaction, too.
Presence of serum in medium is associated with lipid accumulations, mitochondrial impairment, and with low survival rates post-thaw. Major drawback, as using natural protein mixes, is the possibility for the culture medium to get contaminated with pathogenic organisms; also, the wide variability of proteins levels is a problem for reproducibility of results.
Serum contains amino acids, carbohydrates, lipids, inorganic salts, vitamins, hormones, and growth factors boosting/inhibiting oocyte maturation and embryonic development.
Simple serum culture determined block of embryos development in stage 8 to16 cells. Increased number was noted, of lipid droplets in trophectoderm cells in serum-supplemented medium embryos, which also explains embryos sensitivity to deep freezing/thawing. An alternative to serum-enriched media are synthetic media.
Synthetic media, for in vitro embryo fertilization and culture up to blastocyst stage, are a mix of synthetic polymers, such as poly vinyl alcohol (PVA) or poly vinyl pyrrolidone (PVP), which may act as surfactants. Presence of proteins in cumulus is sufficient for capacitating spermatozoa, and for binding thereof to zona pellucida, in hamster, mouse, and humans. Using synthetic media in I VFtechnique in bovine (e.g.: media IVD101, IVM101, and SOF) generated satisfactory results.
Using media with neither fetal serum in bovine, nor albumin bovine serum, for oocytes maturation and embryos culture, also manifested beneficial effects upon blasocyst viability post-thaw. Also, no serum, or low level serum, in culture medium over the first 72 de hours post fertilization, caused more enhanced development of embryos 168 hours post fertilization; however, such embryos manifested lower morphological quality, as against embryos produced entirely in presence of serum.
One more advantage of embryos synthetic culture media, is the comparatively lower occurrence of LOS (Large Offspring Syndrome), results, however, varying. Characteristics of LOS syndrome are as further detailed: dystocic parturition, modifications of development organs; malformations of skeletal muscle; metabolic energy abnormality; high perinatal mortality; increased placental weight due to placental tissue changes; all accompanied by altered expression of a series of genes. To conclude with, media used in all of the in vitro bovine fertilization stages markedly impact embryos growth and development.
Code for stages of bovine embryonic development (Fig 3.11)
1 – non fertilized oocytes;
2 – 2 to 12 cell embryos;
3 – early morula: over 16 cell embryo, clearly outlined, days 5 to 6;
4 – compacted morula: embryo cells compacted into an almost indistinct cell mass, day 6;
5 – early blastocyst: embryo manifests blastocoel cavity less than half the embryo volume, day 7;
6 – blastocyst: embryo manifests wide blastocoel cavity, does not take all of the space delimited by zona pellucida, perivitelline space visible, days 7 to 8;
7 – expanded blastocyst: embryo large enough to take the whole space delimited by ZP, perivitelline space indistinct, ZP thin due to embryo expansion, embryo in rest/ collapse stage, part or total loss of blastocoel fluid, days 8 to 9;
8 – eclosed blastocyst: embryonic stage 7, embryo out of ZP, blastocoel cavity collapsed, no fluid, embryo contour ragged; dark area – the embryoblast; light area – blastocoel cavity collapsed; days 9 to10;
9 – eclosed expanded blastocyst: embryonic stage 8, re-expanded; can be globular or elongated, wide diameter, still elongates as evolves.
Fig. 3.11 Bovine embryonic development stages
Criterions for morphological assessment of embryo
Zona pellucida integrity
regularity of embryo shape and contour;
rate of intact (live) cells;
presence of cells extruded (to perivitelline space);
rate of differing size cells;
cell granulation manifested;
integrity of blastomere membranes.
Classification of embryos by quoted morphology criterions
EXCELENT or GOOD (code 1)
pick up day embryonic development stage;
embryonic mass globular, symmetrical;
embryonic cells even sized, colored and textured;
85% cells intact, at the very least;
no extruded cells manifested;
ZP mooth, no concave surface spot, nor flat;
possibly flawed, e.g. scantily compacted, differing sizing.
Fig. 3.12 Embryonic stage 5, code 1 (Bó and Mapletoft, 2013)
SATISFACTORY (code 2)
embryo moderately uneven, in terms of embryonic mass shape, color, texture and cell sizing;
at least 50% cells viable, wanting in terms of shape (slightly uneven), extruded cells and cell death;
generally, development status belated by 1 to 2 days.
Fig. 3.13 Embryonic stage 5, code 2 (Bó and Mapletoft, 2013)
POOR (code 3)
embryos wanting in terms of embryonic shape outline;
embryonic cells differing sizes, colors, and densities;
at least 25% cells intact;
extruded cells manifest, also few vacuoles, few cellular fragments;
development status belated by over 2 days.
Fig. 3.14 Embryonic stage 4, code 3 (Bó and Mapletoft, 2013)
UNTRANSFERRABLE (code 4)
oocytes non fertilized, embryos degenerated;
major flaws: numerous extruded cells, various size fragmented cells, large and numerous vacuoles, ZP cracked.
Fig. 3.15 Embryonic stage 2, code 4 (Bó and Mapletoft, 2013)
By such techniques embryos go into deep freeze, or else get transferred to receptor cows.
CUMULUS OOPHORUS
Cumulus oophorus, also known as discus proligerus, is a group of (cumulus) cells surrounding oocyte, both in ovarian follicle and post ovulation.
Fig. 3.16 Sow cumulus oophorus expanded, post maturation
In antral follicle, cumulus oophorus can be thought of as an extension of granulosa membrane. The most intimate such cells layer is radiate crown (corona radiata). In response to preovulatory luteinizing hormone (LH) surge, oocyte resumes meiosis, and cumulus cells start producing hyaluronic acid (HA), which is stored in intercellular spaces, and stabilized by accessory proteins. Such process is known as cumulus oophorus, or cumulus cells, expansion (Zhuo and Kimata 2001).
5.1 Functions of cumulus oophorus
5.1.1 Functions of cumulus oophorus during oocyte maturation
In bovine, removing cumulus cells before in vitro maturation (IVM) comes against oocyte maturation. Consequently, cumulus cells are deemed to play a major role in oocyte maturation, as further detailed:
by sustaining ovule in meiotic arrest;
by contributing to meiosis resumption; and
by sustaining cytoplasmic maturation.
Such cumulus oophorus key functions during oocyte maturation are attributed to the complex mesh of gap junctions, being due to specific metabolizing capacity thereof.
5.1.1.1 Cumulus cells keep oocytes under meiotic arrest
Some 70 (80?:) years ago, the follicle was thought to be responsible for meiosis inhibition. In 1935, Pincus and Enzmann proved that mammalian oocytes removed from follicle medium spontaneously resume meiosis. Such breakthrough generated hypothesis that, in mammals, factors originating in follicle fluid, and/or follicle cells, inhibit meiotic oocyte maturation, by a paracrine way. Factor present in folliclular fluid controlling meiosis is actually one low molecular weight peptide which inhibits oocyte maturation, as described for swine, rat and hamster, plus linoleic acid playing the same role in bovine.
Directly transfer of substances, inter cumulus cells and ovule, is also important for oocyte growth and for sustaining meiosis inhibition. Such transfer is mediated by gap junctions allowing for cells to exchange ions and small molecules. A number of substances are supposed to be meiotic inhibitors, e.g. purines (hypoxanthine and adenosine) and cyclic AMP. Cumulus cells are proven to surely control meiosis, just like rat adenylate cyclase activator, forkolin – which stimulates cAMPsynthesis in cumulus-oocyte complex (COC), inhibits nuclear maturation of oocytes enclosed in cumulus (CEO), yet not of cumulus denuded oocytes (CDO). Bovine CEO are much more sensitive to meiosis-inhibiting factors secreted by theca cells, than CDO are. Accompanied by meiotic arrest, oocytes may require sustainment of optimal intracellular cAMP levels, in order to finalize cytoplasmic maturation (Motlik and col., 2000), which is possible only in CEO, as efficient communication is required between cumulus cells and oocyte during entire maturation time (Motlik and col., 2000) to boost oocyte growth and in vitro development.
5.1.1.2. Cumulus cells activate induced meiotic resumption
Only preovulatory follicles have enough LH receptors for the transmission of a signal inducing oocyte maturation. Luteinizing hormones receptors were found in bovine COCs (Baltar and col., 2000) as well as in rat cumulus granulosa cells. Follicle-stimulant hormone (FSH) and LH receptors expressed in mouse oocytes indicate that actions directly exerted by gonadotropins upon oocyte are possible. Various hypotheses were advanced, on the ways gonadotropins may stimulate meiotic resumption in follicle, as further detailed: (1) by direct action upon oocyte, of FSH and LH receptors; (2) by fall of intrafollicular level, thereby inhibiting oocyte maturation; (3) by triggering a signal inducing maturation, transmitted to granulosa cells prevailing in follicle; (4) by blocking, or breaking, gap junctions between cumulus cells and oocyte.
Mattioli and Barboni (2000) state that somatic cells read LH message and send a second messenger to oocyte, by two different mechanisms. Luteinizing hormone may act either upon follicle wall, inducing second messenger secreted by theca and /or granulose cells into follicle fluid; or directly upon gap junctional communication of cumulus cells with ovule.
Cumulus cells respond to LH by Ca level rise diffusing into oocyte by gap-junctions. Shortly after LH peaks, short-term cAMP increases in ovule. Cyclic AMP cannot be viewed as a universal ocyte maturation inhibitor. cAMP regulatory role is based on cAMP levels in cumulus cells and ovule. Bilodeau and col. (1993) suggested that cAMP increase in cumulus cells activates a signal stimulating oocyte maturation, such signal counteracting inhibitory action of high cAMP contents in oocyte. In a few hours of LH stimulation, cumulus cells plasma membranes progressively depolarize. Due to electric binding to such cells, ovules depolarize as well, which leads to second increase in intracellular Ca.
Cumulus-oocyte binding modifies during oocyte maturation. Loss of gap junctions in cumulus oophorus correlates with meiotic resumption in preovulatory follicles, probably by preventing outer cumulus cells meiosis inhibitory signals from reaching oocyte. During maturation second half time, cumulus cells and ovule cooperate, as limited to corona radiata, over which time outer cumulus cells decoupling occurs (Mattioli and Barboni, 2000). Such suppression, of part of gap junctions functions, is a prerequisite for cumulus cells expansion.
Contrariwise to above mentioned aspects, a study run in swine proved that, consecutively to cumulus expansion, cumulus-oocyte projections are disconnected in-between metaphase I and II, while connections cumulus-cumulus stay intact (Suzuki and col., 2000). Such contradictory statements are hard to harmonize, as selective shutting of part of gap junctions cannot be reached by gap junctions inhibitors, such as heptanol (Mori and col., 2000) or by gap junctions decoupling effect of glycyrrhetinic acid. Nevertheless, injecting a fluorescent staining dye, as well as lucifer yellow into in vitro matured pig cumulus-oocyte complexes (COCs) indicates that transfer of staining dye in ovules, at inner cumulus cells layers level, was not interrupted. Thus, Isobe and Terada, 2001, indicate that gap junction allowing for communication between radiate crown cells and oocyte stays intact post meiotic resumption.
Once cumulus cells importance in meiotic maturation is outlined, it may come as a surprise that CDOs, when in a culture medium, can undergo complete meiotic maturation spontaneously. Such is probably a passive oocyte reaction to vanishing of meiosis inhibitory substances.
5.1.1.3. Cumulus cells contribute to oocyte cytoplasmic maturation
Cumulus oophorus is particularly important in oocyte cytoplasmic maturation accomplishment, which is required in developmental capacity for supporting male pronucleus formation, monospermic fertilization, as well as early embryonic development. In bovine, cumulus cells enhance oocyte development, either by secretion of soluble factors which induce developmental competence, or by removal from medium of embryo developmental-suppressive components. Cumulus-oocyte junction must be kept functional, so oocyte growth and in vitro development can be boosted.
Metabolic and protective role of cumulus cells in cytoplasmic maturation is obvious. Cumulus cells lower cystine level in cysteine and boost cysteine absorbtion during IVM of bovine oocytes. As a result, bovine CEOs contain more intracellular glutathione (GSH) than CDOs do (Geshi and col., 2000). GSH stable levels in oocytes matured in vitro cause normally fertilized oocytes number to increase and develop up to blastocyst stage. Post spermatozoon penetration, GSH plays a sperm decondensation into oocyte role, concomitantly activating oocyte, and transforming spermatozoon head within oocyte, into a male pronucleus.
Glutathione also plays a major role in sustaining cellular redox balance, and in protecting cells against oxidative harmful effects. Pig cumulus cells play a critical role as protecting ovule against oxidative stress-induced apoptosis, by increasing GSH contents of ovules (Tatemoto and col., 2000).
Moreover, supplementation of precursors of GSH, such as cysteamine, or cysteine-rich medium, improved IVM efficiency of bovine CDOs, by increasing GSH contents in oocytes. Nevertheless, such approach did not succeed in enhancing developmental competence of pig CDOs. Cumulus cells can stimulate oocyte development, mediated by a series of intracellular pH or Ca changes (Mori and col., 2000).
Glucose is one more sublayer not easily metabolized by oocyte. Cumulus cells metabolize glucose to pyruvate or to intermediates in krebs cycle, which may be transferred to ovule. Consequently, CDOs manifest a high capacity for glucose metabolism, which CEOs do not. Pyruvate may support IVM of mouse origin CDOs, whereas glucose can do the same only when follicle cells are present in culture medium. Mouse cumulus cells may form pyruvate when incubated with glucose and lactate. Successful maturation, fertilization and development were possible when bovine CDOs matured in vitro in presence of pyruvate (Geshi and col., 2000).
One more not entirely explained metabolic effect of cumulus cells is oxygen consumption. Oxygen consumption in human, bovine and mice embryos was measured, by ultramicro fluorescence technique, and by autocorrelation electrode technique. Same techniques could be used for assessment of oxygen consumption by CEOs and CDOs, in order to determine if cumulus cells decreased oxygen tension in oocyte close proximity, and, if so, to determine if oxygen low tension can be proven beneficial for IVM of CDOs.
5.2.1. Cumulus oophorus functions during periovulatory period
During periovulatory period, cumulus cells synthesize a large HA quantity, which organize in-between cells, into a muco-elastico matrix. Such matrix enhances extrusion of oocyte at ovulation, traps oocyte into ciliated infundibular epithelia, next translated to fertilization site. In hamster, granules and filaments in extracellular matrix of cumulus are responsible for binding of oocyte to oviductal cilia. Opposum and other marsupial CDOs are efficiently transported from ovary to oviduct ampulla; which prompts the idea that cumulus oophorus may not always be basic for oocyte trapping.
5.2.2 Cumulus oophorus functions during fertilization
In order to fertilize ovule, capacitated spermatozoa must find a path through extracellular matrix of cumulus oophorum; which is possible by modifying matrix macromolecules structure, and by hyaluronidase activity of sperm protein PH 20, present on of mammalian spermatozoon plasma membranes (Cherr and col., 2001). In several eutherian mammals (manifesting placenta), cumulus oophorus is present in oviduct during fertilization.
Nevertheless, in a number of ruminants, such as ovine and bovine, cumulus oophorus is no more found in interval three to six hours post ovulation, probably being not present during fertilization either. Biochemical changes in cumulus cells, in oocyte close proximity, could generate a specific fluid microenvironment, favorable to in vivo fertilization.
In order to render in vitro matured bovine oocytes surface easily accessible to spermatozoa, a number of the cumulus layers, or all, may be removed prior to IVF, either by mechanical stripping in micropipets, or by vortexing with either a series of enzyme preparations, such as hyaluronidase, or by chemical agents such as sodium citrate.
Such approach is presently run in human IVF, especially because intracytoplasmic sperm injection (ICSI) is the most frequently used technique for treating severe male infertility disorders, unfortunately still causing low fertilization rates in bovine and pig IVF (Suzuki and col., 2000).
Beneficial effects of cumulus oophorus in fertilization can be explained in various ways: (a) by attracting, trapping and /or selecting spermatozoa; (b) by facilitating sperm capacitation, acrosome reaction and /or penetration; and (c) by preventing precocious hardening of zona pellucida.
5.2.2.1 Cumulus cells attract, trap and /or select spermatozoa
Cumulus oophorus could increase number de fecundable spermatozoa in oocyte proximity, by attracting and trapping spermatozoa (Table 3.6). Attracting cumulus oophorus sperm cells could involve a phenomenon similar to chemotaxis. Identification of human testicular receptors mediating sperm chemotaxis sustains the idea that chemical attraction is basic for reproduction. It was suggested that elements involved in ovulation, such as oocytes, cumulus oophorus and follicle fluid, stimulate transport of spermatozoa in oviduct; and that cumulus cells in oocyte proximity secrete a substance, attracting spermatozoa and generating an attractant gradient around cumulus cells.
Sperm chemo-attractants are secreted not only prior to ovulation within follicle, but also post oocyte maturation, outside mature oocyte and cumulus cell follicle. Chemoattractant role of cumulus oophorus is sustained by a study run in vitro in bovine, stating that spermatozoa migrate preferentially to medium containing COCs.
Cumulus oophorus could facilitate fertilization, acting as a sperm trap. Cumulus oophorus spermatozoa trapping may be caused by spermatozoa entanglement in HA (hyaluronic acid) matrix among cumulus cells.
Cumulus oophorus should be able to cause increased number of fecundable spermatozoa in oocyte proximity, a hypothesis apt to be investigated by assessing differences in polyspermy of CEOs (cumulus enclosed oocytes) and CDOs (cumulus denuded oocytes), as penetration of more spermatozoa is more likely to manifest when more spermatozoa are present in oocyte proximity during fertilization. Cumulus oophorus could exert a beneficial effect upon fertilization by selecting morphologically normal spermatozoa, by saving the uncapacitated spermatozoa, as well as by guiding hyperactivated spermatozoa to oocyte surface (Table 3.6).
Hyperactive spermatozoa may penetrate extracellular matrix of viscous cumulus cells more easily than the less motile spermatozoa, as hyperactivation confers a mechanical advantage to spermatozoa in oviduct. Such role of cumulus oophorus could be further investigated, by assessing number of spermatozoa hyperactivated around CEOs and CDOs, by computer-assisted sperm analysis.
Table 3.6 Role of cumulus oophorus in attracting, trapping and selecting spermatozoa.
Cumulus oophorus could generate a micromenvironment facilitating successful capacitation of spermatozoa, acrosome reaction, and /or penetration.
Cumulus cells may act directly upon spermatozoa, by secreting specific factors, or by modulating physical-chemical factors, such as oxygen tension and pH. Sperm penetration requires successful capacitation, sperm-zona pellucida binding, acrosome reaction, sperm-zona pellucida penetration, and sperm-oolemma fusion.
Improvement of spermatozoa capacitation and acrosome reaction are not the main functions of cumulus oophorus. Bovine spermatozoa are already capacitated when released from oviductal isthmus epithelium; while hamster spermatozoa may be capacitated completely in vivo only in presence of female genital tract secretion. Moreover, hamster spermatozoa which undergo acrosome reaction while passing through cumulus oophorus, remain bound to cumulus cells. Acrosome reaction is receptors mediated, directly regulated by agonists from zona pellucida, consequently does not occur within cumulus oophorus.
Cumulus cells produce steroids in vitro (Li and col., 2000). Progesterone may indice acrosome reaction of spermatozoa in humans, bovine, horse, hamster and mouse. Progesterone interacts with specific sperm-binding sites on sperm plasma membranes, inducing substantial increase of cytosolic Ca, which leads to membrane fusion.
A major part of extracellular matrix of expanded cumulus oophorus is made of glycosaminoglycans, such as HA, chondroitin sulfate, dermatan sulfate and heparan sulfate. Heparin may induce in vitro capacitation of bovine spermatozoa. Extracellular matrix of cumulus oophorus composed of HA mainly (Zhuo and Kimata, 2001) can be easily penetrated by spermatozoa, as specific proteins redistributed over acrosomal area during capacitation manifest activity of hyaluronidase.
Hyaluronic acid also binds to plasma membranes protein of spermatozoa, PH-20, resulting in spermatozoa with higher basal Ca level. Consequently, such spermatozoa are more receptive to acrosome induction post binding to zona pellucida (Cherr and col., 2001). In such terms, hyaluronic acid can be a candidate molecule for improvement of sperm capacitation. Moreover, inefficiency of conditioned medium COC in sustaining sperm penetration of bovine CDOs can be explained by HA remaining associated with cumulus cells, after removal of HA from conditioned medium. Other factors which could interfere with sperm capacitation, acrosome reaction and /or penetration, are oxygen tension and reactive oxygen species (ROS). Cumulus oophorus low oxygen levels could reduce chance toxicity of oxygen at atmospheric pressure, which spermatozoa are exposed to when fertilizing CDOs. Oxygen consumption results from generation of ROS. In humans, low levels ROS may boost a redox-regulated cAMP-mediated pathway, which plays a key role in inducing sperm capacitation.
5.2.2.2 Cumulus cells prevent precocious hardening of zona pellucida
Cumulus oophorus may influence fertilization rates of oocyte, by preventing precocious hardening of zona pellucida.
Zona pellucida of horse, rat, and mice oocyte hardens spontaneously when cultivated for IVM in medium not containing serum. Spontaneous hardening of zona pellucida in vitro, possibly followed by premature cortical granules exocytosis, causes rat and mouse oocytes to manifest resistance to sperm penetration. Also, an intact cumulus oophorus during IVM, as well as solubility of pig/mouse zona pellucida, strongly support fecundity rates. In bovine, it was suggested that precocious hardening of zona pellucida does not block penetration of the spermatozoa post IVM, in both CEOs and in CDOs.
5.3 Classification of in vitro matured oocytes
Of late, class I oocytes with homogeneous ovoplasma and cumulus cells compact, were proven to be in good health, and to manifest high developmental potential and capacity for being fertilized. Research (Antosik and col., 2010) compared oocytes classes II-IV with class I.
Table 3.7 Morphological classification of sow COCs, by cytoplasm and cumulus cells aspect (apud Antosik and col., 2010)
The former were described as manifesting low fecundity and developmental potential, due to a series of morphological deficiencies.
Oocytes morphology and quality are determined during ovogenesis and folliculogenesis, being accomplished at final maturation time. Incipient embryonic development is associated with morphology of oocyte-cumulus complex (COC). Correlation would be interesting, of oocytes morphology and protein contents thereof (a study in proteomics) in view of expressing capacity for fecundity.
In 2009, Alvarez and col. assessed imature oocytes quality, based on aspect of cumulus oophorus layer, intending to determine maturation competence of various subpopulations of oocyte-cumulus complex. Six subpopulations were found, as indicated in Fig. 3.17.
Incidence of live oocytes in such population changed markedly, as regards cumulus layer aspect, and only in class D were found oocytes in germinal vesicle stage.
Rates similar to metaphase II were found, in classes A1, A2, B1 and B2 post in vitro maturation, which suggests that nucleus can mature in vitro, in spite of cumulus cells layer varying with each class; while, on the other hand, cytoplasmic maturation rate was proven higher for COCs in class A1, which indicates marked dependence of cytoplasmic maturation on aspects of cumulus cells layer.
Fig. 3.17 Morphological classes of cumulus-oocyte complex – harvested in gilt ovaries (apud Alvarez and col., 2009)
A1 (cumulus dense), A2 (cumulus translucid), B1 (radiate crown), B2 (oocyte partly denuded), C (oocyte denuded) and D (dark layer cumulus oocyte).
Fig. 1.18 Types of cumulus cells expansions post in vitro maturation.
A: cumulus completely expanded. B :cumulus partly expanded. C: oocyte partly denuded. Scale: 100 μm (apud Alvarez and col., 2009)
In order to assess in vitro maturation, study considered several aspects, such as degree of cumulus expansion (only for classes A1 and A2).
Nuclear maturation of COC in classes A1 and A2 was classified based on cumulus cells expansion, post maturation, as indicated in Fig. 2.
In vitro maturation assessment of COCs in classes A1 and A2 was run considering aspects as further detailed:
a) completely expanded: very large, expanded, cumulus cells layer, rich intercellular matrix;
b) partly expanded: cumulus cells layer, less expanded, rich intercellular matrix;
c) partly denuded: only several cells (bound to oocyte) left in cumulus cells layer.
Study conclusion was that quality of COC morphological assessment comes useful for selection of oocytes competent for in vitro fertilization, and embryo production. Such aspect is worth further investigations, in terms of transcription products (transcriptomics) as well as of protein abundance (proteomics).
RESEARCH THEORY AND EXPERIMENT
6. Assessing in vitro maturation of bovine oocytes
Ovaries harvested in slaughterhouse were transported to lab in sterile vessels, containing 0.9% NaCl solution, 33˚C temperature granted. Slaughterhouse to lab transport of ovaries took maximum one hour and a half post harvest. Vessels containing ovaries were set in thermal insulated boxes, so temperature preset can be kept stable.
In vitro bovine oocytes maturation was run in TCM 199 basic medium, to which 10% ECS (estrous cow serum) was added.
On oocyte harvest day, wash and maturation plates were balanced for minimum 4 to 6 hours, in incubator, in CO2 medium, 38.5˚C, maximum humidity.
1% aceto-orcein solution preparation
Orcein is extracted from two lichen varieties, i.e. Rocella tinctoria and Lecanora parella. A synthesis variant is available, yet natural origin orcein is preferred for chromosomes analysis, as yielding better contrast. Orcein is used as 1% solution in 45% acetic acid.
Solution is prepared by pouring 55 ml glacial acetic acid, at boiling point, over 1g orcein powder. Solution is let cool, then 45 ml distilled water is added, and then solution is filtered. Such solution is unstable, so preparation shortly prior to usage is recommend. Aceto-orcein staining allows for determining nuclear maturation.
6.1. Harvesting of cumulus oocyte complexes
Cow cumulus-oocyte complexes (COCs) were harvested, by (2 to 8 mm) follicle puncture and oocyte aspiration, as well as ovaries slicing.
6.1.1. Harvesting by follicular puncture
COCs were harvested from ovaries of cows culled in the slaughterhouse; procedures were punction and aspiration of 2 to 8 mm follicles, by an 18 Gauge needle, attached to a 5 ml bulb-piston syringe, in order to prevent damage to oocytes while handling (Fig. 3.19).
Post puncture, follicular fluid was aspirated concomitantly with COCs existent within each follicle punctioned.
Fig. 3.19 Harvesting COCs in bovine ovaries by follicular puncture
Post harvest by puncture and aspiration of COCs with follicle fluid, such contents was introduced into a 50 ml tapered bottom tube, for sedimenting. After 5 minutes’ rest, COCs were aspirated from tube by means of a 3 ml pipet, a procedure repeated for 3 to 4 times, to make sure that all of the oocytes, and afferent cumulus cells, were aspirated. Next stage was washing the COCs, for which procedure the COCs were translated succesively to 4 plates, with enriched PBS medium, in preparation for maturation.
6.1.2. Classification of oocytes
During the last washing stage, oocytes were classified based on morphological criterions, into three quality categories. Characters considered, identifiable under stereo magnifying glass, are as further detailed:
aspect of zona pellucida
number cumulus oophorus cells layers, and arrangement thereof around oocyte
aspect of cytoplasm
perivitelline space
Ist Category. Oocyte must be compact, surrounded by minimally 3 cumulus cells layers, or more.
2nd Category. Cumulus cells compact, cytoplasm not clearly outlined, fewer cumulus cells layers, as against cells in 1st category; cells must cover all of the zona pellucida, or at least half.
3rd Category: Cumulus cells partly or completely expanded, cumulus dispersed, or oocytes denuded.
Fig. 3.20 COCs on plate, with washing medium: 1- 1st category COCs; 2- 2nd category COCs; 3- 3rd category COCs.
6.2. Oocyte maturation
After the four washing procedures, oocytes (classified in above three categories based on morphological aspect) were introduced into 400 µl TCM 199 enriched with 10% ECS. Maturation was run on plates quality 4 or 5, or glass cups (Fig. 3.21), maturation medium being covered with mineral oil, in order to prevent oxygen action upon oocytes.
Plates thus prepared were introduced in incubator for 24 hours, 38.5˚C and 5% CO2 level, maximum humidity. After 24 hours, cumulus cells expansion degree was examined on heat-plate stereo magnifying glass (Fig. 3.22), such expansion being the first characteristic manifesting maturation degree.
Fig. 3.21 Maturation plate
Fig. 3.22 Assessment of COCs maturation, based on cumulus expansion degree:
1- 1st degree maturation COCs; 2- 2nd degree maturation COCs; 3- 3rd degree maturation COCs.
6.2.1. Denudation of in vitro matured COCs
In vitro matured COCs are transferred onto 30 mm Petri dishes in PBS medium, and then placed on stereo magnifying glass plate. Next, under stereo magnifying glass control, borosilicate glass thinned Pasteur pipets are used (Fig. 3.23), maximum inner heat-thinned tip diameter to around 100 to 110 µm. Repeated aspiration and exhaustion are run, up to accurately denuded oocytes (Fig. 3.24).
Fig. 3.23 Pasteur thinned pipet; inside tip diameter 100 to 110 µm (white arrow); Pasteur pipet not thinned (blue arrow).
Fig. 3.24 Oocytes denuded: in red, totally denuded; in blue, partly denuded.
Denudation of matured COCs can also be run by other methods, such as: extra substances allowing denudation (trypsin, hyaluronidase); or vortexing matured COCs placed in TL-HEPES medium, for 3 minutes.
6.3. 1%aceto-orcein staining
Chemicals required for oocyte preparation for staining, and for staining-proper: PBS; acetic acid: ethanol 1:3; ethanol 70%; 1% aceto-orcein solution; glycerol: acetic acid: water 1:1:3
Lab-prepared TL-HEPES medium: 6.66g NaCL; 0.24g KCl; 0.168g NaHCO3; 0.0476g NaH2PO4 x H2O; 2.4 g HEPES (buffer solution); 1.86mL DL- lactic acid (sodium salt) (60% syrup); 1.0 mL red phenol (0.5% in DPBS); 0.3 g CaCl2x2H2O; 0.1 g MgCl2x6H2O; 3.0 g BSA.
Two lines are drawn in silica paste, 1.6 cm interdistanced, parallel to glass plate shorter edges, about mid plate. One PBS droplet is placed between the two lines drawn. 5 to 10 denuded oocytes are dripped onto PBS droplet. Droplets are carefully covered with 18×18 mm glass plate, pressed slightly, so that oocytes do not break. Glass plates thus prepared are immersed into acetic acid: ethanol 1:3 and kept overnight, so that binding occurs.
On examination day, oocytes are washed in 70% ethanol. Glass plates are set in 90 mm diameter Petri dishes. By 10 to 100 µl pipets, an amount of 1% aceto-orcein solution is aspired and dripped in successive droplets on one of the glass plate edges not obturated with silica paste. By capillarity, staining dye will enter under glass plate, evenly reaching all of the oocytes, and will be let act for 10 minutes. Next, staining dye is removed by a mix of glycerol: acetic acid: water 1:1:3, by same procedure as detailed for aceto-orcein solution application.
Glass plates yielded will be assessed by contrast microscopy (Fig.3.25).
Out of total 20 cow ovaries, 171 COC were harvested, average figures 8.55 COCs/ovary. Research run by Mermillod and col. (1992) indicates that one cow ovary can yield 5 to 10 COC. Zheng-Guang Wang and col., obtained, by puncture (as per a study, in 2007) an average 9.7 oocytes/ovary.
171 COCs were obtained by punction, as further detailed:
in medium supplemented with FSH: 34 COCs, 1st category; 36 COCs, 2nd category; and 30 COCs, 3rd category; post maturation, the yield was a total 29 COCs, 1st category; 27 COCs, 2nd category; and 12 COCs, 3rd category. (Table 3.8)
Fig. 3.25 Final aspect of glass plates with 1%aceto-orcein stained bovine oocytes
in medium not supplemented with FSH: 34 COCs, 1st category; 36 COCs, 2nd category; and 30 COCs, 3rd category; post maturation, the yield was a total 29 COCs 1st category, 27 COCs 2nd category, and 12 COCs 3rd category. (Table 3.9) fix aceleași cifre la ambele amândurora?
Table 3.8. Classification of bovine COCs, before and after maturation, in FSH supplemented medium
Table 3.9. Classification of bovine COCs before and post maturation, in medium with no FSH supplement
COCs quality had a positive impact on expansion rates of cumulus cells, a remark based on morphological aspects visualized post maturation. Administration of medium supplemented with FSH had no marked effect at such stage, an aspect indicated in chart 3.1. In 1st category (C1), 85.34% COCs indicated expansion of cumulus cells (p<0.76); 2nd category (C2), 75% (p<0.61) COCs indicated expansion of cumulus cells; while 3rd category (C3), 43.3% (p<0.03) COCs indicated expansion of cumulus cells.
Chart 3.1 Morphological classification of bovine COCs, before/post maturation in culture medium with/without FSH
In a study run in 2010, Kakkassery and col., figures indicated for COCs with expansion of cumulus cells, are as further detailed: 83.08% for 1st category, 68.29% for 2nd category, and 44.74% for 3rd category.
Similar results were reported for COC in no FSH cultures: 84.03% (p<0.27) for 1st category (C1), 68.06% (p<0.29) for 2nd category, and 33.28% (p<0.09) for 3rd category, COC manifesting cumulus cells expansion.
During 1% aceto-orcein staining, loss of denuded oocytes is limited, an aspect indicated in chart 3.2.
Chart 3.2 Number of oocytes stained vs. number of COCs denuded
Out of 101 COCs matured, by 1% aceto-orcein staining technique, 75 oocytes (74.25%) got stained, 25.75% oocytes loss being recorded during denudation, binding, and staining, respective stages.
In a study by Prentice-Biensch and col. (2012), 31% loss of bovine oocytes was also noted to occur during denudation, binding and staining.
Out of the 75 de oocytes efficiently stained and examined, data visualized as per chart 3.3 resulted.
Chart 3.3 Stages of oocytes maturation +/- FSH
A low oocyte maturation rate in stages MI and MII will be noted (Fig. 3.28. and Fig. 3.29.), based on gamete maturation marker, and no difference between oocytes groups in culture media with, respectively without, FSH. Of total oocytes stained, 7.14% and 9.52% were in MI, respectively in MII, in group FSH in medium; while in group with no FSH in medium, 6.06% and 9.09% were found in MI, respectively MII. Remaining oocytes were found to be at stages GV and GVBD (Fig. 3.26. and Fig. 3.27.). Thus, 54.76%, respectively 28.57% of oocytes matured in medium with FSH were in stage GV, respectively GVBD.
In a study run in 2010 by Kakkassery and col., results yielded for assessment of maturation stages of aceto-orcein stained bovine oocytes, were: 36% oocytes for 1st category; 16% for the 2nd category; and 10% for the stage MII.
Similar results were noted for the lot with no FSH in medium, as further detailed: 54.54% remained in stage GV; and 30.3%, in stage GVBD.
CONCLUSIONS
Presumed low reproduction capacity of culled cows is proven to have a bearing upon the biological material harvested, quality of oocytes thus yielded impacting maturation capacity thereof.
Extra FSH in medium does not interfere with bovine COC maturation. Degree of maturation is determined by assessment of female gamete nuclear stages, during division consecutive to staining by dyes manifesting affinity for nuclear acids.
Staining bovine oocytes with 1% aceto-orcein for assessment of nuclear maturation degree is a method easy to use; yet, by applying such method, oocytes bound and stained cannot be further used for IVF.
Analysis of nuclear maturation is insufficient for thoroughly assessing bovine oocytes maturation; cytoplasmic maturation will have to be considered for the purpose, as well.
7. In vitro fertilization
Assisted reproductive techniques cause obtainment of a large number of conception products. Progress in understanding physiological processes preceding fertilization made possible development of such techniques and standards setting for the stages basic in embryos obtainment. By simplicity and accuracy, in vitro fertilization gained ground and took the floor, as a third generation reproduction biotechniques.
Scope of our research covers determination of optimal methods (given equipment available) for obtainment of bovine embryos by in vitro fertilization.
Materials and methods
Medium required for ovaries transport was 0.9 % NaCl. Basic medium for harvest was PBS, whereas for maturation, basic TCM 199, supplemented with 10% ECS (estrous cow serum) was used. On oocytes harvesting day, washing and maturation plates were balanced for minimum 4 to 6 hours. Balancing is run in a CO2 incubator, at 38.50C, maximum humidity.
7.1. Spermatozoa capacitation and fertilization media
Sperm TALP medium was used for spermatozoa capacitation, and Fert TALP for fertilization medium. 24 hours before fertilization, 10 ml Sperm TALP in medium, and 10 ml Fert TALP in medium, were aspired in syringes and set in incubator, at 38.5 ºC, 5% , maximum humidity. On usage day, 2.2 mg Na pyruvate and 60 mg BSA were added to media. In medium Fert TALP there was also be added 100 µl gentamicin solution, 100 µl heparin solution and 360 µl hypotaurine-epinephrine solution.
Media Sperm TALP and Fert TALP were filtered sterile up to 0.2 µm and set onto 3 plates as further detailed:
plate 1: 4 x 200 µl droplets in Fert TALP (Wash) medium, for washing oocytes off maturation medium, next covered with mineral oil;
plate 2: 4 x 60 µl droplets in Fert TALP (Ready) medium, for oocytes fertilization, next covered with mineral oil;
plate 3: Sperm TALP medium
Plates were let balance in 5% medium, at 38.5 ºC, and maximum humidity, for 3 hours.
7.2. Embryo culture media
For embryos culture basic TCM 199 medium enriched with 20% ECS was used, let balance for 3 hours. Balancing was run before transfer from fertilization medium into fertilized COC culture medium.
COCs harvested by punction were introduced into a 50 ml tapered basis tube, to sediment. After 5 minutes’ rest, COCs were aspirated from tube basis, by a 3 ml pipet, repeating such procedure for 3 to 4 times, to make sure that all of the oocytes were aspirated. Next stage, COCs underwent washing, i.e. successive translation to 4 PBS enriched medium plates, in view of preparation for maturation.
During the last washing stages, oocytes were classified into three quality classes, based on characters found by stereo magnifying glass, i.e. zona pellucida aspect, cumulus oophorus, cytoplasm, and perivitelline space.
1st Class. Set of characteristics such as: oocyte compact, minimum 3 cumulus cells layers, or more;
2nd Class. Set of characteristics such as: thick, normally adhering to granulose, cytoplasm unclear; cumulus compact, few or more several layers, covering all of the zona pellucid, or at least half.
3rd Class. Set of characteristics such as: cumulus cells partly or completely expanded, cumulus dispersed; non-cellular structure with no cumulus cells.
7.3. Oocyte maturation
Oocytes maturation was run as per protocol described at 6.2.
7.4. Oocyte fertilization
Sperm was prepared on fertilization day. Sperm sample was retrieved from liquid nitrogen container and introduced in water, at 350C temperature, for one minute, next ousted, wiped dry, disinfected and wiped again.
Sperm sample contents is equally parted to be set at bottom of two 12 ml tapered basis tubes, later adding 1 ml Sperm-TALP solution.
Tubes thus prepared are set for an hour in incubator, at 38.50C, 5% CO2, 100% humidity, slanting, in order to facilitate spermatozoa reaching medium.
In one more hour, supernatant containing the live spermatozoa was placed into another 12 ml flacon, with 1 ml Sperm TALP medium, to be centrifuged for 10 minutes at 1000 rpm, to later remove supernatant (1 ml).
1 ml fresh Sperm-TALP medium was added onto live spermatozoa left, to be centrifuged again, for 10 minutes, at 1000 rpm. After second centrifuging, supernatant in tube was removed by aspiration, while over the live spermatozoa on tube basis, 40 µl fertilization medium (Fert-TALP) was added, blending by repeated pipeting.
While spermatozoa were left in tube on heating plate, matured COC plates were taken off incubator, then washed in Fert TALP medium (Wash).
After washing, matured oocytes were placed in Fert TALP medium (Ready), and classified based on maturation level. In a fertilization droplet, maximum 20 to 25 COCs were set, later adding 10 µl spermatozoa at a time, in tube left on heating plate.
Fertilization plates (COC-spermatozoa) were then incubated for 22 hours, at 38.50C temperature, 5% CO2, 100% humidity.
7.5. Embryos culture
22 hours post fertilization, all of the oocytes were aspired and set in culture medium for 24 hours, wherefrom were again incubated at 38.50C, 5% CO2, 100% humidity. Thus, after 24 hours, plate was ousted from incubator and examined in order to determine embryos degree of development.
Experiments yielded data as further detailed: out of a total of 52 ovaries, 285 COCs were harvested, 5.4 COC/ovary average figures resulting.
Out of one unique cow ovary, 5 to 10 COCs can be obtained. Various COC harvesting techniques were used, and puncture average figure results 9.7 oocytes/ovary; also, 9.6 oocytes/ovary, by slicing, and 5.8 COC/ovary by OPU technique.
Karadjole and col, in a study run in 2010, record (average figures) 6.5 COC/ovary by OPU technique.
Post ovary punction, we harvested 285 COCs, of which: 90 COCs 1st class (31.5 %); 84 COC 2nd class II (29.4 %); and 111 COCs 3rd class (38.9 %).
A study run in Kerala University, India (Kakkassery and col., 2010), indicates that 356 COCs harvested by puncture yielded 162 COCs 1st class, 116 COCs 2nd class, and 78 COCs 3rd class; while by OPU technique, out of total 288 COCs, the yield was 130 COCs 1st class, 82 COCs 2nd class, and 76 COCs 3rd class.
Out of the 285 COCs we harvested, 24 hours post incubation examination indicated 142 matured COCs (49.8 % ), out of which: 72 COCs 1st class (50.7 % ), 63 COCs 2nd class (44.3 % ), and 7 COCs 3rd class (7.7 % ) (Chart 3.4) .
Fig. 3.30. Embryos in various development stages. A- 2 cell embryos; B- 3 cell embryos.
Fig. 3.31. Embryos in various development stages: C- 4 cell embryo
Chart 3.4. Number of COCs before and post maturation, based on category.
Rate of matured COCs varied based on ovaries lots used. Thus, on 29.10.2013, out of the 33 COCs harvested from 10 ovaries, only 14 (42.4 %) underwent maturation. Similarly, on 30.10.2013, out of 74 COCs harvested from 12 ovaries, 14 matured COCs (18.9 %) were obtained. Yet, on 15.04.2014, out of 83 COCs harvested from 10 ovaries, 72 matured COCs (86.7 %) were obtained (Fig. 3.30).
When oocytes matured in TCM-199 medium supplemented with ECS, FCS or hormone components, maturation rates were comparatively much higher (80.4%, 78.3%, respectively 85.1%), against control group 51.9%.
In 2012, Khandoker and col. harvested, by puncture, 143 COCs from 71 buffalo cow ovaries, out of which 122 COCs (85.3 %) matured.
In co-culture matured COCs-spermatozoa there resulted 24 zygotes (33.3 %), 8 2-cell embryos (11.1 %), 1 3-cell embryo (1.3 %) and 2 4-cell embryos (2.7 %) Fig. 3.31.
During IVF 5th session, out of 72 COCs which underwent various development stages, there resulted 11 COCs, of which 8 2-blastomere embryos, one 3-blastomere embryo, and 2 4-blastomere embryos (Charts 3.5 and 3.6).
Chart 3.5. Number de embryos obtained out of matured COCs total
Chart 3.6. Number de embryos obtained, based on development degree.
In a study run by Clara Larroca and col (2012), after maturation medium was supplemented with FCS and bFF (bovine follicular fluid) yield was as further detailed: out of 181 COCs, post fertilization there resulted 79 embryos, of which 29 reached compact morula stage.
Conclusions
Ovaries retrieved from slaughterhouse contain a heterogeneous oocytes population, which is harvested regardless of follicular dynamics, whereas cow individual characteristics interfere with capacity for development of oocytes (age, physiological state, and breed).
Chances for success of IVF protocols require close to identical embryos maturation, fertilization and development re-created in vivo conditions.
One major role in IVF success is played by how skilled the lab staff are, who must master abilities like fast handling of cells, in quite small amounts of medium, cutting as much as possible on time spent as harvesting/ handling for incubation and beyond.
IVF protocol implementation is a long time process, depending on many factors, related to biological material used and available lab equipment, as well as to personnel expertise.
Sow oocytes culture and genes expression
Our research focused on sow oocytes viability, which we enhanced by cysteine supplemented in culture medium. In such terms, Bcl2 genes expression in sow cumulus cells before and post in vitro maturation was one more target, concomitant with morphological assessment of oocytes post maturation.
What is, actually, Bcl2? Bcl-2 (B cell lymphoma 2), encoded in humans by gene BCL2, is a founder member of regulating proteins Bcl-2 family, which have a bearing on cell death (apoptosis), either by induction (pro-apoptotic) or inhibition (anti-apoptotic) thereof. Bcl-2 is especially seen as a major anti-apoptotic protein, being thus classified as an oncogene. Bcl-2 reads as cell B of lymphoma 2, second member of a proteins gamut described initially in chromosomal translocations involving chromosomes 14 and 18 in follicular lymphoma.
Granulosa cells apoptosis is involved in ovarian folliculogenesis process (Jiang and col., 2003), which is basic for normal ovaries physiology, and plays a basic role in ovary cyclic function. In ovarian follicles, cumulus cells are tightly bound to oocyte, forming cumulus-oocyte complex (COC); also, cumulus cells play a critical role in oocyte maturation and further fertilization, by bi directional signals (Senbon and col., 2003). Prior research related to expression of cumulus cells genes (Mckenzie and col., 2004) and apoptosis thereof (Horn and col., 2005) made it obvious that cumulus cells may express developmental potential of human embryo during IVF. Increased number of cumulus oophorus apoptotic cells was explained as varying with a fall in number of mature oocytes, as equally with low capacity for fertilization, in IVF (Host and col., 2000). BCL2 contents in cumulus cells expresses oocyte competence (Filali and col., 2009).
As per model accepted today (Pepling, 2013), anti apoptotic BCL-2 proteins protect mitochondrial membranes against pro apoptotic proteins. Post activation of programmed cellular death, pro apoptotic proteins (such as BAX) dimerize and enhance mitochondrial membranes permeability, by forming a series of canals, or by interaction with pores existent, in order to generate wider canals; which allows for release of cytocrhome C in mitochondria, and apoptosome formation.
8.1. In vitro maturation stages of sow COCs
We harvested a total 58 ovaries, from 29 culled sows. Ovaries were transported to lab within maximum one hour, in vessels containing physiological serum and added antibiotic (Penstrep 1ml/0.5L NaCl 0.9%) at 35 to 37°C, in a heat insulated box
Oocytes and follicular fluid were harvested together, from ovarian (2 to 6mm) follicles, by punction 5ml syringes, fitted with 18G needles; and were introduced into sterile 50 ml tubes, with PBS. Later, sediment was collected by sterile pipet, and placed on Petri dishes with PBS, for first washing. We harvested oocytes integral with cumulus oophorus, by micropipet (guided by heat-plate stereo magnifying glass) and set on another Petri dish with PBS, for second washing.
8.1.1. COCs maturation
Oocytes on Petri dish with PBS were introduced into TCM maturation medium for third washing; and, finally, in maturation medium with TCM droplets, 50% of samples in medium supplemented with cysteine, and 50% in samples in medium with no extra cysteine. Incubation was at 38.5 °C temperature, 5% CO2 atmosphere air, for 44 hours.
Denuded oocytes were ousted and cumulus cells fluid was translated into another Ependorf tube and centrifuged at 200 g (1060 rpm) for 5 minutes. Sediment was collected, PBS was re-introduced, and same parameters such contents was centrifuged. Supernatant was removed; samples were introduced in liquid azoth for 1 minute, and later into deep freeze, at -80°C, up to running RT-PCR time.
Real time quantitative reverse transcription polymerase chain reaction (real time QRT-PCR)
Real time QRT-PCR is the technique used for quantification and, at the same time, for enhancement of a specific sequence of DNA molecule. DNA is quantified post each enhancement cycle, i.e. aspect real time.
Prerequisites for quantification are as further detailed:
fluorescent dyes binding to double-strand DNA;
modified DNA nucleotides emitting fluorescence when hybridized by complementary DNA.
QRT-PCR is used for quantify RNA as well; QRT-PCR applies as further detailed:
monitoring viral charge;
quantifying genes expression;
allelic discrimination;
testing GMO (genetically modified organisms).
Enhanced product amount varies directly with fluorescence intensity. Method concept proposes that fluorescent molecules monitor progress of enhancement reaction. Hence, fluorescence intensity varies directly with tandem amplicon- multiplication, post each cycle. Ct (cycle threshold) = the first cycle whereat optic system can detect fluorescence, consecutive to amplicon enhancement.
The higher initial DNA quantity in sample, the smaller Ct will be. As per standard curve, soft will compare standards Ct to samples Ct, thereby determining DNA level.
8.2.1. Sample RNA isolating and purifying stages
Cumulus cells lisation:
Washing cells by PBS
Centrifuging at 3000 g to rpm, for 5 minutes.
RNA isolation:
Supernatant removed, RNA lysis buffer added.
Blending by pipeting.
RNA dilution buffer added, introduced in water bath, at 70° C, for 3’.
Centrifuging at 12,000 g to rpm, for 10 minutes, at 20° to 25° C.
Purifying RNA: by centrifugation.
Supernatant separated and introduced in filter columns.
95% ethanol added, blending by pipeting.
Centrifuging at 12,000 g to rpm, for 1 minute.
DNAase I (DNA degrading enzyme by hydrolysis of nucleotide-binding phosphodiesterase ties) added, incubation at 20° to 25°C, for 15 minutes.
Post incubation, DNAase stop solution is added, re-centrifuging at 12,000 g to rpm, for 1 minute.
Washed by RNA Wash solution, centrifuging at 12,000 g to rpm, for 1 minute, re-wash, re-centrifuging for 2 minutes.
Nuclease-free water added on filter membranes, centrifuging at 12,000 g to rpm, for 1 minute.
8.2.2. complementary DNA synthesis
cDNA was synthesized by First Strand cDNA Synthesis Kit (Fermentas)
Table 3.10 Reverse transcription reaction components, in a final 20 μl amount.
Synthesis and enhancement program involved sustaining samples for 60 minutes, at 37°C. Reverse transcriptase activity was stopped by keeping samples at 70°C, for 5 minutes (Table 3.10)
8.2.3. Nuclear acids quality and quantity determination
Nuclear acids quantity and quality were examined by spectrophotometry, on UV-VIS Nanodrop 8000 (Thermo Scientific) spectrophotometer. Based on results yielded for RNA, quantity required by reverse transcription reaction was determined, as well as dilutions required by enhancement reactions, for cDNA.
8.2.4. SYBR Green
Method concept as further detailed.
SYBR GREEN I binds to the minor groove of DNA double helix.
In solution, unbound staining dye emits but slight fluorescence, whereas post binding, fluorescence increases markedly. As SYBR GREEN I is a quite stable staining dye (only 6% activity loss during 30 enhancement cycles) equipment being provided with optic filters, tuned to excitation and emission respective wavelengths; SYBR GREEN I is a selective staining dye, as DNA is quantified (Cherr and col.,2001) – Table 3.12.
Kit used was Maximum SYBR Green/ROX qPCR Master Mix (2X), a ready-made solution optimized for real-time PCR. Kit includes a DNA polymerase (Maximum Hot Start Taq) and dNTPs (nucleotide triphosphates) in buffer optimized for PCR; as well as staining dye, SYBR GREEN I, supplemented with ROX passive reference staining dye.
ROX does not interfere with qPCR, as long as equipment used does not recognize it (e.g. iCycler IQ,), having no say in PCR as emission specter is distinct from SYBR GREEN I specter.
Each sample analysis was twice repeated. Negative control sample for each primer was the sample analyzed with no DNA matrix. (Table 3.11).
Table 3.11 Expression markers (EUROGENTEC, Belgium)
ROX reference staining dye: used as inner reference for SYBR GREEN I signal normalization, when equipment is used which can detect ROX, e.g. Applied Biosystems.
Table 3.12 Enhancement mix for one sample
A multitude of factors interfere with fertilization and with in vitro pig oocytes development. Cysteine is an α-amino acid, precursor of an antioxidant (glutathione), proven beneficial for oocyte maturation and male pig pronucleus formation; while glutathione prevents disintegration of spermatozoa membranes in several species, human included. A study run in 2001 (Jeong and Yang) proved that presence of cysteine in maturation medium markedly improved blastocyst development.
GSH acts as an index of oocyte cytoplasmic maturation, also exerting beneficial effects upon blastocyst formation and quality, assessed by enclosion process and number of cells. GSH induces unbalanced potential cell REDOX, leading to cellular functions alteration, as well as cell cycle changes, such as apoptosis incidence. Apoptosis expresses a biological process along blastocyst formation, a finely tuned process mediated by pro-apoptotic (Bax) and anti-apoptotic (Bcl2-like1) genes. Study of Feugang and col. in 2011 proved that tandem Bcl2-like1/Bax always switches to transcripts Bcl2-like1 direction, an aspect in favor of embryo survival.
In our experiment, oocytes matured in TCM 199 no cysteine medium, and oocytes matured in same medium supplemented with cysteine, indicated no marked differences post maturation, in terms of morphology or number thereof (Fig. 3.32). Quality difference, of oocytes post maturation in cysteine, may become obvious only post IVF, as reaching blastocyst stage.
Fig. 3.32. A. Oocytes matured with no cysteine. B. Oocytes matured with cysteine.
Paradis and col. (2010) made it obvious that bovine oocytes prevent apoptosis in cumulus cells, an effect yielded by modulation of BAX genes expression and of BCL-2, in cumulus cells.
In a study published in 2014, Bogacki and col. indicated that expression of BCL-2 genes was comparatively much higher (p≤0.05) in gilt COCs treated with PMSG/hCG, as against COCs treated with PMSG/hCG+PGF2alpha, and COCs which have expressed spontaneous cycle. Inferior quality oocytes are supposed to be predisposed to apoptosis, while balance of factors pro- and antiapoptotic plays a basic role in cellular apoptosis regulation. Classification of COCs post in vitro maturation is as further indicated in Tables 3.13 and 3.14.
Table 3.13 COCs matured in medium with no cysteine
Table 3.14 COCs matured in medium with cysteine
Results yielded by QPCR technique
mNAR target relation was normalized with β-actin, an expression gene. Number of cycles threshold (Ct) was determined for all of the samples. Method Δ (ΔCt) was used for relative quantification.
As per such method, R (ratio control variant vs. variant enhanced) is computed by relation R = 2-ΔΔCt. Data yielded were statistically processed by ANOVA software. Also, average standard deviance was computed. Bcl2 genes expression varies in various oocyte development stages; also, based on culture media composition, wherein maturation thereof occurred. (Table 3.15)
Bcl2 gene expression increases in mature oocytes cumulus cells, as against in cumulus cells originating in immature oocytes. Also, in cumulus cells yielded by COC in culture medium supplemented with cysteine, Bcl2 expression is close to double expression of cumulus cells in culture media with no cysteine added, even though at morphological-structural level no difference was recorded, between such types cells. (Table 3.16)
Table 3.15 Expression of Bcl2 I: oocytes before maturation; II: oocytes post maturation, no cysteine; III: oocytes post maturation, extra cysteine added.
Conclusions
Extra cysteine in maturation medium does not increase number of oocytes reaching maturity; nor do morphological and structural modifications of oocyte occur, during maturation.
Nevertheless, by quantifying expression of apoptotic Bcl2 genes, markedly increased expression thereof was noted, for COCs in culture medium with cysteine added, as against COCs matured in absence of cysteine.
A high level expression of Bcl2 genes cumulus cells leads to apoptotic inhibition thereof, along a mitochondrial pathway, which inevitably leads to higher oocyte viability and fertilization rates. Comparatively higher fertilization rates are basic for IVF, especially for economicity reasons (oocyte consumption, seminal material, handling and equipment costs). Also, higher fertilization rates cut on work time required.
Table 3.16 Expression of Bcl-2 genes in cumulus cells originating in COC. I: before maturation. II: post maturation no cysteine. III: post maturation, with cysteine.
Based on intervals p (Table 3.17) results yielded can be deemed as statistically marked, and highly marked.
Table 3.17 Reading of yielded data statistics
In terms of such results, further research seems to be in order, on our topic, as further detailed. Extra cysteine action in several culture media could be studied, for other type cases, targeting increased cell viability. Such target is quite likely to be met, with a higher expression level of a series of genes, mainly Bcl2. Also, such genes can be used as molecular markers, in assessment of oocytes viability, implicitly fertilization rates.
DEVELOPMENT TRENDS ANALYSIS
Both Golgi Complex and endoplasmic reticulum are basic research topics, in terms of contributions thereof to embryo development; while the way inositol triphosphate (IP3) interferes with Ca release may indicate the very mechanism whereby oocyte activation can be guided during fertilization.
In bovine oocytes dynamics of cytoskeleton filaments is related to acquisition of nuclear development competency; hence, assessment of intensity and amplitude of modifications undergone by cytoskeleton could indicate potential of such nuclear development competence acquisition.
Starting from studies outlining function of fertiline (dimer glycoprotein binding to oocyte plasma membranes) makes possible investigation of how such fusion process can be induced.
Role of cumulus cells in meiotic maturation could be explained, especially by determining factors which play a role in mechanism of meiotic inhibition.
Embryo oxygen consumption is a high interest topic, quantifiable by various methods, also considering role of cumulus cells in oxygen tension variations, in embryo close vicinity.
Oocyte morphology and quality are defined during ovo- and folliculogenesis, and final during at end of maturation. Incipient embryonic development is associated with morphology of complex oocyte-cumulus (COC). Correlation of oocytes morphology and protein contents (a study in proteomics) would be of interest, as possibly reflecting fecundant capacity.
Morphological quality assessment of oocyte-cumulus complexes is useful for selecting oocytes competent for in vitro fertilization, and embryos production; an aspect worth investigating in terms of transcription products (transcriptomics) as well as of protein abundance (proteomics).
As staining with 1% aceto-orcein yields oocytes no longer viable for IVF, staining dye should be used that does not interfere with such oocyte viability (e.g. Hoechst 33342); also, a series of staining methods should be preferred, that indicate cytoplasmic maturation by staining cellular organelles, or activity thereof.
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