Physiology Of Reproduction
Physiology of reproduction
CHAPTER I
EMBRYONIC DEVELOPMENT OF THE GENITAL TRACT IN DOMESTIC ANIMALS
The sex of the future organism is determined at the fertilization time. If sperm delivers X chromosome a XX pair will be establish and a female will result. If sperm deliver Y chromosome a XY pair will be establish and a male will result.
The development of the genitalia in domestic mammals takes place in early stages, where the differences can be seen clearly and early on. The phase of organogenesis of the future male or female genital apparatus develops co-dependently with the urinary system, keeping close morphophysiological relations the whole life of the animal.
The embryonic germ layers are three layers of tissues that become established during early embryonic development. They give rise to the body tissues. These layers are ectoderm, mesoderm, and endoderm (fig. 1).
Fig. 1. The embryonic germ layers. 1. Endoderm, 2. Mezoderm, 3. Ectoderm
The ectoderm forms from the outer layer of cells. It gives rise to the skin and nervous system. The cells that formed the tube-like structure in the gastrula are endoderm. These cells will form the lining of the gut and the organs derived from the gut. Mesoderm forms between the ectoderm and endoderm. It becomes the muscles, connective tissues, skeleton, kidneys, circulatory and reproductive organs.
The first differentiation occurs in the ventro-median part of the mesonephros (the primitive excretory organ), where the epithelium proliferates and forms the genital ridge. The gonads will be formed from the middle part of the genital ridge and the supporting ligaments from the cranial and caudal part of genital ridge (I. Groza, M. Muntean, 2002)
In the early embryo, when the yolk sac is still present, primordial or the primitive germ cells develop. These cells originate from the inner lining of the yolk sac outside the embryo proper. From there they will migrate progressively towards the intestine, alongside the hind gut and mesentery, using a passive motion following intense divisions or by active migration, using pseudo-pods to finally arrive and colonize the genital ridge. During the time, the primordial germ cells are undergoing intense mitotic divisions and their number increase significantly.
An interpretation confirmed by several in vitro observations reporting that human primordial germ cells, as those of mouse, show several features of motile cells and are able to move actively both on cellular and extracellular matrix substrates. The in vivo time-lapse experiments with confocal microscopy in slices of mouse embryos appeared to definitively confirm that in mammals, primordial germ cells reach the genital ridges by active migration. However, Freman (2003), reinterpreting these and other observations reported above, concludes that morphogenetic movements and local cell divisions rather than active migration are mainly responsible for primordial germ cells displacement in the different regions of the embryo. Even Freman, however, admits that human primordial germ cells might migrate actively to cover a distance of approximately 50 mm separating the preaortic region from the genital ridges (Massimo de Felici, 2013).
Fig. 1.2. Migration of the primordial germ cells (adapt from P.L. Senger, 2000).
Fig. 1.3. Migration of primordial germ cells from the yolk sac into the genital ridge (http://universe-review.ca/).
When the primitive germ cells arrive in the genital ridge they stimulate the proliferation of cells in the region. The proliferation result in the formation of compact strands of tissue called primitive sex cords. This phenomenon causes the genital ridge to enlarge and push toward the mesonephros (primitive kidney). The mesonephros produces urine which is drained by a bilateral pair of ducts called the mesonephric ducts (Wolffian ducts). They extent caudally and empty into the urogenital sinus.
The mesonephros develops a new pair of ducts beside the mesonephric ducts, called the paramesonephric ducts (Müllerian ducts).
The indifferent stage characteristics can be summarised as:
• Gonads begin;
• Pair of ducts capable of male organs (Wolffian ducts);
• Pair of ducts capable of female organs (Müllerian ducts).
• Underdeveloped potential external genitalia (the urogenital sinus).
Fig.1.4. The indifferent stage (http://www.embryology.ch/).
Sexual differentiationDacă gonada indiferentă evoluează spre sexul mascul, deci viitorul organism este posesorul unui cuplu gonozomial XY (heterogametic) și intervine ultimul mesager, care este medularina, cordoanele epiteliale se adâncesc și se dispun paralel; dacă la început ele sunt drepte, spre sfârșitul sexualizării devin sinuoase, se alungesc și ajung în medulara gonadei unde, prin apariția lumenului se transformă în viitori tubi seminiferi
The development of the male reproductive organs.
In males, the indifferent gonad develops into the testis. The sex chromosomes, XY couple (heterogametic) are required and will involve the last messenger, which is medullarine (I. Groza, M Muntean, 2002).
The substance that controls the pathway toward male development is called testis determining factor (TDF) and is controlled by a gene located on the tip of the Y chromosome. TDF is synthesized by the sex cords. The absence of TDF results in the development of the male reproductive system.
The region of the Y chromosome that carries the testis-determining factor contains a gene called SRY (sex region Y). Its product binds to DNA, acting as a transcription factor that is critical for testis production.
Scientists studying sex reversal, a difference between the chromosomal sex and the phenotypic sex, confirmed the importance of SRY. They determined that infertile males who were XX had all acquired a particular snippet of the Y chromosome, which was translocated to X. That small fragment of the Y carries SRY. Conversely, many XY females have a deletion of the part of the Y that includes SRY. Introduction of the mouse SRY gene into an XX mouse causes the formation of testis and the animal develops as a male anatomically; however, it does not produce sperm. Thus, SRY is the testis-determining factor, and is the only gene on the Y chromosome that is essential for development of male genitalia. Some genes required for male fertility are on the Y chromosome, while others are on the X or on autosomal chromosomes. The DAZ genes on the Y are essential for sperm formation; deletion of DAZ results in male infertility.( http://www.learner.org/courses/biology/textbook)
The undifferentiated sex cords begin to align themselves with the small rete tubules (mesonephric tubules which penetrate into the primitive gonad) and their interconnection occurs to provide continuity, via rete testis. These will become seminiferous tubules. Rete testis is a network of tiny ducts that connect the seminiferous tubules to the efferent ducts. The efferent ducts are derived from the mesonephric tubules. The mesonephric duct will give rise to the epididymis and vas deferens.
In the male fetus the paramesonephric duct atrophies due to the effects of the antimüllerian hormone (AMH), which is formed by precursors of Sertol's supporting cells (cells that surround the primordial germ cells and come from the primitive gonadal cords). Despite the effects of this hormone, embryonic remnants of the paramesonephric duct remain behind in males. These are the testicular appendage at the cranial pole of the testis and the prostatic utricle at its caudal pole.
Celulele sexuale mari vor forma spermatogoniile, elementele primordiale ale liniei seminale, iar celulele mici vor deveni celule Sertoli.
The mesenchymal tissue that is found between the newly formed seminiferous tubes will then develop into the interstitial Leydig cells that will secrete testosterone, the masculine hormone.
From the mesonephric duct (Wolff duct) arise:
Epididymis
Deferent duct
Seminal vesicle
Ejaculatory duct
From the endoderm of the urogenital sinus arise:
Urinary bladder
Out of the pelvic part of the definitive urogenital sinus: the prostatic and membranous parts of the urethra, the prostate and the bulbourethral gland (Cowper's).
Out of the phallic part of the definitive urogenital sinus: the spongy part of the urethra and the urethral glands (Littre's)
Fig. 1.5. a,b. The primordial follicles from the fetal ovary. The primordial germ cells (ovogonias) are surrounded by small cells that derive from the germinal epithelium.
The development of the female reproductive organs. These will be influenced by the XX gonosome and the cortexine (I. Groza, M. Muntean, 2002), a substance which induces the differentiation of the female sex organs.
The absence of the Y chromosome will result in lack of TDF production. In the absence of TDF the sex cord fragments into cellular cluster, each enclose a primitive germ cell, which penetrates less deeply into the future ovary. These structures will later form the "primordial follicles”. Rete formation in the ovary is not pronounced and the connection between rete tubules and mesonephric tubules do not occur.
TheseÎn urma acestor transformări, rezultă un cortex primar dens, ce va deveni corticala viitorului ovar, iar zona medulară involuează, rămânând vascularizată dar nepopulată.TheseThe changes will result in a dense primary cortex, which will become the future ovary cortex, while the medullary area involutes, leaving it vascularized but unpopulated. TheCordoanele sexuale primare degenerează și regresează, fiind înlocuite cu o stromă fibro-vasculară. primary sex cords degenerate and regress and are replaced by a fibro-vascular stroma. TheTunica albuginee se formează prin proliferarea țesutului conjunctiv din cordoanele epiteliale. tunica albuginea is formed by proliferation of the connective tissue of the epithelial cords.
Căile genitale femele își au originea în canalele lui Müller, cavitatea uro-genitală, tuberculii genitali și bureleții genitali. The female genital ducts originate in the Müllerian (paramesonephrotic duct), urogenital sinus, and genital tubercle. Din canalele lui Müller se vor forma oviductele, coarnele uterine, corpul uterului, cervixul și vaginul, ultimele rezultând în urma contopirii celor două canale și dispariției peretelui despărțitor. The oviducts, uterine horns, uterine body, cervix and anterior vagina, are formed from the Müller's ducts, with the latter resulting from the fusion of the two ducts, and disappearance of the partition wall.
Fig.1.5. Development of the female gonad
Fig. 1.6. Development of the male gonad
Căile genitale se formează astfel: The external genital organs are formed as follows:
The accessory glands arise from the endoderm of the SUG:
The greater vestibular glands (Bartholini) are paired glands that form from the endoderm of the urogenital sinus. The outflow canals empty sideways in the vaginal vestibule. They correspond to the bulbourethral gland (Cowper) in males.
The lesser vestibular glands (Skene) or paraurethral glands also form from epithelial buds (endoderm) of the SUG and grow into the neighboring mesenchyma. They are distributed over the whole vaginal vestibule and – in males – correspond to the prostate.
din cavitatea uro-genitală: vestibulul vaginal.După formarea aparatului genital femel, elementele rămase din aparatul genital indiferent se atrofiază, formând rudimente care uneori pot fi identificate pe aparatul genital.
After the forming of the female genitalia, the remaining elements from the indifferent genital organ will atrophy, forming outlines that can sometimes be identified along the genital apparatus. Thus, from the Wolffian duct system, remains a vestige found on the broad ligament, called the Rosenmüller organ Wolffian duct system regresses and remains as rudiments in the uterus and are called “Gartner ducts”.
1. 1. Migrația gonadelor The migration of the gonads; clinical importance, migration disorders
Since gonads develop in a different place during embryonic development compared to their position in adults, the gonads must undergo a migration, which is more pronounced in males. Given that the testicles are originally found in the upper abdominal cavity, they must migrate down to the inguinal region, to finally settle in the scrotum.
Această migrare a gonadei mascule se realizează prin intermediul unui pliu peritoneal inserat superior pe peretele dorsal al cavității coelomice, iar inferior în regiunea ingvinală.The male gonad migration is achieved by way of a peritoneal fold, which is dorsally inserted on the dorsal wall of the coelomic cavity and ventrally on the inguinal region. Once the testes are in the inguinal region, they are pulled through the inguinal canal because of regression due to the contraction of gubernaculum testis. Also, it is possible that the pressure associated with visceral growth helps to push the testis or at least hold it near the inguinal ring.
After the gubernaculum penetrates the inguinal ring there is a rapid growth of the distal gubernaculum which results in pulling both the testes and the peritoneum into the scrotum. Rapid growth of the gubernaculum is responsible for mechanically moving the testes into the inguinal canal.
Once the testes are in the inguinal region, it is pulled through the inguinal canal because of regression due to the contraction of gubernaculum testis. Also, it is possible that the pressure associated with visceral growth helps to push the testis or at least hold it near the inguinal ring.
The gubernaculum continues to regress. As this regression occurs, it continues to move the testis deeper into the scrotum and cause a complete encapsulation of the testes by the inner layer of the peritoneum, known as the visceral tunica vaginalis. The outer layer of the peritoneum is the parietal layer of the tunica vaginalis. When the testis has fully descended, the gubernaculum has regressed to a small knot which attaches the testis to the distal scrotum. The vaginal process contributes to the two tunicae of the testis. The inner (visceral) layer covers the testis, epididimis and spermatic cord, and the outer (parietal) layer forms a continuous fold which lies directly adjacent to the visceral tunica vaginalis.
Fig.1.7. Descens of testis. Pig and rat model (Amann R P , and Veeramachaneni D. http://www.reproduction-online.org/).
Fig. 1.8. The genital tubercle in males and females and the ultasound identification
Astfel, testicolul plasat inițial în partea superioară a cavității abdominale, trebuie să coboare până în regiunea ingvinală, fixându-se în pungile testMigrarea gonadelor se face în mai multe etape și se termină la naștere.Gonad migration is performed in several stages and finally ends at birth.
La femele, are loc într-un timp mai scurt, datorită poziției topografice definitive, care este mai apropiată locului de origine.In females, the migration of the gonads takes less time, due to the final topographical position of the genital tract, which is near to the place of origin.
La masculi, dacă până la naștere testicolele nu au ajuns în pungile testiculare, se vorbește de un animal criptorhid uni sau bilateral. Studiul embriogenezei prezintă o importanță deosebită, deoarece explică geneza nașterii indivizilor, atât de sex masculin cât și feminin, precum și apariția anomaliilor.The study of the embryogenesis is of particular importance since it explains the genesis of individuals, both male and female, and where the anomalies occur. Unele anomalii sunt foarte grave mai ales când se produc în fazele diferențierii sexuale, ceea ce determină ca pe același individ, jumătate din organele genitale să fie femele, iar cealaltă jumătate mascule, rezultând un intersex.Some abnormalities are very serious, especially when they happen during the process of the sex differentiation. This will lead to individuals where half of the genitals are female and half male, resulting in intersexuality.
Cele mai frecvente anomalii sunt: uterul dublu, cervixul dublu, atrezia cervixului, brida precervicală, atrezia vaginală și vulvo-vaginală, hipoplazia genitală de origine genetică, uter unicorn, etc.The most common anomalies that are found are double uterus, double cervix, cervix atresia, pre cervical pulley, vaginal and vulvo-vaginal atresia, genital hypoplasia of genetic origin, unicorn uterus, etc.Toate aceste anomalii exclud femela sau masculul de la reproducție, dar adesea acești indivizi sunt descoperiți târziu, la o vârstă înaintată, ceea ce din punct de vedere economic nu este de dorit (Fig.2). All these anomalies exclude the animal from breeding whether the animals is a female or a male, but often these individuals are not discovered until the animal is older, which in economic terms is not desirable.
CHAPTER II
FEMALE REPRODUCTIVE SYSTEM
Fig. 2.1. The female reproductive tract. The cow
The genital system in females consist of:
The ovaries (female gonad)
The oviduct (salpinx)
The uterus (uterine horns and uterine body)
The cervix
The vagina (anterior and posterior)
The vulva
Ciclul ovarian2.1. The Ovary morphophysilogy
In some females, the ovaries are relatively dense in consistency, and can be palpated per rectum, by carefully manipulating the cranial part of the genital tract (cow, mare. Determination of the ovarian status can be made by identification of various structures on the ovary. It is easier to perform in cows comparing with mares. Recent technologies (ultrasound) has enabled to monitor and understand the follicular growth, corpus luteum function in most domestic animals.
Ovarul este componentul principal al aparatului genital femel, fiind responsabil și de controlul potențialului genetic (Fig.15).The ovary is the main component of the female reproductive tract, being responsible for controlling the genetic potential. It has two main functions:
a) dezvoltarea și expulzarea ovulelor apte pentru fecundație (fertile); în cazul monotocicelor de regulă o ovulă este dezvoltată până în stadiul preovulator, iar la politocice „n” ovule, după specie;a) The development and expulsion of the ova (oocyte) that are ready for fecundation (mature); in monotocous species (cow, mare) one follicle is developed to the preovulatory stage, and one oocyte will be expelled while in polytocous species (dog, cat pig) several mature follicles ovulates and more oocytes will be expelled according to the species.
b) funcția endocrină caracterizată prin producerea de estrogeni și androgeni care, deși sunt antagoniști, se susțin reciproc, dictând caracterele sexuale secundare.b) The endocrine function that is characterized by the production of a number of hormones: estrogens and androgens, progesterone, oxytocin, prostaglandins, and inhibine which, although antagonists, are mutually reinforcing, dictating secondary sexual characters and regulates the reproductive activity in most domestic species
Tot la nivelul ovarului, după ovulație se formează și se organizează corpul galben, având ca substrat morfologic componentele rămase din fostul folicul. Also, in the ovary, after ovulation the corpus luteum is formed and organized, with the morphological substrate of the remaining components of the former preovulatory follicle. Corpul galben este o glandă endocrină, care prin celulele luteale secretă progesteronul, iar prin celulele tecii interne secretă progesteron, estrogeni și prostaglandină F2alfa. The corpus luteum is an endocrine gland that by way of the luteal cells secrets progesterone and by way of the internal theca cells, progesterone, estrogen and prostaglandin F2α is secreted.
The ovary is composed of an outer connective tissue surface called the tunica albugineea, covered by single layer of cuboidal cells called the germinal epithelium (with no germinal fuction).
In most species, except the mare, the ovarian cortex is situated under the tunica albugineea.
– corticala în care se găsesc totalitatea foliculilor primordiali și foliculii în diferite faze de dezvoltare, formând „Pool-ul” folicular sau magazia de ovule.The cortex, which contains primordial follicles and follicles in various stages of development (primary, secondary, tertiary, preovulatory), forming the "follicular pool" or the oocyte deposit. Foliculii primordiali se formează la vițea în perioada fetală între ziua a 90-a și 170-a de gestație, când celulele somatice înconjoară ovocitele în stadiul de diploten, formând „Pool-ul” foliculilor primordiali.In calves, the primordial follicles are formed during the fetal stage, between the 90th and 170th day of gestation, when somatic cells surround the oocytes in diplotene stage, forming the primordial follicle pool.
Numărul maxim de celule germinale ajunge în a 110-a zi de gestație la 2,7 milioane de ovule din care vițeaua rămâne la fătare cu 20%.The maximum number of germ cells is reached on the 110-th day of gestation, consisting of 2.7 million ova, but only about 20% will remain to the calf at birth. În corticală se mai găsesc, în afară de foliculi, celule interstițiale, țesut conjunctiv stromal, nervi și vase (artere, vene, limfatice). In the cortical region, in addition to follicles there can also be found interstitial cells, stromal connective tissue, nerves and vessels (arteries, veins, lymphatic vessels) (I.Groza, M. Muntean, 2002).
Celulele interstițiale din corticală provin din mezenchimul gonadei indiferente, din celulele foliculare ale granuloasei și tecii interne ale foliculilor atrezici.The interstitial cells in the cortical region derive from the mesenchyma of the indifferent gonads, the follicle cells of the granulosa and the internal theca of the atretic follicles.Celulele tecii interne cresc în volum, iau o formă poligonală, au o citoplasmă abundentă și devin celule secretoare de androgeni. Internal theca cells will grow in volume, take a polygonal shape, and have an abundant cytoplasm and become androgen-secreting cells.
The ovarian cortex also houses the functional corpus luteum (CL) and degenerating CL (rubrum, nigricans, albicans). The corpus luteum is relatively large and produces progesterone.
In mare, the cortex is situated around the ovulation fossa, an is the only place where the ovulation occurs.
Celulele foliculare din granuloasa foliculilor atrezici se dispun sub formă de cordoane și secretă estrogeni; cu cât numărul lor este mai mare, cu atât ovarul este mai activ.- medulara ovarului este formată din țesut conjunctiv dens, vase sangvine, limfatice și celule interstițiale.The medulla of the ovary consists of dense connective tissue, blood vessels, lymphatic and interstitial cells. În această zonă a ovarului se găsește și o rămășiță din gonada indiferentă denumită „rete ovarii”. In this part of the ovary there is located a remnant of the indifferent gonad called "rete ovarii”, similar to rete testi. Celulele interstițiale ale medularei provin din mezenchimul primar, produc androgeni și prin bogatele rețele vasculare pompează substanțe nutritive din circulația sangvină, deci au și un rol în nutriția acestei zone. The interstitial cells of the medulla originate from the primary mesenchyma, produce androgens and, through rich vascular networks, they deliver nutrients from the blood vessels, being involved in the nutrition of the area as well as the cortex.
2.8. Foliculogeneza 2.2. Folliculogenesis
The folliculogenesis is the process whereby the immature follicles (primordial follicles) develop into more advanced stages and become candidates for ovulation (the expulsion of the ova).
În ovarul puberal activ, sistemul folicular are două funcții principale: de maturare a ovocitelor și de producere a steroizilor ovariani.In the active puberal ovary, the follicular system has two main functions: the maturation of oocytes and the production of hormones. TDezvoltarea ovocitelor, care se găsesc într-o stare latentă și maturarea loc are loc în tot timpul vieții femelei adulte, cele două procese fiind reglate, după unii autori, de numărul de ovocite existente în ovare la naștere.he development of oocytes, which are found in a latent phase (prophase of the first meiotic division), and the maturation take place throughout the life of the adult female, the processes being regulated, according to some authors, depending to the number of existing oocytes in the ovaries at birth (I. Groza, M. Muntean, 2002).
Within the ovarian cortex, several types of follicles can be seen, representing different stages of follicular development, maturity and regression.
Procesul de creștere al ovocitelor se produce în două faze: The growth of oocytes occurs in two phases:
– în prima fază, care se întinde până la formarea foliculului secundar, creșterea ovocitului are loc paralel cu creșterea foliculului. – The first phase, which continues until the secondary follicle formation, where the oocyte growth occurs parallel to the follicle growth.
– în a doua fază, care cuprinde perioada de la formarea foliculului secundar până la cel predehiscent, dezvoltarea foliculului nu mai influențează dezvoltarea ovocitului, care rămâne în stadiul de diploten al profazei meiotice. – In the second phase, that takes place from the formation of the secondary follicle until the formation of the pre-dehiscent stage, the follicular development does not affect the oocyte development, which remains in the diplotene state of the first meiotic division.Odată cu apariția vârfului de LH preovulator, se produce prima diviziune reducțională sau meiotică și separarea primului globul polar. With the occurrence of the preovulatory LH surge, the first meiotic division occurs and the separation of the first polar body takes place. Pentru a avea loc acest proces este necesar ca foliculul sau foliculii să ajungă la o anumită dimensiune, deoarece foliculii mai mici nu reacționează la LH, probabil datorită unor posibilități mai reduse de fixare a hormonuluiFor this process to occur it is necessary for the follicle or follicles to reach a certain size (maturity), because the smaller follicles do not respond to LH surge, probably due to lack of LH recepors.
Blocarea dezvoltării ovocitului, până la apariția vârfului de LH, se datorează prezenței în lichidul folicular a unui „factor de inhibiție a meiozei”, o proteină cu molecula relativ mică și care dispare odată cu creșterea bruscă a LH-lui; maturarea fiziologică completă a ovocitului are loc în 6-8 ore după această creștere.The blocking of the oocyte development, until LH peaks occurs, is determined by the presence of a "oocyte maturation inhibitor ” (OMI) in the follicular fluid, which is a protein with a relatively small molecule that disappears with the LH surge.
The suggested involvement of OMI in regulation of meiosis is based on the following observations: (1) fully grown mammalian oocytes explanted from their follicles undergo meiotic maturation spontaneously, whereas follicle-enclosed ova remain immature until stimulated; (2) co-culture of oocytes isolated from their follicles with follicular granulosa cells, granulosa cell extract and follicular fluid inhibits the spontaneous maturation; (3) the inhibition of oocyte maturation by OMI is reversible and in several of the models employed can be removed by the addition of the physiological trigger of meiosis, luteinizing hormone (LH). (Tsafriri A, Pomerantz S.H., 1986).
Complete physiological maturation of oocytes occurs in the 6-8 hours after the LH preovulatory surge. La maturarea ovocitului, pe lângă hormonii gonadotropi, este necesar să acționeze și estradiolul, hormon steroidic ovarian, care are un rol esențial în maturarea citoplasmatică a acestuia, dar nu este necesar pentru declanșarea meiozei.At oocyte maturation, in addition to gonadotrope hormones, estradiol, an ovarian steroid hormone that plays a key role in its cytoplasmic maturation, must also act, but it is not necessary to promote the first meiotic division.
Perioada în care ovarul este funcțional depinde de rata pierderii foliculilor primordiali, care la vacă începe să scadă după al patrulea an de viață, ajungând la 15-20 de ani, când femela devine sterilă, să mai rămână 100-2000 de foliculi primorThe period during which the ovary is functional depends on the rate of the primordial follicle loss, which in cow begins to decline after the fourth year of life. When the female is 15-20 years and she is sterile, only 100-2000 of primordial follicles remain (I. Groza, M. Muntean).
Fig. 2.4.The ovarian activity. Primordial follicles
Populația de foliculi din ovarul femelelor domestice trece prin următoarele faze de dezvoltare: foliculi primordiali, primari, secundari sau cavitari, maturi sau de Graaf și foliculi dehiscenți (Fig.16).The population of follicles in the ovary of the domestic female passes through the following stages of development: primordial follicles, primary, secondary, mature or Graafian follicles and dehiscence.
Fig. 2.4. The primordial and primary follicles
Foliculul primordial are următoarea structură: ovocitul așezat central, înconjurat de un strat de celule foliculare, ansamblu ce are la exterior o teacă conjunctivă denumită „ovisac”. The primordial follicle (fig.2.4.). At birth, all the surviving oocytes are surrounded by a single layer of flattened follicular epithelial cells, delimitated from the ovarian stroma by a basal membrane (lamina). They form the majority of the follicles within the ovarian cortex. When the primordial follicle is stimulated, it becomes a primary follicle.
Foliculul primar are dimensiuni ceva mai mari, datorită înmulțirii celulelor foliculare, care iau o formă poliedrică și se dispun în mai multe straturi în jurul ovocitului. The primary follicle.The primary follicle is larger in size (but remain microscopic). The oocyte has a central position, and is surrounded by a single layer of follicular cells (cuboidal epithelium). Females are born with a lifetime supply of primordial and primary follicles. These follicles do not divide, but develop into a more advanced secondary follicle or they become atretic. Follicle atresia is the periodic process in which immature ovarian follicles degenerate and are subsequently re-absorbed during the follicular phase of the cycle (From Wikipedia).
The secondary follicle is also microscopic and is characterized by having two or more layers of follicular cells but without an antrum or cavity. In general, the oocyte within the secondary follicle is enlarged, surrounded by a relatively thick translucent layer called zona pellucida. At the end of this stage, the follicular cells start to secrete follicular fluid, which can be seen within the follicular layers as small cavities. The follicular cells divide and become the granulosa cells.
TheFoliculul secundar sau cavitar se caracterizează prin apariția, în interiorul granuloasei, a unor mici cavități umplute cu lichid folicular secretat de veziculele citoplasmatice ale celulelor granuloasei, dar poate fi și un filtrat al plasmei. Tertiary follicle or antral follicle is characterized by the appearance within the granulosa of a single cavity filled with follicular fluid secreted by the granulosa cells, but it may also be plasma filtrate, called Aceste mici cavități confluează, formând o singură cavitate în interiorul granuloasei: „antrul folicular”. follicular antrum.
The folicular fluid fills a single space, which is surrounded by the follicular cells called the membrana granulosa. The granulosa cells that surround the oocyte, and project into the antrum are called as the corona radiata. The oocyte, zona pellucida and the follicular cells surrounding the oocyte (corona radiate) are all expelled at ovulation, and enter the fallopian tube (the oviduct).
There is a basement membrane between the granulosa cells and the theca interna (Slavjansky membrane). The fibrous theca externa merges with the surrounding stroma.
– teaca externă formată din celule fusiforme; – Theca externa composed of connective tissue, forms a transition to the ovarian stroma and contains large blood vessels; It completely surrounds and supports the follicle
– teaca internă formată din celule poliedrice și care este separată de granuloasă, prin membrana Kölliker Slavjanski.- Theca interna; Theca interna is the layer just beneath the theca externa. It is well supplied with capillaries, out of large, lipid-rich cells. Cells of the theca interna are responsible for the production of steroids under the influence of the gonadotrop hormones.Cele două teci sunt bine vascularizate și odată cu formarea lor începe secreția hormonilor steroizi și a acidului hialuronic.
The granulosa cell layer (membrane granulosa) is situated beneath the theca interna. The granulosa cells produces a variety of materials and possess receptors for FSH and LH. These cells are also believed to govern the maturation of the oocyte.
When the tertiaty follicle becomes a dominant preovulatory follicle it is sometime called Foliculul matur sau de „Graaf” – are aspectul unei vezicule ale cărei dimensiuni variază de la specie la specie (15-20 mm la iapă, 12-15 mm la vacă, 7-8 mm la oaie, capră și scoafă, 2-3 mm la cățea și pisică, 0,5-1 mm la iepuroaică).the "Graafian follicle" A small number of antral follicles will evolve into the mature or Graafian follicle, the rest will degenerate (atresia). After the degeneration of the oocyte and the other components, the follicular cavity will be invaded by connective tissue or will develop into a follicular cyst. The preovulatory follicle has the appearance of a vesicle whose size varies from species to species (15-60 mm in the mare, 12-15-20 mm in the cow, 7-8 mm in the ewe, goat and sow, 2-3 mm in the bitch, cat, and 0.5-1 mm in the doe) (I. Groza, M. Muntean, 2002).
Creșterea veziculei se datorează lichidului folicular, care fiind secretat în cantitate din ce în ce mai mare comprimă granuloasa, aceasta subțiindu-se. Teaca internă se hipertrofiază, îi crește vascularizația și apare „ stigma ”, locul pe unde va avea loc ovulația.The increase in size of the vesicle is due to the follicular fluid, which is secreted in large quantities, causing the thinning of the granulosa cell layer. The internal theca becomes hypertrophic, its vascularization increases and the "stigma" appears, which is the place where ovulation will take place.
Fig. 2.7. a,b. Atrezic follicles
Un număr mic din foliculii cavitari evoluează spre foliculul matur sau de Graaf, restul involuează în urma degenerării ovocitului și a celorlalte componente, iar cavitatea foliculară va fi invadată de țesut conjunctiv sau se transformă într-un chist folicular. Foliculul de Graaf devine complet matur odată cu prima diviziune meiotică a ovocitului, iar structura lui are următoarele componente, văzute de la exterior spre interior:
The Graafian follicle is fully mature when the first meiotic division of the oocyte occurs. Its structure has the following components, viewed from the exterior to the interior (Fig. 2.8):
– teaca externă formată din fibre conjunctive și o rețea bogată de capilare, care asigură substanțele nutritive dezvoltării formațiunii foliculare. – External theca composed of connective fibers and a rich network of capillaries that provide the nutrients that help in the formation of the follicular development.
– Internal theca consists of 2-3 layers of rich vascular epithelial cells, loaded with phospholipids, glycerides, cholesterol and esters, which have an endocrine role (estrogen secretion).
– membrana Kölliker-Slavianski separă teaca internă de granuloasă și este formată dintr-un strat subțire de celule.-Kölliker-Slavjianski membrane (basal membrane) separates the internal theca from the granulosa and consists of a thin connective tissue layer.
– granulosa este formată din celule poligonale cu o citoplasmă clară și un nucleu central bogat în ADN; aceste celule sunt dispuse în mai multe straturi o parte înconjurând ovocitul și formând coroana radiată; o altă parte se aglomerează spre polul profund al foliculului și formează „ discul proliger ” sau „ cumulus ooforus ”.
Fig. 2.8. The antral follicle
ovocitul foliculului matur este o celulă mare, de formă sferică, fără mobilitate, formată din citoplasmă și un nucleu sferic, denumit „ vezicula lui Purkinje ”.
The oocyte of the mature follicle is a large, spherical cell, without mobility, consisting of cytoplasm and a spherical nucleus, called the "Purkinje's vesicle”.Nucleul are în structură plasma nucleară, cromozomii și nucleolii. The structure of the nucleus contains nuclear plasma, chromosomes, and nucleoli. La exterior ovocitul are o membrană externă sau „ vitelină ” înconjurată de „ membrana pelucidă ” peste care sunt dispuse mai multe straturi de celule foliculare, ce formează „ coroana radiată”.The oocyte has an external membrane or "vitelline membrane" surrounded by the “membrana pellucida" which is a layer of glycoproteins through which the oocyte and granulosa cells communicate, over which are placed several layers of granulosa cells, which form the "corona radiata". Ovocitul este cea mai mare celulă din organismul femelelor, atingând 160-190 microni la vacă, 150 microni la oaie și capră, 175-200 microni la iapă și 175-190 microni la scroafă.
The Oocyte is the largest cell in the female body, reaching 160-190 microns in the cow. In the ewe and goat it reaches 150 microns, 175-200 microns in the mare and 175-190 microns in the sow.
With the LH peak the following maturation steps are now triggered in and around the oocyte up to ovulation:
In the oocyte:
Termination of the first meiosis with ejection of the first polar body;
Begin of the second meiosis with arrest in the metaphase;
Maturation of the oocyte cytoplasma by preparing molecules and structures that will be needed at the time of fertilization;
In the follicle:
The granulosa cells that sit just outside on the zona pellucida withdraw their processes from the oocyte surface back into the zona pellucida. Those processes were in charge of transferring substances to the oocyte;
The perivitelline space forms between the oocyte and the zona pellucida. This space is necessary for allowing division of the oocyte and for harboring the first polar body formed in the division;
Loosening of the granulosa cells in the vicinity of the cumulus oophorus and proliferation of the granulosa cells;
Increasing the progesterone concentration in the follicle fluid via increased production in the granulosa cell layer.
The results of these processes are:
The correct placement of the uterine tube infundibulum upon the ovarian surface;
The rupture of the follicle wall and the flow of the follicle fluid with the oocyte into the infundibulum;
The inhibition of the maturation of further follicles.
Foliculul dehiscent reprezintă faza finală a evoluției foliculului matur, când el proemină la suprafața ovarului, membrana lui fiind transparentă iar „stigma” este înconjurată de o zonă de congestie capilară roz-roșietică.Follicle dehiscence (ovulation) is the final phase of the follicle development. The follicle will protrude to the ovaries surface, having a transparent membrane called the "stigma", which is surrounded by capillary. Congestion is seen as a reddish-pink area.Înainte de ovulație sau imediat după, ovocitul de gradul I se divide în două celule fiice – ovocitul de gradul II și primul globul polar.
Producerea de steroizi ovarieni 2.3. The production of ovarian steroids
The growth of tertiary follicles is controlled both at the system and local levels. In system level control, the hypothalamus-pituitary-ovarian axis is known to be the main regulator. FSH is the main regulator of recruiting the secondary follicles to grow to tertiary follicles. The actions of FSH are modulated at the system level by tissue receptivity (number and affinity of receptors) and by local regulators (steroids and proteins) that increase or decrease the follicular response to gonadotropins.
LH is critically involved in tertiary follicle growth and maturation. Basal levels of LH are important for recruiting of secondary follicles to grow to become tertiary follicles. LH stimulates androgen production in theca cells. These androgens are transformed into estrogen through the action of the aromatase system. Primary and secondary follicles with intact theca cell layers develop but healthy large tertiary follicles are absent.
Estrogen is synthesized in the granulosa cells of developing follicles. It stimulates granulosa cell growth, increases the synthesis of IGF1 (Insulin Like Growth Factor 1), maintains FSH receptors, induces LH receptor expression, augments aromatase activity and subsequent estrogen production, and attenuates granulosa cell apoptosis. Estrogen stimulates the transition from the secondary to tertiary stage of folliculogenesis and alters the ratio of follicular parenchyma to ovarian stroma in favor of stroma (Yong, 2013).
Leptin is expressed in oocytes, granulosa cells, and theca cells. It increases the meiotic resumption in fully grown follicle-enclosed oocytes. Leptin is highly expressed in the theca cells of antral follicles. Although leptin is expressed in the oocytes and follicle cells, it is thought that non-gonadal leptin can restore fertility. Leptin can suppress ovarian steroid synthesis and IGF1 synergistic effects with FSH on estrogen production in granulosa cells and also inhibits aromatase activity. It has also been suggested that leptin-induced angiogenesis may be important in folliculogenesis (Yong, 2013).
Activins (isoforms: A, AB, and B ) and inhibins (isoforms: A and B) are synthesized in ovarian follicles, the pituitary gland, placenta, and other organs. Inhibin and activin have almost directly opposite biological effects. In the ovaries, activin is synthesized in the granulosa cells of tertiary follicles and is involved in the negative feedback loop of FSH (Yong, 2013).
Activin supports FSH secretion in the pituitary and is involved in the cyclic recruitment of follicles. Activin A inhibits spontaneous luteinization in mature tertiary follicles through the inhibition of LH-induced production of progesterone and oxytocin in granulosa cells. It also attenuates LH-dependent androgen production in theca cells. The effect of activin is under the control of follistatin (Yong, 2013).
Local levels of inhibin and activin change according to the stage of folliculogenesis. It has been proposed that an orderly transition from an inhibin B/activin follicular environment to an inhibin A/follistatin environment is critical for dominant follicle development in women. As follicles reach the size at which the FSH-dependent follicle selection mechanism operates, these follicles produce more activin relative to inhibin, while larger selected follicles secrete proportionally more inhibin. A changing intrafollicular balance between inhibin and activin contributes to granulosa cell proliferation and differentiation, androgen synthesis in theca cells, and oocyte support and development. Interestingly, inhibin A, or its free α-subunit has a negative effect on both oocyte maturation and developmental competence. In contrast, activin A accelerated oocyte maturation and developmental competence (Yong, 2013).
Antimullerian hormone (AMH) is synthesized in the granulosa cells of secondary and early tertiary follicles. The levels of intrafollicular AMH gradually decrease during tertiary follicle growth until selection time. Its expression is under the control of FSH and estrogen. AMH reduces the FSH responsiveness of small tertiary follicles (Yong, 2013).
Lichidul folicular produs de celulele granuloasei ca și prin transudare din sânge se aseamănă cu plasma sangvină, în ceea ce privește conținutul în proteine și electroliți, dar conține în plus hormoni gonadotropi, FSH-ul fiind în concentrații constante iar LH-ul prezintă variații cu creștere bruscă în foliculul preovulThe follicular fluid produced by granulosa cells, similar to blood transudate, resembles blood plasma in terms of its content of proteins and electrolytes, but in addition it contains gonadotrope hormones. FSH has a constant concentration where as LH presents variations causing a sudden growth of the pre ovulatory follicle. Lichidul mai conține și hormoni steroizi dintre care androgenii au valorile cele mai mari.The fluid also contains steroid hormones including androgens, which have the highest values. Astfel androstendionul ajunge la 300-800 nanograme/ml, testosteronul la 15-20 nanograme/ml și dehidropiandrosteronul sulfat la 150-300 nanograme/ml. Therefore, androstenedione can reach 300-800 nanograms / ml, testosterone up to 15-20 nanograms /ml and dehydroepiandrosterone sulphate to 150-300 nanograms/ ml.Estrogenii cresc gradat, fiind absenți în foliculii cavitari foarte mici, iar în foliculul preovulator ajung la 1-2 nanograme/ml. The estrogens increase gradually and are absent in the very small cavitary follicles, where as in the pre ovulatory follicle they are found in a concentration of 1-2 nanograms / ml. Aceștia din urmă vor determina procese de hipermie, hipercongestie și hipersecreție la nivelul uterului, determinând manifestarea caracterelor sexuale secundare (căldurile). They will determine hyperemia, congestion and hyper secretion processes in the uterus, causing expression of secondary sexual characteristics (heat).
A study conducted by Carpintero and col. 2014 concluded that a follicular environment rich in estradiol, progesterone, and testosterone is key to good oocyte development. High rates of progesterone and to a lesser extent, testosterone would be crucial for determining good oocyte quality and key for normal fertilization, as well as an essential step for success in assisted reproduction. Among the oocytes immersed in a follicular environment rich in progesterone and testosterone, those with higher levels of estradiol obtained higher pregnancy rates. In the future, analysis of follicular hormone composition could be considered as an additional tool in oocyte selection.
2.9. Numărul de foliculi dintr-un ovar și categoriile lor 2.4. The Number of follicles in an ovary and their categories
După naștere, în ovarul femelelor se găsește o populație foliculară foarte heterogenă, corticala constituindu-se într-o adevărată „ magazie de foliculi ” (pool-ul folicular).After birth, in the female ovary there lies a very heterogeneous follicular population, where the cortical forms a true deposit of follicles (follicular pool). De aici pleacă o generație de foliculi din care doar un număr extrem de redus ajunge la maturitate și ovulează, ceilalți suferind fenomenul de atrezie foliculară. From here starts a generation of follicles from which only a very small number will reach maturity and will ovulate, while the others will suffer the phenomenon of follicular atresia. La fiecare ciclu, ovarul pierde aproximativ 2000 de foliculi la monotocice, iar la politocice un număr mai mare, astfel încât pe măsură ce femela înaintează în vârstă, populația foliculară se reduce în mod corespunzător.The ovaries will lose about 2,000 follicles in a monotocous heat cycle and in a polytocous heat cycle even more thus, as female’s age, the population of follicles in the ovaries will be reduced. Astfel, la vacă, în jurul vârstei de 15 ani încă mai există în corticala ovarului în jur de 25.Therefore, in a cow, at the age of approximately 15 years in the ovarian cortex there will still be approximately 25.000 de foliculi primordiali, dar 50% din vaci devin sterile nu datorită reducerii populației foliculare, ci pierderii calității lor de ajunge la maturitate și de a ovula.000 primordial follicles, but 50% of cows do not become sterile due to a lack of follicular population, but because of their loss in the ability to mature and ovulate (I. Groza, M. Muntean, 2002).Mărimea foliculilor se poate aprecia doar la animalele mari prin explorație transrectală, când sunt palpați doar cei predehiscenți și dominanți; la vacă, această categorie ajunge la dimensiuni de 15-20 mm, iar la iapă de 20-70 mm.
The follicle size can be evaluated by rectal palpation only in large animals, (cow, mare) where only the cavitry and dominant follicles > 1 cm are palpable; in cows these categories reach approximately 15-20 mm where as in the mare they reach 20-70 mm. ILa animalele mijlocii și mici acest lucru este posibil doar prin laparatomie sau endoscopie, când se pot vizualiza foliculii predehiscenți la oaie și scroafă aceștia au dimensiuni între 8-10 mm, iar la animalele mici de 2-3 mm.n medium and small animals the follicle size can only be evaluated through a laparatomy or endoscopy.
When they are able to be visualized in ewes and sows the predehiscent follicles have a size of approximately 8-10 mm, while the follicles in small animals have a size of approximately 2- 3 mm. Prin examenul microscopic este posibilă observarea tuturor categoriilor de foliculi primordiali, primari, secundari și cavitari. Tehnica ecografică deschide noi orizonturi, fiind posibilă vizualizarea foliculilor, care au peste 5 mm și analiza structurii lor.Ultrasound techniques open new horizons, where follicles and their structures that are over 5 mm, can be viewed and analyzed.
2.10. Activarea ovarului drept și stâng și controlul creșterii foliculare 2.5. The right and left ovary’s and control of follicular growth
The statement is made quite frequently that the right ovary of the bovine ovulates more frequently than the left ovary. However, very meager data are presented in support of the statement. For a review of the earlier reports on the alternating action of the ovaries the reader is referred to Hammond. During the years 1907 to 1915 Stalfors made rectal examinations of pregnant cows for the purpose of determining in which horn of the uterus the fetus was carried. Out Of a total of 923 cows examined, 577 (62.5 per cent) carried the fetus in the right horn of the uterus and 346 (37.5 per cent) in the left horn. One hundred and five of these cows were kept under observation for two successive gestation periods and in 62 cows the fetus was twice carried in the same horn. Hammond observed that in 65 per cent of the animals he examined the follicle ripened in the opposite ovary to that in which the previous ovulation occurred.
Investigators at the Idaho Agricultural Experiment Station reported that out of 166 pregnancies, determined by rectal examination and later proved positive by calf birth, 93 (64 per cent) took place in the right horn of the uterus, and 53 (36 per cent) in the left horn.
Casida, Chapman, and Rupel made an extensive study of the genitalia of heifer calves. The mean weight of the right ovary from 190 calves of Holstein appearance was 1.01 ~ .05 grams, the mean weight of the left ovary was 0.89 ± .04 of a gram. In calves of other breeds the mean weights of the ovaries were: right 1.12 ~ .08 grams; left, 1.03 ± .08 grams. They found a significant difference in the total follicular volume of follicles 4 to 13 ram. in diameter between the right and left ovaries, the right having the greater value.(I. P. Reece and . W. Turner www.journalofdairyscience.org/article)
La femelele speciilor mari, așa cum este vaca, bivolița și iapa, ovarul drept este mult mai activ decât cel stâng, lucru dovedit prin prezența în corticala celui dintâi cu 20% mai mulți foliculi normali, chistici sau corpi galbeni.In the cow, buffalo and mare, the right ovary is more active than the left, quality proven by the presence of 20% more normal follicles, cysts, and corpora lutea in the cortex. Marea majoritate a foliculilor sunt într-o creștere continuă dar un număr redus din această populație ajunge în faza de folicul matur.The vast majority of follicles grow, but only a few reach the stage of a mature follicle. Acest lucru se datorează controlului pe care îl exercită proteinele celulelor granuloasei, care există în lichidul folicular în concentrații diferite. Astfel, se presupune că ar exista un „ inhibitor al maturării ovocitei ” prezent mai ales în foliculii terțiari mici și în cantități mai reduse în cei mijlocii.This is caused by by the granulosa cell proteins that control the process, which exist in the follicular fluid in different concentrations. Thus, it is assumed that there would be an "oocyte maturation inhibition factor" present mainly in small tertiary follicles and in small quantities in the medium ones.El inhibă formarea receptorilor pentru hormonul luteinizant (LH), acțiune ce scade pe măsură ce foliculul se dezvoltă și care cu ajutorul FSH-lui produce estradiol, care la rândul lui favorizează formarea receptorilor pentru LH. This factor inhibits the formation of receptors for luteinizing hormone (LH), an action that decreases as the follicle develops and produces estradiol by help of FSH, which in turn promotes the formation of LH receptors.
La nivelul foliculului matur există substanțe inhibitoare ale secreției de FSH, denumite „ inhibine ” sau „f oliculostatine ” a căror valoare crește foarte mult la începutul estrului inhibând FSH și permițând apariția vârfului de LH urmat de ovulație.In the mature follicle there is a substance which inhibits the FSH, called "inhibins" or "foliculostatins" which is greatly increased in value at the beginning of estrus, inhibiting FSH and allowing LH to peak which then allows the ovulation.
Tot în lichidul foliculilor terțiari mici ai iepei, vacii și scroafei, sa pus în evidență un „ inhibitor al formării corpului galben ”, care scade capacitatea celulelor foliculare din granuloasă de a lega LH-ul, ceea ce favorizează atrezia foliculară. In the fluid of the small tertiary follicles of the mare, cow, and sow, a "corpus luteum formation inhibitor was identified, which decreases the ability of granulosa follicular cells to bind LH, phenomena that promotes follicular atresia. De asemenea, a fost descoperit și un „ activator al formării corpului galben ”, care grăbește formarea lui. It was also discovered that a "corpus luteum forming activator" exists, which speeds up its formation. În reglarea creșterii foliculare, în afară de FSH, LH și estrogeni mai pot avea un rol și alți hormoni ca testosteronul, progesteronul, corticoizii, prolactina și prostaglandinele.In addition to FSH, LH and estrogen, other hormones such as testosterone, progesterone, corticosteroids, prolactin and prostaglandins may also play an important role in the process of regulating follicular growth.
2.11. Atrezia foliculară2.6. Follicular atresia
Based upon the etiomology of the word (from Greek: a= not, tresia=perforated), follicular atresia strictly refers to the failure of a follicle to rupture or ovulate. More broadly, follicular atresia encompasses the fate or demise of all follicles except those destined for ovulation. The process also predominates in the fetal ovary and after birth. Before the time of follicle formation, and upon establishment within the developing ovary, the primordial germ cells become oogonia; while oogonia continue to proliferate, they are also subject to large-scale apoptotic demise.
Around mid-gestation (about 20 weeks of fetal development in human), oogonia undergo transformation into oocytes that enter meiosis, but are later arrested at the prophase stage. This is also the period when oocytes become surrounded by granulosa cells to form primordial follicles.
In the human female fetus, the peak number of oocytes is reached at mid-gestation apr. 7 million cells, but during the last half of gestation at least two-thirds of these are lost, leaving a reserve of 1 to 2 million oocytes at birth. This massive loss of germ cells (named oocyte attrition) results from apoptosis of these cells at all developmental stages
Oocyte attrition also occurs prenatally before follicle formation. Of note is the observation in the bovine that any oocyte that fails to become part of a primordial follicle will be lost.
The loss of germ cells does not end at the time of birth; in the human female, there is an additional 75% loss of oocytes through puberty (with about 400,000 remaining within follicles). In contrast to the prenatal situation, post-natal depletion of oocytes occurs by follicle atresia.
Follicular development is characteristically dynamic throughout the prepubertal stage, with the size of the follicle reserve at puberty being a reflection of the dynamic outcomes of follicular quiescence, growth, or atresia. Throughout reproductive life, about 400 follicles will attain ovulation with an estimated 250,000 follicles lost by atresia at a rate of about 1000 follicles per month.
In woman, the rate of follicular atresia is accelerated in the years preceding menopause (Faddy et al., 1992).
Follicular atresia affects all stages of follicular development, but the proportion of follicles that become atretic is enhanced by increased follicle size. In natural cycles, small antral follicles are particularly prone to atresia.
The adult female mammal has only a finite number of follicles and there is a very high rate of follicular atresia. This suggests follicular atresia is under tight control to ensure oocytes remain available for ovulation throughout the reproductive life of the female.
The regulation of follicular atresia.
Histological descriptions of follicular atresia in the bovine ovary date back nearly 50 years. Among these, two studies in particular established classifications of atresia which differed (Rajakoski, 1960; Marion et al., 1968), and since may have contributed to the misinterpretation of findings by authors of more recent investigations. Irving-Rodgers and coworkers (Irving-Rodgers et al., 2001) re-visited this subject and provided evidence for two basic morphological forms of atresia in cattle: 1) Antral atresia, and 2) Basal atresia. The general histological features of these two forms atresia are summarized below. However, more importantly, Irving-Rodgers and co-workers (Irving-Rodgers et al., 2001) also suggested that more recent studies in which the previous classifications had been implemented to correlate with biochemical or physiological parameters of follicle status should be re-evaluated.
Antral atresia is characterized by the initial elimination of granulosa cells proximal to the antrum. Numerous pyknotic nuclei are evident in these antral layers of the membrane granulosa, and sometimes within the antrum itself. Remnants of mitochondrial and plasma membranes are also seen associated with the pyknotic nuclei (Irving-Rodgers et al., 2001).
The basal granulosa cells, conversely, remain intact and possess many ultrastructural characteristics of healthy cells. Antral atresia is viewed as the classic and most widely-observed form of follicular atresia because it occurs at all stages of follicle development in most species, and it is universally seen in large follicles (> 5 mm in diameter), including the dominant follicle, of monovulatory species (Irving-Rodgers et al., 2001).
Basal atresia entails the destruction of the most basal layer of the follicle, whereas the most antral layers remain intact and healthy (Irving-Rodgers et al., 2001). The basal lamina is often penetrated by macrophages and invading capillaries, and the theca layer of the follicle has additional deposition of collagen. The middle layers of the membrana granulosa exhibit a progression of cellular morphology and ultrastructure from the fragmented, pyknotic cells typical of the basal layers to the healthy, intact cells found in the antral layers.
In the cow/heifer, this form of atresia occurs only in small follicles (< 5 mm in diameter) (IrvingRodgers et al., 2001). Whether or not this form atresia is unique to the bovine is uncertain because, to date, there have been no other reports of its existence in other species.
Apoptosis as a mechanism of follicular atresia Apoptosis is recognized as a hallmark and contributing factor of atresia of antral follicles. It is a cell-specific mechanism of discrete elimination of cells during follicular atresia that ensures regression of the follicle without inciting an overt inflammatory response. During atresia the cells of the follicle undergoing apoptosis are generally scattered throughout the parenchyma, and may or may not include the oocyte. Initiating mechanisms of apoptosis include extrinsic factors, such as the cytokines, and intrinsic factors including oxidative stress, irradiation, and the activation of tumor suppressor genes.
In conclusion, our understanding of the cellular factors influencing the onset of follicular atresia within the ovary is only beginning to emerge. Future research focusing on the mechanisms shared by granulosa cells and oocytes that dictate cell fate (i.e., growth, differentiation, death) and, ultimately, follicular fate (i.e., growth, dominance, atresia), including the molecular, cytoskeletal, and metabolic influences, should provide considerable insight. Examples of these influences include microRNA regulation, cytokeratin filament expression, and oxidative stress control, respectively. These factors, in turn, are of biological and economic significance because they impact other aspects of fertility in both livestock and humans. Once identified, they may hold the key to therapeutic improvements in treating infertility and poor reproductive performance in animals (David H. Townson).
2.12. Ovulația 2.7. Ovulation
Ovulația este un proces biologic ciclic, care constă în eliberarea ovulului apt pentru fecundare, de la nivelul foliculului ovarian matur, proces care începe după instalarea pubertății și dispare înainte de climacterium.Ovulation is the culmination of a complex series of endocrine and biochemical events, within the preovulatory follicle, that finally results in the collapse and expulsion of the oocyte. It is described as a biological process in which the oocyte is released from the ovarian preovulatory follicle, being suitable for fertilization. This process begins after the onset of puberty and disappears before the climacterium (menopause). Acest fenomen complex, în care procesele de integrare și reglare nervoasă joacă un rol fundamental, este dominat de interacțiunea coordonată între „SNC” și sistemul endocriThis complex phenomenon, in which the processes of integration and adjustment of the nervous system plays a fundamental role, is dominated by the coordinated interaction between the central nervous system (CNS) and the endocrine system.
The maturation of the Maturarea „SNC” în determinismul căruia, pe lângă factorii genetici, psihiosenzoriali și de mediu, intervin diferiți hormoni, dintre care sunt cunoscuți în mod cert steroizii ovariani și corticosuprarenali, denotă interrelația strânsă, între cele două componente, de integrare biologică a proceselor de reproducți the maturation of the CNS, in who’s determinism, in addition to genetics, environmental and psycho sensorial factors, are involved different hormones, among which ovarian and adrenal cortical steroids are very well known, showing the close interrelation between the two components of biological integrating of breeding processes.
Ovulația reprezintă un bioritm în cadrul evoluției organitului folicular.Ovulation is a biorhythm in the evolution of the follicular system.
Sistemul de reglare și control al ovulației este un sistem supraetajat, autoreglabil, care conform codului genetic și al interrelațiilor extra și interoceptive, la un anumit moment, este defrenat, antrenând procesele caracteristice bioritmului ovulator.The adjustment and control system of ovulation is a layered and self-regulating system, which according to the genetic code and the extra and interoceptive interrelations, at some point, is broken, developing characteristic ovulatory biorhythm processes. Acest sistem este un complex funcțional, conexiunile supraetajate fiind cortico-hipotalamo-hipofizo-ovariane, hormonale și chimice. This system is a functional complex, the connections being cortico-hypothalamic-pituitary-ovarian, hormonal and chemical (I. Groza, M.Muntean, 2002). În privința modului de acțiune al sistemului nervos vegetativ asupra ovarelor, este posibil ca impulsurile să fie conectate în ciclul funcțional al reproducției prin diencefal. Regarding
Cyclical activity of the ovaries, and ovulation, are influenced by the effects of the sympathetic and parasympathetic nervous system, acting through the internal spermatic plexus, Frakenhauser cords and intramural ganglia. Since the ovaries are vasomotricly innervated, the direct and indirect influences of nerves must be also taken into account. Apoi, prin sistemul port-hipotalamo-hipofizar sunt conectați mediatorii andrenergici și colinergici în elaborarea neurosecrețiilor din nucleii hipotalamici și în secreția hormonilor antehipofizari. Consequently, through the hypothalamic-pituitary-port connection, the adrenergic and cholinergic mediators interact in the development of neurosecretions of the hypothalamic nuclei and in secretion of pituitary hormones.
Participarea sistemului nervos în procesul complex al ovulației este dovedită prin experiențele făcute cu: atropină, morfină, clorpromazină, pentobarbital, farmaconi care blochează căile neurogene pentru secreția LH-ului, întârziind sau blocând ovulația, în timp ce histamina o stimuleThe participation of the nervous system in the complex process of ovulation is proven by experiments made with: atropine, morphine, chlorpromazine, and pentobarbital, products that block the neurogene passageways of the LH’s secretion, which can delay or block ovulation, while histamine stimulates it.
Locul de acțiune al acestor medicamente este probabil eminența mediană și substanța reticulată din sistemul nervos central.La unele specii ovulația poate fi indusă cu doze mici de estrogeni, dar inducția este suprimată prin administrarea unor droguri și numai în prezența unui sistem hipotalamo-hipofizar i The place of action of these drugs is probably the median eminence and the reticulate substance of the CNS. In some species ovulation can be induced with low doses of estrogen, but the induction is suppressed by increased doses.Dacă se secționează tija hipofizară, ovulația nu mai are loc. If the pituitary stalk is sectioned, ovulations can no longer take place. Ovulația are loc atunci când organismul femel beneficiază de condiții de mediu corespunzătoare (lumină, căldură, umiditate, etc) când este furajat cu nutrețuri ce au valoarea biologică ridicată, când este sănătos.Ovulation occurs when the female receives the appropriate environmental conditions (light, heat, humidity, etc.), is well feed with high biological value feed and is healthy.
At puberty (sexual maturity), the female begins a sexual cycle that is based on appropriate interactions between the hypothalamo/pituitary/gonad.
The cycle begins when the hypothalamus secrete GnRH into the hypothalamo-pituitary portal system. GnRH stimulates pituitary cells to secrete follicle stimulating hormone (FSH) and luteinizing hormone (LH). Under their influence, primordial/primary follicles begin growing in the ovary.
A large antral cavity forms in the center of the follicle and a thick layer of collagenous connective tissue forms around its perimeter. During the growing processes the cavitary follicle begins secreting androgens and estrogens. β-estradiol is producing into the follicle. This hormone promotes the expression of gonadotropin receptors on the plasma membranes of follicular cells.
A follicle is said to be mature when it is endowed with an adequate population of gonadotropin receptors. The elevated level of circulating β-estradiol induces a sudden increase in GnRH secretion from the neurosecretory cells of the hypothalamus (cyclic center), and this releasing hormone causes a surge in LH and FSH secretion from the pituitary gland. This surge in gonadotropins initiates the ovulatory process.
Androgen and estrogen secretion is replaced by progesterone synthesis signaling the onset of luteinization of the ovarian follicle.
La speciile domestice, cu excepția pisicii și a iepuroaicei, ovulația este spontană. În cazul femelelor multipare, expulzarea nu are loc simultan ci succesiv, pe măsură ce foliculii se maturează. La toate femelele domestice, cu excepția iepei, ovulația se face pe toată suprafața ovarului. In domestic species, except cats and rabbits, ovulation is spontaneous. In multiparous females, oocyte expulsion does not occur simultaneously but sequentially, as the follicles mature.
In all females, except for the mare, ovulation occurs over the entire ovary. In the mare ovulation take place in the ovulatory fossa.
Durata ovulației este dependentă de raportul cantitativ și în timp, între hormonii FSH și LH (ICSH).The duration of ovulation is dependent, on hormonal balance between FSH and LHCu cât este mai mare cantitatea de FSH și de estrogeni într-un anumit moment al dezvoltării foliculului, cu atât se prelungește perioada de călduri.. The higher the amount of FSH and of estrogen in a certain point of the follicle development, the longer will be the heat. Dehiscența foliculară se produce înainte de terminarea estrului. Follicular dehiscence occurs before the end of estrus in mares, and after the end of the estrus in most domestic animals.
At the tip of a mature follicle, where a stigma forms and the follicle ruptures, there are several different layers of cells:
The surface epithelium, a single-cell layer of cuboidal epithelial cells.
The tunica albuginea, consisting of fibroblasts and collagen.
The theca externa, the follicle’s own capsule of collagenous connective tissue.
The fourth layer consists of the secretory cells of the theca interna.
The basal membrane, situated between the theca interna and stratum granulosum (granulosa cell layer)
Stratum granulosum, from which extends the cumulus mass and its oocyte.
În procesul ovulației se disting două etape : dehiscența foliculară ca urmare a ruperii peretelui folicular la nivelul stigmei, expulzarea ovocitului împreună cu o cantitate de lichid folicular și coroana radiată.In the ovulation process there are two distict phases:
Follicular dehiscence as a result of the rupture of the follicular wall at the stigma level.
The expulsion of an oocyte with a quantity of follicular liquid and the corona radiata. În perioada preovulatorie, la nivelul foliculului dehiscent au loc o serie de procese de ordin morfofiziologic, biochimic, enzimatic, contractil, etc.
During the preovulatory period, a number of morpho physiologic, biochemical, enzymatic and mechanical processes take place in the follicle.
Mecanismul complex al ovulației este explicat prin mai multe teorii: The complex mechanism of ovulation is explained by several theories:
a) „Teoria endosmozei” explică dehiscența și expulzia ovulului prin creșterea presiunii lichidului folicular, fără a putea justifica procesul complex al ovulației. a) The endosmosis theory explained the dehiscence and expulsion of the egg by increased follicular fluid pressure, but this would not justify the complex process of ovulation. Before ovulation,Preovulator, lichidul folicular secretat de granuloasa foliculară crește accelerat, tensiunea intrafoliculară destinde peretele folicular. the follicular fluid secreted by the granulosa cell layer, has an accelerated secretion and the intra follicular pressure tenses the follicular wall. The eventual rupture of a follicle is dependent on degradation of the collagenous connective tissue in the follicle wall and on, intrafollicular pressure of about 20 mm Hg that arises from capillary hydrostatic pressure. At palpation, the preovulatory follicle, in cow and mare, has an increased consistency. Just before ovulation the consistency decreased, and the follicles are fluctuant.
Între membrana bazală și teaca internă apar spații care, cu puțin înainte de ovulație sunt invadate de capilare.Between the basal membrane and the internal theca layer, spaces apear shortly before ovulation, which are invaded by capillaries. La nivelul punctului de minimă rezistență (stigma) se produce ruptura foliculară.At the point of minimum resistance (stigma) the follicular rupture occurs. A follicle will usually rupture within several minutes after the stigma forms. Ovulația este precedată de o congestie ovariană fiziologică și uneori este întovărășită de o mică pierdere de sânge, provenit din ruptura foliculului (hemoragie de ovulație).Ovulation is preceded by a physiological ovarian and follicular congestion and sometimes is accompanied by some loss of blood from the rupture of the follicle (ovulation bleeding).
În pregătirea ovulației intervin activ, sistemul vascular ovarian.Ovarian vascular system is actively involved in the preparation of ovulation. Arterele helicoidale sub acțiunea hormonilor prezintă o proliferare rapidă și o distensie, devenind aproape rectilinii. The helicoidal arteries, under hormonal influence, have a rapid proliferation and distention, becoming almost straight. Are loc o creștere evidentă a circulației intraovariane pentru cel puțin 9 ore, cu un vârf distinct la 4 ore după stimularea gonadotropică a LH-lui. There is a noticeable increase of the intraovarian circulation for at least 9 hours, with a distinct peak at 4 hours after the gonadotropic stimulation of LH.(Fig. 2.18) Intensificarea circulației este asociată de hiperemie, creșterea permeabilității rețelei vasculare de la nivelul foliculului, apariția unui edem folicular, diapedeză eritrocitară, fenomene pe care le putem asemui cu cele determinate de histamină.The increased circulation is associated with hyperemia, increase of the permeability of the vascular network at follicular level, follicular edema, red blood cells diapedesis (outward passage of blood cells through intact vessel walls), phenomena that is closely related to that caused by histamine. Se constată de asemenea, înainte și după ovulație o migrație de bazofile, granulocite și trombocite în foliculul ovulator, care sunt probabil în corelație cu substanțele chemotactice prezente înaintea dehiscenței. There is also a migration of basophils, granulocytes and platelets in the ovulatory follicle, before and after ovulation, which is probably correlated with the chemotactic substances present before dehiscence.În timpul ovulației, fibroblastele tecale migrează în stratul granular, fiind atrase probabil de o substanță chemotactică. During ovulation, fibroblasts migrate to the granular layer, being probably attracted by a chemotactic substance.
b) „Teoria enzimatică” recunoaște o depolimerizare a mucopolizaharidelor prin Hialuronidază, alterându-se astfel peretele folicular, o intervenție a unei enzime asemănătoare colagenazei și a gonadotropinelor, care ar stimula diastaza de la nivelul stromei peretelui folicular. b)The enzyme theory recognizes a depolymerisation of mucopolysaccharids by hyaluronidase, thereby altering the follicular wall, an intervention of an enzyme similar to colagenase and gonadotropins, which stimulates the diastase from the follicular wall stroma.
The enzymes act locally during ovulation, being secreted actively around an area known as the area of ovulation or “stigma”. The mature ovarian follicle contains proteolytic enzymes. The fibrinolysis activity increase in the pre ovulatory follicle, is associated with the larger production of the plasminogen activator. Cyclic AMP and prostagladin E1 și E2, but not PGF2α, are also capable to stimulate the theca cells to produce a plasminogen activator which leads to the plasmin’s activation. In the final stages of the ovulatory process a gradual degeneration of the collagen takes place, resulting in collagenase activation.
The fibroblasts become much more elongated, their cytoplasmic processes exhibit the same type of multivesicular structures, and the extracellular matrix of this layer is less integrated. The fibroblasts of both the theca externa and tunica albuginea are transformed from quiescent, resting cells into active, proliferating fibroblasts.
As these activated cells become motile and begin moving around within the local area of the follicle, they probably secrete proteolytic enzymes that soften the extracellular matrix and facilitate movement of the fibroblasts. In this weakened state, the tissue in the apical area of a follicle begins to separate under the force of a relatively low, but steady, intrafollicular pressure. The result of this dissociation is a gradually thinning of the follicle wall at the site where rupture will eventually occur. Consequently, ovulation and stigma’s rupture happens mainly because of the morphological modifications of the follicle’s wall in this area, denotation that the intrafollicular pressure has a minor role. It is suggested that the oocytes release a deutoplasmatic material, which reacts towards the follicles wall (I.Groza, M. Muntean, 2002).
c) The Hormonal Theory suggests that the modifications of the follicular wall are due to the gonadotropic hormones (Fig.2.11). Thus, under the influence of FSH on the ovarian cortex, the ovarian follicle grows. Once they reach the stage of cavitary follicle they will secrete β-estradiol induces a positive feedback to the hypothalamus Under the influence of hypophyseal FSH/LH, the ovarian follicle becomes mature. If the ratio between the FSH-LH is 1/10, then spontaneous ovulation will occur.
The preovulatory wave of gonadotropins induces a growth of the synthesis of prostaglandin in the mature follicle. While prostaglandin F reaches a maximum level during ovulation, prostaglandin E continues to be produced even a few hours after ovulation.
Fig. 2.11. Endocrine mechanism of ovulation.
The preovulatory growth of prostaglandin is necessary for ovulation. The inhibition of its synthesis will inhibit ovulation, but not luteinisation, though exogen PGF2α has a specific role in the reestablishment of the ovulatory process in animals where the synthesis has been inhibited. There are opinions that prostaglandins, especially PGF2α, cause the rupture of the follicle, since prostaglandins produce a continuation of the follicular hyperemia. The E type of the prostaglandins stimulates, in the pre ovulatory follicle (theca fibroblasts), the forming of cAMP, but by a different mechanism than the LH’s, each effect being separate, but additive. In addition, the follicles which have become refractory to LH, remain completely sensible to prostaglandin. These facts show that LH and prostaglandins act in different areas of the ovary, LH being activated by the secreting cells of the granulosa and internal theca, whereas prostaglandin can stimulate the cAMP formation in the external layer and the adjacent theca tunic.
It is possible that prostaglandins are responsible of the formation of the secondary phase of cAMP during the ovulatory process. Synthetic prostaglandins can induce pituitary LH, which leads to the depletion of the ovarian ascorbic acid. In the physiological process of ovulation, prostaglandins seem to act simultaneously in the synthesis, elaboration and activation of collagenase and the ovulatory enzymes. The type E prostaglandins can stimulate the production of the of plasminogen activator. Even though in the majority of mammals ovulation occurs after a couple of hours after LH surge was either released or injected, the modification of the secretion of the steroids produced by LH intervenes much later.
The factors that decide which of the ovarian follicles will respond to the stimulation of the gonadotropins are still unknown. The localized secretion of estrogen and blood flow distribution seems to be important, as it is known that both factors influence mitosis and the metabolism of ovarian cells. Recent data shows that one of the first responses of the ovaries towards gonadotropins is a growth of intra ovarian pressure. The effects are immediate after i.v. HCG or LH injection and last for 10- 30 minutes.
d) The existence of a neurohormonally controlled fibrilar system on the surface of the ovary. Today it is known that a contractile response can be induced by the activators of the α-adrenergic receptors:ocitocin, and vasopressin, acetylcholine and PGF2α. The response towards these substances confirms the existence of muscular smooth fibers and other structures that contract at the ovarian surface. After the use of stimuli of the smooth muscles fiber on an ovary that was detached during the estrus cycle or after a gonadotrope treatment, not only a response can be seen, but also spontaneous contraction.
Changes in the mechanics associated with the effects of the gonadotropins are necessary for ovulation; also stimulatory action of the PGF2α and norepinephrin on smooth muscles may contribute to follicle rupture.
The ability of the ovary to contract spontaneously and the response of the autonomous agents indicate the presence of some functional muscular activation in the stroma, as well as in the immediate area surrounding the follicle theca. Catecholamines seem to influence the muscular contractions. The adrenergic receptors that are present in the ovary seem to be also implicated in ovulation. It seems that the α-adrenergic nerves and their receptors build up the specific neural component implicated in the process.
Studies about the contractibility of the ovary are still preliminary, further investigation being necessary, especially concerning their role in the endocrine activity and reproduction.
Some of the biochemical aspects in the process of ovulation can be regulated by catecholamine. More recently, there has been evidence that shows that catecholamines seem to affect the eliberation of the prostaglandins. Stimulation of the sympathetic nerve induces a growth of endogen prostaglandin (PGF2α).
Recently, there has been research about the regulation of intra ovarian processes, which goes into detail with the ovulation mechanism.
The pulsation of ovarian tisue is an interesting phenomenon which has led numerous investigators to believe that smoth muscle activity contributes to the proces of mammalian ovulation. However, the functional signifcance of ovarian contractilty has not ben determined. Some researchers continue to uphold the hypothesis that ovarian contractions force the disociation and disruption of thecal tisue at the apex of mature folicles. But, colagen tunics are tenacious, very tenacious and, it is unlikely that even an extensive amount (much les, an indistinct measure) of myoid tisue could provide the necesary force to implement the mechanical rupture of a mature follicle (LAWRENCE L. ESPEY, 1978).
Although some folicles may rupture at the moment when an ovarian contraction exerts moderate stres on an enzymicaly weakened folicle wal, there is stil no evidence that contractions are necesary for ovulation. Others believe that ovarian contractilty is involved in the colapse of the folicle and extrusion of the ovum at the time of rupture. This concept presumes that ovarian smoth muscles give rise to one or more peristaltic-like contractions which begin at the base of a ruptured folicle and push a disloged ovum towards the rupture point (Lawrence l. Espey, 1978).
However, there are no experimental data to show that a mature folicle is surounded by such highly organized myoid tisue. Besides, the folicular antrum in the mammalian ovary is a relatively large cavity, the wals of which would need to colapse against the ovum in order to squeze it out of the folicle. But, there is no available evidence of a suficent decrease in the antral volume during the minutes folowing rupture. It apears more likely that the ovum pasively flows out of the folicle with the stream of folicular fluid and plasma transudate which exude form the site of rupture. Thus, the principal observation of this review is that ovarian contractilty is not necesary for ovulation to take place (Lawrence l. Espey, 1978).
Nevertheles, ovarian contractions are real and they should have some definable role in ovarian physiology. In this interest, this review closes with the following alternative ideas that might be examined as “working hypotheses” in future studies on the function of ovarian contractilty. It should be kept in mind that ovulation is a “physiologicaly traumatic” phenomenon in that it involves the rupture and hemorhage of healthy tisue. Therefore, wound healing and the formation of granulation tisue are necesary steps in the overal proces. Under such conditons, “fibroblasts become not only motile but also contractile” (Lawrence l. Espey, 1978).
The induced (reflex) ovulation requires stimulation on the vagina and/or cervix for ovulation to occur (rabbit, members of the cat family, the ferret, the mink). Camelids (llamas, alpaca, and camels) are also induced ovulators. With the exception of the rabbit, induced ovulators are sustained copulators. The pathway for induced ovulation is illustrated in fig. 2.12.
Females that are reflex ovulators can be induced artificially by using electrical or mechanical stimulation. The stimulation is converted into the action potentials which travel from the vagina/cervix to the spinal cord. Afferent pathways innervate the hypothalamus. The elevated frequency of action potentials in the sensory nerves in the vagina and cervix cause increased firing of hypothalamic neurons, which than results in a preovulatory surge of GnRH. This release causes the release of LH, prompting the cascade of events leading to ovulation. Multiple copulations cause much higher LH surge amplitude than single copulation (P.L. Senger, 2000).
Fig.2.12. Induced ovulation
2.8. Luteogenesis and corpora lutea development
The corpus luteum (yellow body) is an endocrin gland, whose morphology is made up of different components left over from the mature ovule after the ovulation occurs (the oocyte, together with cumulus ooforus, corona radiata and follicular liquid are evacuated). The luteal phase of the estrus cycle begin, and ic characterized by progesterone production.
In the evolution of the corpus luteum (yellow body), there are three stages:
the organization stage;
the efflorescence stage;
rthe regression stage.
a) The organization stage.
When the follicle ruptures at the ovulation, blood vessels also rupture resulting in a bloody-clot-like appearance (corpus hemorrhagicum) which can be observed in the first 2-3 days after the ovulation (fig. 2.14).
The follicle walls collapse into many folds; the theca interna and granulosa cells begin to mix. The basal membrane (Slavjansky) forms the connective tissue substructure of the corpus luteum. The folds, begin to interdigitate, allowing theca interna and granulosa cells to mix uniformly (except humans and othe primate), (P.L. Senger, 2000)
Fig. 2.14. The Organization stage of the corpus luteum in mare
The parenchyma of the new gland is organised firstly from the granulosa cells, which ungergo hipertrophy, accumulates lipids, cholesterol, mucopoli-substances, C vitamin, beta-carotene, xanthophylls and enzymes such as acid and alkaline phosphates, dehydrogenase and lactic dehydrogenase and become large luteal cells (I. Groza, M. Muntean, 2002).
The volume of these cells grows rapidly through the expansion of the cytoplasm; the cellular mitochondrial tubes grow in number and become straight. Also the membranes of the endoplasmic reticulum increase in numbers and the activity of some of the enzymes such as cholesterol esterase begins. This has the role of transforming cholesterol into pregnenolone, and than in progesterone.
The large luteal cells rarely multiply after ovulation. Therefore, the total number of granulosa cells from the follicle may determine the number of steroidogenetic cells and thos the steroidogenetic potential of the corpus luteum.
The internal theca cells become small luteal cells, and they undergo hyperplasia (increase in number), the protoplasm becomes eosynophileic, and these cells secrete progesterone, estrogens, and prostaglandin F2-alfa. Immediately after the corpus luteum begins to form and becomes organized, it also begins to secrete progesterone, starting from 0,1 to 0,5 ng/ml3.
The luteal cells are surrounded by a network of dense capillary network.
The length of this phase is 5-6 days in cows, where the newly formed corpus luteum has a diameter of 6-8 mm, having a champagne screw like shape. This is rectally palpable in 7-8 days after ovulation.
b) The Efflorescence or active stage
This stage lasts 11-13 days, in cow, during which luteal secretory synthesize progesterone from the circulating cholesterol.
Cholesterol is transferred in the smooth reticulum of luteal cells from where it passes into the mitochondria, losing the side chain, and is converted into pregnenolone. The pregnenolone is also incorporated in the smooth reticulum and in progesterone. This mechanism is supported by an enzyme called cytochrome T-450 and 3β hidroxi steroid dehidrogenase.
Fig.2.15. a,b Small (SLC) and large (LLC) luteal cells. (Senger, 2003)
The functionality of the corpus luteum may depend on the degree of vascularity in the cellular layers of the preovulatory follicle and its conent in angiogenic factors. These angiogenic factors may influence the corpus luteum vascularity. The ability of the corpus luteum to vascularize may relate to its ability to synthesize and deliver progesterone.
Sources of mammalian progesterone: in the cow, ewe, sow, and mare corpus luteum is the main source, in the rat, rabbit, cat, the source of progesterone is both the corpus luteum and the interstitial tissue of the ovary. Regarding the plasma progesterone concentration, at a stage it increases gradually reaching 4-6-8 ng / ml plasma.
c) The regression or involution (luteolysis)
Luteolysis indicates the end of the luteal phases in nonpregnant female. It is a process whereby the corpus luteum undergoes irreversibile degeneration, characterized by a dramatic drop in progesterone blood level and decreased of the luteal volume. If the luteolysis do not occur, the animal will remain in a prolonged luteal pahse (anestric).
In the cow this phase takes up to 50-60 days. At this stage, degeneration and successive fibrosis of the corpus luteum takes place in the following sequence: rubrum, nigricans and albicans (fig 1.16).
Fig..2.16. Stages of the corpus luteum in cow: a. organization, b. efflorescence, c. involution (rubrum, nigricans, albicans) (I. Groza, M. Muntean, 2002)
The scar that remains in place of the old corpus luteum is composed of connective tissue in which collagen fibers, elastic tissue and reticular fibers dominate.
2.9. Luteotrop and luteolytic factors
These factors are involved in the organization, evolution, and involution of the corpus luteum. Among the luteotrop factors, the first and the most important in the preatachement periode is LH, or the luteinizing hormone, secreted by anterior pituitary.
Fig . 2.17 Main luteotrophic complexes in some species
The LH is involved also in the ovulation processus; regulate de steroidsythesis of the large and small luteal cells.
During early pregnancy, there are other factors involved in the maintenance of the corpus luteum activity, and prevent luteolisys:
For the early embryo to become an established pregnancy, luteolysis must be prevented (the corpus luteum must be maintained), two major events have to take place:
1) PGF2α synthesis and secretion must be stopped
2) Progesterone secretion must be maintained
The conceptus must provide a timely (before luteolysis) biochemical signal
Conceptus signals its presence to the dam
Signals enable pregnancy to continue
If a signal is not delivered quick enough, luteolysis will occur, progesterone will decline, and the early embryo will die.
In the ewe and the cow:
The blastocyst begins to secrete trophoblastic protein. Both ovine and bovine trophoblastic protein belong to a class of glycoprotein known as interferons (glycoproteins that may possess antiviral action and alter the function of target cells):
Ovine interferon-tau (oIFN τ)
Bovine interferon-tau (bIFN τ)
The trophoblast produces oIFN-τ and bIFN-τ between days 13 to d 21 as the conceptus elongates (spherical to filamentous).
Mechanism of action:
oIFN-τ and bIFN-τ bind to the endometrium and inhibit endometrial oxytocin receptor synthesis pulsatility of PGF2α does not change and therefore luteolysis does not occur. They also promotes protein synthesis thought to be critical to preattachment embryonic survival.
In the sow: The conceptus of the pig produces estradiol between d 11 and 12 after ovulation (coincides with the elongation of the conceptus. β estradiol serves as the signal for maternal recognition of pregnancy. PGF2α is produced by the endometrium and rerouted into the uterine lumen, metabolized, rather than being drained by the uterine veins. Luminal PGF2α has little access to the circulation and can’t cause luteolysis. The sow must have at least two conceptuses in each uterine horn for pregnancy to be maintained.
In the mare: The presence of the conceptus helps to prevent Luteolysis. The equine conceptus does produce proteins; their role in maternal recognition is unknown. The conceptus must migrate within the uterus between 12 to 14 times per day during days 12, 13, and 14 of pregnancy in order to inhibit PGF2α production.
1) This migration appears to be very important because the early embryo does not elongate
2) Conceptus must “touch” enough receptors or secrete “proteins” and place near (on) receptors to maintain pregnancy.
The main luteolytic factor is prostagladin F2α (PgF2α), which is secreted by the endometrial epithelium, especially during late interestrus. From the uterus it is transported to the ipsilateral ovary through a vascular countercourrent exchange mechanism. In ruminants, the PgF2α produced by the endometrium enters the uterine vein and then it diffuses directly into the ovarian artery through the arterial wall. This phenomenon does not happen in mares, where the prostaglandin enters the blood stream and reaches the ovary after the pulmonary passage. It is metabolized in the lungs and only 10% reaches the corpus luteum.
The action mechanism of PgF2α consists in vasoconstriction of the corpus luteum capillaries, leading to a sudden drop in local blood flow; a local anemia appears followed by degenerative processes of luteal cells (apoptosis): picnosis, cariorexis, cariolysis, and ballooning.
The second luteolytic factor is oxytocin. It is locally produced by the corpus luteum (large luteal cells). Luteal oxytocin is stored in secretory granules, when there is no signal of pregnancy, but the largest amount of oxytocin comes from the hypothalamic paraventricular and supraoptic nuclei.
In sheep and cattle, at the end of the estrous cycle, the corpus luteum produces the hormone oxytocin. Oxytocin then stimulates the uterus to produce PgF2α. Uterine PgF2α, in turn, stimulates the CL to produce more oxytocin. This ‘positive feedback’ between luteal oxytocin and uterine PgF2α ultimately causes the regression of the CL.
2.10. Morphophysiology of the genitals
2. 10. 1. 1. Oviduct morphophysiology
The oviduct, or the salpinx, is the first segment of the genital tract and it is very important for fecundation. It is a tiny tube, surrounded and supported by a thin, serous part of the broad ligament known as the mesosalpinx. This delicate subdivision of the broad ligament not only supports the oviducts, but serves as a bursa-like pouch that surrounds the ovary.
Fig. 2.18. The oviduct
The oviduct is divided in three segments:
a) Infundibulum or the segment nearest to the ovary (ovarian end). It partially adheres to the ovary through the utero-ovarian ligament. This opening forms a pocket that captores the oocyte after ovulation. The surface of the infundibulum is covered with finger-like projections (fimbriae) which increase the surface area and cause it to slip over the surface of the ovary at ovulation time. This will increase the chance of capturing the oocyte and transport it toward the ostium (abdominal opening
b) The ampulla or middle segment is the most dilated portion of the oviduct and is particularly important because it is the area where fecundation takes place. It occupies one half of the oviductal lenght
c) The isthmus is the shortest portion of the oviduct and it continues with the tip of the uterine horn. It is smaller in diameter. The junction between ampulla and isthmus is called the ampulo-istmic junction, whereas the junction between isthmus and the uterine horn is called the uterotubar junction (fig.2.18).
The total length of the oviduct varies from species to species. In young cattle suitable for breeding, the length of the oviduct is about 16, 45 ± 2, 9 cm (I. Groza, M. Muntean, 2002).
Oviduct vascularization is ensured by two ovarian artery branches: the uterine branch supplying the ampulla and uterine isthmus, and the ovarian branch irrigating the infundibulum.
From a histological point of view, the oviduct is composed of the endosalpinx (mucosa), myosalpinx (muscularis) and the mezosalpinx (serosa) (Fig. 2.19).
Fig. 2.19. Histology of the oviduct
The endosalpinx or oviductal mucosa is much folded (primary, secondary, tertiary folds), especially in the infundibulum and isthmus segments, decreasing in height toward the isthmus and becoming low ridged in the uterotubal junction. Surface epithelium is simple columnar epithelium, containing two tipes of cells: columnarciliated cells, and nonciliated cells and “spherical” cells, similar to lymphocytes (I. Groza, M. Muntean (Fig 2.23).
Around 20% of epithelial cells are ciliated, the rest are secretor cells that produce the liquid environment of the oviduct and look like a moving forest, the direction of motion being from the uterus to the infundibulum.
Ciliated cells have numerous surface microvili on their luminal surface, mostly in the isthmus, especially in the area between cilia. The rate of beat is affected by the levels of ovarian hormones, being maximal at ovulation or shortly afterward (Fig. 2.21, 2.22).
Non ciliated, secretory cells are more numerous in the ampular area and present cytoplasmic extensions that penetrate the lumen in the early follicular phase and in the luteal phase.
Leukocyte shaped cells are spherical in shape and are prevalent in the early luteal phase of the cycle, when they are migrating from the basal membrane of the epithelium to the lumen. These cells are accompanied by lymphocytes and mast cells, which are more numerous in the isthmus.
Lymphocytes have bactericidal role, while mast cells produce, in their secretor granules, heparin and histamine, which appear to be playing an important role in lipid metabolism, stimulate\ion of blood circulation and increase the permeability of vessels.
– Myosalpinx is composed of smooth muscle, arranged in two layers: an internal circular layer and an external longitudinal layer (Fig. 2.20).
During fertilization, the muscle has a segmental contractile activity, which is progressive and regulates egg transport, denudes the ova and mixes the content of the oviduct. The progression of ova is not continuous, but intermittent, and produced by peristaltic muscle contraction, mainly of the ampulla segment.
The contractile activity of the oviduct’s muscle is dependent on ovarian steroid levels (progesterone and estradiole), being more pronounced at ovulation.
Fig. 2.20. The structure of oviductal musculature
Oviduct musculature form two sphincters: one to the distal abdominal part, „ostium abdominale tubae” and the other in the ampulary-isthmus passage. The latter is rather physiological than anatomical and aims to stop the passage of ova through the ampoullay-isthmus junction. These sphincters are controlled by steroid hormones: estrogen opens them, while gestagens close them and allow entrapment of sperm cells in the isthmus of the oviduct.
At the time of ovulation and after ovulation, when the ova are entering the oviduct, sperm mass is released and reaches the ampulla, where fecundation takes place. The role of the musculature can be sumarised as:
Forming the oviductal jonctions
Facilitate the mixing the oviductal content
Help to denude the ova
Promote fertilization, by increasing egg-sperm contact,
Partly regulate egg transport
– The Mezosalpinx, or the serosa of the oviduct, forms the external layer of the oviduct, which, together with the supporting ligament.
The oviduct’s role is to capture and transport gametes, to ensure the survival of fertilized ova (gametes or zygote) and promote its maturation processes. Through the ciliated formations of the epithelium and smooth muscle contraction, mechanical forces are created by which the egg is propelled to a place of encounter with sperm. The sperm shows a reduced motility until ovulation, but this parameter rises sharply after ovulation.
The oviductal fluid is the result of the activity of secretory cells as well as serum transudation; nutrition is essential for embryo development and maturation. Zygotes can survive only if the liquid contains pyruvate, oxalate, oxal – acetate and lactate, the specific constituent which are absolutely necessary. Nature and composition of the liquid substrates are dependent on ovarian hormone balance, but it seems that the synthesis process of the tubal epithelium may be influenced by the embryos themselves. Oviductal fluid volume is dependent on estrogen and progesterone balance, being highest in estrus and before ovulation, so as to reduce to a minimum in the luteal phase and gestation.
Regarding the direction of movement of tubal fluid in the estrous phase, it is from the ampulla, to the abdominal cavity. However, when the embryo is crossing the utero-tubal junction, a small amount of liquid passes into the uterus. Tubal fluid movement direction is influenced by the position of cilia of ciliated cells, muscular structure of the isthmus, and the existence of the utero-tubal junction.
The role of the oviduct can be sumarise as follow:
Capturing the oocyte (ovum pick-up)
Oocyte passage in the oviduct after ovulation
Oocyte maturation and nutrition
Oocyte progresion tward the fecundation place
Provide optimal environment for fertilization
Sperm transport and capacitation
Sperm nutrition
Development of the early embryo
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2.10.2. The Uterus; morphophysiology
The second segment of the genital tract is the uterus, which, from an anatomical point of view, consists of the uterine horns and uterine body whose size and shape vary from species to species (Fig. 2.24). In bovines, during the fetal stage, the uterus develops in synchrony with the body and is completely formed from the 10th month of life. The uterine horns are hanging from broad ligaments, which are richly vascularized.
From a histological point of view, the uterus is composed of three layers: endometrium (mucosa and submucosa), the myometrium (muscularis) and the visceral serosa or perimtrium, part of the peritoneum, which continues with the broad ligaments.
Histology
The endometrium can be divided into two zones or strata based on their functions and structures. The stratum functionalis is the superficial layer that degenerates partially or completely after pregnancy or estrus. The stratum basalis is the layer that remains after these events. It is from this deeper layer that regenerates the stratum functionalis.
The surface epithelium of the stratum functionalis varies from simple columnar (mare, bitch, queen) to simple or pseudostratified columnar (sows and ruminants). The lamina propria is very cellular. Simple coiled, branched tubular uterine glands are found in the endometrium of most species. The epithelium of these glands is simple columnar and contain both secretory and nonsecretory cells. The height and functions of these cells, as well as the overall morphology of the glands, is hormone dependent.
The uterine glands are formed by the plying of the mucosa in the depths of the endometrium. They are branched, coiled, tubular structures and can reach up to the second layer, the myometrium. Uterine glands secrete uterine mucus. Secretory activity of glandular cells is subjected to hormonal control. Thus, in the estrogen phase of the sexual cycle uterine glands proliferate, are thin, straight, and tubular. High, non-secreting cylindrical cells have a role in sperm-self defense. In the progesterone phase of the cycle, the uterine glands become tortuous and the secretory cells produce a thicker fluid, more loaded with nutrients, forming the "uterine milk" or "embryotrophe”.
In ruminants, thickened regions of the endometrium are present. These regions, called caruncles, are highly vascularized and lack uterine glands. Caruncles represent sites of attachment between the maternal and fetal placenta.
Cyclic Changes in the Uterine Horns and Body
In ruminants:
The endometrium structuere is dependent on the female hormones. It changes during the estrus cycle as the hormones fluctuate. Generally speaking, estrogen is associated with proliferation of the tissues while progesterone is associated with secretory activities.
Proestrus. Under the influence of estrogen from the growing follicle, the endometrium becomes thickened through the proliferation of surface epithelium and the stroma. The tissue becomes congested (containing an abnormal amount of blood) and edematous (containing an abnormal amount of fluid). The uterine glands elongate through cell proliferation. The epithelial cells of the uterus and glands increase in height.
Estrus. Maximum level of estrogen is secreted by the follicle. A LH surge precedes ovulation. The uterine mucosa increases further in thickness through cell proliferation. The uterine glands elongate further and begin to branch, also through cell proliferation. The epithelial cells continue to increase in height as well.
Metrorrhagia, small bleeding, occurs in the zona functionalis shortly before ovulation. The myometrium is highly contractile.
Metestrus. With the formation of the corpus luteum, circulating progesterone increases and estrogen levels decline. The uterine mucosa reaches its maximal thickness with edema and hyperemia (congestion). The uterine glands continue to branch. Bleeding stops around mid-metestrus.
Diestrus. Progesterone levels reach a maximum with the maturity of the corpus luteum at mid-diestrus. The endometrium and glands enter a secretory phase. This activity is greatest during the first portion of diestrus. If pregnancy does not take place, the endometrial line begins to decline as the regressing corpus luteum secretes less and less progesterone. There is involution of the endometrium, regression of the glands and cessation of secretory activities.
In mares:
Anestrus (period of quiescence between estrus cycles). The epithelial lining of the endometrium is made up of simple cuboidal cells. The uterine glands are straight and have cuboidal cells.
Proestrus The height of the epithelial cells in the endometrium increases under the influence of estrogen. The uterine glands begin to coil.
Estrus The cells of the epithelium are tallest during early estrus and then become shorter in late estrus. The endometrium becomes edematous and thickened. The uterine glands proliferate through cell division and become secretory. The myometrium is contractile.
Metestrus This phase is not easily defined histologically in the mare.
Diestrus The endometrial lining is made up of simple columnar cells. Tissue edema tissue decreases. The uterine glands become less secretory.
Carnivores have only one or two estrus cycles per year. Anestrus is the period between the cycles.
Proestrus The endometrium becomes edematous and congested. Bleeding, metrorrhagia occurs in bitches.
Estrus Ovulation occurs shortly after the onset of estrus. Progesterone levels increases after the formation of the corpus luteum. Uterine glands and the stroma begin to proliferate.
Diestrus This may last for 70 days in bitches and is called physiological pseudopregnancy. The corpus luteum continues to produce progesterone and the endometrium resembles that found during pregnancy.
Anestrus The endometrium is thin and regressed, the surface and glandular epithelium are nonsecretory. The uterine glands are straight.
High, non-secreting cylindrical cells have a role in sperm-self defense.
In the progesterone phase of the cycle, the uterine glands become tortuous and the secretory cells produce a thicker fluid, more loaded with nutrients, forming the "uterine milk" or "embryotrophe”.
In the endometrium there are three distinguishing capillary networks: a network in the sub epithelial area, another at the border between the compact and spongy area, and the third between the spongy area and uterine muscle.
Endometrial physiology. Like other parts of the uterus, the endometrium is subjected to hormonal influence, the most important hormones being estrogens and gestagens, which are responsible for all morphological and biochemical changes that occur at this level. In the estrous stage, under estradiol dominance, endometrial cells accumulate large amounts of glucose that can reach up to 40-60 milligrams per 100 grams of endometrial tissue. Glucose accumulated in the cells of the endometrium is converted by the hexokinase enzyme in glucose-6-phosphate and glycogen, resulting energy.
The role of glycogen is very important because it is the metabolic biochemical component of the endometrium; it assures its metabolism, sperm maturation, nutrition, sustainability, and embryo implantation.
The defense mechanism of the uterus is accomplished by cellular factors, and immune-specific antibodies. Cellular factors are brought to the endometrium by blood stream, such as granulocytes, or produced locally by the reticulo-endothelial system: macrophages, plasmacytes, lymphocytes, eosynophiles local histiocytes. Phagocytosis is strong when the endometrium is under the influence of estrogen and is poor under gestagen domination.
Immunologic factors are represented by antibodies, immunoglobulins M and G from blood circulation or local production, such as B-lymphocytes, which agglutinate germs and promote phagocytosis. Unsaturated fatty acids, properdin and flavoproteins are also involved in the defense mechanisms of the uterus.
The Myometrium
Myometrium consists of smooth muscles arranged in two layers:
An internal circular layer.
An external longitudinal layer, blood vessels, and nerves.
The maintenance of the smooth muscles depends on estrogen level. There are numerous gap junctions between the smooth muscle cells. These facilitate coordinated contractions of the myometrium. The smooth muscle cells may enlarge in size ten folds during pregnancy, while the mass of the uterus itself may increase by 20 times. Physiological hyperplasia, increase in cell number and hypertrophy, increase in cell size). During pregnancy, myometrial contraction is inhibited by relaxin. During parturition, uterine contractility is increased by oxytocin from the neurohypophysis.
Under their influence, muscle cells synthesize phospholipids, accumulates glycogen, increase the local RNA, proteins, creatinine, actomyozine and creatinine phosphate synthesis, the last being the main energy donor.
In the estrus, the uterus becomes erectile; the contractile wave goes from the cervix to the oviduct, which stimulates sperm ascension.
The serosa consists of the peritoneal sheath and aims to coat the uterus.AscultațiCitiți fonetic
The role of the uterus
The female uterus connects the cervix and oviduct, insures the male gamete migration, the migration of the zygote, as well as implantation, pregnancy development and parturition.
Also, the uterus plays an endocrine role, by prostaglandine secretion and indirect control of the corpus luteum lifespan.
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