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Ovarian Physiology Series Section 1

OVARIAN PHYSIOLOGY

Ovaries are the generative organs of mammals for the production of oocytes.The oocytes have protective coverings consisting of a few layers of cells which initially surround the female egg called oocyte.

Ovaries perform a dual function.

EXOCRINE

       Under exocrine functions they release oocyte

ENDOCRINE

 Under endocrine function they release ovarian hormones.

OOGENESIS

          It is the name given to the process of egg formation within the ovaries of the mammals. The process is initiated during mammalian foetal life. Its beginning and end differs within different species. (WANI, 1984, wani, 1996, wani, 2009]. The oogenesis begins around 7th month of embryonic life of a female offspring in the mammals

PRIMODIAL FOLLICLE POOL

  Millions of primordial follicles appear in the cortex of the developing foetal ovary. This number of primordial follicles is species specific and progressively diminishes up to menopause in women and with age in other mammals. A foetal lamb or kid [GOAT] has millions but only 85 thousand such follicles are estimated to be present at pubertal oestrus, indicating a loss from foetal to birth and then birth to puberty. A female cow calf has around a 100 thousand such follicle at first oestrus i.e. puberty. These follicles are now reported to be formed from the ovarian surface too and not only the germinal epithelium of the ovary. [WANI AND WANI, 2009]

     PRIMODIAL FOLLICLES IN WOMEN

  The women has 200 thousand at puberty from a birth pool of 500 thousand numbers as they are not seasonal but have poly cyclic receiptivity,which other wise a misnomer or exception in other species. We did confirm the occurrence of gestational heats in small ruminants too [SINHA ET  AL 1980 AND WANI ET AL 1980].The number of these follicles progressively decreases with age, more rapidly with approaching puberty or adolescence.

PUBERTY

The first menarche is indicative of the puberty in women.Simmilarly the first oestrus is the beginning of puberty in other mammals. This may be one of the causes of low fertility in women who decide to marry at an older age.

PRIMODIAL FOLLICLE

         The primordial follicle consists of an oocyte surrounded by a flat stoma cells or somatic cells called granulose cells. This primitive oocyte is called oocyte I. (Guerin, 2009). The granulose cells are covered by a basal membrane known as membrane of Slavjan Ski. The diameter of oocyte I is around 30 µ.

     The primordial follicle changes to primary follicle. The primordial follicle consists of a single  flat layer of germinal cells. When these flat cells change to a cuboidal shape the follicle is named as secondary follicle.

SECONDARY FOLLICLE.

       It still contains oocyte I inside. It has a double layer of germinal cells, cuboidal in shape. The size of oocyte I within it measure around 40 µ.

GRANULOSA CELL GROWTH.

        The number of granulose cell increase and are around 5000 in number. The oocyte grows too and its diameter measure over 60 µ. The follicle is still categorized as secondary follicle and its size may increase with oocyte reaching a diameter of 180 µ inside it. Thus primary to secondary follicle transit involves 6 fold growths in the diameter of the oocyte.

GRANULOSA CELL MULTIPLICATION

         During last stages of secondary follicles isolated islands or space differentiate within multiplying granulose cell layers which differentiate epithelial cells from stoma fibroblasts.

OVARIAN HAEMODYNAMICS

    The blood capillary ramification or what is named ovarian hemodynamic development and described as angiogenic changes in ovarian development of sheep [Wani, 2009, Khatun & Wani, 2007, Khatoon et al. 2006, Goswani et al. 2008].

PRE-ANTRAL FOLLICLE

        The epithelial cells surrounded by encapsulating angiogenic masses give rise to what is known as theca interna or inner layer of the follicle. This secondary follicle in which theca interna is visible is called Pre-antral follicle. The formation of cavity is peculiar recognition of the pre-antral follicle.

THE TERTIARY OR ANTRAL FOLLICLE

        The follicle grows and granulosa cells now differentiate into theca externa and theca interna with a central antrum. The follicle is named as tertiary follicle. The rapid growth and differentiation of follicular (granulosa) cells continues and cells become innumerable around 50 million. The tertiary follicle grows in diameter and reaches a size of about 20 mm or 2 cms.

PRE-OVULATORY FOLLICLE

        The size of the preovulatory follicle is around 20 mm or 2 cms. Any tertiary follicle assuming or attaining this growth is denoted as pre-ovulatory follicle. The differentiation of granulosa cell as theca interna now culminates into another type of cells called theca externa. The oocyte within a particular mass of granulosa cell now invaginates into follicular cavity.

CUMULUS OOPHORUS

  The electron microscopy of these theca cells denoted as cumulus oophurus (theca interna derived cell) has shown that it consists of;

                        Clusters of epitheloid cells

            These cells are morphologically similar to leydig cell which produce male hormones of androgenic nature.          

       

ANGIOGANIC CHANGES 

      The theca externa layer consists of collagen fibres penetrated by numerous blood capillaries. These angiogenic ovarian changes are vivid and transparent. The usual fibroblasts of early follicles change to myo- fibroblasts at this stage

PRE-OVULATORY FOLLICLE

                Preovulatory follicle in women contains oocyte still at the diakinesis stage .With the cytoplasmic growth of the oocyte corresponding   follicle also grows, at this stage oocyte, measure 120 µm in diameter.

OOCYTE CHANGES.

Oocyte too undergoes some changes at preovulatory stage. The cytoplasm and nucleus continues to grow. Cortical granules migrate to the periphery. It is these granules which blast like radioactive nuclear materials and form a firm protective cover around the “fertilized ovum” known as cortical reactions.

Zona reaction

This reaction is displayed when the first sperm strikes the zona pellicuda of the oocyte and is known as Zona reaction. The other inner membrane of the oocyte is called vitteline membrane and its block is called vitelline block.

          

PREVENTION OF POLYSPERMY

   The zona reaction and vitelline block prevents polyspermy at fertilization.The cortical granules are radio-opaque substances which expand like a nuclear mushroom and shadow the wall of the oocyte at fertilization. The cortical granules from cytoplasm of the preovulaty follicle. Shift and merge with plasma membranes.

OOCYTE ACTIVATION

        While these changes occur the nuclear material of oocyte I at blocked level too resume activity but soon its activity is arrested and Block II comes into force. This block effectuates at 2nd metaphase division (Metaphase II). Thus there is now established evidences that oocyte of its own resumes nuclear activity which progresses from Prophase I to Metaphase II well within the ovarian life.

PARTHENOGENESIS

   Is it possible to explore the circumstances, modulators and precursors of this change? More investigation on this topic is needed. This would possibly explain the growth. and maturation of follicle of its own as it happens in parthenogenesis or even after Menopause it would be possible to incite such changes.

BIRTH OF CHIRST-THE HOLLY SPIRIT.

                Once the mechanism triggering this change from Prophase to Metaphase is signalled out, we may explain the process of the birth of Messiah “Haroon” or “Isaac” from unmarried and aged parents as referred to in Holly Quran for the first time in literature in 579 AD.

       This self initiating  change within the oocyte to overcome its nuclear development block can be hypothesis for a broad  new concept  on foliculogenesis, which would fulfil the wishes of infertile and aged couple and give them a new hope for their loved procreation even after death. A more recent research review is presented in following chapters.

LIFE AFTER DEATH IS POSSIBLE

      The post death revival of life in slaughtered sheep has been achieved. (Wani, 1995, Wani, 2001, 2004, 2006, 2008, 2009, 1996 Figures 1-3) The figures of this chapter show our own results of oocyte maturation and fertilization after death (WANI 1995, WANI, 1996, WANI ET AL 1997, 2000).

        The oocyte was recovered, grown and matured invitro within 12-20 hrs of slaughtering the animals. Fertilizing the recovered and matured in-vitro oocytes with semen obtained from the slaughtered Ram epididymus. This innovative research opens a new area of post death revival of life and also explains the truths recorded for the first time in the book of GOD “THE Holy Quran “

 

OVULATION PROCESS

               During ovulation, propelled by the activity of myo fibroblasts in the theca cells, the oocyte I are virtually changed to oocyte II with the above mentioned changes. The released “ovum” in women is thus oocyte II surrounded by cumulus cells. The stage at which oocyte are released in different mammalian species  are  reported (Wani 1996).  And shown in the Figure --1

Primordial Germ Cell Pool

        Primordial follicle  remain in dormancy since its formation during foetal life up to puberty i.e. around 15 years in women, few months 12-15 months in sheep and goats,2-3 years in cattle and buffalos. The puberty age and the status of follicles has been reported and reviewed by [WANI, 1991.wani1984.]

Out of the primordial follicular pool or commonly referred to germ cell pool by many authors including we in our books and thesis (Wani, 1984, 1984 b, 1996, 2006, 2008, 2009). Only a few grow, still fewer mature and  only one  or few ovulates at each cycle.This is species specific and breed specific within the species Table 1

PUBERTY

    The number of the primodial follicles in humans is known to  decrease from 5 million at birth to 2 million at puberty  All these numbers are postulations or calculations on statistical assumption. Only “God’ known the true number However, reviewing the available literature upto 18th of December, 2009.Al literary and investigative figures indicate 5 million at birth and around 2 million at puberty. The significant loss of the germ cell pool occurs ,first between foetal ovarian development , the second cut on the follicles occurs between birth to puberty and progressive cuts and reduction ensues at each oestrus or menstrual cycle with its decrease or near cessation during old age, Women have been seen to have decreased rates at or near menopause.

IS THIS FOLLICULAR LOSS A LOSS IN FERTILITY

   Whether this loss of primordial follicle pool between birth and puberty is similar in fertile and infertile women or there is complete cessation of follicle development after menopause. A chapter on menopause can be reviewed in this book too.

        There are possibly two explanations which could be unveiled in future. In simple terms the answer to the proposed question could be yes. Yes means fertile women have less loss of primordial follicles in their physiological change from childhood to adulthood. This would mean more number of follicles available for ovulation or perhaps a ratio exists within developmental process to have a fixed number of pools to recruit and classify future ovulating follicles. The negative answer will take us to wonder world of Endocrindogy where morphological evidences alone could  not prove the point, but yes one would analyse the degree of success in assisted fertility cases. How and when this would be known is perhaps beyond my vision. At least in my life I do not foresee a revolution of this kind as very few follicular biologists are active on earth. We have tried to review this situation in our forthcoming chapters in this book under nuclear receptors. The purpose of this book is to incite interest among scientists to work on these areas where little work has been initiated.

         Perhaps prioritizing of foetal-maternal interactions by WHO / UNDEP/ USAD/ ICAR can be suggested as has already been advocated by An American science and technology forum. As such other world research organisation may help to provide funds and scholarships to the young and budding scientists. Alternatively, a research donation by an infertile couple (who understand the desperation of millions suffering from infertility) may help  to unveil the hidden potential.

FERTILITY AND FOLLICULAR DEVELOPMENT

As on today the mechanism of folliculogenesis is so inefficient that out of a pool of 7 million prospective eggs (Primordial follicles) hardly 4000 follicles reach preovulatory stage. A new knowledge pool is available on folliculogenesis.

Initial follicular Recruitment

        The foetal  primordial follicle develpment arrested at birth regains the capacity to grow at puberty.This dormancy or the arrest is ascribed to the Local growth promoting factors They are found to be responsible for this wakeup call. Research review shows inhibitory process is responsible for the primordial follicular sleep. This deep sleep can be or is known to broken by stimulatory factors. These inhibitory and stimulatory factors have been researched.

        THE PARACRINE SIGNALLING PATHWAYS

        Here at this stage the oocyte genome is activated. This activation has been reported as transcription of gene. This is what needs further elucidation. What is that nature of this transcribed gene? During this phase of the foliculogenesis a significant development has been searched out. This is what is called paracrine signalling pathway. These paracrine signalling pathways reported  are responsible for communicating signals from follicle or granulosa cells to the inside trapped oocyte. The growth of the oocyte is concomitant and proportionately comparable. This signalling mechanism have increased embryo culture success rate by use of cumulus cells in the co-culture {WANI, 2001, 1996 and others  -1,2,3,4,5,-------------------------}

CO -CULTURE PROCEDURES

        The co-culture with cumulus cells has become a routine procedure  these days. Two receptivity theories have been postulated to explain  co-cultures higher success rate. These  receptivity waves dertermine  the destiny of the primary follicle as follows: 

  Receptivity: is attainted by the follicular wall of few layers thick in the primary follicle. It is found that FSH receptor develop in these follicular cell which make primary follicles responsive to FSH. Thus, we could say that two, receptivity waves formulate the destiny of the primary police. 

 First is Exocrine (EXR):

      Receptivity in which a communication mechanism with “oocyte” is established A communication system between primary follicle cells and oocyte inside.

Second is Endocrine (ENR):

       Receptivity which establishes receptors within primary follicular cells for FSH (Follicle-Stimulating Hormone) now it is established that all follicles do not respond to FSH; it is only primary follicle who respond to it. We may not wonder if Endocrinologist in future renames FSH as PFSH.

Primary Follicle Stimulating Hormone (PFSH):

    During this phase of primary follicle transition, oocyte covering accumulates cortical granules on a glycoprotein polymer. This capsule encircling oocyte comes to be recognized as future Zona-pellucida. This is virtually first separation (physically or morphological) of oocyte from the follicular wall. Is it that this morphological change preludes the future changes and pathways?

Paracrine Signalling Pathway (PSP):

     Does the above described morphological change   initiate the PSP? Future research may show, whether paracrine signalling pathways initiate endocrine response or vies-versa. Similarly the nature of endocrine response towards cortical granule movement and subsequent formation of Zona-pellucida is the singular function of exocrine or endocrine signals or a combination of both.    Thus future research in this era should involve a biologist, embryologist, biochemist, biotech    and an endocrinologist if all hidden secrets of this tiny mass of 01. mm diameter is to be revealed.

RESPONSE TO FSH

    The primary follicles are still single layered cells, the nature of these calls have changed from flat to cubiodal form. Here it is important to note that the old concept of endocrine response to FSH by multilayer follicle is somewhat reformed. The changes for hormonal response to FSH initiate even when primary follicles are single layered but their form has to change from flat to cubiodal shaped cells. The exact mechanism of this control is to be further investigated.

MOHY HYPOTHSIS

• If someone comes out with a follicular switch enhancing response of the follicles to FSH, we may succeed in recovering the otherwise 3 million pools at birth and 2 million losses at puberty. A new biotechnology of follicles is in offering for future folliculogenetists.All the donor agencies the DST, ICMR, ICAR, WHO, FAO need to come with liberal support for these ideas and new research agenda.

SECONDARY FOLLICLE

        The two layered granulosa cell cover around an oocyte makes a secondary follicle. A secondary follicle is a multilayered follicle. The exocrine signalling pathway is a two-way-process communicating signals from oocyte to granulosa cells and vies-versa.

Here within secondary follicle, oocyte secrets signal initiate changes in the follicular wall. The differentiation of follicle outer coat called Lamina Propia or Basal Lamina is the result of these signals.    (REF 67888)

     They further undergo cytological changes and differentiate into THECA EXTERNA and THECA INTERNA CELLS.

      Both these layers and theca’s are interconnected by blood capillaries. During its development a primary follicle is recognized as one with thick zona-pellucida, nine  layer granulosa cells, theca interna, theca externa and a capillary net.     Figures 1-5 wani and wani,1996,2003

 

 

 

 

TERTIARY FOLLICLE

        It is synonymic as Graffian follicle. Graffian follicle  is  on the name of the scientist Von de Graff who first saw a  follicle in a rabbit ovary  and expressed as if a ghost has eluded him. For more details refer (Wani, 1996)

ANTRAL FOLLICLES

        As already described it is marked by;

(a). Antral Cavity or Antrum

(b). Granulosa cells  already differentiated into

      I.      Zona-pellucida

        ii.    Corona Radiata

            iii.    Peri-antral cavity

        Iv.    Cumulus Oophorus  mass

                  These cells have different responses to FSH. Theca cells have Luteinizing Hormone Receptors (LH). Production of androgens by theca cells is in response to LH stimulation. Notable androgens are androstendione. They are aromatized by granulosa cells to produced estrogens i.e. estuarial.

PREOVULATORY FOLLICLE

        Most of the developing follicle in the recruitment pool dies down by a process called Artesia. The Artesia process limits the number of competing follicles to ovulate. A detailed account of atretic process has been described (Wani, 1984). Both granulosa cells and encapsulated oocyte undergoes cells death (Apoptosis). Many relate it  to FSH thrust, but the true mechanism is not known. Many workers in the past have made attempts to utilize the atresia to limit the  over stimulationat superovulation in sheep and goat(Cahill, 1981 & Wani 1984, Wani ,Gelderman,Hahn 1990). Many hormones,enzyme  inhibitors and stimulators play their roles in this process, which may be described elsewhere in this book under endocrine control of folliculogenesis. The hormone of reproduction have  been published (Wani 1996, 2009, 2009 a).

SUMMARY

   For practical demonstration in the lab, teachers the photographic images 1-6 of the following descriptive terms ,to recognize while experimenting in the laboratory.

A) Simple Recognitions

 

1. i.      Primordial Follicle

    Single  layered flat  granulosa cells

1. ii.      Primary Follicle

    Presence of mitotic cells cubiodal granulosa cells

1. iii.      Secondary Follicle

    Presence of theca cells multilayered granulosa cells

1. iv.      Early Tertiary[ pre- antral] Follicle

    Formation of antrum

1. v.      Late Tertiary [Antral] Follicles

        Full formed antrum

1. vi.      Preovulatory Follicles

  Enlarged with differentiation of Zona-pellucida cumulus oophurus and other structure detailed above. All other follicle die down during Artesia only the follicle which escapes the atresia is called as dominant follicle destined to ovulate or preovulatory follicle

 

About the Author

 prof dr dr ghulam mohyuddin wani (G .M.WANI)is Ph.D (Animal Reproduction / Gynaecology), Dr. Med. Vet (Animal Reproduction/ Production),and has been a prolific writer in the field of reproductio.His books on animal biotechnology and reproduction are on the web.About 44 articles on various aspects of animal reproduction are in many free websites.He wishes all his readers to correct the manuscript and give peer reviews too.This series is specialy dedicated to his guide,teacher and philosipher PROF DR KRISHAN LAL SAHNI HEAD ANIMAL REPRODUCTION AND DIRECTOR CIRG,WHO IS NO MORE.This is my little trbute to noblest of the men I have seen in my life.I am unfortune not have participated in his funureal as I never knew of his death till I went to CIRG IN JANUARY,2010,May GOD BLESS THE PERSON WITH HEAVEN.