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Ovarian Life Cycle

Eli Y. Adashi, M.D.
Professor and Chairman
Department of OB/GYN
U of U College of Medicine

Objectives

Definitions

Outline

Take Home Points






Objectives

  1. To appreciate the processes of germ cell autogeny; folliculogenesis; ovulation and corpus luteum activity; endocrine, paracrine and autocrine regulators; and their biological significance.

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Definitions

Dominance:  a term assigned to the status of the follicle destined to ovulate given its presumed key role in regulating the size of the ovulatory quota.

Established ovarian regulators:  a group of mostly endocrine but at times autocrine principles, the indispensability of which to ovarian function has been repeatedly and convincingly demonstrated. Pituitary gonadotropins represent the most striking example of an established ovarian regulator.

Putative intraovarian regulator:   a group of generally peptidergic principles the highly regionalized and exquisitely timed expression of which is presumed to account for the physiologic phenomena that cannot be fully attributed to conventional endocrine concepts. In this connection, it is hoped that improved understanding of putative intraovarian regulators may provide the necessary clues to elucidate the precise cellular mechanism(s) responsible for the apparent differential fate of distinct follicular populations.

Recruitment:  the process wherein the follicle departs from the resting pool to begin a well- characterized pattern of growth and development. Recruitment, although obligatory, does not guarantee ovulation. Stated differently, recruitment is necessary but not sufficient for ovulation to occur.

Selection:   the final winnowing of the maturing follicular cohort by atresia down to a size equal to the species-characteristic ovulatory quota.

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Outline

  1. Introduction

  2. Germ Cell Ontogeny

  3. Folliculogenesis

  4. Follicular Recruitment

  5. Follicular Selection

  6. Follicular Dominance

  7. Ovulation

  8. Corpus Luteum Formation and Demise

  9. Signaling Systems

  10. Estrogen Biosynthesis

  11. Progestin Biosynthesis

  12. Androgen Biosynthesis

  13. The Climacteric Ovary

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Outline

  1. Introduction

    The ovary, an ever-changing tissue, is a multicompartmental organ with a broad range of distinct biologic properties. Responding to cyclic pituitary gonadotropin secretion, the various follicular compartments interact in a highly integrated and seemingly programmed manner. All of this, of course, is designed to subserve a single central objective; i.e., the generation of a mature fertilizable ovum for the consequent preservation of the species. At the heart of the ovarian life cycle is the follicle recognized as the fundamental functional unit of the ovary since the middle of the sixteenth century.

  2. Germ Cell Ontogeny

    The ovarian life cycle begins at the most unlikely location; i.e., the wall of the yolk sac and the ventral wall of the hindgut near the origin of the allantoic evagination. It is here that the primordial germ cells originate, either from or among the primitive endodermal cells some time toward the end of the third week of gestation. This, in turn, is followed by migration of the germ cell elements to the primitive gonadal folds during weeks 3 to 5 of life.

    This remarkable translocation is accompanied by a steady increase in cell number through mitotic divisions. Locomotion is thought to be accomplished by ameboid movements, the use of pseudopodia, and most certainly, some form of chemotactic guidance.

    Upon arrival at the genital ridge by the fifth week of gestation, the premeiotic germ cells, now referred to as oogonia, continue to multiply as they settle down. During the subsequent two weeks of intrauterine life, weeks 5 to 7 of gestation (often referred to as the "indifferent stage"), the primordial gonadal structure constitutes no more than a bulge on the medial aspect of the urogenital ridge.

    From this point on, the oogonial endowment is subject to three simultaneous ongoing processes: mitosis, meiosis, and atresia (degeneration). Meiosis not only converts oogonia into primary oocytes well before actual follicle formation, but unlike mitosis, also provides temporary protection from oogonial atresia, thereby allowing the germ cells to invest themselves with granulosa cells and to form primordial follicles. As a result of the combined impact of mitosis counterbalanced by atresia, the number of germ cells peaks at 6 to 7 x 106 by 20 weeks of gestation, at which time two-thirds of the total germ cells are intrameiotic dictyate primary oocytes while the remaining third can still be viewed as oogonial. Some of the former have in the interim invested themselves with a single layer of spindle- shaped (non-cuboidal) primordial (pre)granulosa cells, thereby giving rise to primordial follicles, the formation of which begins around 16 weeks of gestation.

    From midgestion onward, however, relentless and irreversible attrition progressively diminishes the ovarian germ cell endowment by way of follicular (rather than oogonial) atresia, which begins around month six of gestation and continues throughout life. Ultimately, some 50 years later, what has been referred to as the oocytic "gene bank" is finally exhausted. Consequently, newborn female infants enter life, still far from realizing reproductive potential, having lost as much as 80% of their germ cell endowment. By the onset of puberty, virtually 95% of all follicles have been lost, only 400 to 500 of which (i.e., < 1% of the total) will in fact ovulate in the course of a reproductive life span.

  3. Folliculogenesis

    While little information exists at this time regarding the morphogenic principles responsible for follicular organization, it is quite certain that formation of primordial follicles (to end no later than six months postpartum), the first step in follicular development, is entirely gonadotropin-independent. Although other factors are undoubtedly at play, it is virtually certain that even the earliest phases of follicular development beyond the primordial follicle stage ar gonadotropin-dependent. Once recruited, primordial follicles migrate towards the medullary region of the ovary for further development.

    The next phase in follicular development, the so-called slow growth phase, is concerned with the conversion of primordial follicles 60 um in diameter to primary follicles and eventually to mature secondary but still preantral follicles 120 um in diameter. The process begins when the spindle-shaped granulosa cell precursors of some primordial follicles differentiate into a single layer of cuboidal cells surrounding a primary oocyte, thereby yielding primary follicles. Thereafter, proliferation of primary follicular granulosa cells gives rise to multiple cellular layers, thereby yielding a preantral and ultimately antral "secondary follicle," the maximal granulosa cell endowment of which is estimated at 600. It is at this point that the granulosa cells become physiologically coupled by gap junctions. The resultant electrical coupling yields an expanded, yet integrated and functional, syncytium concerned with metabolic examination change and the transport of diffusible low molecular weight substances, thereby compensating for the otherwise avascular intrafollicular environment. Moreover, the granulosa cells extend cytoplasmic processes to form gestational gap-junction-like unions with the plasma membrane of the oocyte. Undoubtedly, it is this latter communication system which is responsible in large measure for the tight control exerted by the cumulus granulosa cells on the resumption of meiosis by the enclosed primary oocyte.

    Although the early "theca interna" has in fact been acquired at the end of the primary follicle stage, the "theca externa" is a characteristic of the secondary follicle forming only as the follicle expands and compresses surrounding stroma. Whereas the theca interna cells assume an epithelioid appearance and characteristics of steroidogenic cells, the theca externa, in turn, retains its spindle-shaped configuration, thereby merging with adjacent stromal cells.

    It is at this juncture that the secondary, still pre-antral follicle embarks on an 85-day journey, spanning three ovulatory cycles, during which a secondary follicle, 120 um in diameter, will be converted into a Graafian preovulatory follicle, 20 mm in diameter. The first leg of the journey, the so-called accelerated growth phase, constitutes the folliculogenic segment wherein preantral secondary follicles 120 um in diameter are converted into antral follicles, 2 mm in diameter. This growth phase is characterized by a 600-fold increase in granulosa cell endowment concurrent with a greater than 15-fold increase in overall follicular diameter. This overall increase in follicular size is accomplished not only through granulosa cell proliferation but also through progressive enlargement of the antrum (central follicular fluid-filled cavity), thereby establishing secondary antral follicles.

  4. Follicular Recruitment

    The term recruitment is employed here to indicate that a follicle has entered the final growth trajectory, a well-characterized pattern of growth and development. It is the luteal pool of 2 mm secondary antral follicles which constitutes the launching pad from which the follicles destined to ovulate in the next cycle will be recruited. Indeed, follicles must go through the terminal exponential growth phase during which time 2 mm follicles achieve preovulatory (Graafian) status (and a 20 mm diameter) at which point the oocyte occupies an eccentric position, surrounded by several layers of cumulus granulosa cells.

  5. Follicular Selection

    Importantly, it is during this last phase of folliculogenesis that follicular selection is completed. This term implies the final winnowing of the maturing but not quite yet dominant follicular cohort, by atresia, down to a size equal to the species-characteristic ovulatory quota. In the human, follicular selection is presumed to occur during the first five days of the cycle at a time when the leading follicular diameter is 5 to 10 mm.

  6. Follicular Dominance

    The term dominance refers to the status of the follicle destined to ovulate, given its presumed key role in regulating the size of the ovulatory quota. It is generally agreed in the human that a selected follicle becomes dominant about a week before ovulation; i.e., as early as days 5 to 7 of the cycle at a time when follicular diameter is around 10 mm. Only the dominant follicle can at this point in time boast detectable levels of FSH in its follicular fluid. Expectedly, this same follicle also displays significant follicular levels of estradiol.

  7. Ovulation

    In the end, it is the dominant follicle which, under the influence of the midcycle LH surge, undergoes dramatic transformations designed to effect further oocyte maturation as well as follicular rupture. All told, it is the rupture of the follicle which concludes the life cycle of the follicle proper while initiating the life cycle of its successor, the corpus luteum. As midcycle approaches, a dramatic rise is noted in the circulating levels of estradiol followed in turn by an LH (and to a lesser extent FSH) surge, the ability of which to trigger follicular rupture is well established. It is the midcycle gonadotropin surge that marks the end of the follicular phase of the cycle and precedes actual rupture by as much as 36 hours. For reasons not well understood but possibly because of unique microenvironmental circumstances, one (rarely, more than one) follicle ovulates and gives rise to a corpus luteum during each menstrual cycle. Mechanically, ovulation consists of rapid follicular enlargement followed by protrusion of the follicle from the surface of the ovarian cortex. Ultimately, rupture of the follicle results in the extrusion of an oocyte-cumulus complex. Fortuitous endoscopic visualization of the ovary around the time of ovulation reveals that elevation of a conical "stigma" on the surface of the protruding follicle precedes rupture. Rupture of this stigma is accompanied by gentle, rather than explosive, expulsion of the oocyte and antral fluid, suggesting that the latter is not under high pressure.

  8. Corpus Luteum Formation and Demise

    After ovulation, the dominant follicle reorganizes to become the corpus luteum. Thus, following rupture of the follicle, capillaries and fibroblasts from the surrounding stroma proliferate and penetrate the basal lamina. This rapid vascularization of the corpus luteum may be guided by angiogenic factor(s) readily detected in the follicular fluid. Concurrently, the mural granulosa cells undergo morphologic changes collectively referred to as "luteinization." These latter cells, the surrounding theca-interstitial cells, and the invading vasculature intermingle to give rise to a corpus luteum. Clearly, it is this endocrine gland which is the major source of sex steroid hormones secreted by the ovary during the postovulatory phase of the cycle. An important aspect of this phenomenon is the penetration of the follicle basement membrane by blood vessels, thereby providing the granulosa/luteal cells with circulating levels of LDL.

    Normally, the functional span of the corpus luteum is 14 + 2 days. Thereafter, the corpus luteum spontaneously regresses, to be replaced (unless pregnancy occurs) at least five cycles later by an avascular scar referred to as the "corpus albicans." The mechanisms underlying luteolysis remain unclear. However, there is little doubt as to the central role of LH in the maintenance of corpus luteum function. Thus, withdrawal of oligohydramnios support under a variety of experimental circumstances has virtually invariably resulted in luteal demise. However, in the event of an intervening pregnancy, hCG secreted by the fetal trophoblast maintains the ability of the corpus luteum to elaborate progesterone, thereby enabling the maintenance of early gestation until the luteoplacental shift.

  9. Signalling Systems

    Preantral granulosa cells are predominantly targeted by FSH. Indeed, a negligible number of LH receptors is observed in preantral granulosa cells. At that point the binding of LH is confined to theca- interstitial cells. Importantly, however, granulosa cells of antral follicles appear capable of binding both LH and FSH. Thus, in contrast to the presence of FSH receptors in granulosa cells from follicles of all sizes, LH receptors are found only in granulosa cells of large preovulatory follicles. These observations are in keeping with the notion that the ontogenetic acquisition of LH receptors is under the influence of FSH. Both LH and FSH hormonal action appears to require the intermediacy of the membrane-associated enzyme adenylate cyclase. Indeed, it is generally accepted that gonadotropin-mediated stimulation of adenylate cyclase results in the conversion of intracellular ATP to cAMP. The latter, in turn, is thought to bind to the regulatory subunit of a protein kinase (commonly referred to as A-kinase) whereupon the catalytic subunit of the enzyme is activated and dissociated. The latter, in turn, phosphorylates key intracellular proteins central to the signal transduction sequence. However, the exact nature of the proteins involved remains unknown at this time.

  10. Estrogen Biosynthesis

    Granulosa cells are the cellular source of the two most important ovarian steroids, estradiol and progesterone. Although the granulosa cells and their luteinized counterparts are capable of producing progesterone independent of other ovarian cell types, the biosynthesis of estrogens requires cooperation between the granulosa cells and their thecal neighbors. The participation of these two cell types and of the two gonadotropins (FSH and LH) in ovarian estrogen biosynthesis underlies the concept of the two cell/two gonadotropin hypothesis, an integrative process required for ovarian estrogen biosynthesis. According to this view, theca-derived, LH-dependent, aromatizable androgens (androstenedione and testosterone) are acted upon by FSH-inducible granulosa cell aromatase activity. A broader view of this concept could and probably should allow its extension to include intercellular exchanges of other steroidogenic substrates (e.g., C21 progestins).

  11. Progestin Biosynthesis

    The granulosa (like the theca-interstitial) cell is amply endowed to carry out progestin biosynthesis. Central to this process is the availability of abundant supplies of cholesterol which serves as the starting material for the steroidogenic cascade. Recent studies have shown that cholesterol used for steroid hormone production is derived primarily from circulating serum low-density lipoprotein (LDL) rather than from de novo cellular biosynthesis from acetate. LDL particles are known to bind to specific membrane receptors, the LDL-receptor complexes entering the cell by receptor-mediated endocytosis. The resultant free cholesterol is re-esterified and is stored in the cytoplasm in lipid droplets. Faced with steroidogenic demands, the cholesterol ester is hydrolyzed and the free cholesterol transported to mitochondria for standard steroidogenic processing. Accordingly, cholesterol is converted to pregnenolone by way of the rate-limiting mitochondrial enzyme cholesterol side chain cleavage. The subsequent conversion of pregnenolone to progesterone occurs relatively readily by virtue of the relative abundance of the cytoplasmic enzymes 3 beta-hydroxysteroid dehydrogenase/D5,D4-isomerase.

  12. Androgen Biosynthesis

    There is little doubt that the granulosa and theca-interstitial cells are capable of elaborating a large number of proteins as assessed by gel fractionation analysis. It is equally clear that the identity of most of the proteins elaborated remains a mystery at this time. On the other hand, a measurable number of readily identifiable proteins has been studied. Many, such as steroidogenic enzymes and cell surface receptors, are quite self-evident. Yet others are briefly discussed below.

    1. Inhibin, an FSH-inducible (32kDa) protein, constitutes a unique granulosa cell marker, the functional role of which in reproductive physiology is under active investigation. Structurally, inhibin is a hetero dimer comprised of a common alpha-subunit (18kDa) but different beta-subunits (14kDa). Both forms (a/BA and a/BB) of inhibin (A and B respectively) possess similar physiological properties. Although inhibin is likely to play an endocrine role by inhibiting pituitary gonadotropin release, recent studies indicate that inhibin may also play a local intraovarian role.

    2. Activin, unlike inhibin, is comprised of dimers of the beta subunits of inhibin (BA/BB or BA/BA). Although possibly active at the level of the hypothalamic-pituitary unit, granulosa cell-derived activin has also been shown to enhance the FSH-supported induction of granulosa cell LH receptors.

    3. Follistatin, which is a recent addition, constitutes a single-chain polypeptide (315 amino acids) originally isolated from porcine follicular fluid. Although structurally distinct from both inhibin and activin, this FSH-inducible granulosa cell-derived polypeptide appears to suppress the release of pituitary FSH, but not LH, in a manner reminiscent of inhibin. The potential relevance of follistatin to ovarian physiology, if any, remains unknown at this time.


  13. The Climacteric Ovary: Gonadotropin-Dependent Androgen Production

    Despite its exposure to high circulating levels of gonadotropins, the postmenopausal ovary is an atrophic, yellowish, lusterless structure with a wrinkled surface weighing less than 10 gm. Microscopically, the cortex is thin and usually devoid of follicles (11.25; formerly 11.14). Although devoid of follicles, the menopausal ovary is not a defunct endocrine organ. Indeed, analysis of peripheral and ovarian vein blood from postmenopausal women indicates that the postmenopausal ovary secretes predominantly androstenedione and testosterone. In fact, the concentrations of testosterone and androstenedione in ovarian venous effluents of postmenopausal women are 15 and 4 times higher, respectively, than their peripheral venous levels.

    Occasionally, the postmenopausal ovarian cortex shows evidence of stromal hyperplasia. When stromal hyperplasia is florid, the ovary may be enlarged, consisting almost entirely of hyperplastic stromal nodules. In such cases, the lipid-rich luteinized cells of the hyperplastic stroma resemble the theca interna cells of the follicle. Thus, ovaries with stromal hyperthecosis may produce enough androgens to result in circulating testosterone levels within the male range, hirsutism, and virilization.

    The medulla of the postmenopausal ovary is large in relation to the cortex, comprising corpora albicantia and candicantia traversed by sclerosed blood vessels. Functionally, the most important medullary component may well be the hilar cell comprising groups of large epithelioid cells closely connected to bundles of non-myelinated nerve fibers and small vessels. Histochemically similar to the interstitial cells of the testes, hilar cells are generally assumed to display considerable steroidogenic potential.

    Rarely do hilar cells give rise to functional neoplasms; i.e., hilus cell tumors. The latter usually produce excess amounts of androgens leading to signs and symptoms of virilism. However, signs and symptoms of estrogen excess may also be evident in circumstances characterized by significant peripheral aromatization.

    Given the inevitable hypoestrogenic state consequent to the cessation of ovarian function, several key complications may ensue. These include urogenital atrophy, hot flashes, osteoporosis, and increased cardiovascular morbidity and mortality. These complications, most of which are partly, if not fully, traceable to estrogen deficiency, are most appropriately managed by the provision of estrogen replacement therapy. This, for the most part, can be done by providing estrogen for some or all of the calendar month. Estrogen, in turn, may have to be supplemented by a course of progestin for women with an intact uterus in whom protection of the endometrial lining is essential. If unchecked, persistent unopposed estrogenic stimulation may lead to endometrial hyperplasia and even endometrial cancer. There is little doubt that in the absence of contraindications to estrogen replacement therapy, all estrogen-insufficient menopausal individuals are in principle eligible for estrogen replacement therapy. Although the latter is associated with small, albeit uncertain, risks, current consensus favors the notion that the benefits far outweigh whatever risks may be associated with this therapeutic approach.


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Take Home Points

  1. Optimal ovarian estrogen biosynthesis is contingent upon the cooperation of the two gonadotropins (LH and FSH) and the two ovarian somatic cell types (granulosa and theca). In contrast, progesterone biosynthesis is primarily LH-dependent and is carried out at the level of the granulosa-lutein cell.

  2. The pronounced progestational capabilities of the corpus luteum reflect its highly vascular nature, a phenomenon due to the breaching of the follicular basement membrane at the time of ovulation along with neovascularization of the former follicular apparatus.

  3. FSH reception appears to constitute an early feature of the granulosa cell. Consequently, it is the FSH receptor that provides the granulosa cell with a window to the outside world through which other signaling systems can be acquired.

  4. Contrary to conventional wisdom, the indispensability of estrogens to intraovarian physiology is now being challenged. A strong body of evidence would suggest that primate/human follicle may not depend on estrogen for growth and maturation.

  5. The ovary itself may, in fact, play a zeitgeber(German - "timegiver") role during the menstrual cycle. A time-keeping function subserved by the activities of the cyclic structures of the dominant ovary. The 28-day menstrual cycle is thus the result of the intrinsic life span of the cyclic ovarian dominant structure and not the result of time changes dictated by the brain or pituitary. The dominant follicle thus determines the length of the follicular phase; the corpus luteum determines the length of the luteal phase.

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