The Sexual Cycle and Ovulation

At sexual maturity (puberty), the female begins a sexual cycle (Le.. a menstrual cycle in bumans) that is besed on rhythmic interaction between the hypothalamo/hypophyseal axis and the ovaries. A given cycle is initiated when the hypothalamus begins secreting gonadotropin-releasing hormone (GnRH) into the hypothalamo-hypophyseal portal system where it travels to the anterior hypophysis (pituitary) and stimulates gonadotropes to secrete follicle stimulating hormone (FSH) and luteinizing hormone (LII).

Under the influence of these two gonadotropic hormones from the pituitary, primordial follicles begin growing in the ovary.

During this developmental process known as folliculogenesis, a large antral cavity forms in the center of the follicle and a thick layer of collagenous connective tissue forms around its perimeter. As the follicle grows, it begins secreting androgens and estrogens.

Ovarian b -estradiol 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 ganadotropin receptors that are responsive to LII and/or FSH.

At this stage of the sexual cycle, the elevated level of circulating b - estradiol induces a sudden increase in GnRH secretion from the neurosecretoly cells of the hypothalamus, and this releasing hormone causes a surge in LII and FSH secretion from the pituitary gland. This surge in gonadotropins initiates the ovulatory process.

During the next several hours, androgen and estrogen secretion is replaced by a marked increase in ovarian progesterone synthesis. The rise in this progestin signals the onset of luteinization of the ovarian follicle. In addition, the elevation in circulating progesterone inhibits further secretion of GuRN, LH and FSH.

The hypothalamo/hypophyseal axis begins to secrete these hormones once again to initiate the next sexual cycle only after the corpus Interim begins to deteriorate (i.e., undergo luteolysis) and progesterone secretion is diminished.

Rupture of the Ovarian Follicle

Mammalian ovulation is a unique biological phenomenon in that it requires the physical disruption of healthy tissue at the surface of the ovary.

Initially, during the first several hours after a mature ovarian follicle has been stimulated by an ovulatory surge in pituitary gonadotropins, there is no conspicuous change in the appearance of an ovulatory follicle.

However, 4-6 hours into the ovulatory process, a follicle will begin to blush. There is clear evidence that the capillaries in the follicle wall have dilated, and the tissue has become hypcremic. There is negligible other macroscopic, or microscopic, evidence of pending rupture until 1-2 hours before the follicle wall will actually burst. As the time of rupture nears, the apex of a mature follicle protrudes more and more above the surface of the ovary and the follicle wall itself gradually becomes thinner.

Eventually, the apical most portion of the follicle becomes translucent and rapidly balloons above the normal curvature of the follicle wall to form a stigma This nipple-like bleb may not form in all species of mammals, and will not occur if the vascular supply to the ovary has been impaired. However, a follicle will usually rupture within several minutes after the stigma forms. The eventual rupture of a follicle is dependent on adequate degradation of the collagenous connective tissue in the follicle wall and on a modest, but essential, intrafollicular pressure of about 20mm Hg that arises from capillary hydrostatic pressure.

After the follicle wall bursts, the oocyte and surrounding cumulus cells are usually extruded within 1-2 minutes. Ovulation is complete when the egg-bearing cumulus mass is expelled from the ovary.

Anatomy of Ovulation

At the apex of a mature follicle, where a stigma forms and the follicle ruptures, there are five different layers of cells (see Figure 1 ). The outemost layer is the surface epithelium, a single-cell layer of cuboid epithelial cells.

The second layer is the tunica albuginea, consisting of fibroblasts and collagen that form a tenacious sheath around the entire ovary. The third layer is the theca externa, the follicle’s own capsule of collagenous connective tissue which delineates its boundary. The fourth layer consists of the secretory cells of the theca interna just inside the theca extends. The fifth and innemost layer is the stratum granulosum, from which extends the cumulus mass and its oocyte.

This section examines the morphophysiological changes that occur in these layers of the follicle wall during the hours preceding the release of the egg. Although the details of the changes are based on ultrastructual studies of ovulatory follicles from the rabbit, most reproductive physiologists agree that the basic anatomy of mammalian follicles are comparable from one species to the next, and it is generally thought that the mechanism of follicular rupture is basically the same in all species of mammals.

In the case of the rabbit, the ovulatory process normally requires about 10 hours. Therefore, this analysis will examine the morphophysiological aspects of the follicle wall at 10 hours before follicular rupture (i.e.. at the beginning of the ovulatory process) (figure 1 ), at approximately !4-i hour before rupture (Figure 2), and at about 1-5 minutes before rupture (figure 3).


10 Hours before Follicular Rupture

1. Surface Epithelium: In mature ovarian follicles, the surface epithelium is a single layer of cuboidal cells that are loosely attached to a thin basal lamina at the surface of the connective tissue (the tunica albuginnea) surrounding the ovary (Figure 1). The most conspicuous feature of these cells is the dense cytoplasmic spheres that are common on the basal side of the cells. The composition of these dense granules is unknown, but it is unlikely that they are involved in the ovulatory process, because the surface epithelium can be gently scraped from the surface of a mature follicle, yet the follicle will still ovulate in response to adequate stimulation by gonadotropin(s). The other interesting feature of the surface epithelium is that the cells contain polymoipbous nuclei that somewhat resemble the nuclei of polyinorphonuclear leukocytes. It is possible that the surface epithelium functions as a first line of defense to protect the vital procreative elements of the ovary.

2. Tunica Albuginea: Beneath the ovarian surface epithelium is the layer of dense collagenous connective tissue known as the tunica albuginea.. This layer, which surrounds the entire ovary, consists almost entirely of fibroblasts, along with extracellular collagen and related ground substance (Figure_I ). The collagen usually is not readily visible by transmission electron microscopy unless the tissue is treated with 1% phosphotungsric acid, or some other stain that increases the electron density of collagen.

The fibroblasts In this thecal tissue give the appearance of spindle-shaped smooth muscle cells if the follicle is sectioned on a plane perpendicular to the apical follicle wall. However, if one cuts thin sections of a follicle on a plane that is tangential to (he surface of the ovary, then the cells In this layer appear round, or ovoid, and it is quite obvious that they are platter-shaped flbroblasts that produce substantial amounts of collagen.

3. Theca Externa: The follicle itself is surrounded by its own layer of collagenous connective tissue called the theca externa. This tissue is quite similar to the tunica albuginea, and these two layers of thecal tissue are so contiguous at the apex of a follicle that it is difficult to distinguish them from one another (Figure 1). The theca externa usually contains a few more fibroblasts than the tunica albuginea, but the outer tunic of connective tissue contains more collagenous exiracellular material. There is not a conspicuous difference in the cellular composition of the theca externa at the apex of a follicle (where rupture will occur) versus the base of the follicle (which is surrounded by ovarian stromal tissue).

4. Theca Interna: This highly differentiated thecal tissue is a thin layer of steroidogenically active cells (hat are supplied by a number of large capillaries which collectively comprise the bulk of the ovarian circulation (Figure 1). Fibroblasts and collagen are sparse in this thin layer. The secretory cells of the theca interna are characterized by large oval nuclei with prominent nucleoli; and, like most steroid-secreting cells, their cytoplasm is dominated by lipid droplets, numerous mitochondria, and Golgi networks that are distributed throughout their smooth endoplasmic reticulum.

These cells are sometimes referred to as “interstitial cells” in the current literature. The interior border of the theca interna is clearly delineated by a thin, but conspicuous, basal lamina called the membrana propria. This basal lamina has been erroneously referred to as a double membrane because of its close association to the plasma membranes of the granulosa cells that adhere to its inner border.

5. Stratum Granulosum: The gramilosa layer at the inner most surface of the follicle wall arises from a single layer of epithelial cells which surround the oocytes of primordial follicles. The granulosa cells that are attached to the membrana propria extend in a columnar pattern from this basement membrane (Fisure 1). The remaining cells toward the follicular antrum are more cuboidal and are distributed inward for a total depth of 3-10 cells, depending on the species of animal The cells of the granulosa layer are metabolically integrated by an extensive labyrinth of gap junctions that couple this layer into a syncytium with the cumulus oophorus.

In the vicinity of the tight junctions between granulosa cells it is common to observe invaginations from one cell to the other that become pinched-off and form phagocytic-Iike vesicles within one or the other of the abutting cells. These vesicles constitute the transfer of cytoplasm from one granulosa cell to another, and their contents can include mitochondria, lipid droplets, or other large areas of cytoplasm. The cumulus mass, which includes the oocyte, consists of granulosa-like cells that protrude inward from any portion of the stratum granulosum, i.e., from either the apical or the basal region of this innermost layer of the follicle. This random morphological arrangement positions the oocyte toward the center of the follicular antrum and probably facilitates its dislodgement and expulsion from the follicle at the time of ovulation. Also, this central location of the fragile germ cell may serve to protect it from the degradative events that occur within the thecal layers of the follicle wall during the ovulatory process.


4-1 Hour before Follicular Rupture

1. Surface Epithelium: Within the last several hours of the ovulatory process, conspicuous morhological changes take place in the epithelial cells on the apical surface where rupture is destined to occur. The cells of the surface epithelium develop numerous vacuoles within their cytoplasm, and the cells appear to be necrotic (figure 2). There is no evidence that the muci-like dense granules in these cells release their contents into the thecal layers of the follicle, or contribute in any way to the mechanism of ovulation. During the final hour of the ovulatory process, these cells begin to slough from the stigma area of the follicle, and they are usually absent at the time of rupture.

2. Tunics Atbuginea: The tunica albuginea that covers the ovarian surface of most preovulatory follicles consists of quiescent fibroblasts. However, conspicuous changes occur in this collagenous connective tissue during the final several hours prior to follicular rupture. The fibroblasts begin to project long cytoplasmic process from their central mass, and on their longitudinal plane these cells become as much as 100 microns in length (Figure 2). The tips of these cytoplasmic processes oftentimes consist of unusual muhivesicular structures that probably contain bioactive agents that contribute to the decomposition of the extracellular collagenous elements during the final stages of the ovulatory process. In addition to these cellular changes, the extracellular matrix of collagen and ground substance begins to dissociate during the hour preceding follicular rupture.

3. Theta Externa: As a follicle approaches the moment of rupture, the collagenous connective tissue of the theca externa undergoes changes similar to the overlying tunica albuginea
(Figure 2). The flbroblasts become much more elongated, their cytoplasmic processes exhibit the same type of multivesicular structures, and the extracelhilar 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 of 15-20mm Hg. The result of this dissociation is a gradually thinning of the follicle wall at the site where rupture will eventually occur.

4. Theta Interns: The principal ovulatory changes in the theca interna occur in the extensive network of capillaries that is characteristic of this area of the follicle wall. Within 4-6 hours after initiation of the ovulatory process there is a measurable increase in ovarian blood flow and the follicles become hyperemic. Follicles become visibly redder as their capillaries dilate, and their blood content increases as much as 5-fold. In addition, the permeability of the thecal capillaries increases significantly during ovulation.

These marked changes in the vasculature result in occasional extravasation of blood and the formation of petechiae in the walls of some follicles. Also, there is an increase in the number of polymorphonuclear leukocytes in the patent blood vessels (picture 2). However, macrophages, or other derivatives of leukocytes are rarely observed in the thecal tissues outside the vascular compartment prior to follicular rupture. Therefore, although some investigators believe that leukocytic cells may contribute the proteolytic enzymes that are thought to degrade the follicular connective tissue during ovulation, it is probably more likely that leukocytes begin to accumulate in ovulatory follicles in response to leukotactic agents that are generated by an acute inflammatory reaction that activates the thecal fibroblasts and initiates ovulatory decomposition of the follicular wall prior to infiltration of the area by leukocytes.

5. Stratum Granulosum: During the last hour preceding follicular rupture, the cells of the granulosa layer become less firmly attached to one another. As the apical area dissociates and the follicle wall becomes thinner, the innermost granulosa cells begin to slough from the wall and become dispersed in the follicular fluid (Figure 2). Those cuboidal cells that remain attached to the membrana propria usually contain an increasing number of lipid droplets that were not present a few hours earlier. Thus, the granulosa cells become steroidogenically active during the hours preceding follicular rupture. Otherwise, there are negligible changes in the ultrastucture of the granulosa cells at the apex of an ovulatory follicle.


1-5 Minutes before Follicular Rupture

it has been possible to obtain electron micrographs of rabbit follicles only a few minutes before rupture (figure 3). Shortly before the follicle wall breaks, it balloons out to form a stigma at the apical most area where it will rupture. Only traces of the surface epithelium remain clinging to the disintegrated tunica albuginea and theca externa, Extravasated erythrocytes appear more frequently in the vicinity of the theca interna, and most of the stigmal region is void of capillaries. Essentially all of the granulosa cells have sloughed into the follicular fluid, or have retracted toward the base of the stigma as the membrana propria disintegrates and the follicle wall undergoes its final thinning before rupture. Rupture ultimately occurs at the apical most area of the follicle simply because this is, morphologically, the thinnest (i.e., the weakest) site along the ovarian surface.

Biochemistry of Ovulation

A. Historical Background

1. The 1960’s: In the 1960’s, it became apparent that ovarian follicles do not rupture as a consequence of any significant increase in intrafollicular pressure. A variety of evidence revealed that rupture is probably due, instead, to the action of proteolytic enzymes that decompose the collagenous connective tissue in the thecal layers of the follicle wall and the ovarian tunic. By the end of the 1960’s, it was also apparent that ovarian steroid metabolism changes markedly in response to an ovulatory surge in gonadotropin. Progesterone synthesis increases within several hours after initiation of the ovulatory process, while ovarian estrogens and androgens decline in a reciprocal pattern.

2. The 1970’s: In the early 1970’s, it was discovered that there was a significant increase in ovarian prostanoid synthesis during ovulation. This information, together with considerable evidence that anti-inflammatory agents like indomethacin can inhibit ovulation, led to the general assumption that prostaglandins E2 and F2a are essential for ovulation, yet other data raises questions about the role of ovarian prostanoids in the mechanism of ovulation. In that same decade it became apparent that ovarian plasminogen activator also increases in response to most gonadotropic hormones. It has been hypothesized that this serine protease might contribute to the ovulatory process by digesting connective tissue components of the follicle wall, or by activating a procollagenase. however, targeted deletion of the genes for several types of plasminogen activator has not yielded an anovulatory phenotype. Therefore, the precise role of plasminogen activator in ovulation remains unclear.

3. The 1980’s: By the 1980’s, more attention was being given to the fact that ovulatory follicles are hiperemic, and that such a vascular response is a cardinal sign of inflammation. In addition, it became evident that a wide variety of non-steroidal anti-inflamniatoiy drugs can inhibit ovulation. This information, along with other supporting data, led to the hypothesis that an ovulatory dose of gonadotropin initiates an inflammatory-like response in mature ovarian follicles. Subsequently, it was demonstrated that ovarian kallikrein activity increases, and that kinin formation might contribute to the ovarian vascular changes during ovulation. As this decade ended, there were reports that cytokines, platelet-activating factor, growth factors, and metalloproteases might also influence the inflammatory response that occurs in ovarian follicles during ovulation.

B. Current Knowledge about the Biochemistry of Ovulation

To augment the above knowledge about the biochemical events of ovulation, several laboratories have
characterized the hormonal regulation of genes for enzymes involved in the synthesis of steroids, eicosanoids, proteases, and other agents that have been implicated in the ovulatory process.

1. Steroid Metabolism: Several members of the cytochrome P450 family of enzymes are now known to be expressed in ovarian tissue in a pattern that is consistent with what is presently known about ovarian progestin and estrogen synthesis during ovulation. Transcription of the gene for cytochrome P450 side chain deavage enzyme, which increases progesterone synthesis by increasing the rate of conversion of cholesterol to
pregnenalone, is up-regulated in ovarian follicles (mainly in the theca interna and stratum granulosum) several hours after the ovulatory process has been initiated by gonadotropins. Conversely, the gene for cytochrome P450
aromatase, which converts testosterone into 17b -estradiol, is concomitantly down-regulated in a pattern parallel to the decline in ovarian estmgen synthesis at the time of ovulation.

2. Ecosanoid Metabolism: Two prostaglandin synthase genes have been identified in ovarian follicular tissues. Prostaglandin synthase-l is constituitively expressed and has been localized to thecal cells and intent cells. Prostaglandin synthase-2 is rapidly and transiently induced by the ovulatory surge in luteinizing hormone and it is localized inclusively in the granulosa cells of those follicles destined to ovulate. The expression of these genes leads to enzymatic activity that causes 50-100-fold increases in prostaglandins E2 and F2a in foflicular tissue during ovulation. In addition to these two eicosanoids there is more recent evidence that lipoxygenase enzymes cause marked increases in 12- and 15-hydroxyeicosatetranoic acids associated with bioactive agents such as the lipoxins.

3. Expression of Other Bioactive Factors: Recent work at the molecular level has revealed ovarian increases of mRNAs for nerve growth factor, oxytocin, tissue inhibitor of metalloproteinase (TIMP), kallikrein, and vascular endothelial growth factor/vascular permeability factor (VEGIPF) after the stimulation of follicles by gonadotropin. Regarding NOF, both the growth factor, itself and the receptor for NGF are expressed by thecal fibroblasts of ovulatory follicles. Most of the evidence indicates that oxytocin mRNA is expressed lathe granulosa layer, but the function of this neuropeptide in the ovary is uncertain. TIMP is also expressed in the granulosa layer, and this inhibitor may modulate ovarian proteolytic activity in the vicinity of the oocyte during ovulation. Ovarian kallikrein activity produces kinins that promote vasodilatation and contribute to the hyperemic reaction in ovulatory follicles. The increase in VEG/PF causes follicular capillaries to become more permeable and promotes angiogenesis during the luteinization of ovulatory follicles.

4. Detection of Novel Biochemicals in Ovulation by Differential Display: It is likely that innumerable other mediators of the ovulatory process remain to be elucidated. The new molecular protocol known as ‘differential display” is a valuable method that is now being used to isolate and identify mRNAs of genes that are uniquely expressed in the ovary during ovulation. This molecular technique, which was developed by P. Liang and AB. Pardee at Harvard University in 1992, is based on the display of differentially expressed mRNA/cDNA by electrophoresis on an acrylamide gel following rtPCR amplification of subpopulations of gene transcripts from different groups of experimental tissues. This method has been used recently to discover the unique expression during ovulation of genes for a carbonyl reductase with 20b -hydroxysteroid dehydrogenase activity, a long interspersed nucleotide element (LINE) that is highly repeated in mammalian genomes, and a nerve growth factor-induced substance (NGFI-A). This latter substance, NGFI-A, is usually expressed concomitantly with the proto-oncogene c-fos and the metalloproteinase saromelysin-1, and therefore it is quite likely that lire transcription of genes for these factors is also up-regulated during ovulation. Thus, in the future, the differential display procedure has the potential of elucidating many other biochemical agents that are involved in the ovulatory process.

Current Hyphotesis on Ovulation.

The above background information on ovulation, along with the current literature on this topic, serve as the basis for the following “working hypothesis” on the mechanism of mammalian ovulation: The ovulatory surge in LII (or, exogenous hCG) Initiates acute changes in steroid and eicosanoid metabolism in the granulosa cells of mature follicles. The ensuing increases in local prostaglandins, lipoxins, kinins, platelet-activating factor, VEG/PF, and other vasoactive agents collectively cause substantial dilatation of the capillaries in the theca interim of the follicle wall. This significant change in the capillaries of ovulatory follicles results in a 4-fold increase in the volume of the ovarian vascular compartment.

Concomitant with this hyperemic response, the permeability of the thecal capillaries increases to the extent that serum proteins are exuded into the interstitial spaces of the Ibilicle in a manner characteristic of inflamed tissues. Since blood serum is well known for its ability 10 activate fibroblasts, it can be predicted that the exuded serum stimulates the quiescent fibroblasts in the theca externa and tunica. albuginea of an ovulatory follicle and causes them to transform into proliferating cells.

The activated thecal
fibroblasts begin secreting a metalloproteinase (perhaps stromolysin-1, which is regulated by NGF) that. degrades the extracellular matrix of collagenous connective tissue in the follicle wall. Ultimately, the follicle wall loses its tensile strength and eventually ruptures under the force of a modest, but effective, intrafollicular pressure.