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.