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The mRNA transcripts for trout ovulatory proteins (TOPs) are dramatically up-regulated at the time of ovulation. Previous studies indicated that TOPs were produced by the ovaries and were also present in the coelomic fluid that bathes ovulated eggs. In the present study, Western analysis indicated that TOPs were not present in the coelomic fluid prior to ovulation and therefore must be secreted into the coelomic fluid in large quantities during and after ovulation. Using in situ hybridization and immunocytochemistry, TOP mRNA and proteins were localized to the granulosa cell layer of the postovulatory follicle. A whole-follicle in vitro incubation system was used to look at the effects of various mediators on TOP mRNA and protein levels. Results of several different secondary messenger agonists suggest that TOPs are regulated through a G protein-mediated pathway that does not involve cAMP but may involve the activation of protein kinase C. Other agonists that had significant effects on TOP RNA and/or protein included transforming growth factor alpha (TGF-alpha), serine proteases, corticosteroids, bacterial lipopolysaccharide, and the nitric oxide generator SNAP ([+/-]-S-nitroso-N-acetylpenicillamine). Overall, while several compounds caused significant effects, none were able to reproduce the increase in TOP RNA and protein that occurs in vivo, suggesting that the natural mediator of TOPs may still be untested, or that a combination of mediators may be involved. Finally, coelomic fluid inhibited the growth of the Gram negative bacterium, P. aeruginosa, and this inhibition was lost following immunoprecipitation of TOPs. This suggests that one function of TOPs may be to protect ovulated eggs from bacterial infection.

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Pituitary cell lines (GGH3) expressing the GnRH receptor (GnRHR) were used to investigate the effect of GnRHR concentration on the ability of a GnRH agonist to activate second messenger systems. Four different strategies were utilized to generate cells expressing functionally different concentrations of receptors: (1) transient transfection with different concentrations of wild type GnRHR into GH3 cells, (2) utilization of two cell lines derived from a common stably transfected line expressing high (4,209 +/- 535 receptors/cell) or low (1,031 +/- 36 receptors/cell) concentrations of GnRHR, (3) co-incubation of GGH3-1' cells with a GnRH agonist (Buserelin) and a GnRH antagonist to compete for binding sites, and (4) photo-affinity binding to GnRHR with a GnRH antagonist to change effective receptor concentration. A range of receptor concentrations (1,000-8,000 receptors/cell) were generated by these techniques. Inositol phosphate (IP) and cAMP accumulation were quantified to assess the effect of receptor concentration on receptor-effector coupling. Under all four paradigms, the efficacy and potency of Buserelin stimulated IP production was dependent on receptor concentration. In contrast, Buserelin stimulated cAMP release was relatively unchanged at varying concentrations of GnRHR. This suggests that the cellular concentration of GnRHR affects the induction of cell signaling pathways. These results demonstrate that a single ligand-receptor-complex can differentially activate second messenger systems and present a mechanism by which multiple physiological endpoints can be differentially regulated by a single hormone/receptor interaction.

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A central question in endocrinology is how a single ligand interacting with a single receptor can mediate multiple responses. GnRH interaction with receptor offers a prime example, leading to the regulation of synthesis and release of at least three molecules, regulation of target cell responsiveness and receptor number. The present study suggests a molecular model consistent with extant data that provides a mechanism by which this may occur and, further, which allows for coordinate regulation.

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The authors have briefly discussed the molecular structure, regulation, and function of progesterone receptors in the mammalian ovary. Particularly important is the contrast in the regulatory mechanisms of PR induction in the ovary (gonadotropins/membrane receptor mediated) and other well-known progesterone target tissues, such as the uterus and mammary gland (estrogen/nuclear receptor mediated). Future research will focus on how the PR gene responds to these hormonal regulatory signals in this cell-specific manner. Equally important in this discussion has been the mounting evidence indicating that PRs are an essential component of the ovulatory process. The observation that PR-/- knockout mice are incapable of undergoing ovulation, even in response to gonadotropin challenge, further supports the previous physiological evidence indicating that PRs in preovulatory follicles are induced before, and are necessary for, ovulation. Further studies are required to determine the identity of PR-regulated target genes during the periovulatory period. Although our knowledge of PR structure, regulation, and function has increased dramatically during the past decade, many exciting questions remain related to the regulation and function(s) of PRs in the ovary and other tissues.

PIP: Research into the structure, function, expression, and regulation of progesterone receptors (PRs) in the mammalian ovary has the potential to refine understanding of ovulatory processes. A consistent finding of animal studies is the periovulatory expression of the PR gene in the granulosa cells of preovulatory follicles. The presence of PRs in these follicles in the transition from the preovulatory to periovulatory periods may be crucial to successful ovulation given the finding that the blockage of progesterone action at the level of the ovary substantially decreases ovulation rates in hamsters and rats. Estrogen is the primary regulator of PR gene expression in uterine epithelial cells, but not in the ovary. On the other hand, estrogen action is important for preparing granulosa cells for gonadotropin induction of PR gene expression. This cell-specific pattern of gonadotropins/membrane receptor-mediated PR induction in the ovary and estrogen/nuclear receptor mediation in the uterus and mammary gland requires further study. Also needed are studies to determine the identity of PR-regulated target genes during the periovulatory period. The overall thrust of the existing literature, however, indicates that PRs are an essential component of the ovulatory process.

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