Martin R. Clark

Human chorionic gonadotropin stimulation of immunoreactive prostaglandin synthase in the rat ovary

Follicular prostaglandins (PG) increase markedly in the hours after the preovulatory gonadotropin rise in the rat. The present investigation was performed to determine if the increased prostaglandins result from elevation of the amount of the principal enzyme in the conversion of arachidonic acid to prostaglandins, i.e. PG synthase. PG synthase was purified from sheep seminal vesicles and rat ovaries for the preparation of monoclonal antibodies. A monoclonal antibody was utilized in a competitive, microtiter plate-based enzyme immunoassay to quantitate PG synthase protein. Follicular development was stimulated in 26-day-old rats by injection of 20 IU of PMSG, and 51 h later 20 IU of hCG was injected. Ovaries were removed from rats before and 8 h after the hCG injection for quantitation of PG synthase by enzyme immunoassay. PG synthase immunologic activity was increased three-fold by hCG stimulation. These findings support the hypothesis that the preovulatory gonadotropin rise causes increased PG synthase protein in the rat ovary.

The role of protein synthesis in the stimulation by LH of prostaglandin accumulation in rat preovulatory follicles in vitro.

Preovulatory follicles isolated from immature rats, treated in vivo with pregnant mares serum gonadotropin, were incubated in vitro and the accumulation of prostaglandin E measured. The addition of luteinizing hormone (5 mug/ml) increased this accumulation, after a lag period of 3 hours. This delay suggested the involvement of macromolecular synthesis in the mechanism of prostaglandin stimulation by luteinizing hormone. When the synthesis of protein was inhibited by the addition of puromycin (100 muM), the luteinizing hormone stimulation of prostaglandin E in these follicles was completely abolished. This inhibition was not seen with an analogue of puromycin, which does not inhibit protein synthesis, puromycin amino-nucleoside. These data suggest that concomitant protein synthesis is required for the luteinzing hormone stimulation of prostaglandin accumulation in rat follicles.

Localization and in vitro synthesis of prostaglandins in components of rabbit preovulatory Graafian follicles

Graafian follicles obtained 9 hours after the injection of human chorionic gonadotropin (hCG) into mature rabbits were dissected into a follicular fluid component, a granulosa cell-oocyte component, and a residual wall component, (the latter containing mostly theca tissue with a small and variable amount of adhering granulosa cells). The amounts of PGE and PGF were determined for each component. The follicular fluid contained approximately 4-10 times more PGE and PGF than either the granulosa cell-oocyte component or the residual wall component. The latter two components contained approximately equal amounts of these prostaglandins. The in vitro biosynthesis of PGE and PGF was also studied and it was found that the granulosa cell-oocyte component had about 4 fold the capacity of the residual wall, and that the follicular fluid synthesized no prostaglandins. There was no significant effect of LH on either PGE or PGF synthesis in any of the components.

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused in vitro.

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused in vitro were measured in order to compare PG changes in this model system with those that occur in vivo and in isolated, LH-treated follicles in vitro. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 microgram/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17 beta. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement. Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other in vivo and in vitro models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.

Ovarian prostaglandin synthase: Immunohistochemical localization in the rat

Ovarian prostaglandin synthase is stimulated by the luteinizing hormone surge resulting in the preovulatory increase in prostaglandins. In the present study, the ovarian cellular localization of prostaglandin synthase was identified by immunohistochemistry. Ovaries were collected from 27-day-old rats at the time of stimulation with pregnant mare serum gonadotropin (8 IU) (zero hours), 48 hours later, or 8 hours after administration of human chorionic gonadotropin (5 IU). At zero hours prostaglandin synthase immunostaining was present in the theca of larger follicles and interstitial regions. The number and intensity of immunostained cells in the theca increased between zero and 48 hours. The granulosa cell layer adjacent to the basement membrane in large antral follicles exhibited immunostaining. A similar staining pattern was observed 8 hours after human chorionic gonadotropin. These observations indicate that in the rat, the theca, interstitium, and granulosa contain prostaglandin synthase immunoreactivity and may contribute prostaglandins during follicular development and ovulation.

Stimulation of prostaglandin synthetase activity in rat granulosa cells by gonadotropins in vivo.

Abstract The effect of in vivo exposure to gonadotropin on prostaglandin synthetase activity in rat granulosa cells was examined in two experimental settings. The first setting was immature rats treated with pregnant mares serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The second was mature rats on the day of proestrus. In the experiments using immature rats, the administration of hCG (20 I.U.) at noon of the second day after the PMSG (20 I.U.) injection led to large (more than 5 fold) increases in granulosa cell prostaglandin synthetase activity 5 and 10 h later. Follicular fluid PGE levels were also markedly increased at 5 and 10 h after hCG. Similar results were also found in experiments performed with mature proestrus rats. Granulosa cell prostaglandin synthetase activity was elevated at approximately 4 and 8 h after the endogenous LH surge (about 4 p.m. on proestrus), in comparison with the activity at midnight of diestrus, or noon and 4 p.m. on proestrus. In these experiments the changes in prostaglandin synthetase activity (10 fold) also paralleled the increases in follicular fluid PGE concentrations. Thus the exposure to gonadotropin in vivo produced essentially the same effect as we had reported earlier for isolated granulosa cells incubated with LH in vitro . The stimulation of prostaglandin synthetase activity must therefore be ascribed an important role in the physiological regulation of granulosa cell prostaglandin synthesis by LH.

Acute stimulatory effects of luteinizing hormone-releasing hormone (LHRH) and LHRH analogues on the preovulatory rat follicle

Publisher Summary This chapter discusses acute stimulatory effects of luteinizing hormone-releasing hormone (LHRH) and LHRH analogues on the preovulatory rat follicle. In a study described in the chapter, immature Sprague–Dawley rats, 26 days old, were treated with 10 IU PMSG. This treatment resulted in the ovulation of 12–18 ova early on the morning of day 29. Ovarian follicles or granulosa cells were isolated on the morning of day 28. The follicles were incubated in Krebs–Ringer bicarbonate buffer with glucose and 1% bovine serum albumin. Granulosa cells were cultured in Eagles minimal essential medium with Hepes and 10% fetal calf serum. For the in vivo experiments, the rats were hypophysectomized on the morning of day 28. It was found that mammalian oocytes remain arrested in the prophase stage of the first meiotic division until meiosis is resumed in the preovulatory follicle following the LH surge. Oocyte meiosis can also be initiated in vitro by the addition of gonadotrophin to isolated preovulatory rat follicles.