Effect of luteinizing hormone on the pattern of steroid production by preovulatory follicles of pregnant mare's serum gonadotropin-injected immature rats.

Preovulatory follicles were explanted on the day before ovulation from immature rats given a single injection of Pregnant Mare's Serum gonadotropin (PMS) 2 days earlier. The follicles were incubated for 4 h in modified Krebs bicarbonate buffer containing glucose and albumin in absence or presence of ovine luteinizing hormone (NIH-LH-S18; 0.1-10 mug/ml). The accumulation of progresterone, androstenedione and 17beta-estradiol in the medium was determined by radioimmunoassay. As in indicator of LH exposure the meiotic stage of the follicle-enclosed oocyte was determined at recovery by interference contrast microscopy. The first group of follicles were explanted in the morning, before the endogenous gonadotrophin surge. In hormone-free medium the oocytes remained in the dictyate stage, whereas addition of LH induced oocyte maturation. These follicles, when incubated in hormone-free medium, secreted predominantly androstenedione and estradiol and only low amounts of progesterone. In the presence of LH the secretion of all steroids was enhanced. The second group of follicles were explanted in the evening, 2-4 h after the endogenous gonadotrophin surge. After incubation in hormone-free medium the follicle-enclosed oocytes had matured. The steroid secretion by the follicles was different from that of the first group. In hormone-free medium they secreted predominantly progesterone and low amounts of androstenedione and estradiol. Addition of LH to the medium caused further enhancement of progesterone secretion, but had no effect on androstenedione and estradiol secretion. The third group of follicles were explanted in the evening from rats in which the preovulatory gonadotrophin surge had been prevented by Nembutal treatment. Oocyte maturation and steroid secretion did not differ from that found for the first group of follicles explanted in the morning. The results are compatible with the hypothesis that LH, after a transitory stimulation, inhibits androgen and estrogen secretion and stimulates progesterone secretion by the preovulatory ovarian follicle.

Involvement of cyclic AMP and protein synthesis in the desensitization of the prepubertal rat ovary.

Luteinizing hormone (LH) stimulates cyclic AMP (cAMP) production in ovarian cells. After the initial stimulation there is a development of refractoriness (desensitization) to a subsequent LH stimulation. The present investigation was designed to study the role of cAMP in this desensitization process and the influence of protein-synthesis inhibitors. Ovaries from 23-day-old rats were preincubated in buffer, dibutyryl-cAMP, 8-bromo-cAMP or LH. After a washing period in buffer, the ovaries were incubated in the presence of LH. Those ovaries preexposed to the cAMP analogues showed a reduced cAMP-production to LH. This reduction was found to be both dose- and time-dependent, but was never as marked as after preincubation with LH. Desensitization developed more slowly after preincubation with cAMP than with LH. Inhibitors of protein synthesis (puromycin, cycloheximide) completely abolished the cAMP-induced desensitization but only partially prevented the LH-induced desensitization. These results show that processes, dependent and independent of cAMP, are involved in the desensitization after LH stimulation of the prepubertal rat ovary.

Sphingosine and psychosine, suggested inhibitors of protein kinase C, inhibit LH effects in rat luteal cells.

The possible involvement of protein kinase C on luteinizing hormone (LH) effects in dispersed rat luteal cells was investigated using two substances that have been reported to be protein kinase C inhibitors, sphingosine and psychosine. Sphingosine efficiently inhibited protein kinase C activity both in brain and luteal cytosol fractions. Both substances inhibited LH-stimulated cyclic adenosine monophosphate (cAMP) accumulation in a dose-dependent fashion with an LD50 at 3-7 microM (sphingosine) and 40 microM (psychosine). LH-stimulated progesterone production was also inhibited with an ID50 at 6-10 microM (sphingosine) and 40-100 microM (psychosine). The inhibition was not due to an increased phosphodiesterase activity since IBMX (3-isobutyl-1-methylxanthine, 0.1 mM) and RO 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, 0.1 mM) did not abolish the inhibitory effect of sphingosine. To study the mode of action of sphingosine, forskolin and cAMP analogues were tested. The effect of these substances on steroidogenesis was inhibited, as well as the forskolin-induced cAMP accumulation, by sphingosine. This study demonstrates a clear inhibition of LH-stimulated effects by sphingosine and psychosine. LH action in rat luteal cells is discussed in relation to protein kinase C and the possible mode of sphingosine action.

Induction by PGF2a of 20alpha-hydroxysteroid dehydrogenase in first generation corpora lutea of the rat.

20alpha-Hydroxysteroid dehydrogenase (20alpha-OH-SDH) activity was determined in the first generation corpora lutea from prepubertal rats injected with 10 I.U. of pregnant mare's serum gonadotrophin (PMSG) on day 30. The enzyme was not detectable in 1-9-day-old corpora lutea but a significant activity was seen on day 10. Enzyme activity increased during day 11 and day 12. In vivo administration of prostaglandin F2a (PGF2a) induced the enzyme in rats with corpora lutea older than 3 days. When prolactin was given concurrently with PGF2a, the corpus luteum activity of 20alpha-OH-SDH was lower than when PGF2a was given alone. It is concluded that the present "corpus luteum model" is suitable for further analysis of the cellular mechanisms of the luteolytic effect of prostaglandins (PGs) as well as of the role of gonadotrophins in the luteolytic process.

Adenosine as substrate and receptor agonist in the ovary.

In the present study the possible dual effects of adenosine as substrate and adenosine receptor agonist in rat granulosa cells, cumulus-oocyte complexes, luteal cells and ovarian membranes are discussed. Adenosine is an indispensable compound in cell energy metabolism, as precursor to cofactors, second messenger and nucleic acids. Adenosine is also an agonist to adenosine receptors. The adenosine receptor can either inhibit (A1) or stimulate (A2) adenylate cyclase. Alternatively, in some cells adenosine receptor activation is linked to other cellular events like inhibition of Ca2+ fluxes. Adenosine is taken up by isolated preovulatory granulosa and luteal cells from pregnant mare serum gonadotropin-treated immature rats, but follicle stimulating hormone (FSH) decreases the uptake by granulosa cells. Adenosine, but not the non-metabolizable adenosine analogs 5'-(N-ethyl)carboxamide-adenosine (NECA), 2-chloro-adenosine (2-Clado), N6-(R-phenyl-isopropyl)-adenosine (R-PLA) and N6-(S-phenyl-isopropyl)-adenosine (S-PLA), increase granulosa cell ATP levels. FSH and luteinizing hormone (LH) decrease granulosa cell ATP levels in the presence or absence of adenosine. It has previously been shown that FSH and LH decrease oxygen consumption by cumulus-oocyte complexes and increase their lactate production. These effects have been suggested to be due to a competition of cofactors (e.g. ADP) common to glycolysis and the respiratory chain. The fact that adenosine reverse the gonadotropin-induced effects on oxygen consumption and lactate production support this theory. Adenosine and its analogs increase cAMP accumulation in luteal and granulosa cells only in the presence of gonadotropins, and this effect is antagonized by the adenosine receptor antagonist 8-phenyl-theophylline (8-PHT). Furthermore, adenylate cyclase is stimulated by adenosine analogs in membranes from non-luteinized and luteinized ovarian membranes and in luteal cell homogenates. The effect of NECA is antagonized by 8-PHT. In the membranes, the rank order of potency was NECA greater than 2-Clado greater than R-PLA greater than S-PLA, suggesting adenosine A2 receptors. In summary, it is suggested that adenosine can act both as a substrate to intracellular metabolism and as an adenosine A2 receptor agonist in granulosa and luteal cells. A paracrine short loop positive feedback model is proposed where extracellular adenosine, derived from a gonadotropin-induced extracellular increase in cAMP and a decrease in cellular ATP, enhances gonadotropin stimulation in granulosa and luteal cells.