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LHRH and LHRH agonists have been reported to exhibit somewhat paradoxical antireproductive effects. The current studies were undertaken to examine the actions of these substances on one parameter of ovarian function which is required for ovulation, i.e. prostaglandin production. Granulosa cells were obtained from immature rats 48 h after injection of 20 IU PMSG and incubated for up to 5 h in vitro. LHRH, [D-Ala6, des-Gly-NH2(10)]LHRH-ethylamide (Analog I) and [D-Leu6, des-Gly-NH2(10)]LHRH-ethylamide (Analog II) were observed to stimulate prostaglandin accumulation by granulosa cells. Analog I, for example, at 100 ng/ml increased PGE from 0.19 +/- 0.06 in the control to 7.7 +/- 2.01 ng/2 x 10(6) cells (n = 7; P less than 0.01) after 5 h. TRH, on the other hand, had no effect on prostaglandin accumulation. When added with LH or FSH, the stimulation by maximal concentrations of Analog I and the gonadotropins appeared additive. Although the stimulation of prostaglandin accumulation by LH appears to be mediated by cAMP, no effect of Analog I on the amount of cAMP could be detected. cAMP was determined in cells plus medium at times of 1.5 min to 5 h and at concentrations of 10-2000 ng/ml Analog I, but did not change from the control. LHRH and LHRH agonists, therefore, stimulate the accumulation of ovarian granulosa cell prostaglandins and do so in a manner apparently distinct from that of LH or FSH.

Incubation of bovine corpus luteum slices at 37 C with luteinizing hormone showed that 10.0 microng LH/ml caused a maximum rise in tissue cyclic AMP content within 15 min. Slices incubated with 1.0 or 0.1 microng LH/ml showed a much more gradual accumulation of this nucleotide. In the absence of added LH, a marked decline in the amount of cyclic AMP was observed during the first 60 min. The possible role of cyclic AMP in the action of LH was reexamined by studying the dose-response effect of LH on the stimulation of progesterone synthesis, cyclic AMP-dependent protein kinase activation, and cyclic AMP accumulation. After a 2-h incubatin, the results showed that the dose required to elicit a minimal significant stimulation of steroidogenesis was 0.01 microng/ml LH. At this and higher concentrations of LH, a concomitant stimulation of protein kinase activity and progesterone synthesis was also consistently observed. However, significant accumulation of cyclic AMP became consistently detectable only at 0.1 microng/ml LH. This report is the first to show a positive correlation between the activation of cyclic AMP-dependent protein kinase and the stimulation of steroidogenesis in the corpus luteum at the same minimal effective level of LH. These results indicate that cyclic AMP and the cyclic AMP-dependent protein kinase probably play important intermediary roles in the stimulation of steroidogenesis by LH in the bovine corpus luteum.

This investigation uses electron microscopy to examine the effect of prostaglandins on follicular tissue during the ovulatory process. The ultrastructure of follicles from indomethacin-treated rabbits was compared to the ultrastructure of normal ovulatory follicles in order to determine the morphological differences between follicles with negligible and normal prostaglandin synthesis, respectively. The most obvious difference between the two groups of follicles was that the tissue at the apex of normal follicles began to thin out significantly by 9 h post coitus (near the time of ovulation), whereas the follicles from indomethacin-treated animals showed no signs of thinning, even as late as 12 h post coitus. It appeared that the fibroblasts in the follicles with limited prostaglandin synthesis failed to undergo the normal ovulatory transformation from a quiescent to a proliferative state. Otherwise, the prostaglandin-deficient follicles had a number of morphological features similar to those which usually occur in ovulatory tissue. There was detectable loosening of the connective tissue elements and some indication of edema at the apex of the mature follicles. Also, granulocytes became localized in the vascular compartment of these follicles. In addition, there tended to be an increase in the multivesicular structures which protrude from the fibroblasts, as well as an increase in the dense granules which accumulate in the cytoplasm of the surface epithelial cells. Collectively, these data suggest that normal prostaglandin synthesis in ovulatory follicles may be important in the connective tissue decomposition and ultimate thinning of the follicle wall.

The present study was conducted to examine the effect of LH on phospholipid metabolism in corpora luteal tissue. Collagenase-dispersed cells obtained from bovine corpora lutea of early pregnancy were incubated with 32PO4 in the presence or absence of LH and examined for their ability to incorporate this label into phospholipids. LH (1 microgram/ml) significantly increased 32P incorporation into total lipid extracts, with a time course similar to that of progesterone synthesis. This stimulation of 32P incorporation was dependent on the concentrations of LH, and this dose-response relationship correlated well with the dose response of LH-induced progesterone production. Bovine serum albumin and ACTH had no apparent effect on 32P incorporation into phospholipids or progesterone production. Separation of luteal cell phospholipid extracts by thin layer chromatography revealed that LH stimulated the incorporation 32PO4, mainly into phosphatidic acid and phosphatidylinositol, with small increases occurring in the polyphosphoinositide fraction. The LH-induced labeling of these individual phospholipids also appeared to be temporally and dose-related to the LH-induced increases in progesterone synthesis. LH had no effect on the labeling of phosphatidylcholine, phosphatidylserine, sphingomyelin, or cardiolipin. These results indicate that LH has selective effects on phospholipid metabolism in bovine luteal cells which may be a part of the mechanism of action of LH on steroidogenesis.

The effects of several steroid hormones on progesterone synthesis and cAMP accumulation in isolated bovine corpora luteal cells were investigated in an attempt to determine if any of the steroids would affect the basal level of these processes or their response to gonadotropin. Isolated bovine corpora luteal cells responded to LH with a significant (P less than 0.05) increase in progesterone synthesis and cAMP accumulation when incubated at 37 C for up to 1 h. Exogenous cAMP and analogs of cAMP also significantly stimulated steroidogenesis in these incubated cells. Stimulation of progesterone synthesis by 1 microgram/ml LH was significantly suppressed (P less than 0.05) in the presence of 5--10 microgram/ml estradiol. This inhibition appeared to be largely specific for 17beta-estradiol, in that other steroids such as estrone, estriol, 17alpha-estradiol, cortisol, and dihydrotestosterone were not inhibitory. Testosterone was found to be inhibitory, but it is uncertain if this effect was due to the androgen itself or to its conversion to estradiol. Estradiol did not affect the increase in endogenous cAMP caused by LH in these cells, but did inhibit the effect of exogenous dibutyryl cAMP on progesterone synthesis. The magnitude of this inhibition of the effect of dibutyryl cAMP was not, however, equal to the estradiol inhibition of the stimulation of progesterone synthesis by LH. These data indicate that estradiol, a possible physiological luteolytic agent, has a direct inhibitory action on the corpus luteum and produces its suppression by blocking the stimulatory effect of LH at a step after cAMP.

LH stimulates an increase in prostaglandins in vitro in preovulatory follicles from rats pretreated with PMS gonadotropin. The role of cAMP in this action of LH was examined by incubating preovulatory follicles with various substances and determining the resultant prostaglandin (PG) E accumulation by radioimmunoassay. LH (5 microgram/ml) increased PGE accumulation to approximately 4 times the control (181 +/- 23 to 886 +/- 83 pg/follicle). The addition of 20 mM cAMP also stimulated PGE accumulation, and the addition of 20 mM cAMP in the presence of 0.5 mM 1-methyl-3-isobutylxanthine was as effective as LH. Other nucleotides such as ATP, ADP, 3'-AMP, 5'-AMP, cGMP, and O2'-monobutyryl-cAMP did not stimulate PGE accumulation. On the other hand, (Bu)2cAMP, 8-bromo-cAMP, and N6-monobutyryl-cAMP produced an increase in PGE accumulation similar to that observed with LH. In addition, 10 microgram/ml cholera toxin was shown to increase both cAMP and PGE accumulation in preovulatory follicles. These results indicate that the prostaglandin response of follicles is specific for cAMP-like nucleotides or substances capable of increasing intracellular cAMP. The data support the concept that cAMP mediates the effect of LH on PGE accumulation in preovulatory follicles in the rat.