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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.

The relationship between LH-induced steroidogenesis and the production of cyclic AMP and cyclic GMP was studied as a function of LH dose and time in isolated luteal cells from pregnant cows. Submaximal steroidogenic concentrations of LH caused a transient but significant rise in cyclic AMP that peaked after incubation for 5 min. A consequent rise in progesterone occurred at 30 min even though cyclic AMP had returned to the basal level at that time. Higher steroidogenic doses of LH elicited a maximum increase of cyclic AMP at 5 min and this was sustained for up to 1 h; the related progesterone production was significantly raised at 15 min and reached a maximum plateau at 30 min. The corresponding levels of cyclic GMP did not appear to be altered by any of the LH concentrations used. The present study has provided direct evidence that even at very low doses of LH, cyclic AMP plays an intermediary role in the stimulation of steroidogenesis in a mixed population of cells isolated from the bovine corpus luteum. Cyclic GMP, on the other hand, did not appear to play a role in the action of LH on the same population of luteal cells.

The subcellular localization of adenylate cyclase (ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1) in bovine corpus luteum was studied using isotonic and hypotonic homogenization and fractionation conditions. All fractions prepared were assayed for adenylate cyclase, marker enzymes and DNA. Only plasma membrane marker enzyme, 5'-nucleotidase paralleled the distribution of adenylate cyclase under both isotonic and hypotonic conditions (conditionsoth isotonic and hypotonic conditions (coefficient of correlation = 0.95). Two main fractions prepared under hypotonic conditions were subfractionated by discontinuous sucrose gradient centrifugation. The highest amount of adenylate cyclase was found in a fraction having a density approximately equal to 1.13 g/cm3. The specific activity of this fraction was 4--6 times higher than that of the homogenate. The electron microscopic study of this fraction revealed the presence of a single type of particulate material consisting of small vesicles exhibiting a typical unit membrane structure. It is concluded that this adenylate cyclase is primarily localized in the plasma membranes. Basal adenylate cyclase activity of plasma membranes was stimulated 2--3 times by luteinizing hormone (10 mug/ml), 3--4 times by prostaglandin E2 (10 mug/ml), 4--6 times by NaF (0.01 M) and two times by methanol (0.2%).

The purpose of this study was to determine whether estrogens exerted a direct inhibitory effect on progesterone synthesis in isolated human luteal cells in vitro. It was found that hCG stimulated progesterone synthesis by luteal cells, obtained from corpora lutea of the menstrual cycle, whereas cells isolated from corpora lutea of pregnancy were unresponsive to exogenous hCG. Estradiol markedly inhibited (P less than 0.001) this hCG effect in luteal cells of the menstrual cycle, and this inhibition was dose dependent. Estradiol did not block the stimulation of cAMP accumulated by hCG in the luteal cells of the cycle but did inhibit the stimulatory effect of dibutyryl cAMP on progesterone synthesis. These data suggest that estrogens may directly cause functional luteolysis in the human and that its site of action may be after the accumulation of cAMP.

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.

Estradiol has been previously reported to be a potent inhibitor of hCG-stimulated progesterone accumulation in isolated cells from human corpora lutea of the menstrual cycle. We assessed the role of prostaglandin (PG) in this in vitro estradiol-induced inhibition by measuring PGF and adding a blocker of PG synthesis (indomethacin) to the incubation medium. The inhibition by estradiol of hCG-stimulated progesterone accumulation occurred without any increase in PGF accumulation in the incubations. Furthermore, PGF accumulation was markedly reduced (P less than 0.05) at all concentrations of indomethacin testes (0.1, 1, and 10 microgram/ml), but the inhibitory effect of estradiol on hCG-stimulated progesterone accumulation was not prevented. These data suggest that the inhibitory effect of estradiol observed in vitro is not mediated by PGs.

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.