Progesterone Output Research Articles

Effect of a calcium antagonist (ICI 109,081) upon follicular steroidogenesis and oocyte maturation in the golden hamster.

AbstractThe effect of a calcium antagonist (a derivative of dihydropyridazinone: ICI 109,081), upon oocyte maturation, and steroid production by pre‐ovulatory hamster follicles was determined. Sexually immature golden hamsters were injected with 40 iu PMSG, and pre‐ovulatory follicles (591–740 μm) were isolated after 72–78h. The culture of intact follicles in medium containing 1 μg/ml ICI 109,081 showed an increased output of progesterone (p<0.05). However, in the presence of LH, ICI 109,081 was seen to cause significant decreases in the output of progesterone, 17α‐hydroxyprogesterone and androstenedione (all p<0.05) with a significant increase in the production of oes‐tradiol (p<0.05). The culture of oocytes isolated 72h after PMSG showed significant reductions in the number of oocytes resuming meiosis when compared with controls (94%) for ICI 109,081 concentrations of 0.1 μg/ml (58%, p<0.05) and 1 μg/ml (32%, p<0.05). Smilarly, the pre‐treatment of animals with 3 mg ICI 190,081 lh before injection of 25 iu hCG, caused the ovulation of 96/98 ova with an intact germinal vesicle. The results suggest a role of calcium in the regulation of oocyte maturation, and as a mediator in the action of LH upon steroidogenesis by pre‐ovulatory follicles.

The effects of ionophores on steroidogenesis and morphology of avian granulosa cells.

Granulosa cells, isolated by collagenase digestion from the mature ovarian follicle of laying hens, were incubated in the presence of two ionophores, lasalocid (X537A) and ionomycin, to determine their effects on basal and stimulated steroidogenesis, as well as their effects on various cell parameters including DNA, RNA, and protein synthesis. Both ionophores caused a dose-dependent inhibition of agonist-promoted progesterone production and, in the presence of calcium, a small but significant increase in basal output of progesterone. Whereas the conversion of pregnenolone to progesterone was unaffected by the ionophores, the activity of cholesterol side-chain cleavage enzyme was inhibited in a dose-related manner. Both ionophores decreased cellular levels of ATP and inhibited the incorporation of radioactively-labeled precursors into DNA, RNA, and proteins. Morphologically, ionophore-treated cells showed swelling of the rough endoplasmic reticulum. Similar morphological changes were also observed in cells treated with oligomycin, a known metabolic inhibitor. These results suggest that the ionophores lasalocid and ionomycin impair release of energy and thereby exert the principal cause of the inhibited steroidogenic response by granulosa cells to a variety of agonists.

Seasonal variations in semen quality of bulls: correlations with environmental temperature

Seventeen bulls were used for a 12-month survey of semen quality and for the estimation of plasma progesterone and testosterone and semen testosterone concentrations. Sperm output showed two minima, in mid-winter and late summer, and the percentage of abnormal sperm was highest and their ability to survive freezing was lowest at the summer minimum. Plasma progesterone concentration was negatively correlated with the total ejaculate content of testosterone, and positively with the local average maximum daily temperature. Temperature showed a quadratic relationship with the percentage of abnormal sperm ejaculated one month later, with the minimum percentage occurring at 14.5 degrees C. Temperature also showed a quadratic relationship with the numbers of sperm in semen ejaculated two months later, with the maximum number occurring at 17 degrees C. These relationships may reflect the impaired or enhanced survival of the temperature sensitive meiotic prophase and alterations in the output of testosterone and progesterone by the testis.

Evidence for the preferential utilization of esterified cholesterol in progesterone production by rabbit corpora lutea.

Our previous studies show that lipoproteins stimulate progesterone secretion by rabbit luteal cells in vitro and that estradiol modifies this effect. This study examines the relationship between estradiol and serum lipoproteins for progesterone production by rabbit corpora lutea in vivo. Using morphometric analysis, we determined that estrogen treatment of hysterectomized pseudopregnant (E-hyst) rabbits increased luteal lipid volume by mid-pseudopregnancy without altering serum progesterone levels. Treatment of E-hyst rabbits with 4-amino-3,4,pyrazolo pyrimidine (APP) during early to mid-pseudopregnancy reduced serum cholesterol levels without decreasing serum progesterone concentrations. However, 3-hydroxy-3 methyl glutaryl-CoA reductase activity was increased. Thus, in the presence of exogenous estrogen, serum cholesterol is esterified and stored rather than converted directly into progesterone. APP-treatment of E-hyst rabbits during late-pseudopregnancy, when estrogen receptor levels are low, increased serum progesterone levels and reduced intracellular lipid content. Thus, stored lipid is the primary source of cholesterol for progesterone synthesis. In addition, estrogen, via estrogen receptor, is important in maintaining steady progesterone output despite fluctuations in serum lipoprotein levels. A working model for cholesterol utilization by rabbit luteal cells is presented, which suggests that stored cholesterol esters, derived from both endogenous and exogenous sources, is the key source or cholesterol for progesterone production. Furthermore, we propose that estradiol regulates the uptake and storage of cholesterol and its rate of metabolism into progesterone.

Diurnal progesterone rhythms in the female mouse.

The patterns of peripheral progesterone concentrations were investigated in a number of murine models on a 13 h light: 11 h darkness lighting regime. The pattern in the intact mouse at dioestrus was compared with that in the ovariectomized mouse. A diurnal pattern was recorded in both, maxima occurring around the end of the light period; no conspicuous nadir was recorded, levels of progesterone remaining relatively constant over a 14-h period. Adrenalectomized mice displayed no such rhythm, indicating that the adrenal is responsible for any diurnal rhythm in peripheral plasma progesterone concentrations at dioestrus. At pro-oestrus in intact animals a similar rhythm was observed, but the maximum levels of progesterone were approximately five times greater than at dioestrus and, moreover, persisted in adrenalectomized mice, indicating that the rhythm of adrenal secretion of progesterone is masked by ovarian secretion. Ovariectomized mice with implants of oestradiol-17 beta displayed a similar rhythm to that of intact mice at dioestrus, but had significantly higher plasma progesterone levels around the time of the maxima although not over the total 24-h period. An s.c. injection of oestradiol benzoate superimposed on oestrogen levels produced by implants had no significant effect on plasma progesterone levels. Also at pro-oestrus the pattern of peripheral LH concentration was investigated in both the intact and the adrenalectomized mouse. For both, maxima were recorded just before darkness, at 19.00 h, in advance of the progesterone surge. In adrenalectomized mice this surge at 19.00 h was attenuated. The possible role of adrenal progesterone in ovulation and the mechanisms by which endogenous oestrogens might enhance adrenal progesterone output are considered.

Fertility in the Afrikaner cow. 3. Once a day suckling and its effect on the pattern of resumption of ovarian activity and conception rate in early lactation

Earlier studies in the Afrikaner cow indicated that suckling exerts a profound influence on the resumption of cyclic ovarian activity and conception post partum. The aim of this study was to assess the effect of once a day suckling on the resumption of ovarian activity and conception within 100 days of calving. Partial weaning significantly ( P < 0.01) reduced the number of cows which were anovulatory for 100 days post partum from 13 32 of normally suckled (NS) cows to 2 33 in partially weaned (PW) cows. Conception rates were significantly ( P < 0.001) improved from 16 32 in NS cows to 30 33 in PW cows. Within the cows which ultimately ovulated, reduced suckling intensity did not significantly shorten the post-partum interval to first rise (0.27 ng/ml) in plasma progesterone concentration (55 ± 5 days in NS cows to 52 ± 4 days in PW cows). Considering all cows in the study, partial weaning reduced ( P < 0.01) the mean post-partum interval to first ovulation by at least 20 days. Within cows which conceived, the mean interval to conception was 72 ± 5 days in NS cows and 69 ± 3 in PW cows; once again if all cows are considered in the analysis, partial weaning shortened ( P < 0.01) the mean post-partum interval to conception by a minumum of 21 days. The probability of conception appeared to be highest 55–65 days post partum, regardless of whether or not this was preceded by an oestrous cycle of normal duration. Total progesterone output during a shortened luteal phase was estimated to be 10% of the output during a normal luteal phase (5.9 ng/ml·days ± 1.1 vs. 51.8 ng/ml·days ± 5.8, respectively). A reduction in the suckling stimulus had no significant effect on any parameter of luteal function measured in this study. However, the resumption of ovarian activity in Afrikaner cows appears to be suppressed to varying degrees by the constant attentions of a suckling calf. In this study post-partum anovulation was the primary cause of conception failure within 100 days of calving.

Evidence for granulosa-theca interaction in the secretion of oxytocin by bovine ovarian tissue.

We have examined the ability of granulosa cells, from carefully selected preovulatory bovine follicles, to secrete oxytocin in vitro. Although cells from 83% of follicles underwent functional luteinization (greatly increased progesterone secretion) in serum-supplemented culture, only 69% had cells capable of oxytocin secretion. Secretion followed a similar time course in all cultures, with the peak appearing on day 3. Oxytocin, but not progesterone, output could be consistently increased by addition of pieces of theca interna tissue, or theca conditioned medium, to the cultures. The effect could be achieved by exposure to theca tissue at any time prior to peak output without altering the time course of secretion. Oxytocin could not be detected in follicular fluid from any of the selected follicles, nor in medium from theca cultured alone. We conclude that the potential for oxytocin secretion is a feature of follicular maturation which is lost during atresia and that the stimulus to secretion is associated with luteinization but not with progesterone output. Finally, the intermixing of follicular cells during corpus luteum formation may provide a mechanism for the enhancement of oxytocin secretion within a predetermined time frame.

Effects of prostaglandins E 2 and F 2α on progesterone metabolism by rat granulosa cells

Rat granulosa cells were cultured with or without PGE 2 and/or PGF 2α. Accumulation of endogenous progesterone and 20α-hydroxy-4-pregnen-3-one was determined. Additionally, [4- 14C]progesterone metabolism was assessed. PGE 2 increased progesterone accumulation, in part, by decreasing progesterone catabolism to 20α-reduced progestins. In contrast, PGF 2α stimulated 20α-hydroxysteroid dehydrogenase activity, thus increasing progesterone catabolism. Combined treatment with PGE 2 and PGF 2α augmented progesterone accumulation to levels above controls but below those attained with PGE 2 alone. These data indicate that PGE 2 and PGF 2α exert opposite effects on progesterone production and catabolism and that the ratio of PGE 2 to PGF 2α in the local granulosa cell milieu may be of importance in determining overall progesterone output.

Effects of steroids on progesterone output by explants of human chorion.

Human chorion can synthesize and metabolize progesterone, and changes in progesterone synthesis by chorion at term might be important in the processes leading to parturition. We examined whether other steroids present within the maternal compartment and amniotic fluid during late pregnancy influence progesterone output by explants of chorion. We also sought differences in steroid effects on progesterone output in association with labor. Explants were prepared from chorion collected after the spontaneous onset of labor and vaginal delivery and chorion collected after cesarean section without active labor. To study the short-term effects of steroids on progesterone output by chorion, explants were incubated for 4 h with 3 microM pregnenolone and 3 microM of a potential interacting steroid. Other explants were preincubated for 24 h with steroid, then rinsed and incubated for 4 h with 3 microM pregnenolone and 3 microM of the same steroid as during preincubation. Under these conditions, dehydroepiandrosterone and androstenedione inhibited progesterone output by explants of chorion obtained at spontaneous labor and at cesarean section. Testosterone also inhibited progesterone output, but only in cesarean section chorion. If explants were preincubated for 24 h with steroid and then rinsed and incubated for 4 h with pregnenolone only, progesterone synthesis returned to control values. This finding indicates that the mechanism of action of these inhibitory steroids is likely through an effect on 3 beta-HSD activity and not due to a change in the rate of enzyme synthesis. We also noted apparent stimulatory effects of steroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of 20 alpha-dihydroprogesterone on progesterone output by human chorion explants.

The output of progesterone by explants of chorion incubated without exogenous precursor was increased in the presence of 20 alpha-dihydroprogesterone (20 alpha-diHP). We have now examined the nature of the 20 alpha-diHP effect. Explants of chorion were incubated with [3H]progesterone and 3 microM non-radiolabelled 20 alpha-diHP to examine the effect of 20 alpha-diHP on progesterone metabolism. Significantly more radioactivity remained as unmetabolized [3H]progesterone compared to chorion incubated in the absence of non-radiolabelled 20 alpha-diHP. There was also a decrease in the output of [3H]-20 alpha-diHP by the explants but no change in the output of a second metabolite of progesterone [3H]-5 alpha-pregnanedione. When explants were incubated with 20 alpha-diHP in the presence of the 3 beta-hydroxysteroid dehydrogenase inhibitor, trilostane, the output of progesterone was significantly greater than with explants incubated with trilostane but without addition of 20 alpha-diHP. Radiochemically pure [3H]progesterone was produced by explants of chorion incubated with [3H]-20 alpha-diHP. We conclude that 20 alpha-diHP may serve as substrate for chorionic progesterone production through 20 alpha-hydroxysteroid dehydrogenase. 20 alpha-diHP, formed from progesterone, may also exert product feedback inhibition on this enzyme.

In vitroGnRHa (HOE766) effects on ovarian steroid‐output in non mammalian vertebrates

Abstract Direct effects of gonadotrophin releasing hormone analogs (GnRHa) on gonadal steroidogenesis have been shown, either in vitro or in vivo, in male and female mammals, in the granulosa cell of fowls, and in the testis of amphibians. No information is available on the effect of GnRHa on steroid‐output in the other classes of vertebrate. Here we report preliminary results on in vitro effects of a GnRHa (HOE766) in the female of Torpedo ocellata, Gobius paganel‐lus, Rana esculenta and Podarcis sicula sicula. No effect has been observed in fishes (perhaps due to the experimental conditions). In the edible frog and in the Italian wall lizard, GnRHa treatment exerted a direct effect on progesterone output.

Steroidogenic effect of 17 beta-estradiol on rabbit luteal cells in vitro: estrogen-induced maintenance of progesterone production.

Previous studies have established that 17 beta-estradiol is the principal luteotropic hormone in the rabbit. However, a direct effect of 17 beta-estradiol on rabbit luteal cell progesterone production has been difficult to show in vitro. The goal of this study was to develop a system in which the effect of estrogen on luteal cell progesterone production could be studied in vitro. To that end, a dissociated rabbit luteal cell preparation was developed using collagenase and the resultant isolated cells were studied using a perifusion system. Optimization of the cell digest procedure revealed that: inclusion of 2% bovine serum albumin in our optimal dissociation medium increased cell yield; and animals killed by cervical dislocation maintained more stable levels of progesterone during a 7-h perifusion compared to animals killed with barbituate-induced euthanasia (euthobarb). When dissociated luteal cells were perifused with medium, stable progesterone output (greater than 80% of initial levels) was observed for 5-6 h, after which medium progesterone concentrations declined. The inclusion of 17 beta-estradiol (10(-8) M) in the perifusion medium maintained progesterone output at control levels for up to 15 h. However, the maintenance of progesterone was not noted until after 5 h of perifusion, suggesting that the effect of estradiol may be time dependent. Thus, this investigation describes a rabbit luteal cell dissociation technique and perifusion system that may be used to examine the mechanism through which estradiol acts to maintain rabbit luteal progesterone production.

Modulation by cortisol of adrenocorticotropin-induced activation of adrenal function in fetal sheep.

We examined the hypothesis that cortisol (F) modulates the activation of adrenal function induced by treating fetal sheep in vivo with pulsatile ACTH (P-ACTH). Chronically catheterized sheep fetuses were infused in utero for 100 h between day 127 and day 131 of pregnancy with P-ACTH; P-ACTH plus metopirone; P-ACTH plus metopirone plus F; P-ACTH plus metopirone plus dexamethasone, or saline (controls). After 100 h, basal and ACTH-stimulated output of 11-desoxycortisol (S), F, and progesterone from collagenase-dispersed fetal adrenal cells was measured. Adrenal cells from fetuses treated with P-ACTH in vivo had significantly greater basal and stimulated (delta) outputs of F and S in vitro than controls. These effects were attenuated in fetuses pretreated with P-ACTH plus metopirone. Concurrent in vivo treatment with ACTH plus metopirone plus F restored basal and delta outputs of F and S to values that were not significantly different from those after P-ACTH alone. In vivo treatment with dexamethasone in addition to P-ACTH plus metopirone significantly raised basal outputs of F and S, but the cells were unresponsive to ACTH in vitro. Basal output of progesterone was significantly greater after in vivo P-ACTH plus metopirone plus dexamethasone, but no treatment raised delta progesterone output over controls. These results support a role for glucocorticoids in modulating ACTH-induced activation of adrenal function in late gestation fetal sheep.

The role of catecholestrogens in placental steroidogenesis

The effect of the catecholestrogen, 2-hydroxyestrone (2-OHE1), on placental steroidogenesis was studied by incubating 2-OHE1 with placental explants for 24 hours and measuring the output of estradiol (E2) and progesterone (P4). 2-OHE1 stimulated the accumulation of E2 and P4 in the media. This effect was inhibited by the alpha-adrenergic blocker, phenoxybenzamine, and the beta-adrenergic blocker, propranolol. We conclude that 2-OHE1 affects placental steroidogenesis and that this effect could possibly be mediated through adrenergic receptors.

Influence of high-density lipoprotein on estradiol stimulation of luteal steroidogenesis.

The aim of this investigation was to determine whether luteal cells utilize cholesterol derived from high-density lipoprotein (HDL) for steroidogenesis and whether estrogen enhances luteal utilization of exogenous sterol. Incubation of Day 15 corpora lutea (CL) with different doses of human HDL resulted in a dose-dependent increase in progesterone production. HDL in vitro enhanced the overall steroidogenic capacity. However, the percentage of increases in 17 alpha-hydroxyprogesterone, testosterone and estradiol were significantly less than that of progesterone. Day 12 hypophysectomized and hysterectomized pregnant rats were treated with either estradiol, testosterone or vehicle for 72 h. Serum pregnenolone and progesterone were markedly increased by the steroid treatment, yet in vitro production of progesterone by CL in all the groups was similar. However, in the presence of HDL in the media, only luteal tissues from steroid-treated rats increased their progesterone output. The reduced production of progesterone by luteal cells of vehicle-treated rats was not due to an accumulation of pregnenolone but to an overall reduction in exogenous sterol utilization. In summary, results of this investigation suggest 1) luteal cells of pregnant rats effectively utilize cholesterol from HDL for maximal steroidogenesis, and 2) estradiol may stimulate luteal steroidogenesis, at least in part, by affecting the incorporation or utilization of cholesterol from HDL into the cell.

Effect of PGF-2α on progesterone production in swine luteal cells at different stages of the luteal phase

Suspensions of luteal cells were prepared by enzymatic dispersion of pig corpora lutea obtained at specific times during the estrous cycle. Luteal cells from early corpora lutea produced more progesterone (4.73 ± 0.84 nmol/10 6 cells; day 3) than those from late diestrus (0.73 ± 0.04 nmol/10 6 cells, day 15); (P < 0.05). Bovine LH enhanced progesterone production in a dose dependent manner particularly in cells from 9 to 15 day corpora lutea. Also PGF-2α enhanced progesterone output in cells from mid-late corpora lutea. PGF-2α did not exert any antigonadotropic effect since it further increased the progesterone production induced by LH. Luteal cells produced PGF-2α with levels ranging between 1.6 and 2.7 pmol/10 6 cells throughout the whole luteal phase. The cellular content of cAMP was markedly increased by LH (556 ± 60%) while it was not affected by PGF-2α. Plasma membrane receptors for PGF-2α were not detected in the analyzed tissue.

Steroidogenesis in granulosa cells during follicular maturation: evidence for desensitization-resensitization during the ovulation cycle.

Output of progesterone, the end product of the steroidogenic pathway, was studied in isolated chicken granulosa cells in relation to follicular maturation and during the ovulation cycle with particular reference to the period between the LH peak and ovulation. The evidence gathered from a series of experiments conducted during the past 2-3 years in the authors' laboratory indicate that the steroidogenic capacity of granulosa cells during follicular maturation is regulated not so much by receptor-coupled adenylate cyclase activity as has been proposed by other investigators, but by the activity of key steroidogenic enzymes, particularly the cholesterol 20,22 desmolase. Furthermore, granulosa cells undergo cyclic sensitization following the endogenous LH surge reaching maximal responsiveness about 1 hr before oviposition. This is followed by a rapid desensitization shortly before ovulation. This desensitization extends to the second and subsequent developing follicles probably in proportion to the evolving LH receptors. It is suggested that granulosa cells remain in such a desensitized state for several hours postovulation, during which time progesterone responses to LH are attenuated and consequently ovulation does not occur prematurely. It is proposed that this intraovarian mechanism is an important component of the physiological events that regulate the ovulation cycle in the domestic fowl.

High correlation between prolactinemia, 125-I hLH binding and progesterone secretion by an experimental luteoma

Autoimplantation of an ovary, containing fresh corpora lutea, into the spleen of an ovariectomized rat is followed by strong luteinization and size increase of the grafted gonad. Thus, large amounts of luteal tissue for biochemical studies, and their histological controls are available. Furthermore, progesterone secretion can be easily determined in samples collected from the portal vein. Since prolactin has been implicated in the control of luteal tissue, the role of this hormone on hLH binding and progesterone secretion was determined. Different levels of endogenous serum prolactin were achieved by pharmacological treatments with neurotropic agents. Scatchard plots of 125-I hLH binding data derived from luteoma particulate fractions revealed the presence of one type of binding site with high affinity. At the same time as binding increased, prolactinemia augmented, with a high correlation (R:0.99) between prolactinemia and LH binding. Moreover, progesterone secreted by the luteoma increased as LH binding sites augmented (R:0,97). It is concluded that a high correlation between prolactinemia and LH binding, as well as between this last parameter and progesterone output exists in the experimental luteoma.

Role of lipoproteins and prolactin in luteal function in the ferret.

This study investigated luteal function in vitro during early pregnancy and pseudopregnancy in the ferret. Corpora lutea taken from animals on Day 13 following the ovulatory stimulus (mating or gonadotropin treatment) were dissociated with collagenase and incubated with ovine prolactin (Prl), ovine luteinizing hormone (LH), total lipoprotein fraction from canine serum, canine high-density lipoproteins (HDL), canine low-density lipoproteins (LDL) or combinations of Prl, LH, HDL, and LDL. Total lipoproteins produced statistically definable increases in progesterone accumulation in incubation media at 5 microliter (approx. 50 micrograms protein) through 25 microliter (250 micrograms protein) of the total lipoprotein solution. LDL in doses of 1 or more microgram protein stimulated progesterone accumulation in 2-h incubations and a similar stimulation was observed in the presence of 60 or more micrograms HDL. Prl, LH or the combination of Prl and LH had no apparent stimulatory influence on progesterone accumulation in vitro. Prl in combination with LDL further stimulated progesterone output by luteal cells in short-term incubation relative to LDL alone. Prl and LH together with LDL produced an increase in stimulation over LDL alone, but, for the most part, this augmentation did not exceed that recorded in the presence of the combination of Prl and LDL. No interactions between HDL and luteotropic hormones were present. The results indicate that lipoproteins increase progesterone output from ferret luteal cells, presumably by providing substrate for steroid hormone synthesis. No direct role for LH in ferret luteal function emerged from these experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of guanine nucleotides and divalent cations on forskolin activation of rabbit luteal adenylyl cyclase: evidence for the existence of an inhibitory guanine nucleotide-binding regulatory component.

The effects of guanine nucleotides and divalent cations on the activation of rabbit luteal adenylyl cyclase by the diterpene forskolin were investigated. Saturating concentrations of forskolin elicited 10- to 15-fold stimulation of adenylyl cyclase activity in the absence of added guanine nucleotide. No lag was observed in the time course of forskolin-induced activation. Addition of 10 microM guanosine triphosphate (GTP) and guanyl-5'-yl imidodiphosphate [GMP-P(NH)P] inhibited forskolin activation by 10-15% and 30-40%, respectively, in the presence of 3.0 mM MgCl2. GMP-P(NH)P was more potent than GTP in inhibiting forskolin activation of adenylyl cyclase having an IC50 of 36 nM compared to 610 nM for GTP. In contrast, the Kact for stimulating adenylyl cyclase activity by both GMP-P(NH)P and GTP were similar, 1.00 and 0.86 microM, respectively. GMP-P(NH)P-induced inhibition of the forskolin-activated enzyme was not due to the hysteretic nature of GMP-P(NH)P activation of luteal adenylyl cyclase, as addition of GMP-P(NH)P to an enzyme that had been treated 5 min earlier with forskolin resulted in the immediate inhibition of enzymatic activity. Addition of GMP-P(NH)P to a concentration-effect curve for forskolin increased the Kact value for forskolin form 7.18 to 26.8 microM. There was a different MgCl2 concentration requirement for maximal stimulation of luteal cyclase by GMP-P(NH)P (8 mM MgCl2) and maximal inhibition of forskolin-stimulated activity by GMP-P(NH)P (0.5-0.6 mM MgCl2). Further, MnCl2 concentrations above 1.0 mM completely abolished the inhibitory action of GMP-P(NH)P on forskolin activation of luteal cyclase. In fact, at 2.0 mM MnCl2, adenylyl cyclase activity in the presence of GMP-P(NH)P plus forskolin was greater than that of forskolin alone. Thus taken together, these findings suggest that the rabbit corpus luteum contains an inhibitory guanine nucleotide-binding regulatory component in addition to a stimulatory regulatory component. Further, these components demonstrate differing requirements for both guanine nucleotides and divalent cations in order to interact with the catalytic moiety of adenylyl cyclase. The existence of such an inhibitory component suggests the presence of an inhibitory receptor in the corpus luteum which could negatively regulate adenylyl cyclase resulting in the inhibition of cAMP production and reduced progesterone output from the corpus luteum under normal physiological conditions.