Stimulated Progesterone Secretion Research Articles

Lactate stimulates progesterone secretion via an increase in cAMP production in exercised female rats

The effect of exercise on the production of ovarian progesterone was examined in female rats. During in vivo experiments, diestrous rats were catheterized via the right jugular vein (RJV), and blood samples were collected before and after 10, 15, 30, and 60 min of swimming. In addition, blood samples were collected from the RJV before and 2, 5, 10, 15, 30, 60, and 120 min after 10 min of infusion of lactate (13 mg.kg-1.min-1) through the left femoral vein. To explore if lactate modulates progesterone secretion by acting directly on rat ovary or on anterior pituitary gland (AP), an in vitro experiment that mimicked the in vivo condition was performed. The ovarian tissue was challenged with lactate (0.01-10 mM) or porcine follicle-stimulating hormone (1 microgram/ml) and 3-isobutyl-1-methylxanthine (1 mM) for 60 min, and the AP was challenged with lactate ranging from 0.1 to 10 mM or 10 nM gonadotropin-releasing hormone for 30 min. The postexercise levels of plasma glucose, lactate, and progesterone at 10, 15, and 30 min were significantly higher than the corresponding basal levels. Plasma luteinizing hormone (LH) did not change after exercise. An elevation of plasma lactate and progesterone was found at 15 and 30 min subsequent to 10 min of infusion of lactate. Lactate ranging from 0.01 to 10 mM significantly increased ovarian adenosine 3',5'-cyclic monophosphate (cAMP) and progesterone production in a dose-dependent manner. LH concentration in plasma was not changed subsequent to lactate infusion. LH level in media samples was not altered after incubation of AP with lactate. These results suggest that the increase of plasma progesterone level in rats during exercise is independent of LH secretion and at least in part is due directly to a stimulatory effect of lactate on the production of ovarian cAMP.

Intracellular regulation of progesterone secretion by the superoxide radical in the rat corpus luteum.

In this study we examined the prospect that the superoxide radical (SOR) is involved in the mechanism by which LH stimulates progesterone secretion in the rat corpus luteum (CL). Treatment of dispersed CL cells with low doses of LH or a SOR-generating system (xanthine-xanthine oxidase) resulted in a significant increase in progesterone release and SOR production. High doses of each treatment were inhibitory. SOR generation also decreased hCG binding. To determine whether SOR may be required for progesterone secretion, dispersed cells were electroporated with antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)] and treated with either low (50 ng) stimulatory or high (20 micrograms) inhibitory doses of LH. At 50 ng LH, insertion of SOD or CAT dose-dependently inhibited progesterone secretion. However, at high doses of LH (20 micrograms), which are associated with high levels of SOR, electroporation of SOD or CAT produced the opposite response. This stimulatory response of SOD or CAT on progesterone release was also dose related. These results indicate that SOR may be involved in the mechanisms that stimulate as well as those that inhibit progesterone release. The effect on progesterone secretion appears to be dose related, with small increases associated with stimulation and high levels involved in inhibition of secretion.

Effect of endothelin-1 on bovine luteal cell function: role in prostaglandin F2alpha-induced antisteroidogenic action.

Endothelin-1 (ET-1) a vasoactive peptide, is synthesized and secreted by endothelial cells. In the bovine corpus luteum (CL), endothelial cells constitute a major proportion (53.5%) of total CL cells. This study was designed to examine the effects of ET-1 on bovine luteal cell functions and its involvement in the action of PGF2alpha. To better define the cells implicated in this process, we used CL slices, whole CL-derived cells, and steroidogenic large (LLC) and small (SLC) luteal-like cells. High affinity binding sites for ET-1 (K(d), approximately 0.3 x 10(-9)) were present in both steroidogenic luteal cells. The binding affinity of ET-1 was 3 orders of magnitude higher than that of ET-3, and a selective ETA receptor antagonist (BQ123) competed similarly to ET-1, suggesting the presence of ETA receptors. The lack of effect of ET-3 on CL-derived cells further supported this conclusion. Both basal progesterone secretion and bovine LH (5 ng/ml)-stimulated progesterone secretion from CL-derived cells were significantly inhibited by ET-1 in a dose-dependent manner, whereas preincubation of these cells with ETA receptor antagonist prevented the inhibitory effect of added ET-1. Incubation of LLC with 10(-8) M ET-1 inhibited their progesterone secretion (114.8 vs. 176.7 ng/10(5) cells-20 h; P < 0.05). On the other hand, ET-1 did not affect progesterone production from SLC despite the presence of ET-binding sites. PGF2alpha only inhibited LH-stimulated progesterone secretion by luteal slices. This antisteroidogenic effect of PGF2alpha could be prevented by the addition of a selective ETA receptor antagonist. Luteal tissue and microvascular endothelial cells isolated from bovine CL produced ET-1; in contrast, the peptide was undetectable in the culture medium or in cell extracts of either LLC or SLC. These data support the concept that ET-1 may play a paracrine regulatory role in bovine luteal function and propose a novel role for this peptide in mediating PGF2alpha-induced luteal regression.

Differential effects of insulin-like growth factor-I and follicle-stimulating hormone on proliferation and differentiation of bovine cumulus cells and granulosa cells.

Effects of gonadotropins and peptide growth factors on 3H-thymidine incorporation, as an indicator of proliferation of granulosa cells and cumulus cells from bovine ovarian follicles during in vitro culture, were determined. Incorporation of 3H-thymidine in cumulus cells during 24-h culture in protein-free medium was about 100-fold greater than in granulosa cells from small follicles. Small but significant (p < 0.05) increases in incorporation of 3H-thymidine in cumulus cells (2.5-fold), but not in granulosa cells, resulted from addition of FSH, LH, or epidermal growth factor to culture media. Much larger, highly significant (p < 0.01) increases in 3H-thymidine incorporation resulted from exposure of both cumulus cells (18-fold) and granulosa cells (6-fold) to insulin-like growth factor-I (IGF-I). Addition of IGF-I together with FSH resulted in additive increases in 3H-thymidine incorporation in granulosa cells over a wide range of each agonist. In contrast, addition of FSH markedly decreased the stimulatory effect of a maximally effective concentration of IGF-I on incorporation in cumulus cells. Addition of estradiol, either alone or with IGF-I, had no consistent effect on 3H-thymidine incorporation in either cumulus cells or granulosa cells. Differentiated functions of cumulus and granulosa cells, including cumulus expansion (an indicator of hyaluronic acid secretion) and progesterone secretion, were investigated under the same culture conditions. FSH was highly effective in stimulation of cumulus cell expansion, with maximal expansion occurring at the lowest concentration tested (0.04 micrograms/ml). LH was effective only at considerably higher concentrations consistent with degree of contamination with FSH. IGF-I was ineffective in inducing cumulus expansion, either alone or together with FSH. FSH and IGF-I by themselves each stimulated progesterone secretion by both cumulus and granulosa cells, with further additive stimulatory effects by the two agents in combination. On a per cell basis, cumulus cells produced considerably more progesterone than granulosa cells under both basal and hormone-stimulated conditions. The results suggest that IGF-I has a dual role in follicle regulation--that it acts additively with FSH as an autocrine regulator of granulosa cell growth and, in the absence of FSH, as a paracrine regulator of cumulus cell proliferation in small antral follicles. The effect of FSH, by inducing cumulus expansion, may terminate further cumulus cell proliferation before ovulation.

Differential effects of oxytocin on steroid production by bovine granulosa cells

Oxytocin (OT) and its mRNA are expressed at very low levels in granulosa cells from bovine preovulatory follicles isolated before the LH FSH surge and increase dramatically between the surge and ovulation. We have shown previously that OT stimulates progesterone secretion by granulosa cells obtained before, but not after the gonadotropin surge, suggesting that OT may be involved in the follicular/luteal phase shift in steroidogenesis from estradiol/androgen to progesterone. One objective of this study was to determine if OT affects estradiol as well as progesterone production by utilizing culture conditions that maintain estradiol secretion in vitro. A second objective was to determine if OT regulates steroidogenesis by effects on the levels of mRNA for the steroidogenic enzymes involved in progesterone synthesis, cytochrome P450 side-chain cleavage (P450 scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), or in estradiol production, cytochrome P450 aromatase (P450 arom). Granulosa cells were isolated from bovine preovulatory follicles and cultured for 3 days with or without OT in medium supplemented with either insulin (1 μg/ml) + 1% fetal calf serum (FCS), which maintains basal estradiol secretion, or low doses of FSH (1 and 2 ng/ml) + 1% FCS, a culture condition that maximizes effects of FSH on estradiol secretion. After the first day of culture, OT stimulated progesterone ( P < 0.01) and inhibited estradiol production ( P < 0.01) in both control and FSH-treated cultures. In contrast, OT had only a small stimulatory effect on the levels of mRNA for P450 scc and 3β-HSD and no effect on mRNA for P450 arom. These findings suggest that: (1) OT plays an autocrine role in regulating the follicular luteal phase shift in steroidogenesis by both increasing progesterone and inhibiting estradiol production and (2) the differential effects of OT on steroid production are not mediated primarily by effects on levels of mRNA for steroidogenic enzymes.

Effects of growth hormone, prolactin, insulin-like growth factors, and gonadotropins on progesterone secretion by porcine luteal cells.

Growth hormone (GH) and IGF-I have receptors within the corpus luteum (CL) and stimulate CL function. Our objective was to investigate the effects of GH, prolactin (PRL), IGF-I, IGF-II, LH, and FSH on progesterone secretion by porcine luteal cells during mid-pregnancy. Gilts (crossbred Yorkshire/Landrace) were slaughtered on d 44 of pregnancy and CL were collected. Large and small luteal cells (LLC and SLC, respectively) were obtained from dissociated CL and separated by elutriation. Luteal cells were incubated with 0, 1, 10, or 100 ng/mL of GH, PRL, IGF-I, IGF-II, LH, and FSH or combinations of 10 ng/mL of these reagents for 24 or 48 h. Culture media were harvested and concentrations of progesterone analyzed by radioimmunoassay. Growth hormone, PRL, and IGF-I increased (P < .05; 100 ng/mL dose) concentrations of progesterone in media of LLC. Insulin-like growth factor-II, LH, and FSH had no effect on progesterone in LLC cultures. In SLC cultures, GH, PRL, IGF-I, IGF-II, and FSH failed to stimulate progesterone secretion, whereas LH increased progesterone secretion (linear effect of dose; P < .05). Combinations (10 ng/mL each hormone) of GH and IGF-I or PRL and IGF-I increased progesterone secretion by LLC compared with control, GH, PRL, or IGF-I alone (P < .05). Similar combinations of GH or PRL with IGF-I had no effect on SLC. Conclusions are that GH and PRL are stimulatory to progesterone secretion by LLC (location of GH receptor) and SLC are responsive to LH during mid-pregnancy. Both GH and PRL are synergistic with IGF-I for increased progesterone secretion.

Progesterone Release of Bovine Corpus Luteum in Response to Oxytocin in Different Culture Systems.

The aim of the present study was to investigate the effect of oxytocin (OT) on progesterone secretion by bovine corpus luteum (CL) in different culture systems. When luteal cells were cultured for 1, 2, 4 and 15 h at a density of 6 × 104 cells/ml, OT (10-7 M) did not affect progesterone secretion. On the other hand, bovine LH strongly stimulated progesterone secretion by the cells 2-15 h after incubation (P<0.01). In another experiment, a novel luteal slice culture system was used, in which luteal cells maintained cell-to-cell contact. Three slices (15-20 mg/a slice) from a CL were pre-incubated for 1 h, and then each slice was incubated for 1 h in a culture tube with a medium (3 ml) supplemented with and without bovine LH (100 ng/ml) or OT (10-7 M). Slices were replaced every 1 h in a fresh medium during the 5 h incubation experiment. A 1-h stimulation with bovine LH and OT induced significant but different effects on the release of progesterone. OT acutely stimulated progesterone release at doses 10-7 M (155%; P<0.05), and bovine LH significantly increased progesterone release during and after stimulation (223%; P<0.01). These data support the hypothesis that OT plays a role in modulating the mechanisms of progesterone secretion as an intraluteal regulator. Differences observed between the two systems might be due to the presence or absence of cell-to-cell contact.

Functional luteolysis in response to hydrogen peroxide in human luteal cells.

To evaluate the effect of reactive oxygen species in human corpus luteum function, we investigated whether hydrogen peroxide (H2O2) affects the in vitro luteal cell production of steroids. H2O2 treatment (1.0-100 microM) of mid and late luteal cell cultures elicited a dose-dependent decrease in basal progesterone production. However, treatment of mid luteal cells with a low concentration of H2O2 (0.01 microM) significantly stimulated progesterone secretion (P < 0.05). In addition, H2O2 (100 microM) markedly inhibited human chorionic gonadotropin (hCG)-stimulated progesterone and estradiol secretion. cAMP production was enhanced (2.4-fold, P < 0.05) by hCG treatment of luteal cells. The addition of H2O2 (0.1-100 microM) to hCG-stimulated luteal cell cultures elicited a decrease in cAMP concentration (P < 0.05) and in the specific binding of radiolabeled hCG by luteal cells. Progesterone and estradiol production stimulated by dibutyryl cAMP were significantly inhibited by H2O2 (P < 0.05). These findings suggest that H2O2 interferes with basal steroid production and, in hCG-stimulated conditions, it may inactivate the gonadotropin-receptor complex. The anti-steroidogenic action of H2O2 therefore raises the possibility of a modulatory role of H2O2 in human luteal steroidogenesis.

Stimulation of progesterone secretion in dispersed cells of rat corpora lutea by antioxidants

Antioxidants were used to investigate the role of free radicals in control of luteal steroidogenesis. Corpora lutea from pseudopregnant rats were enzymatically dispersed, the cells were incubated with antioxidants, and progesterone production was measured. Addition of the antioxidants nordihydroguaiaretic acid (NDGA), butylated hydroxytoluene (BHT), and the gonadotropin luteinizing hormone (LH) resulted in a dose-dependent increase in progesterone secretion. However, the response pattern to these treatments differed with the age of the corpora lutea, and unlike LH neither NDGA nor BHT treatment resulted in an increase in the intracellular second messenger cAMP. Nevertheless, LH and antioxidant-induced progesterone stimulation could be blocked by the addition of either aminoglutethimide or ketoconazole, cytochrome P450 side-chain cleavage (cytochrome P450 SCC) enzyme inhibitors, which prevent the conversion of cholesterol to pregnenolone and thus block steroid hormone synthesis. Also, unlike exposure to LH, exposure to antioxidants resulted in an additional increase in progesterone production in luteal tissue saturated with 25 hydroxycholesterol, a soluble cholesterol analog which serves as a substrate for cytochrome P450 SCC. This study suggests that the site of antioxidant action in affecting progesterone secretion may be at the cytochrome P450 SCC enzyme. Based on these results and on studies in other steroid hormone-producing cells, it appears that free radicals may be involved in regulating synthesis by modulating activity of cytochrome P450 SCC enzyme in rat luteal tissue.

Metabolism of modified LDL by cultured human placental cells

All major classes of normal circulating lipoproteins can be metabolized by human placental cells. However, the metabolism of abnormal or modified lipoproteins has been little studied. We therefore investigated whether placental cells metabolize acetylated low density lipoprotein (ac-LDL) or oxidatively-modified LDL (ox-LDL), both of which are metabolized by scavenger receptors, and if so, whether modified LDL stimulates progesterone secretion as does normal LDL. Placental macrophages and trophoblasts were isolated on a 40% Percoll gradient after enzymatic digestion. The cellular uptake and degradation of [ 125I]-ac-LDL was 20-fold higher than [ 125I]-LDL in both macrophages and trophoblasts. Both cell types demonstrated high affinity and saturable degradation. Similarly, increased esterification of [ 14C]-labelled oleic acid to cholesterol was observed when cells were incubated with ac-LDL vs. LDL. Uptake of ac-LDL by trophoblasts also was demonstrated by colocalization of fluorescently labelled ac-LDL and fluorescent antibodies specific for trophoblasts. Similar colocalization of fluorescent ac-LDL and fluorescent anti-macrophage specific epitopes was seen in macrophages. Uptake and degradation of [ 125I]-ac-LDL by placental cells was inhibited by increasing concentrations of unlabelled ac-LDL or fucoidin but not LDL, indicating uptake by a scavenger receptor. Both unlabelled ac-LDL and ox-LDL inhibited uptake of [ 125I]-labelled ox-LDL, suggesting uptake by a common mechanism. Although secretion of progesterone by trophoblasts was stimulated by incubation with LDL, progesterone secretion by trophoblasts was not stimulated by ac-LDL and only minimally stimulated by ox-LDL. Conclusions: Scavenger receptors are present in human placental trophoblasts as well as macrophages. Scavenger receptor activity greatly exceeds that of LDL receptor activity in both cell types. However, cholesterol assimilated via the scavenger receptor pathway appears to be disconnected from endocrine steroidogenesis in trophoblasts. Thus, we hypothesize that scavenger receptors function in trophoblasts to degrade modified lipoproteins and prevent toxic effects on placental cellular function and fetal growth and development.

Tumour necrosis factor-alpha differentially alters progesterone and prostaglandin F2 alpha production by porcine luteal cells.

This study examined the effects of tumour necrosis factor-alpha (TNF alpha) on basal and stimulated progesterone secretion, as well as prostaglandin F2 alpha production, by small, large and mixed porcine luteal cells and assessed the action of TNF alpha in the presence and absence of indomethacin. Corpora lutea were isolated from gilts on days 8-9 of the oestrous cycle and enzymatically dissociated. Luteal cells were either subjected to elutriation to isolate small and large cells or were separated from erythrocytes by a polysucrose gradient to serve as the mixed luteal cell group. Then 24-well culture plates were seeded with 150,000 small, 30,000 large and mixed (30,000 large+100,000-250,000 small) luteal cells suspended in 1 ml medium 199 media supplemented with 5 micrograms insulin/ml, 40 ng cortisol/ml and 50 micrograms low-density lipoproteins/ml. Cells were cultured for up to 24 h in a humidified incubator at 37 degrees C with 5% CO2 in air. TNF alpha time- and dose-dependently suppressed (P < 0.05) LH-induced, but not basal, progesterone secretion by small luteal cells. Moreover, TNF alpha inhibited (P < 0.05) forskolin-mediated, but not cyclic AMP-mediated, progesterone secretion by small luteal cells. The LH-stimulated progesterone secretion by small luteal cells was not affected by TNF alpha in the presence of indomethacin. Progesterone secretion by large and mixed luteal cells was not affected by TNF alpha. Prostaglandin F2 alpha production by small and mixed, but not large, luteal cells was enhanced (P < 0.05) by TNF alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism controlling generation of superoxide radical in luteinized rat ovaries during regression.

The mechanism regulating the luteolytic release of superoxide radical (SOR) was examined in prostaglandin F2 alpha (PGF2 alpha)-treated rats. Tail vein injection of PGF2 alpha caused a rapid increase in SOR in mitochondria and plasma membrane samples prepared from luteinized rat ovaries. The peak in the mitochondria preceded that in the plasma membrane, and both occurred before progesterone concentrations decreased in the blood. The amount of SOR produced was greater when samples from the plasma membrane, mitochondria, and cytosol were combined. In plasma membrane samples, SOR generation was lowered by inhibitors of intracellular signaling pathways, but not by cyanide, which blocks electron transport in respiratory enzymes. In mitochondria samples, however, SOR was blocked by cyanide, but not by inhibitors of intracellular signaling enzymes. The addition of phospholipase-A2, phorbol myristate acetate (protein kinase-C activator), or arachidonic acid stimulated SOR production in plasma membrane samples from ovaries of control rats, and phorbol myristate acetate and arachidonic acid inhibited LH-stimulated progesterone secretion in dispersed rat luteal cells. Also, when mitochondria prepared from ovaries of PGF2 alpha-treated rats were added to dispersed corpus luteum cells, there was an increase in SOR generation and an inhibition of LH-stimulated cAMP formation and progesterone secretion. These results indicate that SOR production in the corpus luteum after PGF2 alpha treatment is generated by several subcellular components. Formation in the plasma membrane may be initiated by SOR generation from the mitochondria and regulated by intracellular signaling pathways. Our results indicate that formation of SOR may lead to the disruption of LH stimulation of progesterone secretion.

Prostaglandin F 2a stimulates progesterone secretion by porcine luteal cells in vitro throughout the estrous cycle

In this study we examined the stimulatory effects of PGF 2α on progesterone secretion by procine luteal cells on different days of the estrous cycle, and the effects of PGF 2α, A23187 and PMA on progesterone secretion by isolated large and small luteal cells, in vitro. Corpora lutea were obtained from cycling pigs (days 6–16), collagenase dispersed and luteal cells incubated in medium 199 in the absence or presence of increasing doses of PGF 2α, A23187, and PMA. Progesterone concentrations in spent media were measured by RIA. PGF 2α stimulation of progesterone secretion by mixed luteal cells did not vary significantly throughout the estrous cycle. Progesterone secretion by large, but not small, luteal cells was increased (p<0.05) in a dose-dependent fashion by PGF 2α. A23187 also caused a dose-dependent increase in progesterone secretion by large luteal cells but inhibited small luteal cells. Progesterone secretion by both large and small luteal cells was significantly increased by increasing doses of PMA. We conclude that the stimulatory response of luteal cells to PGF 2α in vitro did not correlate with PGF 2α receptor concentrations (not measured in this study), and we speculate that calcium/protein kinase C may be involved in mediating the stimulatory action of PGF 2α on luteal cell progesterone secretion.

Human atrial natriuretic peptide infusion and ovarian venous progesterone secretion in Yucatan micropigs

Chronic indwelling catheters were implanted in the ovarian and external jugular veins of Yucatan micropigs during the luteal phase. Infusion of hypertonic saline (0.4 mol NaCl l-1) increased the concentration of porcine immunoreactive atrial natriuretic peptide in ovarian venous blood plasma by 0.61-1.81 fmol ml-1 more than that of the saline control group. Conversely, endogenously released porcine immunoreactive atrial natriuretic peptide was associated with low ovarian venous progesterone concentrations. Infusion of human atrial natriuretic peptide induced a dose-dependent increase in the progesterone concentration in ovarian venous blood. The results reported here suggest a duality in the ovarian response to atrial natriuretic peptide: a decrease in progesterone secretion in response to low concentrations and stimulated progesterone secretion in response to high concentrations of this peptide.

Luteotrophic and luteolytic actions of ovarian peptides.

Corpora lutea of all species investigated so far, including the human, produce oxytocin and a variety of other regulatory peptides. The role of these peptides is largely unknown. The subtypes of large luteal cells are able to produce tumour necrosis factor (TNF) and at the end of the luteal phase TNF-producing macrophages invade the aged corpus luteum, indicating that this cytokine may be involved in the process of luteolysis. The present contribution reviews briefly the known functions of oxytocin and substance P in the corpus luteum and then elaborates the possible involvement of luteal and macrophage TNF during luteolysis. Oxytocin applied to intact corpus luteum stimulates the secretion of progesterone and oestradiol. The stimulation of progesterone secretion by oxytocin is due to the stimulated oestrogen production. TNF, when tested in vitro, inhibits both luteal cell progesterone and oestradiol production. The TNF-mediated inhibition of aromatase activity therefore prevents the luteotrophic effects of a variety of peptides including oxytocin. This appears to be the mechanism by which TNF induces luteolysis.

Stimulation of progesterone secretion by recombinant follistatin-288 in human granulosa cells.

The effects of recombinant human follistatin (follistatin-288) on basal and hCG-stimulated progesterone secretion were examined in cultured human granulosa cells. Follistatin increased progesterone secretion in a dose-dependent manner. However, follistatin did not augment hCG- or cAMP-stimulated progesterone secretion. Time-course analysis revealed that follistatin increased progesterone secretion after 24 h of incubation. Follistatin also enhanced basal, but not hCG-stimulated, 20 alpha-hydroxyprogesterone accumulation, indicating that the increase in progesterone accumulation was not due to a blockade of the 20 alpha-hydroxylase metabolic pathway. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, follistatin significantly increased intracellular cAMP accumulation to levels comparable to those induced by 1 IU/ml hCG. These results provide the first evidence of a stimulatory action of follistatin on progestin secretion in the human ovary, which is accompanied by an increased accumulation of intracellular cAMP levels. Follistatin may well be another potential regulator of steroid hormone production in human granulosa cells during the periovulatory period.

Stimulation of progesterone secretion by recombinant follistatin-288 in human granulosa cells

The effects of recombinant human follistatin (follistatin-288) on basal and hCG-stimulated progesterone secretion were examined in cultured human granulosa cells. Follistatin increased progesterone secretion in a dose-dependent manner. However, follistatin did not augment hCG- or cAMP-stimulated progesterone secretion. Time-course analysis revealed that follistatin increased progesterone secretion after 24 h of incubation. Follistatin also enhanced basal, but not hCG-stimulated, 20 alpha-hydroxyprogesterone accumulation, indicating that the increase in progesterone accumulation was not due to a blockade of the 20 alpha-hydroxylase metabolic pathway. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, follistatin significantly increased intracellular cAMP accumulation to levels comparable to those induced by 1 IU/ml hCG. These results provide the first evidence of a stimulatory action of follistatin on progestin secretion in the human ovary, which is accompanied by an increased accumulation of intracellular cAMP levels. Follistatin may well be another potential regulator of steroid hormone production in human granulosa cells during the periovulatory period.

The in vitro perifused rat ovary: IV. Modulation of ovarian steroid secretion by insulin.

Insulin has been implicated as a regulatory factor in ovarian steroidogenesis. To assess this issue, we examined the role of insulin on steroid secretion by whole ovaries from pregnant mare serum gonadotropin (PMSG)-pretreated rats, using an in vitro perifusion ovarian model. Three concentrations of insulin were perifused in the absence and presence of gonadotropin pulse-stimulation. Bioactive insulin concentrations after perifusion of ovarian tissue were 4, 40 and 400 mIU/ml. In the absence of gonadotropin-pulse stimulation, acute perifusion with insulin had no effect on ovarian steroid secretion. During stimulation with luteinizing hormone (LH) plus follicle stimulating hormone (FSH), insulin significantly decreased the secretion of testosterone and estradiol but increased the output of progesterone. This effect was only evident after 180 min perifusion and with the highest concentration of insulin. We conclude that acute elevation of insulin concentration has no effect on basal ovarian secretion of progesterone, testosterone, or estradiol in gonadotropin-pretreated rats. However, insulin has a moderate effect on steroid secretion in ovaries exposed to LH/FSH pulsatile stimulation, resulting in a decrease in estradiol and testosterone release, and stimulation of progesterone secretion.

Cholinergic stimulation of progesterone and estradiol secretion by human granulosa cells cultured in serum-free medium.

Cholinergic effects on hormone secretion by human granulosa cells (GCs) are not well characterized. The aim of this study was to explore the direct action of acetylcholine and carbachol on progesterone and estradiol secretion of human GCs cultured in serum-free medium. Granulosa cells were obtained from 26 women undergoing in vitro fertilization and embryo transfer. Progesterone and estradiol production was measured in the presence and absence of acetylcholine, carbachol, or atropine using radioimmunoassays; statistical analysis of the data was performed by ANOVA. Acetylcholine significantly stimulated progesterone secretion by GCs in a dose-related manner. Estradiol secretion was also stimulated by acetylcholine, but this effect did not show dose dependency. Carbachol showed a similar stimulatory effect, but to a lower degree; both effects can be blocked by acetylcholine. The results suggest that cholinergic action on steroid production by human GCs is mediated through the muscarinic route, and cholinergic neurotransmission may have a physiological significance in the intra-ovarian regulatory pathways.

Direct stimulation of bovine ovarian progesterone secretion by low concentrations of alpha-interferon.

The ruminant conceptus secretes proteins during early pregnancy which maintain the corpus luteum. These trophoblast proteins are related to the alpha II-interferons and prevent luteolysis indirectly by disrupting the secretion of endometrial prostaglandin. Although trophoblast interferons appear to be largely confined to the uterine lumen, it remains possible that they also act peripherally. This report describes in vitro studies which suggest that interferon may influence hormone secretion by the ovary directly. The study employed i) a well defined serum-free culture model in which bovine granulosa cells secrete the luteal hormones progesterone and oxytocin, and ii) serum-free and serum-supplemented cultures of cells from early CL. Dose-response experiments were performed using bovine recombinant alpha-interferon (brIFN). Progesterone and oxytocin secretions were measured over 4-5 days of culture and DNA content was also determined. Low concentrations of brIFN (10(-15) mol/l to 10(-11) mol/l) stimulated progesterone secretion by granulosa cells by up to three fold, without significantly affecting oxytocin concentrations or culture DNA content. Concentrations of 10(-10) mol/l to 10(-1) mol/l suppressed progesterone secretion in a log dose-related manner (r = 0.97) with evidence of toxicity (lower oxytocin concentrations and significantly reduced DNA compared with controls). Progesterone secretion by luteal cells in serum-free culture was stimulated in the presence of 10(-15) mol/l brIFN, whilst high concentrations again caused inhibition. The data show that ovarian cells can respond directly to low concentrations of interferon-like proteins.(ABSTRACT TRUNCATED AT 250 WORDS)