The non-benzodiazepine anxiolytic drug etifoxine causes a rapid, receptor-independent stimulation of neurosteroid biosynthesis.

Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism.

Expression of aldo-keto reductase 1C23 in the equine corpus luteum in different luteal phases.

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3ß-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.

I. Levels of 5α-reduced progesterone metabolite in the midbrain account for variability in reproductive behavior of middle-aged female rats.

At middle-age, the reproductive capacity of female rats begins to decline. Whether there are consequences for social and reproductive behaviors related to changes in estradiol (E(2)), progesterone (P(4)) and its 5α-reduced metabolites, dihydroprogesterone (DHP) and 5α-pregnan-3α-ol-20-one (3α,5α-THP), is of interest. In Experiment 1, 1-year-old female breeder rats that had "maintained their reproductive status" (having 4-5 days estrous cycles, > 60% successful pregnancies after mating, > 10 pups/litter) or their age-matched counterparts with "declining reproductive status" were assessed in social interaction, standard mating, and paced mating when in proestrus. Rats that maintained reproductive status tended to have higher levels of proceptivity, and significantly reduced aggression, towards males, compared to rats with declining reproductive status. Basal midbrain E(2) and DHP levels accounted for a significant proportion of variance in lordosis. In Experiment 2, 1-year-old, age-matched, female breeders that had maintained reproductive status or were in reproductive decline were compared to three-month old, nulliparous females that had regular (4-5 days) or irregular estrous cycles. Age did not influence paced mating but younger rats had greater diencephalon E(2) than did middle-aged rats. After mating, rats with declining/irregular reproductive status had higher P(4) and DHP levels in midbrain than did rats with maintaining/regular reproductive status, albeit differences in midbrain 3α,5α-THP were not seen. Middle-aged rats that maintained reproductive function had greater 3α,5α-THP formation in diencephalon compared to other groups. Thus, age-related changes in central progestogen formation in midbrain or diencephalon may contribute to some variability in expression of reproductive behaviors.

Oxidative stress-induced inhibition of adrenal steroidogenesis requires participation of p38 mitogen-activated protein kinase signaling pathway.

Previous studies from this laboratory identified excessive oxidative stress as an important mediator of age-related decline in steroid hormone production. Here, we investigated whether oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. To accomplish these studies, we employed a highly responsive mouse adrenocortical cell line, Y1-BS1 cells that secrete large quantities of steroids when stimulated with lipoprotein plus hormone. Treatment of these cells with superoxide, H(2)O(2) or 4-hydroxy-2-nonenal (HNE) significantly inhibited steroid production and increased phosphorylation and activation of p38 MAPK. None of the treatments altered the phosphorylation of either extracellular signal-regulated kinases or c-Jun N-terminal kinases (JNKs). Pretreatment of Y1-BS1 cells with MnTMPyP, a cell-permeable superoxide-dismutase/catalase mimetic reactive oxygen species (ROS scavenger), completely prevented the superoxide- and H(2)O(2)-mediated inhibition of steroid production. Likewise, antioxidant N-acetylcysteine completely blocked the HNE-induced loss of steroidogenic response. Incubation of Y1-BS1 cells with either MnTMPyP or NAC also upregulated Bt(2)cAMP and Bt(2)cAMP+hHDL(3)-stimulated steroid synthesis, indicating that endogenously produced ROS can inhibit steroidogenesis. Inhibition of p38 MAPK with SB203580 or SB202190 upregulated the basal steroid production and also prevented the oxidant-mediated inhibition of steroid production. mRNA measurements by qPCR indicated that Y1-BS1 adrenal cells predominantly express p38 MAPKalpha isoform, along with relatively low-level expression of p38 MAPKgamma. By contrast, little or no expression was detected for p38 MAPKbeta and p38 MAPKdelta isoforms in these cells. Transfection of Y1-BS1 cells with either caMKK3 or caMMK6 construct, the upstream p38 MAPK activators, decreased steroidogenesis, whereas transfection with dnMKK3 or dnMKK6 plasmid DNA increased steroidogenesis. Similarly, transfection of cells with a dnp38 MAPKalpha or dnp38 MAPKbeta construct also increased steroid hormone production; however, the effect was less pronounced after expression of either dnp38 MAPKgamma or dnp38 MAPKdelta construct. These results indicate that activated p38 MAPK mediates oxidant (excessive oxidative stress)-induced inhibition of adrenal steroidogenesis.

Influence of 17beta-estradiol on the synthesis of reduced neurosteroids in the brain (in vivo) and in glioma cells (in vitro): possible relevance to mental disorders in women.

Brain neurosteroids modulate gamma-aminobutyric acid type A (GABAA) receptor activity, thereby playing a role in mood disorders. Alterations in 17beta-estradiol (E2) and progesterone (P) are also known to play a significant role in psychopathology in women. The aim of the present study was to evaluate the synthesis of dihydroprogesterone (DHP), tetrahydroprogesterone (THP), and the activity of 5alpha-reductase (5alphaR) which regulates the reduction of P to DHP on exposure to supraphysiological levels of E2 in vitro (C6 glioma cells) and in vivo (mouse brain). The results showed that supraphysiological levels of E2 induced a decrease in the accumulation of both neurosteroids, probably by decreasing the activity of 5alphaR. We hypothesize that the high levels of E2 in pregnancy attenuate the increase in the conversion of P to THP in the brain and that the ratio of E2/P modulates the sedative effect of THP. This process may be relevant to psychopathological disorders that are ascribed to drastic alterations in estrogen levels, such as premenstrual syndrome, pregnancy-related mental disorders, and postpartum "blues".

Fundulus heteroclitus gonadotropins.5: Small scale chromatographic fractionation of pituitary extracts into components with different steroidogenic activities using homologous bioassays.

Fractionation and characterization of gonadotropins (GtH) from Fundulus heteroclitus pituitary extracts were carried out using a biocompatible liquid chromatographic procedure (Pharmacia FPLC system). Chromatographic fractions were monitored for gonadotropic activities (induction of oocyte maturation and steroid production) using homologous follicle bioassays in vitro. Size-exclusion chromatography eluted gonadotropic activity in one major protein peak (Mr approximately 30,000). Anion-exchange and hydrophobic-interaction chromatography (HIC) yielded two distinct peaks of 17beta-estradiol (E2)- and 17alpha-hydroxy,20beta-dihydroprogesterone (DHP)-promoting activity with associated oocyte maturation. Two-dimensional chromatography (chromatofocusing followed by HIC) resolved pituitary extracts into two active fractions; both induced E2 synthesis, but one was relatively poor in eliciting DHP and testosterone production. Thus, using homologous bioassays, at least two quantitatively different gonadotropic (steroidogenic) activities: an E2-promoting gonadotropin (GtH I-like) and a DHP-promoting gonadotropin (GtH II-like), which has a lower isoelectric point but greater hydrophobicity than the former, can be distinguished from F. heteroclitus pituitaries by a variety of chromatographic procedures. This study complements previous biochemical and molecular data in F. heteroclitus and substantiates the duality of GtH function in a multiple-spawning teleost.

Placentation in the African elephant, Loxodonta africana. I. Endocrinological aspects.

Placental and fetal tissues were recovered from the uteri of 59 pregnant elephant that ranged in estimated age from day 18 to month 21 of gestation. Incubation of placenta and fetal gonad, alone or in combination, with tritium-labelled cholesterol, pregnenolone and androstenedione failed to yield any labelled progestagens or oestrogens from placenta, but did produce small amounts of labelled progesterone and 5alpha-dihydroprogesterone from fetal gonad. Immunochemical staining of tissues with four antisera specific for enzymes involved in the steroidogenic pathway revealed no staining in sections of placenta but positive labelling for P450 side chain cleavage enzyme (SCC450) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) of the interstitial cells that comprise the bulk of the enlarged fetal gonads during the second half of gestation. Saline extracts of placental tissue showed no activity in three different gonadotrophin assays. In view of this endocrinological inactivity in the zonary elephant placenta and the probable reliance on maternal luteal sources of progestagens for maintenance of the pregnant state, the argument is advanced that uncomplicated abortion would probably follow a single administration of a PGF analogue given at any stage of pregnancy. If so, the treatment might constitute an efficacious method for controlling population increases in elephants maintained in enclosed game parks in Africa.

Pathogenesis of diabetic neuropathy--do hyperglycemia and aldose reductase inhibitors affect neuroactive steroid formation in the rat sciatic nerves?

The activation of the polyol pathway through aldose reductase (AR) might be involved in diabetic neuropathy. A considerable structural similarity exists between AR and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) (both belonging to aldo-keto reductase superfamily); 3alpha-HSD forms 5alpha-reduced-3alpha-hydroxylated steroids, possibly possessing neurotrophic functions. Aim of these experiments was to test "in vitro" in rat sciatic nerves, whether glucose concentrations in the diabetic range might affect the capacity of 3alpha-HSD to transform dihydroprogesterone (DHP) into tetrahydroprogesterone (THP), a steroid proved to possess neurotrophic effects. The capability of AR inhibitors, drugs used to avoid diabetic complications, to decrease THP formation was also assessed. 3alpha-HSD activity was evaluated by the conversion of labelled DHP into THP (in a single case dihydrotestosterone was used as substrate, and the corresponding 3alpha-hydroxylated metabolite was evaluated). Freshly prepared rat sciatic nerve homogenates were used as source of the enzyme. Whole brain, liver and prostate served as "control" tissues. The results show that glucose added up to a concentration of 400 mg/dL (well above the euglycemic upper level) does not affect the 3alpha-HSD activity in the sciatic nerve and in the other tissues considered. Similarly, when the enzyme was challenged by two AR inhibitors, tolrestat and sorbinil, added in a concentration about 10 times higher than their IC50 for AR, no significant changes were observed. Analogous results were achieved when DHT was used in presence of glucose (400 mg/dL) and sorbinil. We conclude that hyperglycemia or the administration of the AR inhibitors do not affect 3alpha-HSD activity in peripheral nerves and therefore do not reduce the formation of steroid metabolites possibly endowed with neurotrophic action.

Biosynthesis of the neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha hp), a specific inhibitor of FSH release.

The gonadal steroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) is a neuroactive steroid with anxiolytic and analgesic actions. In addition, 3 alpha HP has been shown to inhibit GnRH activity on gonadotropes and selectively suppress FSH release from pituitary cells, without an effect on LH. The enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD) has been presumed to be the enzyme responsible for the conversion of progesterone to 3 alpha HP, but this has never been confirmed in vitro or in vivo. We have now determined the mechanism of 3 alpha HP synthesis in vivo using specific enzyme inhibitors and in vitro using recombinant proteins. Incubation of [(3)H]progesterone with purified recombinant rat and human 3 alpha HSD isoforms showed that both the rat 3 alpha HSD and the human type 2(brain) 3 alpha HSD converted progesterone to 3 alpha HP. Age-dependent 3 alpha HP production was demonstrated in pituitary and cortex. Incubation of both tissues with indomethacin, a known 3 alpha HSD inhibitor, decreased the conversion of progesterone to 3 alpha HP by at least 70%, indicating that 3 alpha HSD was responsible for this conversion. As human type 2 3 alpha HSD is expressed in a region-specific fashion in the brain, 3 alpha HP may only be made in specific regions of the brain. Furthermore, the data suggest that the pituitary has the capacity for 3 alpha HP production, which may provide an additional mechanism for regulation of GnRH action.

Dependence on prolactin of the luteolytic effect of prostaglandin F2alpha in rat luteal cell cultures.

Luteal regression is a multistep, prolonged process, and long-term luteal cultures are required for studying it in vitro. Cell suspensions from ovaries of superovulated rats were enriched with steroidogenic cells, seeded on laminin or fibronectin, and maintained in defined medium for up to 10 days. Progesterone secretion was much lower than that of 20alpha-dihydroprogesterone, a product of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). Prolactin added throughout the incubation period gradually increased the percent progesterone out of total progestins to fourfold, while reducing 20alpha-HSD mRNA by 73%. Luteinizing hormone accelerated the establishment of higher percent progesterone by prolactin but by itself had no effect. Prolactin did not increase total progestin production or cytochrome P450 side-chain cleavage (P450(scc)) mRNA. Cell viability was unaffected by prolactin and/or LH. Prostaglandin F2alpha (PGF2alpha) was added 7-8 days after seeding. In prolactin-treated cells, PGF2alpha reduced steroidogenesis after 4-45 h, and at 45 h total progestins and P450(scc) mRNA were reduced by 45%. At 8-45 h PGF2alpha reduced the percent progesterone out of total progestins, and at 45 h 20alpha-HSD mRNA was doubled. In contrast, in prolactin-deprived cultures, PGF2alpha had little effect on total progestins or 20alpha-HSD mRNA but doubled P450(scc) mRNA. Phospholipase C activity was stimulated by PGF2alpha regardless of prolactin. Thus, when prolactin-treated, our cultures are a good model for mature corpora lutea challenged with PGF2alpha; the finding that without prolactin PGF2alpha has an alternative set of actions could help in identifying the signaling pathways of PGF2alpha responsible for its luteolytic effects.

Effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary.

We examined the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on progestin biosynthesis in cultured luteal cells from rat ovary. Luteal cells from immature rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were cultured in the absence or presence of ovine luteinizing hormone (LH) (100 ng/ml) and PACAP-38 (10, 100 and 1000 ng/ml). Following 48 hours of incubation, the levels of progesterone, 20 alpha-hydroxy-4-pregnene-3-one (20 alpha-OH-P) and adenosine 3',5'-monophosphate (cAMP) were measured in the culture media. PACAP alone significantly stimulated the production of progesterone and 20 alpha-OH-P in a dose-dependent manner (p < 0.01 and 0.05, respectively, ANOVA). LH-induced production of progesterone and accumulation of cAMP were significantly decreased by increasing concentrations of PACAP (p < 0.05 for each, ANOVA). Conversely, LH-stimulated 20 alpha-OH-P production was enhanced by PACAP in a dose-dependent manner (p < 0.05). Since PACAP decreased the ratio of progesterone to 20 alpha-OH-P production in LH-stimulated cells, PACAP-mediated inhibition of the stimulatory action of LH on progesterone production may be involved in the initiation of luteolysis. PACAP-38 also suppressed increases in LH receptor content in cultured luteal cells. These results suggest that PACAP regulates the effects of LH on luteal cell function and that PACAP might be closely linked to reproduction.

Cytotoxic effects of cigarette smoke alkaloids inhibit the progesterone production and cell growth of cultured MA-10 Leydig tumor cells.

Inhibition of corpus luteum progesterone synthesis by cigarette smoke alkaloids might, in part, explain the generally poorer outcome of pregnancy in smoking women. The present experiments evaluate the effects of cigarette smoke alkaloids on progesterone biosynthesis and cell growth. Studies were based using the MA-10 Leydig tumor cell line. The steroid pathway in MA-10 cells has only two specific enzymatic steps. The cholesterol passes to the cholesterol side-chain cleavage enzyme and then metabolizes the resulting pregnenolone to progesterone and partly to 20alpha-dihydroprogesterone. Incubation of MA-10 cells with nicotine, cotinine, anabasine, all of these alkaloids, or an aqueous extract of cigarette smoke resulted in dose-dependent inhibition of progesterone and 20alpha-dihydroprogesterone synthesis. The number of cells in the treated dishes seemed less than the control. This latter finding prompted experiments evaluating the short-term effects of the alkaloids on cell growth. Growth of MA-10 cells influenced with alkaloids or smoke extract was also inhibited. All of the inhibitory effects of nicotine, cotinine, anabasine and cigarette smoke extract on MA-10 cells were explained by cytotoxicity. The cytotoxic effect could reduce the fertilization, implantation, and early human development. This mechanism entails the consequence of impaired placental growth, disorder in the placental vascular architecture and placental circulation, and small-for-date babies.

The proestrous prolactin surge is not the sole initiator of regressive changes in corpora lutea of normally cycling rats.

During the estrous cycle, secretion of prolactin is largely restricted to a surge on proestrus. We investigated whether this proestrous prolactin surge initiates regression of the corpora lutea of the preceding cycle. Adult rats were killed prior to the prolactin surge (Proestrus group), following the prolactin surge (Estrus group), after chemical blockade of the prolactin surge with bromocryptine (Estrus+BRC group), and after blockade of the prolactin surge and administration of prolactin (Estrus+BRC+PRL group). Corpora lutea of the current (proestrus) or preceding (estrus) cycle were dissected out, weighed, and sectioned for immunohistochemistry or cultured for examination of in vitro progestin production. Numbers of luteal monocytes/macrophages, differentiated macrophages, and apoptotic nuclei per high-power field were greater for Estrus and Estrus+BRC+PRL than for Estrus+BRC, which in turn had greater numbers than Proestrus (P< 0.05). In contrast, BRC completely reversed the decline in luteal weight observed between Proestrus and Estrus (P<0.05). Number of major histocompatibility complex II-positive cells was not different between groups (P>0.05). Finally, progestin production by corpora lutea in vitro was lower for Proestrus than for the other groups (P<0.05). The results indicate that the prolactin surge alone is not responsible for initiation of apoptosis or immune cell infiltration in regressing corpora lutea of the estrous cycle, although prolactin increases these markers of regression. Prolactin does cause a decline in luteal weight; however, the corpora lutea retain the capacity for steroidogenesis. We conclude that although prolactin has a role in luteal regression, it is not solely responsible for the initiation of this process.

Leptin modulates the glucocorticoid-induced ovarian steroidogenesis.

Leptin regulates food intake and other activities through its hypothalamic receptor. Leptin receptors are also found in other organs, including the ovary. Direct effects of leptin in ovarian steroid production were studied in primary rat granulosa cells and in rat and human granulosa cell lines. Leptin (0.6-18 nM) suppressed ovarian steroid synthesis costimulated by FSH and dexamethasone. Production of pregnenolone, progesterone, and 20alpha-hydroxy-4-pregnen-3-one was inhibited by leptin. This inhibition was due at least in part to reduced expression of adrenodoxin, a component of the P450scc system enzyme. Costimulation of progesterone production by forskolin and dexamethasone was also inhibited by leptin, whereas the forskolin-induced cAMP production was not affected. We find that leptin induces c-Jun expression and attenuates the transcriptional activity of the glucocorticoid receptor (GR) in granulosa cells. Elevation of c-Jun expression by other means, e.g. 12-O tetradecanoyl-phorbol-13-acetate or transfecting with a c-Jun expression vector, abolished the transcriptional activity of the GR. A leptin-induced elevation of c-Jun modulates the transcriptional activity of the GR, possibly leading to the observed attenuation of steroidogenesis. It was recently shown that glucocorticoids stimulate leptin expression in vivo, which in turn, inhibits cortisol synthesis. A direct action of leptin on the ovary is an additional element of a regulatory network that maintains the homeostasis of steroid production.

Synthesis, metabolism and levels of the neuroactive steroid, 3alpha-hydroxy-4-pregnen-20-one (3alphaHP), in rat pituitaries.

The neuroactive steroid, 3a-hydroxy-4-pregnen-20-one (3alphaHP), is a metabolite of progesterone and a precursor of 3alpha-hydroxy-5alpha-pregnan-20-one (5alphaP3alpha; allopregnanolone). In addition to analgesic and anxiolytic effects by interaction with the GABA(A) receptor complex, 3alphaHP regulates pituitary FSH secretion by rapid non-genomic interaction with the Ca2+-driven cell signaling mechanisms. Since gonadectomy and adrenalectomy do not result in elimination of 3alphaHP, and since there is the possibility of paracrine and/or autocrine regulation of FSH release, the capacity of pituitary cells to regulate levels (by synthesis, metabolism, and storage) of 3alphaHP was examined. Anterior pituitaries from random cycling female rats were incubated, either as fragments or as cultured cells, for 1, 4 or 8 h with 3H- or 14C-labeled progesterone. The steroid metabolites were identified by thin-layer chromatography, autoradiography, high pressure liquid chromatography (HPLC), derivatization and GC/MS. Pituitary cells actively converted progesterone to 3alphaHP along with 5alphaP3alpha, 5alpha-pregnane-3,20-dione, 20alpha-hydroxy-5alpha-pregnan-3-one, 3beta-hydroxy-5alpha-pregnan-20-one, 5alpha-pregnane-3alpha(beta), 20alpha-diols, 20alpha-hydroxy-4-pregnen-3-one, and 4-pregnene-3alpha(beta), 20alpha-diols. The results indicate the presence of the following steroidogenic enzymes in anterior pituitary cells: 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO), 20alpha-HSO, 3beta-HSO, and 5alpha-reductase. The activities of 5alpha-reductase and 3alpha-HSO were approximately equal and greatly exceeded those of the other enzymes. After 8 h of incubation with 100 ng progesterone per pituitary, about 20% of the progesterone was metabolized and 3.18 ng of 3alphaHP had been formed. The accumulation of 3alphaHP increased approximately linearly with the time of incubation. Metabolism studies using [1,2,6,7-(3)H]3alphaHP showed that pituitary cells convert about 29% and 8% of the 3alphaHP to progesterone and 5alphaP3alpha, respectively, in 2 h. Specific radioimmunoassays determined 11.6 and 7.5 ng of 3alphaHP per pituitary, respectively, in 25- and 40-day-old non-cycling female rats; these concentrations of 3alphaHP were about 2-3-fold greater than those of progesterone in the same pituitaries. In older (80-100 days old) cycling rats, the levels of 3alphaHP were about 9.4 and 18.6 ng/pituitary at 13.00 h and 22.00 h, respectively, on the day of proestrus, while the concomitant circulating levels were 13.7 and 5.4 ng/ml. The results indicate a marked capacity of rat pituitary cells to synthesize the neuroactive and FSH regulating steroid, 3alphaHP, from progesterone, and in turn to metabolize 3alphaHP to the neurosteroid, allopregnanolone, and to progesterone. The studies suggest cyclic biosynthetic and metabolic pathways for 3alphaHP and other steroids in the pituitary. They also indicate that the regulation of FSH secretion by 3alphaHP may be (in part, or in whole) via paracrine or autocrine mechanisms.

Localization of epidermal growth factor (EGF) receptor in the rat corpus luteum, and EGF and transforming growth factor-alpha stimulation of luteal cell steroidogenesis in vitro.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) have potent mitogenic effects on granulosa and theca cells. However, their effects on steroidogenesis by these cells is controversial, and there is limited information regarding their effects on luteal cell steroidogenesis. The present study investigated the cellular distribution of the EGF receptor (EGF-R) in the rat corpus luteum (CL) by immunocytochemical staining, and the effects of EGF and TGF-alpha on progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) production in cultures of luteal cells. Using a primary antibody directed against the human EGF-R peptide, specific EGF-R staining was obtained in the CL. Both small and large luteal cells had EGF-R staining. In initial cell culture experiments, treatment of freshly isolated luteal cells with EGF or TGF-alpha (0.5-50 ng/ml) for 24 h had no effect on progesterone and 20 alpha-OH-P accumulation. Addition of LH (250 ng/ml) alone caused a 3.5-fold increase in both progestins, but co-treatment with EGF or TGF-alpha produced no further enhancement of progestin accumulation. However, when cells were seeded overnight and the attached cells were washed prior to growth factor treatment for 3 days with media change every 24 h, both EGF and TGF-alpha caused dose-dependent increases in progesterone accumulation/24 h period (up to 2-fold at 50 ng/ml growth factor) on days 1 and 2 but not day 3 of treatment. 20 alpha-OH-P accumulation was similarly stimulated (up to 2.5-fold) by EGF and TGF-alpha under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of luteal steroidogenesis by an LHRH antagonist (Nal-Lys antagonist: antide) in vitro during early pregnancy in the rat.

LHRH and its analogues are known to exert direct effects on the ovary. Herein we have described a direct inhibitory effect of an LHRH antagonist (Nal-Lys antagonist: antide) on the basal progesterone (P4) and pregnenolone (P5) production by luteal cells obtained from the day-8 pregnant rat. Luteal cells incubated with two doses of antide (10(-4) and 10(-7) M) for 24 or 48 h showed suppression of P4 production. P5 production was suppressed by both doses of antide within 12 h of incubation. Neither dose of antide interfered with P5 production when the duration of incubation was extended beyond 12 h. The 20 alpha-dihydroprogesterone yield from the luteal cells treated with these doses of antide remained unaffected. We estimated the activities of the cholesterol side-chain cleavage (P450scc) enzyme (which is a key enzyme involved in the conversion of cholesterol to P5) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) (which catalyses the conversion of P5 to P4) in the luteal cells treated with different doses of antide. Both doses of antide suppressed the activity of the P450scc enzyme after 12 h of incubation and the 3 beta-HSD content of the luteal cells after 48 h of incubation. These observations indicate that antide exerts a direct inhibitory effect at the level of the corpus luteum, that differential suppression of P5 and P4 during different periods of incubation with antide is due to a defect in either the P450scc or the 3 beta-HSD enzyme system, or both.

Inhibition of steroidogenesis by luteal cells of early pregnancy in the rat in response to in vitro administration of a gonadotropin-releasing hormone agonist.

Previous studies from this laboratory have demonstrated that the administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo in early or mid-pregnancy to rats induces antifertility effects by suppressing the luteal production of progesterone (P4) within 24h with a concomitant increase in luteal lipid droplets and decreases in the luteal cytochrome P450 side chain cleavage (P450scc) enzyme and its mRNA content. These observations suggest a direct inhibitory effect of GnRH-Ag on the corpus luteum. Here we demonstrate a suppressive effect of GnRH-Ag in vitro on the basal P4, pregnenolone (P5) and 20 alpha-dihydroprogesterone (20 alpha-DHP) production by luteal cells obtained during early pregnancy in rats. We further studied its effect on two key enzymes, namely P450scc and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), which participate in the conversion of cholesterol to P5 and conversion of P5 to P4, respectively. We observed that two doses of GnRH-Ag, 10(-4) and 10-7 M, suppress the basal P4 production in vitro after 12 h of incubation by luteal cells; P4 remained suppressed after 48 h of incubation. Basal P5 production was also suppressed after luteal cells were incubated for 12 h with 10(-4) M and 10(-7) M GnRH-Ag, but incubation for 48 h with GnRH-Ag failed to alter P5 production by these cells. 20 alpha-DHP production was suppressed after incubating the luteal cells with both doses of GnRH-Ag for 12 h. GnRH-Ag inhibited P450scc activity after 12 h of incubation and 3 beta-HSD protein content at all time periods measured. These results suggest that GnRH exerts a direct inhibitory effect on luteal steroidogenesis. This inhibition is due to its suppressive effect on P450scc and/or 3 beta-HSD and not due to an increase in P4 metabolites.

Synthesis of the allylic regulatory steroid, 3 alpha-hydroxy-4-pregnen-20-one, by rat granulosa cells and its regulation by gonadotropins.

The production of the allylic regulatory steroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) in the rat ovary was examined and compared to progesterone levels through use of specific RIAs that had been validated by capillary gas chromatography-mass spectrometry (GC/MS). Results showed that serum levels of 3 alpha HP are comparable to levels of progesterone at all ages examined. In the 4-day cycling rat, serum levels of 3 alpha HP were highest during diestrus and lowest during proestrus and estrus, while serum FSH levels were highest during proestrus/estrus and lowest during diestrus. Hypophysectomy resulted in decreases in ovarian and serum 3 alpha HP. Treatment of hypophysectomized rats with eCG, but not hCG, increased ovarian and serum 3 alpha HP, while serum progesterone was elevated by treatment with hCG. Ovariectomy resulted in a 55-60% reduction in serum 3 alpha HP, indicating that ovaries are a substantial, but not exclusive, source of 3 alpha HP in serum. As further evidence, cultures of preparations consisting primarily of either granulosa cells or granulosa/theca "shells" produced 3 alpha HP in time-dependent amounts comparable to those of progesterone. Granulosa cells in culture showed a significant increase in accumulation of 3 alpha HP (and progesterone) due to treatment with FSH, but not LH. In contrast to the granulosa-only cell cultures, follicle shells consisting of theca and granulosa cells responded to either LH or FSH treatment with marked increases in 3 alpha HP; increases resulting from combined treatment (FSH + LH) were significantly greater than those due to each hormone alone, but the increases were not additives.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of atrial natriuretic peptide on rat ovarian granulosa cell steroidogenesis in vitro.

PROBLEM: The localization and biological effects of atrial natriuretic peptide (ANP) are not limited to cardiac tissue but extend to a number of extra-atrial tissues and organs, including the ovary. The objective of the present study was to determine the effects of ANP on granulosa cell steroidogenesis. Hence, the direct effects of ANP on the production of progesterone, 20 alpha-hydroxpregn-4-en-3-one (20 alpha-OH-P), and estrogen by undifferentiated and differentiated rat ovarian granulosa cells were examined in vitro. METHOD: Undifferentiated granulosa cells obtained from the ovaries of diethylstilbestrol-primed, immature rats were treated with increasing doses (10(-12) to 10(-6) M) of rANP for 48 h. RESULTS: ANP evoked a two- and threefold increase in progesterone and 20 alpha-OH-P production relative to untreated controls, respectively. Increasing doses of ANP in combination with porcine FSH (125 ng/well) resulted in a biphasic response in progesterone production above FSH-treated controls. Specifically, a maximal inhibition of 35% in progesterone production was achieved at 10(-9) M ANP, followed by a stimulation to levels comparable with FSH-stimulated controls at higher doses examined. Increasing doses of ANP evoked a twofold increase in FSH-stimulated 20 alpha-OH-P production over respective controls. Following pretreatment of granulosa cells with FSH for 48 h to evoke differentiation, ANP caused a significant dose-dependent inhibition in basal progesterone production that resulted in a 76% suppression at the highest dose examined. In contrast, ANP evoked a 2.8-fold increase in 20 alpha-OH-P production when compared with controls. Finally, in FSH-stimulated differentiated granulosa cells, ANP evoked a threefold increase in progesterone production and a 65% inhibition in 20 alpha-OH-P production. ANP exerted no significant effects on estrogen production by either undifferentiated or differentiated granulosa cells in the presence or absence of FSH. CONCLUSIONS: These data demonstrate that ANP can modulate directly progestin steroidogenesis in both undifferentiated and differentiated rat ovarian granulosa cells in vitro and, therefore, may play an important role in granulosa cell differentiation and follicular maturation.