Mechanism of gonadotropin action in amphibia: involvement of mitochondria.

Luteinizing hormone (a pituitary gonadotropic hormone) stimulates Delta(5)-3beta- hydroxysteroid dehydrogenase activity in the microsomal fraction of frog testes when incubated together with the mitochondria; incubation together with the nuclei instead of the mitochondria does not result in increased, Delta(5)-3beta-hydroxysteroid dehydrogenase activity. The increase is not, induced by adenosine triphosphate, it appears to be hormone-specific, and it is sensitive to puromycin and actinomycin D. These data suggest that the mitochondrial DNA may be involved in mediating the action of luteinizing hormone in amphibian steroidogenesis.

Regulation of estrogen receptor (ER) levels in MCF-7 cells by progesterone metabolites.

Estradiol-17beta (E2) may participate in carcinoma of mammary cells containing estradiol receptors (ER) at sufficient levels. Hence, the regulation of ER levels may be important for the progression of estrogen-dependent mammary carcinomas. Our previous findings that the progesterone metabolite, 5alpha-pregnane-3,20-dione (5alphaP), exhibits marked mitogenic and metastatic properties, whereas the progesterone metabolites, 4-pregnen-3alpha-ol-20-one (3alphaHP) and 4-pregnen-20alpha-ol-3-one (20alphaHP), oppose these actions, prompted examination of the possible effects of these progesterone metabolites on ER concentration in MCF-7 breast cancer cells. Cells were exposed for 24h to 0 (control) or 10(-10) to 10(-6)M E2, 5alphaP, 3alphaHP, 20alphaHP or combinations of these steroids, and ER concentrations were determined for intracellular estrogen receptors by specific binding of [(3)H]E2. The total ER number (nuclear plus cytosolic) in control samples was 2551+/-164 per cell. E2 and 5alphaP resulted in significant dose-dependent increases in total ER numbers ( approximately 1.6-fold and approximately 2.2-fold at 10(-6)M, respectively). In combination, E2+5alphaP resulted in additive increases in ER numbers. Individually, 3alphaHP and 20alphaHP each resulted in dose-dependent decreases (43% and 54% at 10(-6)M, respectively) in total ER numbers and inhibited the E2- or 5alphaP-induced increases in ER levels. In combination, 3alphaHP+20alphaHP resulted in dose-dependent additive suppression of ER levels. Treatment with cycloheximide or actinomycin D indicated that both transcription and translation are involved in 5alphaP and 3alphaHP action on ER numbers. Real time RT-PCR showed increases in expression of ERalpha transcripts due to 5alphaP and increases in expression of ERbeta due to 3alphaHP; expression levels of either ERalpha or ERbeta were not significantly altered when cells were treated with 5alphaP+3alphaHP. The results are the first to show that the pro- and anti-cancer progesterone metabolites also have marked selective (up or down) regulatory effects on ER levels in MCF-7 breast cancer cells.

Dutasteride affects progesterone metabolizing enzyme activity/expression in human breast cell lines resulting in suppression of cell proliferation and detachment.

Recent evidence indicates that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that breast carcinoma and tumorigenic breast cell lines have higher 5alpha-reductase and lower 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO activities and mRNA expression levels than normal tissue and non-tumorigenic cell lines. The 5alpha-reduced progesterone metabolites such as 5alpha-dihydroprogesterone (5alphaP) promote both mitogenic and metastatic activity in breast cell lines in culture, whereas the 4-pregnene metabolites, 4-pregnen-3alpha-ol-20-one (3alphaHP) and 4-pregnen-20alpha-ol-3-one (20alphaHP) have the opposite (anti-cancer-like) effects. The 5alpha-reductase inhibitor dutasteride has been shown to inhibit 5alpha-reduction of testosterone to 5alpha-dihydrotestosterone in prostate tissue, resulting in decreased prostate volume. The aim of this study was to determine if dutasteride is an effective inhibitor of progesterone 5alpha-reduction in human breast cell lines and if such inhibition reduces mammary cell proliferation and detachment. The effect of dutasteride on progesterone metabolizing enzyme activities and mRNA expression were examined in tumorigenic MCF-7 and non-tumorigenic MCF-10A human breast cell lines. Dutasteride (10(-6)M) inhibited progesterone conversion to 5alpha-pregnanes by >95% and increased 4-pregnene production. The results indicated that effects of dutasteride on the progesterone metabolizing enzymes are due to direct inhibition of 5alpha-reductase activity and to altered levels of expression of 5alpha-reductase and HSO mRNAs. Treatment of cells with progesterone without medium change for 72 h resulted in significant conversion to 5alpha-pregnanes and increases in cell proliferation and detachment. The increases in proliferation and detachment were blocked by dutasteride and were reinstated by concomitant treatment with 5alphaP, providing proof-of-principle that the effects were due not to progesterone but to the 5alpha-reduced metabolites. This study provides the first evidence that dutasteride is a potent progesterone 5alpha-reductase inhibitor and that such inhibition may be beneficial in breast cancer.

Modification of steroidogenesis in rat adrenocortical cells transformed by Kirsten murine sarcoma virus.

Adrenal fibroblasts from adult rats acquire some adrenocortical parenchymal characteristics as a consequence of transformation in early passage with Kirsten murine sarcoma virus. To further define the effects of Kirsten murine sarcoma virus-induced transformation on the steroid enzymes of these cells, we investigated the capacity of Kirsten murine sarcoma virus-transformed and untransformed adrenocortical fibroblasts to convert progesterone to C19 and C21 steroid metabolites. Over 95% of metabolites produced were identified and quantitated, and rates of enzyme activities over 24 h were calculated. The transformed and untransformed cells exhibited 5 alpha- and 5 beta-reductase, 3 alpha-, 3 beta-, and 20 alpha-hydroxysteroid dehydrogenase; 11 beta-, 17-, and 21-hydroxylase (HY); and C17-20-lyase activities. Viral transformation resulted in several metabolites not found in untransformed cells, significantly increased 5 beta-reductase, 3 beta-hydroxysteroid dehydrogenase, and C17-20-lyase activities, and significantly decreased 5 alpha-reductase, 3 alpha- and 20 alpha-hydroxysteroid dehydrogenase, and 21-HY activities. The 11 beta-HY and 17-HY activities remained unchanged. The results support previous data suggesting that adrenocortical fibroblasts express some characteristics of adrenocortical parenchymal stem cells. In contrast to other experimental systems, viral transformation of adrenocortical fibroblasts did not cause a generalized reduction of differentiated functions. Instead, specific increases and decreases in individual enzyme activities, with persisting synthesis of fetal and adult adrenocortico-specific steroids, resulted in an altered steroid profile that may have unique effects on the biology of the malignant cells.

The 4-pregnene and 5alpha-pregnane progesterone metabolites formed in nontumorous and tumorous breast tissue have opposite effects on breast cell proliferation and adhesion.

Progesterone is required for the full proliferative activity of the breasts and may be directly or indirectly involved in either stimulating or inhibiting breast cancer. To determine whether the effects on breast cancer are attributable to progesterone metabolites, we compared the capacity of nontumorous and tumorous breast tissue to convert progesterone and then tested the effects of these metabolites on breast cell proliferation and anchorage. Tissues from the operated breasts of six patients with infiltrating duct carcinomas were incubated with [14C]progesterone for 2, 4, and 8 h, and the metabolites were identified and quantified. The identified metabolites (equal to >95% of recovered radioactivity) can be divided into those that retain the double bond of progesterone in the carbon-4 position of ring A (4-pregnenes) and those that are 5alpha-reduced (5alpha-pregnanes). The results show that tumorous breast tissue has elevated 5alpha-reductase activity, which results in significantly higher total levels of 5alpha-pregnanes, especially 5alpha-pregnane-3,20-dione (5alphaP), whereas normal (nontumorous) breast tissue produces more 4-pregnenes, especially 3alpha-hydroxy-4-pregnen-20-one (3alphaHP). 5alphaP and 3alphaHP are each one enzymatic step removed from progesterone, resulting from the action of either 5alpha-reductase or 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO), respectively. The ratio of 5alpha-pregnanes:4-pregnenes is >5-fold greater and the ratio of 5alphaP:3alphaHP is nearly 30-fold greater in tumorous than nontumorous breast tissue incubates. In vitro studies with three breast cell lines (MCF-7, MCF-10A, and ZR-75-1) show that 3alphaHP dose dependently inhibits, whereas 5alphaP significantly stimulates, proliferation. Additional studies with MCF-7 and MCF-10A cells indicate that each of the 4-pregnenes isolated from breast tissue suppresses, whereas each respective 5alpha-reduced product stimulates, cell proliferation. Studies of cell anchorage were conducted using MCF-7 cells and various concentrations of 5alphaP or 3alphaHP. The number of cells attached to the substrate was significantly (P<0.05) decreased by treatment with > or =30 nM 5alphaP and increased by treatment with > or =50 nM 3alphaHP. Conversely, the number of cells detached from the substrate after partial trypsin exposure was significantly increased by treatment with > or =40 nM 5alphaP and decreased by treatment with > or =30 nM 3alphaHP. The results suggest that a change in in situ progesterone metabolism, resulting in an increased 5alpha-pregnane:4-pregnene (especially 5alphaP:3alphaHP) ratio, may promote breast cancer by promoting increased cell proliferation and detachment, whereas increases in 4-pregnenes may retard these tumorigenic processes. These studies suggest that endogenous progesterone metabolites may provide a new hormonal basis for breast cancer.

Membrane 5alpha-pregnane-3,20-dione (5alphaP) receptors in MCF-7 and MCF-10A breast cancer cells are up-regulated by estradiol and 5alphaP and down-regulated by the progesterone metabolites, 3alpha-dihydroprogesterone and 20alpha-dihydroprogesterone, with associated changes in cell proliferation and detachment.

Previous studies have shown that the progesterone metabolite, 5alpha-pregnane-3,20-dione (5alphaP), exhibits mitogenic and metastatic activity in breast cell lines and that specific, high affinity receptors for 5alphaP are located in the plasma membrane fractions of tumorigenic (ER/PR-positive) MCF-7 cells. The aim of this study was to determine the effects of the mitogenic (estradiol; 5alphaP) and anti-mitogenic (3alpha-hydroxy-4-pregnen-20-one, 3alphaHP; 20alpha-hydroxy-4-pregnen-3-one, 20alphaHP) endogenous steroid hormones on 5alphaP receptor (5alphaP-R) numbers and on cell proliferation and adhesion of MCF-7 and MCF-10A cells. Exposure of MCF-7 cells for 24h to estradiol or 5alphaP resulted in significant (p < 0.05-0.001) dose-dependent increases in 5alphaP-R levels. Conversely, treatment with 3alphaHP or 20alphaHP resulted in significant (p < 0.05-0.01) dose-dependent decreases in 5alphaP-R levels. Treatment with one mitogenic and one anti-mitogenic hormone resulted in inhibition of the mitogen-induced increases, whereas treatment with two mitogenic or two anti-mitogenic hormones resulted in additive effects on 5alphaP-R numbers. Treatments with cycloheximide and actinomycin D indicate that changes in 5alphaP-R levels depend upon transcription and translation. The non-tumorigenic breast cell line, MCF-10A, was also shown to posses specific, high affinity plasma membrane receptors for 5alphaP that were up-regulated by estradiol and 5alphaP and down-regulated by 3alphaHP. Estradiol binding was demonstrated in MCF-10A cell membrane fractions and may explain the estradiol action in these cells that lack intracellular ER. In both MCF-7 and MCF-10A cells, the increases in 5alphaP-R due to estradiol or 5alphaP, and decreases due to 3alphaHP or 20alphaHP correlate with respective increases and decreases in cell proliferation as well as detachment. These results show distribution of 5alphaP-R in several cell types and they provide further evidence of the significance of progesterone metabolites and their novel membrane-associated receptors in breast cancer stimulation and control. The findings that 3alphaHP and 20alphaHP down-regulate 5alphaP-R and suppress mitogenic and metastatic activity suggest that these endogenous anti-mitogenic progesterone metabolites deserve considerations in designing new breast cancer therapeutic agents.

The role of progesterone metabolites in breast cancer: potential for new diagnostics and therapeutics.

Proliferative changes in the normal breast are known to be controlled by female sex steroids. However, only a portion of all breast cancer patients respond to current estrogen based endocrine therapy, and with continued treatment nearly all will become unresponsive and experience relapse. Therefore, ultimately for the majority of breast carcinomas, explanations and treatments based on estrogen are inadequate. Recent observations indicate that 5alpha-pregnane and 4-pregnene progesterone metabolites may serve as regulators of estrogen-responsive as well as unresponsive human breast cancers. The conversion of progesterone to the 5alpha-pregnanes is increased while conversion to the 4-pregnenes is decreased in breast carcinoma tissue, as a result of changes in progesterone metabolizing 5alpha-reductase, 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO activities and gene expression. The 5alpha-pregnane, 5alpha-pregnane-3,20-dione (5alphaP) stimulates, whereas the 4-pregnene, 3alpha-hydroxy-4-pregnen-20-one (3alphaHP), inhibits cell proliferation and detachment, by modulation of cytoskeletal and adhesion plaque molecules via the MAP kinase pathway and involving separate and specific plasma membrane-based receptors. The promotion of breast cancer appears to be related to changes in in situ concentrations of cancer-inhibiting and cancer-promoting progesterone metabolites. New diagnostic and therapeutic possibilities for breast cancer are suggested.

Steroidogenesis in rat Leydig cells: effect of gonadotropins on the activity of 5-ane and 5-ene 3alpha- and 3beta-hydroxysteroid dehydrogenases during sexual maturation.

The in vivo influence of gonadotropins on the activities of oxidoreductases of androst-5-ane and androst-5-ene steroids and pregnenolone was examined in testes from young rats. Animals were given daily injections of human CG for 5 days starting at 20 days of age and the testicular 12,000 X g supernatants were assayed for steroid oxidoreductase activities. Marked increases (up to 8-fold) were noted in the rate of oxidation of the 3beta-hydroxyl of 3beta-hydroxy-5beta-androstan-17-one, 3 beta-hydroxy-5alpha-androstan-17-one, 5alpha-androstane-3beta,17beta-diol, dehydroepiandrosterone, and pregnenolone, and in the 3-keto reduction of 17beta-hydroxy-5alpha-androstan-3-one, 17beta-hydroxy-5beta-androstan-3-one, 5beta-androstane-3,17-dione, and 5alpha-androstane-3,17-dione. The hormone response required a certain amount of time as no response was detected until 72 h after the first injection. As little as 1 IU hCG/injection resulted in significant increases in 3beta-oxidoreductase (3beta-HSD) activities. FSH and TSH gave no significant increases and 25 microgram NIH-LH-S18 resulted in increases only when the hormone was suspended in a sesame oil-beeswax mixture. Hormone treatments did not result in increased 5-ane-3alpha-HSD activities. Rats receiving chronic human CG treatment starting at 66 days of age showed less marked increases in 5-ane-3beta-HSD activities than the younger rats and no significant enhancement in 5-ene-3beta-HSDs. It is suggested that during sexual maturation the testicular biosynthesis of active 5-ane androgen(s) proceeds via 5-ane precursors with the help of age and gonadotropin-dependent 5-ane 3beta-oxidoreductase.

Steroidogenesis in rat leydig cells: changes in activity of 5-ane and 5-ene 3beta-hydroxysteroid dehydrogenases during sexual maturation.

The activities of hydroxysteroid dehydrogenases of 5-ane and 5-ene steroids were examined in interstitial tissue from testes of rats at different ages. The enzyme reactions were localized in the Leydig cell cytoplasm of isolated cells and in frozen tissue slices. Relative reaction velocites of the NAD-linked hydroxysteroid dehydrogenases were obtained spectrophotometrically with 17 steroid substrates using the 12,000 X g supernatant of isolated interstitial cells from 28-29 day old rats; the rate of 3(alpha,beta) dehydrogenation of 5-ane-3beta steroids was markedly (10 to 20X) higher than that of 5-ene-3beta steroids and 5-ane-3alpha steroids. The hydroxysteroid dehydrogenase activities of testes from 124 rats between the ages of 15 and 138 days were determined using as substrates, 3beta-hydroxy-5beta-androstan-17-one, 3beta-hydroxy-5alpha-androstan-17-one, 3beta,17beta-dihydroxy-5alpha-androstane, dehydroepiandrosterone and pregnenolone. Between the ages of 15 and 32 or 34 days the gonads grow in size more rapidly than the body and the 5-ane-3beta-hydroxysteroid dehydrogenase activities show very marked increases; changes in the 5-ene-3beta-hydroxysteroid dehydrogenases are much less pronounced, so that at 34 days the activity of 3beta-hydroxy-5beta-androstan-17-one dehydrogenase is approximately 20 X that of dehydroepiandrosterone dehydrogenase. After 34 days, the 5-ane-3beta-hydroxysteroid dehydrogenase activities decline. It is suggested that during sexual maturation the testicular biosynthesis of active 5-ane androgens may proceed via 5-ane precursors with the help of age-dependent 5-ane-3beta-hydroxysteroid dehydrogenases.

Suppression of follicle-stimulating hormone by the gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one involves actions at the level of the gonadotrope membrane/calcium channel.

We have previously shown that the gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) suppresses FSH release in cultures of anterior pituitary cells. We undertook exploration of the mechanisms of this suppression by examining the possible sites of 3 alpha HP action in isolated anterior pituitary cells of rats. The specific objective of this study was to determine if 3 alpha HP suppresses FSH by action at the level of the gonadotrope membrane and/or calcium channels. Pituitary cells from adult randomly cycling female rats were precultured for 72 h and then treated for 4 h with 10 nM GnRH and 0.1 nM 3 alpha HP with or without Ca2+ channel agonists or antagonist. In other experiments, cells were treated with BSA-conjugated 3 alpha HP, progesterone, or 3 beta HP (the stereoisomer of 3 alpha HP). Levels of FSH were determined by RIA in media and cells. GnRH-stimulated FSH release and the total FSH (released plus cellular) were significantly suppressed by 3 alpha HP. The Ca2+ ionophore A23187 induced FSH release and 3 alpha HP significantly suppressed both released and total FSH in its presence. In combination with a high dose (100 microM) of the dihydropyridine-sensitive Ca2+ channel antagonist nifedipine, 3 alpha HP suppressed FSH secretion to a greater extent than the antagonist alone. Cellular content of FSH was also decreased by nifedipine (100 microM) and was further suppressed in the presence of 3 alpha HP. The phenylalkylamine-sensitive Ca2+ channel antagonist methoxyverapamil (D600) suppressed GnRH-induced FSH release, and 3 alpha HP significantly potentiated the suppression. Released and cellular FSH were increased by the dihydropyridine-sensitive agonist BAYK 8644, whereas 0.1 nM 3 alpha HP suppressed this agonist-induced FSH to a greater extent than the maximum dose (100 microM) of nifedipine. In order to test for direct action at the level of the gonadotrope membrane, 3 alpha HP was conjugated to BSA (3 alpha HP-BSA) and administered to cultured pituitary cells. The 3 alpha HP-BSA conjugate (but not progesterone-BSA or 3 beta HP-BSA) significantly suppressed release of FSH. The results of the study suggest that 3 alpha HP may be interacting with the Ca2+ channel component of the GnRH signal transduction mechanism; in addition, 3 alpha HP may also suppress FSH release (and possibly synthesis) through direct action at the level of the gonadotrope membrane.

Rat sertoli cells: a rapid method for obtaining viable cells.

A method is described for obtaining populations of viable Sertoli cells from rat testes. Minced whole testes from rats of 15 to 29 days of age are sequentially treated with collagenase and pancreatin. The resulting suspension of cells is sedimented through a sucrose density gradient. Preparations are produced consisting of from 60% to 82% Sertoli cells, an enrichment of 2 to 5 times the proportion of Sertoli cells in whole testes of these ages. The preparations are free of interstitial cells, are essentially free of peritubular cells and contain reduced numbers of germinal cells; the main contaminating cell types are spermatogonia and spermatocytes. The Sertoli cells are considered to be 95% viable by their ability to exclude trypan blue and by subsequent culturing in vitro. The entire procedure requires 3 h. Maintenance of the Sertoli-enriched fraction in modified Eagle's minimal essential medium temporarily at 41 C allows preparations of Sertoli cell monolayer cultures consisting of 95%-98% Sertoli cells within 3 days.

Selective suppression of follicle-stimulating hormone by 3 alpha-hydroxy-4-pregnen-20-one, a steroid found in Sertoli cells.

Previous reports have not identified a naturally occurring steroid that selectively inhibits FSH secretion without also inhibiting LH secretion. The effect of 3 alpha-hydroxy-4-pregnen-20-one (3-HP), a steroid produced in Sertoli cells, on gonadotropin secretion in intact and castrate male and female, prepubertal and adult rats and in cultures of anterior pituitary cells was investigated. Intact prepubertal male rats were treated with a single sc injection of 0.2 mg/kg 3-HP, and castrate male and female rats were given a daily sc injection of 0.2 mg/kg 3-HP for 4 days. Serum FSH levels were suppressed by 26-44% (P less than 0.001-0.05), with no similar effect on serum LH levels. The acetyl derivative of 3-HP (3-HPA), administered to castrate prepubertal and adult rats for 4 days (0.625 mg/kg), resulted in significant decreases (P less than 0.001) in serum FSH to 45% and 19% of castrate control levels, respectively, without a significant effect on LH levels. Treatment of castrate prepubertal male rats with various doses of 3-HPA (0.001-0.625 mg/kg X day) for 4 days resulted in a dose-related suppression of serum FSH. Similar results were obtained with chronic (14-day) treatment of intact male rats with 3-HPA. Treatment of young (15-day-old) intact males with either 3-HP or 17 beta-hydroxy-5 alpha-androstan-3-one (DHT) for 14 days showed that DHT resulted in significant increases in prostate and seminal vesicle weights, while 3-HP showed no apparent androgenic activity. The effects of treatment with 3-HP, 3 beta-HP, 17 beta-estradiol, and DHT (0.025-0.625 mg/kg X day) were compared. Treatment with 3 beta-HP resulted in significant increases in serum FSH levels; 17 beta-estradiol and DHT suppressed both gonadotropins (at the higher doses administered), while 3-HP suppressed only FSH. 3-HP (3.16 X 10(-11) M) and/or LHRH (3 X 10(-8) M) were employed in primary cultures of anterior pituitary cells. Addition of LHRH resulted in 6- to 8-fold increases in the secretion of FSH and LH, while 3-HP suppressed basal (P less than 0.05) and LHRH-stimulated (P less than 0.001) FSH secretion by 26% and 77%, respectively. We conclude that 3-HP selectively suppresses FSH secretion and may be involved in the normal regulation of FSH secretion in the male.

Selective suppression of follicle-stimulating hormone secretion in anterior pituitary cells by the gonadal steroid 3 alpha-hydroxy-4-pregnen-20-one.

The effect of 3 alpha-hydroxy-4-pregnen-20-one (3HP), a Sertoli cell steroid, on the secretion of gonadotropins from rat anterior pituitary cells in culture was examined and subsequently compared with the action of other gonadal steroids and steroids structurally related to 3HP. Pituitary cells from randomly cycling, sexually mature female rats were isolated and maintained in culture 72 h before use. On the day of treatment, medium was changed, steroids (10(-16)-10(-4) M) and/or LHRH (10(-8) M) were added, and cells were allowed to incubate for a further 24 h. Medium was then examined for gonadotropin content by RIA. 3HP treatment of anterior pituitary cells resulted in a significant reduction of both basal and LHRH-induced FSH secretion, while LH secretion was unaffected. The lowest effective dose of 3HP (10(-16) M) significantly decreased basal FSH secretion to 65.6% of control levels. The lowest effective dose of 3HP that significantly inhibited (by 31%) LHRH-induced FSH secretion was 10(-14) M 3HP. Maximum suppression by 3HP of basal FSH secretion occurred between 10(-10)-10(-8) M, and maximum suppression of LHRH-induced secretion occurred at 10(-12) M. None of the other gonadal steroids tested (progesterone, testosterone, 5 alpha-dihydrotestosterone, 17 beta-estradiol, 20 alpha-hydroxy-4-pregnen-3-one, and 5 alpha-pregnane-3,20-dione) had a similar selective effect on FSH secretion; progesterone, testosterone, and 17 beta-estradiol actually resulted in increased FSH release, and 5 alpha-pregnane-3,20-dione resulted in significant increase in basal LH. A number of metabolites and structural variations of 3HP were examined in this in vitro system at concentrations of 10(-12)-10(-6) M, and none exhibited a similar selective FSH-suppressing activity as 3HP. The data suggest that the selective FSH-suppressing effect of 3HP seen previously in vivo and here in vitro is due to 3HP itself and not the result of a metabolite of this molecule.

Suppression in gonadotropes of gonadotropin-releasing hormone-stimulated follicle-stimulating hormone release by the gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one involves cytosolic calcium.

The gonadal- and neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) suppresses FSH release in cultures of anterior pituitary cells. In a previous report, we showed that this suppression is achieved at least in part by an interaction at the plasma membrane level. We undertook to examine the possible interaction of 3 alpha HP at the level of intracellular Ca2+. Anterior pituitary cells from adult randomly cycling female rats were treated for 4 h with 10 nM GnRH and 0.1 nM 3 alpha HP with or without protein kinase C activator (SC10), antagonist (H-7), intracellular Ca2+ chelator (TMB-8), and intracellular Ca2+ mobilizer (glutamate), and with or without EGTA and Ca2+ in the medium. FSH content in media and cells was determined by RIA. The protein kinase C (PKC) activator, SC10, increased basal levels of secreted FSH. 3 alpha HP suppressed (P < 0.05) SC10-stimulated basal FSH release. The PKC inhibitor, H7, decreased GnRH-induced FSH release; FSH was further suppressed (P < 0.05) by 3 alpha HP in the presence of H7. These results were interpreted to indicate that 3 alpha HP may act in part at the level of PKC and also at another site(s). The intracellular Ca2+ chelator, TMB-8, suppressed released and cellular GnRH-stimulated FSH to the same extent as 3 alpha HP; FSH was not further decreased by 3 alpha HP in the presence of TMB-8. 3 alpha HP suppressed glutamate-stimulated FSH release in Ca(2+)-free medium (P < 0.01). Moreover, GnRH-induced release of FSH was suppressed to the same degree by 10(-10) M 3 alpha HP as by 10(-4) M EGTA. In pituitary cell suspensions, the GnRH-induced [Ca2+]i elevations were significantly (P < 0.05) attenuated by 3 alpha HP. From these and previous results, a model is proposed for the action of 3 alpha HP. The model suggests that 3 alpha HP may interact with gonadotropes at the level of the PKC cell signaling pathway and intracellular Ca2+ mobilization, in addition to the plasma membrane/calcium channel. The interaction effects a decrease in intracellular Ca2+, leading to decreases in FSH release from those pituitary gonadotropes that are responsible for FSH. The consistent decrease in total FSH (released plus cellular content) by 3 alpha HP suggests that this neurosteroid may also suppress FSH synthesis.

Antinociceptive effects of the neuroactive steroid, 3alpha-hydroxy-5alpha-pregnan-20-one and progesterone in the land snail, Cepaea nemoralis.

Results of investigations with vertebrates have implicated neuroactive steroids and in particular 5alpha-reduced metabolites of progesterone such as 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP/3A5P and originally allopregnanolone) in the rapid modulation of diverse functions including that of nociceptive sensitivity. These effects have been indicated to involve modulation of GABA receptors. Results of recent phylogenetic studies have revealed the presence of GABA receptors in invertebrates that may also be subject to modulation by steroids and neuroactive steroids. The present study examined the effects of the neuroactive steroid, 3alpha-hydroxy-5alpha-pregnan-20-one, as well as progesterone on aversive thermal (nociceptive) responses in a mollusc, the land snail, Cepaea nemoralis. 3alpha-Hydroxy-5alpha-pregnan-20-one had significant dose-related (0.01-1.0 microg) antinociceptive effects in Cepaea increasing the latency of response to a 40 degrees C surface, with maximum effects being evident 15-30 min after administration. These effects of 3alpha-hydroxy-5alpha-pregnan-20-one were stereospecific, with the stereoisomer 3beta-hydroxy-5alpha-pregnan-20-one (3B5P) failing to affect nociceptive responses. Progesterone also had significant dose-related (0.10-10 microg) antinociceptive effects that, however, were delayed in onset and relatively prolonged (60-120 min), suggestive of the formation of active metabolites. The presence of endogenous progesterone (12.36+/-0.17 ng/g tissue) was ascertained by a radioimmunoassay further supporting a functional role for steroids in Cepaea. The antinociceptive effects of 3alpha-hydroxy-5alpha-pregnan-20-one and progesterone were blocked by the GABA antagonists, bicuculline and picrotoxin, while being relatively insensitive to opioid and N-methyl-D-aspartate antagonists. These results suggest an early evolutionary development and phylogenetic continuity of neuroactive steroid and GABA involvement in the mediation of nociception.

Progesterone metabolism by guinea pig intrauterine tissues.

Progesterone metabolism by guinea pig amnion, chorion, myometrium, and endometrium was studied at the following gestational stages. Day 45 represents mid-gestation, about 5 days before strong chorion interaction between the entire surface of the chorion and the uterus; days 57-58, 1-2 days after chorion attachment, and 2-3 days before the onset of pubic symphysis relaxation; days +1-+6, 1-6 days after the onset of pubic symphysis relaxation, i.e. within 1 week of parturition. The high metabolic activity of chorion exceeded that by amnion at all stages. Metabolism by endometrium and myometrium was always low. Conversion of progesterone by amnion significantly decreased (P < 0.05) between days 57-58 and days +1-+6. Progesterone metabolites produced by chorion and amnion were identified by TLC, HPLC, and capillary GC/MS. Both tissues converted progesterone to three major products during 60-min incubations. These were 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and 3 beta-hydroxy-5 alpha-pregnan-20-one. The metabolite pattern differed between the two tissues. Three-minute incubations with chorion resulted in a significantly higher proportion of 3 alpha-hydroxy-4-pregnen-20-one (P < 0.01) and 5 alpha-pregnane-3,20-dione (P < 0.025) than at 60 min. The production of 3 beta-hydroxy-5 alpha-pregnen-20-one by chorion decreased (P < 0.05) between days 50-51 and 57-58. The ratio of 3 alpha-hydroxy-5 alpha-pregnan-20-one to 3 beta-hydroxy-5 alpha-pregnan-20-one increased (P < 0.05) between days 45 post-relaxation. The marked conversion of progesterone by chorion, or the formation of one or more of its metabolites, may serve to influence uterine function prior to delivery.

A radioimmunoassay for the regulatory allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP).

The allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), found in gonadal and brain tissues by radiotracer and chemical methods, had been shown to play a role in gametogenesis, gonadotropin secretion and brain excitability. Since no simple assay was available, a radioimmunoassay for 3 alpha HP was developed using [3H]3 alpha HP and an antiserum raised against 3 alpha HP-20-CMO conjugated to bovine serum albumin. The specificity of the assay for the 3 alpha allylic configuration of 3 alpha HP was confirmed by examining 32 other steroids; cross-reaction with steroids containing different configurations (including metabolites of 3 alpha HP such as progesterone) was less than 0.9%. A Scatchard plot indicated a Ka of 1.56 X 10(9) M-1. Inter- and intra-assay coefficients of variation were 13.1 and 4.5%, respectively. The sensitivity of the assay was 6 pg and the 50% intercept of the standard curve was approx. 123 pg. The measurement by RIA of 3 alpha HP from standard solutions and HPLC purified tissue extracts was confirmed qualitatively and quantitatively by GC/MS methods. The RIA method was employed to determine 3 alpha HP levels in cultured Sertoli cells and in serum of intact and ovariectomized adult rats. Although for most uses, chromatography would not be necessary, two possible methods are presented to enable the separation of 3 alpha HP from other interfering steroids prior to RIA.

Metabolism of progesterone by avian granulosa cells in culture.

Previous studies have demonstrated that progesterone is the primary product of steroidogenesis in avian granulosa cells during short-term incubation. However, during more prolonged culture, lasting several days, the progesterone content in the medium was found to decrease progressively, indicating in vitro metabolic conversion. In the present study we have isolated and identified a number of progesterone metabolites. Granulosa cells, isolated from mature ovarian follicles of laying hens, were cultured in medium 199 supplemented with fetal calf serum and containing [14C]progesterone. After 4 days in culture, cells + media were extracted and the radioactive metabolites separated and identified by TLC, HPLC and GC-MS. Several of the metabolites were further characterized by derivatization and crystallization to constant specific activity. A total of 24 radioactive substances was detected. Of these, 15 have been positively identified, 5 tentatively and the remaining 4 are unidentified. The principal metabolite, representing more than 45% of the total radioactivity, was identified as 3 alpha-hydroxy-5 beta-pregnan-20-one. In addition, significant amounts of 3 alpha-hydroxy-5 alpha-pregnan-20-one (5.76%), 5 beta-pregnane-3,20-dione (3.05%), and 5 alpha-pregnane-3,20-dione (2.95%) were detected and identified. The results indicate that avian granulosa cells possess 3 alpha-hydroxy-steroid dehydrogenase (3 alpha-HSD), 17 beta-HSD, 20 alpha-HSD, 20 beta-HSD, 17 alpha-hydroxylase, C17-20-lyase and 5 alpha- and 5 beta-reductase activities. These enzyme activities may convert progesterone to biologically inactive or less active metabolites. However, a functional role for some of these metabolites cannot be ruled out.

Analgesic effects of the putative FSH-suppressing gonadal steroid, 3 alpha-hydroxy-4-pregnen-20-one: possible modes of action.

The effects of intracerebroventricular (i.c.v.) administrations of the putative follicle stimulating hormone (FSH) suppressing gonadal steroid, 3 alpha-hydroxy-4-pregnen-20-one (3A4P) on the nociceptive responses of male mice were examined. This allylic steroid elicited significant, dose-dependent (0.001-1.0 micrograms) analgesic responses for 90-150 min after injection. These analgesic effects of 3A4P were stereospecific, the stereoisomer, 3 beta-hydroxy-4-pregnen-20-one (3B4P) failing to affect the nociceptive responses. The analgesic effects of 3A4P were blocked by peripheral administrations of the GABA antagonists, bicuculline and picrotoxin, and reduced by the benzodiazepine antagonist, Ro 15-1788. The exogenous opiate antagonist, naloxone, and the putative endogenous opioid antagonist, Tyr-MIF-1 (Pro-Leu-Gly-amide), also reduced 3A4P-induced analgesia, while i.c.v. administration of 3A4P (0.001 and 0.01 micrograms) itself attenuated the analgesic effects arising from peripheral administrations of opiate receptor agonist, morphine. In addition, the calcium channel antagonists, nifedipine and verapamil, enhanced 3A4P-induced analgesia but had no evident effects on the actions of 3B4P. These results suggest that the central analgesic effects of the FSH-suppressing steroid, 3A4P, arise via benzodiazepine--GABA--opiate mechanisms and calcium channels. These findings also suggest possible central modes of action whereby 3A4P may elicit selective suppression of FSH.

Analgesic effects of the progesterone metabolite, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and possible modes of action in mice.

The effects of intracerebroventricular (i.c.v.) administrations of the progesterone metabolite, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3A5P), on the nociceptive responses of male mice were examined. 3A5P elicited significant, dose-dependent (0.001-1.0 microgram) analgesia for 90-120 min after administration. These effects of 3A5P were significantly more potent than those of progesterone. The stereoisomer, 3 beta-hydroxy-5 alpha-pregnan-20 one (3B5P), failed to affect the nociceptive responses, indicating that the analgesic effect of 3A5P is stereospecific. The analgesic effects of 3A5P were blocked by peripheral administrations of the GABA antagonists, bicuculline and picrotoxin, and reduced by both the opiate and benzodiazepine antagonists, naloxone and Ro 15-788, respectively. The calcium channel antagonists, nifedipine and verapamil, enhanced 3A5P-induced analgesia but had no evident effects on the actions of 3B5P. These results suggest that the central analgesic effects of the progesterone metabolite, 3A5P, may arise via mechanisms involving calcium channels, the GABA-benzodiazepine-chloride complex and endogenous opioid systems.