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.

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.

The endogenous progesterone metabolite, 5a-pregnane-3,20-dione, decreases cell-substrate attachment, adhesion plaques, vinculin expression, and polymerized F-actin in MCF-7 breast cancer cells.

Tumorous human breast tissue readily converts progesterone to 5alpha-pregnane-3,20-dione (5alphaP), and this metabolite has been shown to stimulate proliferation and to decrease adhesion of MCF-7 breast cancer cells. To determine the mechanisms of action of 5alphaP on cell adhesion, MCF-7 cells were grown without or with 5alphaP (10(-9)-10(-5) M), and the effects on cell and nuclear morphology, adhesion plaques, vinculin and actin expression, actin polymerization, and microfilament distribution were examined by immunohistochemistry, morphometry (using confocal microscopy and digital computer imaging analysis), and Western blotting. Treatment of cells with 10(-9)-10(-6) M 5alphaP resulted in dose-dependent decreases in cell area, cell-to-cell contacts, and attachment to the substratum, and increases in variation in nuclear area. These changes in the 5alphaP-treated cells were accompanied by decreases in vinculin-containing adhesion plaques, vinculin expression, polymerized actin stress fibers, and decreases in insoluble and increases in soluble actin fractions. The results suggest that the observed decreases in adhesion and increases in cell proliferation following 5alphaP treatment may be owing to depolymerization of actin and decreased expression of actin and vinculin. We conclude that the endogenous progesterone metabolite, 5alphaP, may be involved in promoting breast neoplasia and metastasis by affecting adhesion and cytoskeletal molecules.

Glycerol disrupts tight junction-associated actin microfilaments, occludin, and microtubules in Sertoli cells.

Intratesticular injections of glycerol have been shown to result in a marked and prolonged reduction of spermatogenesis, accompanied by increased permeability of the blood-testis barrier. Because the permeability of the blood-testis barrier is regulated by Sertoli cell tight junctions, and tight junction organization is regulated by the cytoskeleton, we undertook to examine the effects of glycerol treatment on cytoskeletal actin microfilaments and microtubules, and on the tight junction protein, occludin, in Sertoli cells. Adult rats received a single intratesticular injection of either saline (controls) or a 10% glycerol solution. At 24 hours and 7, 15, and 21 days after injection, testes were collected and prepared for routine histology, cryosectioning, or whole seminiferous tubule immunohistochemical staining; and the preparations were viewed by light and confocal microscopy. In saline-injected testes, Sertoli cells had a cytoskeletal and junctional organization that resembled that of normal testes. F-actin microfilaments, located in the basal region, were arranged in regular bundles or chords that circumscribed the perimeter of each Sertoli cell at the level of the tight junction. Occludin colocalized with tight junction-associated actin filament distribution and microtubules formed a geometric array associated with spermatogenic cells. In contrast, in glycerol-treated Sertoli cells, microfilament and microtubule organization and occludin distribution were partially or completely disrupted. From these results we conclude that glycerol treatment either directly or indirectly disrupts tight junction-associated F-actin and occludin and tubulin organization in rat Sertoli cells. Perturbation of the tight junction-associated proteins could explain the increase in permeability of the blood-testis barrier observed after glycerol treatment. Impaired spermatogenesis following glycerol treatment is likely a consequence of a leaky blood testis barrier and disrupted Sertoli cell cytoskeleton. Glycerol injections may serve as a useful tool in studying the relationship between cytoskeletal organization and the stabilization of Sertoli-Sertoli cell junctions.

Plasma membrane receptors for the cancer-regulating progesterone metabolites, 5alpha-pregnane-3,20-dione and 3alpha-hydroxy-4-pregnen-20-one in MCF-7 breast cancer cells.

Recent observations indicate that the progesterone metabolite, 5alpha-pregnane-3,20-dione (5alphaP), which is produced at higher levels in tumorous breast tissue, promotes cell proliferation and detachment, whereas 3alpha-hydroxy-4-pregnen-20-one (3alphaHP), which is produced at higher levels in nontumorous breast tissue, suppresses proliferation and detachment of MCF-7 breast cancer cells. The objective of the current study was to determine the presence and characteristics of binding sites for these endogenous putative cancer-regulating steroid hormones. Radiolabeled 5alphaP and 3alphaHP were used in radioligand binding assays on MCF-7 cell (membrane, cytosolic, and nuclear) fractions. Binding of [(3)H]5alphaP and [(3)H]3alphaHP was observed only in the plasma membrane fraction, whereas estradiol binding sites were confirmed in the cytosolic and nuclear fractions. The respective membrane binding sites exhibited specificity for the 5alphaP and 3alphaHP ligands with no appreciable displacement at 200- to 500-fold excess by other steroids. The association rate constants were calculated as 0. 107/min and 0.0089/min and the dissociation rate constants were 0. 049 9 and 0.011 for 5alphaP and 3alphaHP, respectively. Saturation analyses indicated single classes of molecules with dissociation constants of 4.5 and 4.87 nM and receptor densities of 486 and 629 fmol/mg protein, respectively, for 5alphaP and 3alphaHP. Exposure of MCF-7 cells to estradiol for 1, 24, 48, and 72 h resulted in 2.3, 4. 2-, 2.99-, and 1.7-fold increases, respectively, in 5alphaP receptor density. 3alphaHP resulted in partial suppression of the estradiol-mediated increase in 5alphaP receptor density. This is the first report of receptors for the progesterone metabolites, 5alphaP and 3alphaHP, of their occurrence in breast cancer cell membranes, and of the induction of 5alphaP receptors by estradiol. The results provide further support for the potential importance of progesterone metabolites in breast cancer.

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.

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.

Brief exposure of mice to 60 Hz magnetic fields reduces the analgesic effects of the neuroactive steroid, 3alpha-hydroxy-4-pregnen-20-one.

Relatively weak, extremely low frequency (ELF), magnetic fields have been shown to exert a variety of biological effects, although the modes of action remain to be established. Neuroactive steroids and neurosteroids have been shown to produce a diverse range of rapid centrally mediated behavioral and physiological effects that are reported to be sensitive to magnetic fields. Here we show that brief exposure of male mice to an ELF magnetic field (30 min, 60 Hz, 141 microT peak) significantly reduces the analgesic effects arising from intracerebroventricular (i.c.v.) administration of the centrally produced allylic neuroactive steroid, 3alpha-hydroxy-4-pregnen-20-one (3alphaHP) and that the dihydropyridine (DHP) calcium channel antagonists, diltiazem and nifedipine, block the inhibitory effects of the 60 Hz ELF on 3alphaHP-induced analgesia. These results indicate that exposure to 60 Hz ELF affects the analgesic effects of neuroactive steroids such as 3alphaHP through alterations in calcium channel function. These findings raise the possibility that ELF magnetic fields may, in part, exert their actions through effects on diverse neuroactive steroid modulated processes.

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.

Acute, nongenomic actions of the neuroactive gonadal steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), on FSH release in perifused rat anterior pituitary cells.

We have previously shown that the gonadal and neurosteroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), can selectively suppress gonadotrophin-releasing hormone (GnRH) induced follicle-stimulating hormone (FSH) release from static cultures of anterior pituitary cells during a 4-h incubation period. The actions appeared to be at the level of the gonadotroph membrane and the cell signaling pathway involving Ca2+ and protein kinase C (PKC). In order to investigate further if the effects of 3 alpha HP on FSH release are generated by nongenomic mechanisms, we monitored the short-term effects of 3 alpha HP using dispersed anterior pituitary cells in a low dead-volume perifusion system with short (< or = 5 min) exposures to the steroid. Pulses of GnRH (10(-8) or 10(-7) M) lasting 2-5 min resulted in marked peaks of FSH release, and the variation in FSH amounts released from the cells in a particular column were minimal if the interval between successive GnRH pulses was at least 3-4 h. A 5-min pulse of 3 alpha HP (10(-9) M) administered simultaneously with the GnRH pulse suppressed GnRH-induced FSH release. On the other hand, similar treatment with the stereoisomer 3 beta-hydroxy-4-pregnen-20-one (3 beta HP), had no effect, but progesterone and estradiol pulses augmented the GnRH-induced FSH release. Pretreatment of cells with a 5-min pulse of 3 alpha HP, at 120, 60, or 30 min prior to a GnRH pulse suppressed the GnRH-induced FSH release. The suppression of GnRH-induced FSH release by 3 alpha HP was only partial if the start of the 3 alpha HP pulse occurred 0.5 or 1.0 min after the start of the GnRH pulse, and no suppression occurred if the start of the 3 alpha HP pulse was delayed by 2-5 min. The FSH release elicited by 5-min pulses of the Ca2+ ionophore A23187, the Ca2+ agonist BAY K8644, the PKC activator phorbol 12-myristate 13-acetate (PMA), or phospholipase C (PLC) was suppressed by simultaneous pulses of 3 alpha HP. The suppression of FSH release by 3 alpha HP appeared to be stereospecific, since no suppression was observed with 5 alpha-pregnane-3,20-dione (5 alpha P) or 3 alpha-hydroxy-5 alpha-pregnan-20-one (5 alpha P3 alpha). In separate experiments, cells were treated with pulses of BSA conjugates of 3 alpha HP, 3 beta HP, or progesterone; the 3 alpha HP-BSA, but not the 3 beta HP-BSA or the progesterone-BSA, suppressed the GnRH-induced release of FSH. The results of this study provide the first evidence that 3 alpha HP exerts immediate (nongenomic) and direct effects on GnRH-induced FSH release by interacting at the level of the pituitary gonadotroph membrane and the phosphoinositol cell signaling cascade involving Ca2+.

Nongenomic actions of steroids on gonadotropin release.

The release of gonadotropins is effected by GnRH and regulated by steroids. The classical mechanism of steroid hormone action, which implies the binding of hormone receptor complexes to regulatory elements of nuclear genes, is derived largely from the well-studied and familiar steroids such as progesterone, testosterone, and estradiol. Their effects on gonadotropin release generally have been examined following hours or days of exposure and therefore cannot account for the rapid effects of steroids on gonadotropin release. Moreover, tissues such as gonad, pituitary, and hypothalamus can produce a variety of hormonally active steroids in addition to these well-studied, traditional ones. The recently discovered allylic steroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), is readily interconverted from/to progesterone and is found at appreciable levels in serum, gonads, pituitary, hypothalamus, and other tissues. 3 alpha HP has provided the "missing link" in the progesterone biosynthetic/ metabolic pathways, allowing cyclical 4-pregnene and 5 alpha-pregnane pathways to be described for steroidogenic tissues. Among the functions ascribed to 3 alpha HP is the ability to selectively and rapidly (within seconds or minutes) suppress GnRH-provoked FSH release. In vitro studies using pituitary gonadotropes in culture and in perifusion paradigms suggest that suppression of FSH release by 3 alpha HP occurs as a result of nongenomic mechanisms of action. These mechanisms are discussed and include interaction at the level of receptors in the gonadotrope membrane and the cell-signaling pathway involving protein kinase C, phospholipase C, or IP3-induced Ca2+ mobilization and Ca2+ channels. This may be the first evidence of a gonadal steroid regulating gonadotropin release by nongenomic mechanisms of action. In order to understand the critical role of steroids in the rapid regulation of secretory (and bence, circulating) levels of gonadotropins, other gonadal steroids will need to be examined for their nongenomic action on gonadotropes.

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.

Long-term alterations in the permeability of the blood-testis barrier following a single intratesticular injection of dilute aqueous glycerol.

The mechanism whereby glycerol exerts its antispermatogenic action is not known. The objective of this study was to determine, with the use of [3H]inulin and [125I]albumin by in vivo and in vitro methods, whether glycerol exerts its effect by altering the permeability of the blood-testis barrier (BTB). Adult male rats received a single intratesticular injection of either glycerol (10% or 20%; treated) or saline (control), and 2, 4, 8, 26, and 56 weeks after treatment, either [3H]inulin or [125I]albumin was administered either by infusion or directly to the testicular tissues. Radioactivity was measured in testicular tissue, rete testis fluid, and seminiferous tubular fluid. Following in vivo administration, the uptake of [3H]inulin by seminiferous tubules, rete testis fluid, and seminiferous tubule fluid was significantly greater in the treated than in the control testes at all times after treatment. Radiolabeled inulin, injected into isolated testes or added to medium in which isolated tubules were incubated, accumulated at significantly higher levels in the seminiferous tubule compartment of treated than control tissues. Rete testis fluid from treated testes, collected by micropuncture following efferent duct ligation, contained about 5- to 13-fold more radioactivity than fluid from controls. Following infusion of 50 microCi of [125I]albumin into the jugular vein, the accumulated radioactivity was significantly greater in testicular and epididymal tissues and in the seminiferous tubule fluid from treated than from control animals. In all experiments the significant differences between treated and control were maintained during the period of 2-56 weeks following glycerol treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Male preference for the odors of estrous female mice is enhanced by the neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP).

The effects of the centrally produced allylic neurosteroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), on the responses of male mice to the odors of estrous female mice were examined in an odor preference test. Control untreated mice displayed a significant preference for the odors of an estrous female, spending more time in a Y-maze in the vicinity of the odors of an estrous than a non-estrous female. Intracerebroventricular (i.c.v.) administrations of 3 alpha HP enhanced male preference for the odors of estrous females, causing a significant dose-related (0.01-1.0 microgram) increase in the amount of time spent in the proximity of the odors of the estrous female, while having no significant effect on the responses to the non-estrous female odors. These effects of 3 alpha HP were stereospecific, with the stereoisomer, 3 beta-hydroxy-4-pregnen-20-one (3 beta HP), having no significant effects on odor preferences. The analgesic, morphine, also had no significant effects on the responses to female odors suggesting that the enhanced preference for estrous female odors were unlikely to be directly due to any analgesic effects of 3 alpha HP. The effects of 3 alpha HP were significantly reduced by peripheral administrations of the GABAA antagonists, bicuculline and picrotoxin, but were unaffected by either the benzodiazepine antagonist, Ro 15-1788, or the opiate antagonist, naloxone. These results suggest that the neurosteroid 3 alpha HP has facilitatory effects on olfactory mediated male sexual interest or motivation that involve interactions with the GABAA receptor.

Suppression of spermatogenesis by low-level glycerol treatment.

Previous studies have shown that a single intratesticular injection with 70% glycerol results in rapid and long-term suppression of spermatogenesis. Because of the extensive testicular damage in the form of focal destruction and large numbers of acellular seminiferous tubules, it was difficult to examine the possible chemical mechanisms of antispermatogenic action. Our objective was to explore regimens of treatment that would result in significant suppression of spermatogenesis without significant testicular damage. Sexually mature Sprague-Dawley rats were injected intratesticularly with various glycerol concentrations (0, 10, 20, 40, or 70%) and with volumes of either 200 or 350 microliters per testis. At times between 1 and 36 weeks after the injection, the effect on weights of testes and accessory sex organs, testicular histology, sperm numbers, serum hormonal levels, and gonadotropin-receptor binding was examined. An injection of 350 microliters/testis of a 10% glycerol solution led to a significant decrease in weights of testes and epididymides. The treatment resulted in an overall suppression of spermatogenesis, with about 90% fewer sperm in the epididymides than in control animals. Histologically, the treatment decreased the number of normal tubules from 97% (control) to 16% and resulted in testes in which about 75% of the tubules were aspermatogenic (containing only Sertoli cells and spermatogonia). The number of acellular tubules (tubules without cytological detail) was generally less than 5% of the total, and there was negligible focal destruction. Serum levels of gonadotropins and androgens were not altered significantly by the treatment, and Sertoli cell glutamyl transpeptidase activity appeared normal. An equi-osmolar solution of glucose also resulted in significant suppression of spermatogenesis, but the effect of glycerol was significantly greater, suggesting a mechanism in addition to hyperosmolarity. This study, therefore, is further evidence for the specificity of glycerol actions and for its potential as an antispermatogenic agent.

Reduction of predator odor-induced anxiety in mice by the neurosteroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP).

The effects of the centrally produced allylic neurosteroid, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP), on the responses of male mice to an aversive, anxiety-inducing, predator (cat) odor were examined in an odor preference test. Control untreated mice displayed an anxiogenic response to the cat odor, spending a minimal amount of time in a Y-maze in the vicinity of the cat odor. Intracerebroventricular (i.c.v.) administrations of 3 alpha HP had an anxiolytic action, resulting in significant dose-related (0.01-1.0 micrograms) increases in the amount of time spent in the proximity of the cat odor. These anxiolytic effects of 3 alpha HP were stereospecific, with the stereoisomer, 3 beta-hydroxy-4-pregnen-20-one (3 beta HP) having no significant effects on odor preferences. The analgesic, morphine, also had no significant effects on the response to cat odor indicating that the anxiolytic actions of 3 alpha HP were unlikely to be related to any analgesic effects. The effects of 3 alpha HP were significantly reduced by peripheral administrations of the GABAA antagonists, bicuculline and picrotoxin, but were unaffected by either the benzodiazepine antagonist, Ro 15-1788, or the opiate antagonist, naloxone. These results indicate that the allylic neurosteroid 3 alpha HP has anxiolytic actions involving interactions with the GABAA receptor.

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)

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.