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Migraine is a very painful, disabling and extremely common disorder among the world's adult population, especially women, and it is associated with a variety of comorbidities. Neuroactive steroids exhibit pleiotropic actions on the nervous system. Alterations in their peripheral and central levels could be involved in the pathogenesis, still not fully understood, of migraine and its comorbidities. The purpose of our exploratory study was to determine and compare the serum levels of dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), 5α-dihydroprogesterone (DHP) and pregnenolone (PREGNE) between women suffering from migraine without aura (MO group, n = 30) and age-matched non-headache women as controls (C group, n = 30). Correlations with age, migraine years and frequency were also analyzed. The patients were enrolled at a headache center; controls were patients' contacts. Calibrators and serum samples were spiked with the internal standards (ISs) solution and treated to deplete proteins and phospholipids. The obtained extracts were evaporated to dryness, derivatized and analyzed by LC-MS/MS in multiple reaction monitoring mode. Analytes' levels were determined by interpolation on the regression curves, generated from the analyte quantifier ion peak area to the corresponding IS. MO group presented significantly lower levels of DHEAS, DHEA and DHP compared to C group (P 0.05, Student't-test) and the neurosteroid levels negatively correlated with years of migraine and migraine days/3 months (P 0.05, linear regression analysis). These results parallel to previous studies showing reduced serum levels of allopregnanolone and pregnenolone sulfate in women with migraine. The low serum levels found for both excitatory and inhibitory neurosteroids suggested that women with migraine might suffer from inadequate neuroprotection, anti-inflammation activity and pain modulation. These deficits might underlie the migraine chronification process and represent the link between migraine and its various comorbidities.

Objective The present study investigated the anti-seizure effects of 5α-dihydroprogesterone (DHP) in an animal model of human drug-resistant focal seizures with impaired awareness. DHP was administered via the intravenous (IV) route. Methods Female Wistar rats were implanted with an electrode in the right basolateral amygdala. They were then kindled to 15 stage 5 seizures, stability tested, and cannulated in the jugular vein. Multiple doses of IV DHP were tested against focal electrographic seizures and secondarily generalized convulsions. The time-course of DHP’s action was also examined. Results The dose-response study, done at 5 min after injection, showed a dose-dependent suppression of both generalized and focal seizures, with an ED50 of 1.69 mg/kg for the generalized convulsive seizures and an ED50 of 3.48 mg/kg for the focal electrographic seizures. Ataxia, as rated by the Loscher ataxia scale, was also seen, with a TD50 of 3.57 mg/kg. The time-response study, done at the ED75 for focal seizure suppression, showed suppression of both generalized and focal seizures from immediately after injection to about 60 min post-injection. Significance DHP has demonstrated anti-seizure effects in a drug-resistant model of human focal seizures with impaired awareness. Its analogs might be developed as new anti-seizure drugs.

In vivoandin vitroevidence indicates that the bioactive, 5α-reduced progesterone metabolite, 5α-dihydroprogesterone (DHP) is synthesized in the placenta, supporting equine pregnancy, but its appearance in early pregnancy argues for other sites of synthesis also. It remains unknown if DHP circulates at relevant concentrations in cyclic mares and, if so, does synthesis involve the non-pregnant uterus? Jugular blood was drawn daily from cyclic mares (n = 5). Additionally, ovariectomized mares (OVX) and geldings were administered progesterone (300 mg) intramuscularly. Blood was drawn before and after treatment. Incubations of whole equine blood and hepatic microsomes with progesterone were also investigated for evidence of DHP synthesis. Sample analysis for progesterone, DHP and other steroids employed validated liquid chromatography–tandem mass spectrometry methods. Progesterone and DHP appeared a day (d) after ovulation in cyclic mares, was increased significantly by d3, peaking from d5 to 10 and decreased from d13 to 17. DHP was 55.5 ± 3.2% of progesterone concentrations throughout the cycle and was highly correlated with it. DHP was detected immediately after progesterone administration to OVX mares and geldings, maintaining a relatively constant ratio with progesterone (47.2 ± 2.9 and 51.2 ± 2.7%, respectively). DHP was barely detectable in whole blood and hepatic microsome incubations. We conclude that DHP is a physiologically relevant progestogen in cyclic, non-pregnant mares, likely stimulating the uterus, and that it is synthesized peripherally from luteal progesterone but not in the liver or blood. The presence of DHP in pregnant perissodactyla as well as proboscidean species suggests horses may be a valuable model for reproductive endocrinology in other exotic taxa.

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.

Progesterone is commonly considered as a female reproductive hormone and is well-known for its role in pregnancy. It is less well appreciated that progesterone and its metabolite allopregnanolone are also male hormones, as they are produced in both sexes by the adrenal glands. In addition, they are synthesized within the nervous system. Progesterone and allopregnanolone are associated with adaptation to stress, and increased production of progesterone within the brain may be part of the response of neural cells to injury. Progesterone receptors (PR) are widely distributed throughout the brain, but their study has been mainly limited to the hypothalamus and reproductive functions, and the extra-hypothalamic receptors have been neglected. This lack of information about brain functions of PR is unexpected, as the protective and trophic effects of progesterone are much investigated, and as the therapeutic potential of progesterone as a neuroprotective and promyelinating agent is currently being assessed in clinical trials. The little attention devoted to the brain functions of PR may relate to the widely accepted assumption that non-reproductive actions of progesterone may be mainly mediated by allopregnanolone, which does not bind to PR, but acts as a potent positive modulator of γ-aminobutyric acid type A (GABA(A) receptors. The aim of this review is to critically discuss effects of progesterone on the nervous system via PR, and of allopregnanolone via its modulation of GABA(A) receptors, with main focus on the brain. Fil: Schumacher, M.. Inserm; Francia Fil: Mattern, C.. MetP Pharma AG ; Suiza Fil: Ghoumari, A.. Inserm; Francia Fil: Oudinet, J. P.. Inserm; Francia Fil: Liere, P.. Inserm; Francia Fil: Labombarda, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Sitruk Ware, Régine. The Rockefeller University; Estados Unidos. Population Council. Center for Biomedical Research ; Estados Unidos Fil: de Nicola, Alejandro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Guennoun, R.. Inserm; Francia

Progesterone is a known anticonvulsant, with its inhibitory effects generally attributed to its secondary metabolite, 5α,3α-tetrahydroprogesterone (THP), and THP's enhancement of GABAA receptor activity. Accumulating evidence, however, suggests that progesterone may have non-genomic actions independent of the GABAA receptor. In this study, we explored THP/GABAA-independent anticonvulsive actions of progesterone in a mouse model of hippocampal kindling and in mouse entorhinal slices in vitro. Specifically, we examined the effects of progesterone in kindled mice with or without pretreatments with finasteride, a 5α-reductase inhibitor known to block the metabolism of progesterone to THP. In addition, we examined the effects of progesterone on entorhinal epileptiform potentials in the presence of a GABAA receptor antagonist picrotoxin and finasteride. Adult male mice were kindled via a daily stimulation protocol. Electroencephalographic (EEG) discharges were recorded from the hippocampus or cortex to assess "focal" or "generalized" seizure activity. Kindled mice were treated with intra-peritoneal injections of progesterone (10, 35, 100 and 160mg/kg) with or without finasteride pretreatment (50 or 100mg/kg), THP (1, 3.5, 10 and 30mg/kg), midazolam (2mg/kg) and carbamazepine (50mg/kg). Entorhinal cortical slices were prepared from naïve young mice, and repetitive epileptiform potentials were induced by 4-aminopyridine (100μM), picrotoxin (100μM) and finasteride (1μM). Pretreatment with finasteride did not abolish the anticonvulsant effects of progesterone. In finasteride-pretreated mice, progesterone at 100 and 160mg/kg decreased cortical but not hippocampal afterdischarges (ADs). Carbamazepine mimicked the effects of progesterone with finasteride pretreatments in decreasing cortical discharges and motor seizures, whereas midazolam produced effects similar to progesterone alone or THP in decreasing hippocampal ADs and motor seizures. In brain slices, progesterone at 1μM inhibited entorhinal epileptiform potentials in the presence of picrotoxin and finasteride. We suggest that progesterone may have THP/GABAA-dependent and independent anticonvulsive actions in the hippocampal-kindled mouse model.

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/β-HSDs and 5α/β-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17β-HSD type 2 (HSD17B2). Also, 3α-HSD and 3β-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5β-reductase (AKR1D1) are responsible for the formation of 5α- and 5β-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/β-HSD activities of AKR1C1, AKR1C2 and AKR1C3.

This study tested the hypothesis that the Src/Raf/MAPK signaling pathway is involved in the facilitation of the lordosis and proceptive behaviors induced by progesterone (P) and its ring A-reduced metabolites in ovariectomized, estradiol-primed rats. Intraventricular (icv) infusion of PP2 (7.5, 15 and 30 microg), a Src kinase inhibitor, significantly depressed P-dependent estrous behavior (lordosis and proceptivity) in estradiol-primed rats. Icv infusion of 30 microg of PP2 also significantly attenuated estrous behavior induced by the ring A-reduced P metabolites 5 alpha-dihydroprogesterone (5 alpha-DHP) and 5 alpha-pregnan-3alpha-ol-20-one (allopregnanolone). PP2 did not inhibit estrous behavior induced by administration of high doses of estradiol alone to ovariectomized rats. We also assessed if the ventromedial hypothalamus (VMH) is one of the neural sites at which progestins activate Src signaling to facilitate estrous behavior. Bilateral administration of 15 microg of PP2 into the VMH inhibited the stimulation of both lordosis and proceptive behaviors elicited by subcutaneous P administration to estradiol-primed rats. These results suggest that progestins act through Src/Raf/MAPK signaling to initiate estrous behaviors in estrogen-primed rats. This event is one component of the cellular pathways leading to the display of estrous behaviors induced by P and its ring A-reduced metabolites in female rats.

Progesterone has long been linked to breast cancer but its actual role as a cancer promoter has remained in dispute. Previous in vitro studies have shown that progesterone is converted to 5a-dihydroprogesterone (5aP) in breast tissue and human breast cell lines by the action of 5 a-reductase, and that 5aP acts as a cancer-promoter hormone. Also studies with human breast cell lines in which the conversion of progesterone to 5aP is blocked by a 5a-reductase inhibitor, have shown that the in vitro stimulation in cell proliferation with progesterone treatments are not due to progesterone itself but to the metabolite 5aP. No similar in vivo study has been previously reported. The objective of the current studies was to determine in an in vivo mouse model if the presumptive progesterone-induced mammary tumorigenesis is due to the progesterone metabolite, 5aP. BALB/c mice were challenged with C4HD murine mammary cells, which have been shown to form tumors when treated with progesterone or the progestin, medroxyprogesterone acetate. Cells and mice were treated with various doses and combinations of progesterone, 5aP and/or the 5 a-reductase inhibitor, finasteride, and the effects on cell proliferation and induction and growth of tumors were monitored. Hormone levels in serum and tumors were measured by specific RIA and ELISA tests. Proliferation of C4HD cells and induction and growth of tumors was stimulated by treatment with either progesterone or 5aP. The progesterone-induced stimulation was blocked by finasteride and reinstated by concomitant treatment with 5aP. The 5aP-induced tumors expressed high levels of ER, PR and ErbB-2. Hormone measurements showed significantly higher levels of 5aP in serum from mice with tumors than from mice without tumors, regardless of treatments, and 5aP levels were significantly higher (about 4-fold) in tumors than in respective sera, while progesterone levels did not differ between the compartments. The results indicate that the stimulation of C4HD tumor growth in BALB/c mice treated with progesterone is due to the progesterone metabolite 5aP formed at elevated levels in mammary cells as a result of the 5a-reductase action on progesterone. The results provide the first in vivo demonstration that stimulation of breast cell tumorigenesis and tumor growth accompanying progesterone treatment is due to the progesterone metabolite 5aP, and that breast tumorigenesis can be blocked with the 5a-reductase inhibitor, finasteride. Fil: Wiebe, John P.. University of Wenstern Ontario. Department of Biology; Canadá Fil: Rivas, Martin Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Mercogliano, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Elizalde, Patricia Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Schillaci, Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina

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.

Allopregnanolone (3alpha,5alpha-TH PROG) and 5alpha-dihydroprogesterone (5alpha-DH PROG), the two most important neuroactive steroids synthesized in the brain, potently modulate neuronal activity by allosterically regulating GABA action at GABA(A) receptors or by changing specific GABA(A) receptor subunit gene expression, respectively. We recently reported [Proc. Natl. Acad. Sci. USA 95 (1998) 3239] that in patients with severe depression there is a decrease in the CSF levels of 3alpha,5alpha-TH PROG, which is normalized by treatment with drugs (i.e. fluoxetine) that improve psychopathology. The mechanism by which fluoxetine and other selective serotonin reuptake inhibitors normalize 3alpha,5alpha-TH PROG CSF levels appears to involve a direct stimulation of 3alpha-hydroxysteroidoxidoreductase (3alpha-HSD), an enzyme that catalyses the reduction of 5alpha-DH PROG into 3alpha,5alpha-TH PROG. Here, we propose the use of socially-isolated mice that have a downregulation of 3alpha,5alpha-TH PROG and of 5alpha-DH PROG expression to establish a model to study the behavioral consequences of this deficiency. After 4-6 weeks of isolation, these mice exhibit increased anxiety and aggressive behavior and also a decreased response to the administration of GABA-mimetic drugs. In these mice, the decrease in 3alpha,5alpha-TH PROG is selectively normalized by the use of fluoxetine in doses that reduce behavioral abnormalities. In addition, the expression of 5alpha-reductase Type I mRNA and protein was lower in socially-isolated mice than that in group-housed mice whereas 3alpha-HSD mRNA expression remained unchanged. The results of these studies may enable us to design drugs that specifically affect neurosteroidogenic enzymatic activities and may provide an efficacious treatment for the psychopathologies associated with psychiatric disorders.

Because of its low levels in late pregnancy, the relationship of progesterone to pregnancy maintenance in Equidae is not obvious. This study investigated the levels of progesterone (4-pregnane-3,20-dione; P4) and 5α-dihydroprogesterone (5α-DHP) during pregnancy in zebras in relation to reproductive state. Blood samples from female zebras (Equus burchelli, E. zebrahartmannae, E. grevyi) were taken at Dvur Kralove Zoo. Progesterone and 5α-DHP were separated by high-performance liquid chromatography techniques and detected by cross-reacting antibodies. Identification of progestins was achieved by comparing the identity of peaks of the samples with a standard. In E. z. hartmannae progesterone, values reached 50 ng/mL at the beginning of pregnancy and dropped to levels below 1 ng/mL during the second half of pregnancy. In contrast, 5α-DHP increased up to 123 and 183 ng/mL during late pregnancy in E. z. hartmannae and E. burchelli, respectively. In E. grevyi, 5α-DHP levels of 368 ng/mL were obtained during pregnancy, whereas progesterone values were similar in pregnant and non-pregnant individuals. These marked differences in the course of progesterone and 5α-DHP levels point to the importance of 5α-DHP for pregnancy maintenance in zebras. Zoo Biol 18:325–333, 1999. © 1999 Wiley-Liss, Inc.

Introduction Of the nearly 1.4 million new cases of breast cancer diagnosed each year, a large proportion is characterized as hormone receptor negative, lacking estrogen receptors (ER) and/or progesterone receptors (PR). Patients with receptor-negative tumors do not respond to current steroid hormone-based therapies and generally have significantly higher risk of recurrence and mortality compared with patients with tumors that are ER- and/or PR-positive. Previous in vitro studies had shown that the progesterone metabolites, 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP), respectively, exhibit procancer and anticancer effects on receptor-negative human breast cell lines. Here in vivo studies were conducted to investigate the ability of 5αP and 3αHP to control initiation, growth, and regression of ER/PR-negative human breast cell tumors. Methods ER/PR-negative human breast cells (MDA-MB-231) were implanted into mammary fat pads of immunosuppressed mice, and the effects of 5αP and 3αHP treatments on tumor initiation, growth, suppression/regression, and histopathology were assessed in five separate experiments. Specific radioimmunoassays and gas chromatography-mass spectrometry were used to measure 5αP, 3αHP, and progesterone in mouse serum and tumors. Results Onset and growth of ER/PR-negative human breast cell tumors were significantly stimulated by 5αP and inhibited by 3αHP. When both hormones were applied simultaneously, the stimulatory effects of 5αP were abrogated by the inhibitory effects of 3αHP and vice versa. Treatment with 3αHP subsequent to 5αP-induced tumor initiation resulted in suppression of further tumorigenesis and regression of existing tumors. The levels of 5αP in tumors, regardless of treatment, were about 10-fold higher than the levels of 3αHP, and the 5αP:3αHP ratios were about fivefold higher than in serum, indicating significant changes in endogenous synthesis of these hormones in tumorous breast tissues. Conclusions The studies showed that estrogen/progesterone-insensitive breast tumors are sensitive to, and controlled by, the progesterone metabolites 5αP and 3αHP. Tumorigenesis of ER/PR-negative breast cells is significantly enhanced by 5αP and suppressed by 3αHP, the outcome depending on the relative concentrations of these two hormones in the microenvironment in the breast regions. The findings show that the production of 5αP greatly exceeds that of 3αHP in ER/PR-negative tumors and that treatment with 3αHP can effectively block tumorigenesis and cause existing tumors to regress. The results provide the first hormonal theory to explain tumorigenesis of ER/PR-negative breast tissues and support the hypothesis that a high 3αHP-to-5αP concentration ratio in the microenvironment may foster normalcy in noncancerous breast regions. The findings suggest new diagnostics based on the relative levels of these hormones and new approaches to prevention and treatment of breast cancers based on regulating the levels and action mechanisms of anti- and pro-cancer progesterone metabolites.

Coupling high performance liquid chromatography with gas chromatography- mass fragmentography has made it possible to simultaneously measure subpicomolar concentrations of allopregnanolone and its precursors, pregnenolone, progesterone and 5 alpha-dihydroprogesterone (5 alpha- DHP) in various brain areas. Allopregnanolone was measured in the brain of adrenalectomized/castrated (ADX/CX) rats in nanomolar concentrations long after peripheral sources of allopregnanolone were removed. A partial decrease (approximately 30%) in the content of allopregnanolone was found in the brains of ADX/CX rats compared to sham-operated rats. Moreover, the content of allopregnanolone in brains of sham-operated as well as ADX/CX rats was nonuniformly distributed (olfactory bulb > striatum > cortex > hippocampus) and was one to two orders of magnitude higher than in plasma or liver. Infusion of pregnenolone sulfate in ADX/CX rats elicited a fourfold increase in 5 alpha-DHP and progesterone content and a seven- to eightfold increase in the content of allopregnanolone in brain but not in liver or plasma. Furthermore, the content of allopregnanolone in brain increased to the same extent in both sham-operated and ADX/CX rats following pregnenolone sulfate infusion. The 5 alpha-reductase inhibitor, (17 beta)17[[bis(1- methylethyl)amino]carbonyl] androstane-3,5-diene-3-carboxylic acid (SKF 105111), reduced the brain content of allopregnanolone and blocked the increased formation of allopregnanolone in brain following pregnenolone sulfate infusion. The results clearly demonstrate that the synthesis of allopregnanolone from 5 alpha-DHP and progesterone occurs in the brain and that a significant amount of allopregnanolone is synthesized locally in brain from its precursors. These experiments suggest that the brain, like adrenals and gonads, is a steroidogenic organ which produces allopregnanolone as one of its own most important physiologically relevant steroids.

Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) enzymic activities (NADH-linked and NADPH-linked) were measured in anterior pituitaries (AP) from aged female rats during three stages of reproductive senescence (constant estrus: CE; repeated pseudopregnancies: PSP; and anestrus: AN). To assess ovarian influence on these enzymes during these stages of reproductive aging, we also determined enzyme levels from ovariectomized rats from each stage treated with estrogen or vehicle. Progesterone 5 alpha-reductase and NADH-linked 3 alpha-HSOR activities were 2-fold higher in pituitaries of CE rats as compared to those of PSP and AN rats. NADPH-linked 3 alpha-HSOR levels did not differ among the three stages. All three enzyme levels were elevated 2- to 5-fold as compared to the corresponding enzyme levels from young cycling rats. After ovariectomy (10 days), 5 alpha-reductase activity in PSP and AN rats was elevated 3- to 4-fold relative to mean levels in intact PSP and AN rats. Ovariectomy had no effect on 5 alpha-reductase levels in CE rats. Under similar conditions, young cycling rats exhibit a 10-12-fold increase. Treatment of ovariectomized PSP and AN rats for 3 days with estradiol benzoate (10 micrograms/day) restored 5 alpha-reductase levels. Ovariectomy had no effect on the NADPH-linked 3 alpha-HSOR levels in CE, PSP or AN animals which is similar to that observed with young rats. Ovariectomy also had no effect on the NADH-linked 3 alpha-HSOR levels except for the CE group. The ovariectomized CE rats exhibited reduced pituitary NADH-linked 3 alpha-HSOR levels (30%). In contrast, young rats exhibit elevated pituitary NADH-linked 3 alpha-HSOR levels after ovariectomy (4- to 5-fold). These changes suggest the possibility that altered processing of progesterone and its 5 alpha- and 3 alpha-reduced products may be one means by which the effectiveness of progesterone is reduced during aging. The results also suggest an altered ovarian role in the regulation of these enzymes.

Previous work from our laboratory has shown that progesterone causes a rapid reduction of pituitary nuclear estrogen receptors. This effect is accompanied by a loss of estrogen action such as estrogen-induced progesterone receptor synthesis and prolactin release. Recent work has demonstrated that the effect of progesterone on estrogen receptor depletion is dose-related. Therefore the purpose of this study was to examine the effect of different doses of progesterone upon estrogen-induced prolactin release. Progesterone exhibited a multiphasic effect on estrogen-induced prolactin release. The 0.8 mg/kg body wt and 3.2 mg/kg body wt doses of progesterone significantly inhibited estrogen-induced prolactin release, whereas the intermediate dose, 2.0 mg/kg, was without effect. Progesterone is rapidly metabolized to 5alpha-dihydroprogesterone in the pituitary, and this metabolite has recently been shown to deplete pituitary estrogen receptors similar to progesterone. Thus the effect of 5alpha-dihydroprogesterone onstrogen-induced prolactin release was also examined. 5alpha-dihydroprogesterone was found to exhibit a multiphasic effect similar to progesterone in inhibiting estrogen-induced prolactin release. The 0.4 mg/kg and 2.0 mg/kg doses of 5alpha-dihydroprogesterone significantly inhibited estrogen-induced prolactin release, whereas the 0.8 mg/kg dose was ineffective. The effect of 5alpha-dihydroprogesterone required estrogen priming and was blocked by the antiprogestin, RU486, suggesting progesterone receptor mediation.

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.

Among the species investigated, the rate of spontaneous cancers of mammary glands is highest in humans and dogs (Hamilton, 1974; Owen, 1979). In addition, spontaneous mammary tumours are known to occur in strains of mice (Webster & Muller, 1994), rats (Russo et al. 1990; Sukumar, 1995) and cats (Hamilton, 1974; Kessler & von Bombard, 1997). Although both oestrogen and progesterone are known to be involved in normal mammary development as well as in the proliferative changes that occur during the oestrous or menstrual cycle, pregnancy and lactation (Going et al. 1988), only the role of oestradiol has been extensively investigated. The role of progesterone in mammary cancer is not understood and, although progesterone metabolism is known to occur in mammary tissue, the potential autocrine/paracrine role of the in situ metabolites has only recently begun to be explored in human breast tissues and cell lines (Wiebe et al. 2000).

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.

Summary: Purpose: Progesterone has been shown to be anticonvulsant in several animal seizure models. The purpose of the present study was to investigate the anticonvulsant actions of progesterone and its primary metabolite 5α-dihydroprogesterone in the amygdala kindling model. Methods: Female Wistar rats were implanted in the right basolateral amygdala with a long-term, bipolar electrode. The subjects were kindled to 30 stage 5 seizures and stability tested. Multiple doses of progesterone and 5α-dihydroprogesterone were then tested for anticonvulsant activity against focal electrographic and generalized convulsive kindled seizures. The time course of progesterone's anticonvulsant action also was examined. Results: Progesterone had a median effective dose (ED50) of 103 mg/kg against generalized convulsions at 15 min after injection. Subjects were not sedated at the time of seizure testing, although sedation developed later (40–60 min after injection). In time-course experiments, it was found that 120 mg/kg of progesterone caused complete suppression of the generalized convulsion from 20 to 160 min after injection. Suppression of the focal discharge also was seen in some animals between 20 and 160 min. 5α-dihydroprogesterone had an ED50 of 2.9 mg/kg against generalized kindled convulsions and an ED50 of 4.3 mg/kg against focal afterdischarge 15 min after injection. 5α-dihydroprogesterone did not produce sedation 15 min after injection, or at any later time interval. Conclusions: Progesterone is anticonvulsant only at high doses when tested against amygdala kindled seizures. 5α-dihydroprogesterone is considerably more potent than progesterone. At low, nonsedative doses, it was effective against both the kindled amygdala focal afterdischarge and the generalized convulsion.

This study was conducted 1) to ascertain whether the high levels of plasma 5 alpha-dihydroprogesterone (5 alpha DHP) during the luteal phase of the human ovarian cycle and pregnancy are attributable to high rates of production or, alternatively, low rates of clearance, and 2) to estimate the relative distribution of the irreversible metabolism of 5 alpha DHP, i.e. hepatic compared with extrahepatic clearance of plasma 5 alpha DHP. The concentration of 5 alpha DHP in plasma of women during the luteal phase of the ovarian cycle and pregnancy is very high, viz. 12-40% that of progesterone. Thus, a potentially large source of steroid precursor is available for the formation of bioactive 5 alpha-pregnanolone metabolites. We found that the MCR of 5 alpha DHP in women and men is 4187 +/- 312 L plasma/24 h (range, 3181-5506; n = 6). The MCR of 5 alpha DHP as a function of body surface area was 2406 +/- 240 L/24 h.m2. The MCR of 5 alpha DHP, therefore, is the greatest of any steroid reported, except for that of the catechol estrogens, which are metabolized intravascularly by erythrocyte catechol-O-methyltransferase. Based on estimated rates of liver plasma flow (1500 L/24 h) and hepatic extraction (75-85%) of lipophilic steroids that are not specifically bound in plasma with high affinity to binding proteins as determined by other investigators (e.g. 5 alpha DHP), we estimate that approximately 1200 L plasma/24 h are cleared of 5 alpha DHP by liver and approximately 2800 L plasma/24 h are cleared of 5 alpha DHP (70%) by metabolism in extrahepatic tissues. Thus, 5 alpha DHP can serve as a precursor for bioactive 5 alpha-pregnanolone(s).

The purified cytosolic 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) from female rat pituitary which catalyzes the reversible conversion of 5 alpha-dihydroprogesterone (5 alpha-DHP) to 3 alpha, 5 alpha-tetrahydroprogesterone (3 alpha, 5 alpha-THP) has been characterized in terms of its steroid substrate specificity, dihydrodiol dehydrogenase activity and inhibition by drugs such as medroxyprogesterone and indomethacin. The purified enzyme has a strong preference for the C21 progestin steroid substrates, 5 alpha-DHP and 3 alpha, 5 alpha-THP, over the corresponding C19 androgenic steroid substrates, 5 alpha-dihydrotesterone (5 alpha-DHT) and 3 alpha, 5 alpha-tetrahydrotestosterone (3 alpha, 5 alpha-THT). The apparent Km for 5 alpha-DHP (80 nM) is about 250 times lower than the Km for the androgenic steroid, 5 alpha-DHT (21 microM). In the oxidative direction, the apparent Km for 3 alpha, 5 alpha-TP (1.4 microM) is about 3-fold lower than the Km for the androgenic steroid, 3 alpha, 5 alpha-THT (4.2 microM). A number of other naturally occurring 3-keto- and 3 alpha(beta)-hydroxy-steroids were assessed for their ability to act as inhibitors (alternate substrates) of the 3 alpha-reduction of 5 alpha-DHP catalyzed by the purified 3 alpha-HSOR. None of the 3 beta- or 5 beta-isomers had any effect. Of the other 3-keto and 3 alpha- steroids tested, only deoxycorticosterone and the ovarian progestins showed any significant inhibition. These may be acting as inhibitors since there was little, if any, direct 3 alpha-reduction of progesterone to 3 alpha-hydroxy-4-pregnen-20-one. Unlike the liver cytosolic 3 alpha-HSOR, the pituitary enzyme has no associated dihydrodiol (quinone) dehydrogenase activity. This enzyme is similar to other cytosolic 3 alpha-HSORs from liver and brain in that it is potentially inhibited by indomethacin and by medroxyprogesterone.

Progesterone and its metabolite 5 alpha-dihydroprogesterone (5 alpha-DHP) have been shown to bring about gonadotropin release in the estrogen-primed ovariectomized rat. One of the actions of progesterone is the decrease in occupied estrogen receptors (E2Rs) in the anterior pituitary and uterus. This study attempted to determine if 5 alpha-DHP had a similar effect on pituitary and uterine E2Rs. Estrogen-primed ovariectomized mature female rats were injected with either vehicle, or 0.2, 0.8 or 2 mg/kg body weight (BW) of 5 alpha-DHP, 2 h before sacrifice and 1 h before the last estradiol injection (2 micrograms/rat). Nuclear E2Rs were determined by Scatchard analyses in the uterus and anterior pituitary. Total nuclear E2R levels of both tissues showed a 2-fold increase in the number of estradiol-binding sites after estradiol administration, as compared to control groups. In estradiol-primed rats, 5 alpha-DHP produced a significant decrease in total nuclear E2R levels in a tissue-specific manner. In the pituitary, there was a maximal and significant decrease in nuclear E2Rs with 0.2 and 2.0 mg/kg BW of 5 alpha-DHP as compared to estradiol alone; the intermediate dose of 0.8 mg/kg BW of 5 alpha-DHP induced a smaller nonsignificant change in nuclear E2Rs. In the uterus, 5 alpha-DHP showed a dose-dependent decrease in nuclear E2Rs. The 5 alpha-DHP effect in both tissues was due to a specific reduction in the occupied form of nuclear E2Rs levels. The unoccupied form of E2Rs was unaffected by 5 alpha-DHP administration. 5 alpha-DHP did not have any effect in the absence of estrogen priming.(ABSTRACT TRUNCATED AT 250 WORDS)

A radioimmunoassay for the simultaneous determination of progesterone (P) and 17-hydroxyprogesterone (17-OHP) in human plasma (0.02–1.0 ml) from various conditions is described. The procedure involves addition to plasma samples of [3H]-P and [3H]- 17-OHP (45,000 d.p.m.) serving as both tracers to calculate losses and competitive steroids in the immunological reaction, and extraction by diethyl ether. Purification is achieved by chromatography on Sephadex LH 20. Two antisera raised against P-11-α-hemisuccinate-bovine serum albumin and 17-OHP-3-(O-carboxymethyl)-oxime-bovine serum albumin in rabbits are used. The unbound steroids are removed by toluene extraction. The criteria of precision, accuracy, sensitivity and specificity are evaluated in some detail. Plasma P from normal men is similar to that from children (infants and prepubertal children) and that from women in the follicular phase. Plasma 17-OHP from men, by contrast, is higher than that from children, and that from women in the follicular phase. This suggests an origin of 17-OHP from adult testis. Plasma P and 17-OHP from pregnant women from 11th to 40th post-ovulatory days are significantly higher than that from women in the last luteal phase (26–30 days of cycle). This indicates that serial determinations of these two steroids, after the 13th post-ovulatory day can contribute to the diagnosis of pregnancy.

Progestin binding, which may represent receptor binding, was examined in female rat anterior pituitary cytosol. Specific progesterone, 5α-dihydroprogesterone (5α-DHP) and R5020 binding, as well as cortico-steroid binding globulin (CBG)-like complexes, were assayed in cytosol from five animal models: estradiol benzoate primed-ovariectomized-adrenalectomized (EB-OVX-ADX), ovariectomized-adrenalecto-mized (OVX-ADX), estradiol benzoate primed-ovariectomized (EB-OVX), ovariectomized (OVX) and intact rats. With all three progestins, estrogen priming consistently and significantly enhanced specific binding levels, and cytosol from EB-OVX-ADX rats had the greatest amount of binding. In contrast, presumptive CBG-like levels increased with adrenalectomy but not with EB treatment. Specific progesterone and 5α-DHP complexes dissociated more rapidly during Sephadex LH-20 elution than specific R5020 and CBG-like binding complexes. Maximal values for progesterone and 5α-DHP binding were rapidly attained at 4°C and the binding was decreased in the presence of protease, but not DNase or RNase. 5α-DHP binding had an apparent K d of 0.9 to 2 × 10 −9 M and the number of binding sites increased with EB treatment in the OVX-ADX rat. The 5α-DHP binding exhibited greater specificity for progestins (particularly progesterone and R5020) than for androgens, glucocorticoids or estradiol.

A 200 mg dose of micronized progesterone (P) was administered orally to six healthy volunteers. Serial blood samples were collected and plasma P was assayed according to three methods of different specificity. The results were compared to those obtained with a reference technique based on gas chromatography-mass spectrometry. The interference of P metabolites particularly 5 alpha- and 5 beta-dihydroprogesterone was shown to be important when no or inadequate chromatography was included in the method. Radioimmunoassay after chromatography on a Celite column yielded results similar to those of the reference method. After oral administration, plasma P reached a peak at 3.00 +/- 0.44 h (mean +/- SEM; range: 1-4 h). The mean peak level 4.70 +/- 1.15 ng/ml (mean +/- SEM; range: 2.40-10.10 ng/ml) was at the lower limit of the range defined for a normal functional corpus luteum. The levels returned to initial values by 24 h after the single oral dose. The separation of P from its metabolites by an adequate chromatographic system appears mandatory after oral administration of micronized P.

In gravid women who are destined to develop pregnancy-induced hypertension (PIH), normal pregnancy-associated refractoriness to the pressor effects of administered angiotensin II (A-II) is lost several weeks before the onset of hypertension. From a study of the determinants of A-II pressor responsiveness in normal gravid women, it appears likely that the loss of resistance to A-II is principally unrelated to plasma renin activity or to plasma A-II levels. However, it recently has been shown that the vascular refractoriness to A-II in normal women can be reduced significantly by the administration of the prostaglandin synthetase inhibitors, indomethacin or aspirin. In seven women who had developed PIH and who had lost their refractoriness to A-II, the infusion of 5alpha-pregnan-3,20-dione (5alpha-DHP) was associated with restoration of refractoriness to the pressor effects of A-II. Moreover, in five normotensive gravid women beyond 28 weeks' gestation in whom the refractoriness to A-II was reduced by the administration of indomethacin, the intravenous infusion of 5alpha-DPH was associated with restoration of refractoriness to the pressor effects of A-II. These observations are consistent with the view that a progesterone metabolite(s) may be important in the maintenance of normal blood pressure during human pregnancy.

The binding of 5α-dihydroprogesterone (DHP)1 to proteins in pregnancy serum has been investigated and compared with the binding of progesterone. Characteristic properties of the DHP-serum protein interaction were unsaturability, low affinity and poor complex stability. Fractionation of serum using a variety of protein separation techniques, revealed that DHP interacts with several proteins. At low temperature (0–4°C) albumin appeared to be the principal binding component whereas higher temperatures seemed to favour binding to β-lipoproteins and α2-macroglobulin. Binding to specific binding proteins such as the corticosteroid binding globulin (CBG) and the sex hormone-binding globulin, (SHBG) were detectable but appeared to be quantitatively unimportant. Progesterone showed a similar multicomponent interaction but differed from DHP in the extent of binding to CBG. Binding of either hormone to the α1-acid-glycoprotein was negligible. The present study shows that the high endogenous DHP levels present in pregnancy sera are caused by factors other than high affinity protein binding.

Three days after ovariectomy adult female rabbits were injected intramuscularly with either 2 mug estradiol/kg b.w., 0.5 mg progesterone/kg b.w., 2 mug estradiol plus 0.5 mg progesterone/kg b.w., or oil vehicle daily for 5 days. On the sixth day the animals were anesthetized and given a continuous infusion of [14C]progesterone and [3H]5alpha-pregnane-3,20-dione (5alpha-DHP) for 4 h via the femoral vein. Head extractions, calculated from the difference in blood concentrations of radioactive steriods between the femoral artery and jugular vein, were in the range of 60-75% for both progesterone and 5alpha-DHP and were unaffected by the hormone treatments. Nine brain areas and the pituitary were analyzed for [14C]progesterone, [3H]5alpha-DHP and [14C]-5alpha-DHP. Generally, the brain and pituitary retention and distribution of [14C]progesterone and [3H]5alpha-DHP and the brain and pituitary metabolism of [14C]progesterone were unaffected by hormone treatments. In the cerebellum, progesterone treatment increased [14C]progesterone retention as compared to oil treatment. All tissues contained 2-7 times more [14C]progesterone than arterial blood concentrations. [3H]5alpha-DHP tissue: arterial blood concentrations ratios were much lower ranging from 0.9 to 1.78. [14C]progesterone and [3H]5alpha-DHP showed similar brain distribution with highest concentrations in the pons, pons reticulum and midbrain reticulum. All tissues converted progesterone to 5alpha-DHP, but only the cerebellum contained more [14C]5alpha-DHP than [14C]progesterone.

In mammals, progesterone has diverse effects which could result from the action of progesterone itself and/or specific metabolites. In neuro-endocrine tissues, progesterone is rapidly metabolized principally to 5α-dihydroprogesterone and 3α-hydroxy-5α-pregnan-20-one, and it is unclear whether progesterone and/or metabolite.(s) mediate cellular responses. Below we report on the selective tissue accumulation of progesterone and certain of its metabolites. [ 3 H]-progesterone or [ 3 H]-5α-dihydroprogesterone (5α-DHP) was injected intravenously (i.v.) into ovariectomized rats for 10 or 30 min or into oestrogenprimed ovariectomized rats for 10 min. Isotopic dilution analyses of the accumulated tritium in plasma and selected tissues were made for progesterone, 5α-DHP, 3α-hydroxy-5α-pregnan-20-one, 20α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one and 5α-pregnane-3α, 20α-diol. Significant differences between tissues were noted in terms of which steroidal compounds were accumulated above plasma and non-target tissue levels. The pituitary accumulated large amounts of [ 3 H]-5α-DHP from either injected steroid but progesterone levels did not differ from plasma and non-target tissues. The hypothalamus accumulated significant amounts of both 5α-UHP and progesterone in all groups. The midbrain-tectum accumulated 5a-DHP comparable to hypothalamus and pituitary levels in all groups after injection of either steroid. The pineal had the greatest accumulations of progesterone in all groups but there were no accumulations of 5α-DHP above non-target tissue levels following [ 3 H]-progesterone injections. The pineal also accumulated some 20α-metabolites. In uterus there was significant accumulation of progesterone only with oestrogen priming and little accumulation of 5α-DHP. These results and others suggest that the diverse effects of progesterone may result from progesterone per se , from metabolites or from combinations.

Blood samples were collected from eight mares for ten days before and two days following parturition. Progesterone, 5 alpha-dihydroprogesterone and hydroxy-5 alpha-pregnanones were extracted from serum, separated by Sephadex LH-20 column chromatography and quantitated by radioimmunoassay. Progesterone levels ranged from 8.5 to 4.1 ng/ml among mares with normal parturition and decreased to 1.2 ng/ml after parturition. One mare with a retained placenta maintained the prepartal levels of progesterone into the postpartum period. 5 alpha-dihydroprogesterone increased to a level of 133 ng/ml at 80 hrs before parturition and began decreasing and reached a level of 9.2 ng/ml in the postpartum period. Levels of hydroxy-5 alpha-pregnanones were variable with a high of 1690 ng/ml and declined to a postpartum level of 60 ng/ml. The timing of the decline varied among the mares. The possible source and significance of these progestins remain speculative.

We have examined the distribution of progesterone 5 alpha-reductase activity in minces of discrete brain areas from two strains of adult ovariectomized mice known to be differentially responsive to the behavioral effects of exogenously administered 5 alpha-pregnane-3,20-dione (5 alpha-DHP). Minces of freshly dissected midbrain tegmentum (TEG), hypothalamus-preoptic area (HPOA), hippocampus (HIP), and parietal cortex (CTX) were incubated with [4-14C]progesterone in a Krebs-Henseleit bicarbonate buffer solution containing 10 mM glucose for 2 h at 37 degrees C. The [14C]5 alpha-DHP produced was identified by comparison with known standards in paper and thin-layer chromatography systems, derivative formation, and recrystallization to a constant 14C/3H ratio. We found a 6-fold difference in the extent to which various brain areas formed 5 alpha-DHP. The amount of conversion was similar for both strains of mice. When expressed as picomoles of 5 alpha-DHP formed per 2 h per 20 mg of tissue, the following order of activity was observed: TEG greater than HPOA greater than HIP greater than CTX.

The specific binding of 5 alpha-dihydroprogesterone (5 alpha-DHP), progesterone and R5020 to anterior pituitary nuclear extracts was studied using ovariectomized rats treated with estradiol benzoate and progesterone. The binding equilibrium association constant for 5 alpha-dihydroprogesterone with different preparations of nuclear extract ranged from 4.0 +/- 0.54 microM-1 to 59 +/- 10 microM-1. The association constants for progesterone and R5020 were 0.39 +/- 0.81 nM-1 and 1.5 +/- 0.15 nM-1, respectively. The binding of 5 alpha-DHP was specific in that it could be competed only by R5020, progesterone and 5 alpha-DHP and not by other progesterone metabolites and other hormonal steroids tested. With [3H]-progesterone and [3H]R5020 as ligands the most efficient competitors also were R5020, progesterone and 5 alpha-DHP. Estrogen priming of ovariectomized rats consistently and significantly increased the number of binding sites for all three progestins and subsequent progesterone treatment enabled their detection at higher levels in the nuclei.

An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5 alpha-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5 alpha-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent Km(progesterone) = 88.2 +/- 8.2 nM; apparent Kia(NADPH) = 7.7 +/- 1.1 microM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinate which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent Kis = 7.8 +/- 1.0 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 9.85 +/- 2.1 microM and apparent Kii = 63.2 +/- 12.5 microM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.

Progesterone (P), deoxycorticosterone (DOC), estradiol-17 beta (E2) and cortisol (F) were determined simultaneously in the peripheral and the ovarian veins in 13 patients. Blood was collected either by direct sampling during laparotomy (12 patients) or by selective catheterization (1 patient). In all ovarian effluents P and E2 levels were significantly higher than in the peripheral vein. This was also true for DOC except in one ovarian effluent. The gradient was higher on the side of the corpus luteum-bearing ovary. P and E2 levels were correlated in ovarian as well as in peripheral veins. In ovarian effluents, DOC gradients were only significantly correlated with P levels (r = 0.63; P less than 0.01) suggesting a metabolic relationship between the two steroids.