Alterations of 20 alpha-hydroxysteroid dehydrogenase activity in cultured rat granulosa cells by follicle-stimulating hormone and testosterone

The effect of follicle-stimulating hormone (FSH) and testosterone (T) on rat granulosa cell progestin metabolism was investigated by incubation of the cells for 24 h with FSH and/or T and subsequent reincubation with an appropriate rabiolabeled steroid for 3 h. Exposure to varying concentrations of FSH (8-1000 ng/ml) and T (4-500 nM) decreased overall 4-[14C] progesterone utilization and accumulation of 20 alpha-reduced metabolites of progesterone in a dose-related manner. The accumulation of 5 alpha-reduced metabolites was not markedly changed by FSH and T treatments. Treatments with FSH and/or T decreased utilization of all progestins studied: progesterone by 30-50%, 20 alpha-hydroxy-4-pregnen-3-one by 23-31%, 3 alpha-hydroxy-5 alpha-pregnan-20-one by 41-64%, and 5 alpha-pregnane-3 alpha,20 alpha-diol by 26-34%. The greatest effects were observed following FSH + T treatments. Decreased utilization of substrates was associated with the decrease of 20 alpha-hydroxy-steroid dehydrogenase activity; the conversion of progesterone to 20 alpha-hydroxy-4-pregnen-3-one was decreased by 44-62%, the conversion of 20 alpha-hydroxy-4-pregnen-3-one to progesterone was decreased by 41-61%, the conversion of 3 alpha-hydroxy-5 alpha-pregnan-20-one to 5 alpha-pregnane-3 alpha,20 alpha-diol was decreased by 42-69%, and the conversion of 5 alpha-pregnane-3 alpha,20 alpha-diol to 3 alpha-hydroxy-5 alpha-pregnan-20-one was decreased by 53-60%. The incubation of granulosa cells with cyanoketone (10(-6)M), an inhibitor of delta 5,3 beta-hydroxysteroid dehydrogenase, virtually eliminated de novo progesterone production but did not alter the inhibitory effect of FSH and T on radiolabeled progesterone utilization and accumulation of 20 alpha-reduced metabolites, indicating that the observed effects are not influenced by endogenous production of progesterone. It was concluded from these studies that both FSH and testosterone inhibit the 20 alpha-hydroxysteroid dehydrogenase activity and consequently decrease progesterone catabolism by granulosa cells.     

Structural requirements for progesterone binding to the hepatic endoplasmic reticulum in the female rat

Microsomes isolated from the liver of the female rat specifically bind progesterone. The progesterone-microsomal complex shows highly specific characteristics. The binding is probably associated with the carbonyl groups at positions C-20 and C-3. Other steroids compete for microsomal binding sites less effectively. Competition for progesterone binding sites by other steroids in percentages: testosterone 33; testosterone propionate, 9; 17-methyltestosterone, 23.2; cortisol, 6.4; estradiol-17 beta, 1.8; 17 alpha-ethynyl estradiol, 4.7; mestranol, 1.0; norethynodrel, 4.5; ethisterone, 7.1; lynestrenol, 4.3; medroxyprogesterone, 23.3; medroxyprogesterone acetate, 15.2; 5 alpha-pregnane-3,20-dione, 47.6; 5 beta-pregnane-3,20-dione, 20.7; pregnenolone, 14.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8%; 20 beta-hydroxy-4-pregnen-3-one, 2.8; 3 beta-hydroxy-5 alpha-pregnan-20-one, 5.2; 4-pregnene-3 beta, 20 beta-diol, 2.1; 11 alpha-hydroxyprogesterone 21.0; 16 alpha-hydroxyprogesterone, 7.9; 17-hydroxyprogesterone, 26.7; 16 alpha, 17-epoxyprogesterone, 2.7; 16 alpha-methylprogesterone, 3.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8; promegestone, 27.0. 3 beta-Hydroxy-5 beta-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 5-pregnene-3 beta,20 beta-diol, 5-pregnene-3 beta, 20 alpha-diol; 5 alpha-pregnane-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol diacetate, 5 beta-pregnane-3 alpha, 20 beta-diol, 3 alpha, 17-dihydroxy-5 beta-pregnan-20-one, 17-hydroxypregnenolone, 6-methyl-17-hydroxypregnenolone, pregnenolone-16 alpha-carbonitrile, dihydrotestosterone and cholesterol show no competition at all. The varying degree of competition by different steroids is unrelated to their lipid solubility.     

Saturated metabolites of progesterone in human myometrium during pregnancy.

A method for the separation and quantitative determination of epimeric 3alpha/beta-hydroxy-5alpha/beta-pregnan-20-ones by gas liquid chromatography and electron capture detection is presented. Reliability of the method and applicability to biological material was tested. In one total human uterus and 20 samples of myometrium the concentration of epimeric pregnanol-ones was determined. 3beta-hydroxy-5alpha-pregnan-20-one and 3alpha-hydroxy-5alpha-pregnan-20-one were present in similar quantitative range as progesterone and 20alpha-hydroxy-4-pregnen-3-one. A correlation between progesterone and concentration of metabolites could not be established. 3beta-hydroxy-5beta-pregnan-20-one was not detected in the tissue.     

Quantitative changes in the metabolism of 20alpha-hydroxy-4-pregnen-3-one by rat hypothalamus and pituitary during proestrus.

The in vitro conversion of 20alpha-hydroxy-4-pregnen-3-one (20alpha-DHP) by medial basal hypothalamus and anterior pituitary was investigated throughout the day of proestrus in the 4-day cyclic rat. Reverse isotopic dilution analysis was utilized to quantitate the substrate remaining and three metabolic products: 20alpha-hydroxy-5alpha-pregnan-3-one, 5alpha-pregnane-3alpha,20alpha-diol and progesterone. Serum levels of 20alpha-DHP, progesterone, LH and FSH were measured by radioimmunoassay. Conversion of 20alpha-DHP to its 5alpha-reduced metabolites (20alpha-hydroxy-5alpha-pregnan-3-one and 5alpha-pregnane-3alpha,20alpha-diol) by the pituitary was constant throughout proestrus except for a significant decrease at 1600 h, near the end of the critical period. Although 5alpha-reduction of 20alpha-DHP by the hypothalamus fluctuated, it was relatively high at 1600 h and was lowest at 1400 h. Small amounts of progesterone (less than2%) were formed but there was not variation with time. The decrease in pituitary enzymic activity coincided with the time when serum levels of LH, FSH and progesterone were increasing but not with later times when the elevated serum levels were maintained. Thus, there may be endogenous regulation of 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase activity in rat pituitary and perhaps hypothalamus on the afternoon of proestrus. The regulation and subsequent effects of quantitative changes in 5alpha-reduction of 20alpha-DHP by pituitary and hypothalamus remain to be elucidated.     

Progesterone metabolism in human endometrial stromal and gland cells in culture.

Metabolism of progesterone by human endometrium has been described, but the rapidity and extent of progesterone metabolism is incompletely documented in cellular fractions of normal endometrium. Therefore, we evaluated progesterone metabolism in separated stromal and gland cells in culture obtained from normal human endometrium by thin-layer chromatography. We find that in both cell types, the most abundant metabolite is 3beta-hydroxy-5alpha-pregnan-20-one (70%), followed by 5alpha-pregnane-3,20-dione (15%), and 3alpha-hydroxy-5alpha-pregnan-20-one (10%). A small amount is metabolized to 5alpha-pregnane-3alpha/3beta,20alpha-diols and to 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one. The metabolism of progesterone in cultured endometrial cells occurs rapidly; 70% of progesterone is metabolised in 8 h, and 90% by 24 h. We conclude that when in vitro experiments are conducted utilizing progesterone treatment, the rapidity and the extent of the metabolism of this steroid should be taken into account.     

Secretion of pregnane compounds from polycystic ovaries of androgen-sterilized rats

Progesterone, 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH), 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-DHP), 20 alpha-hydroxy-5 alpha-pregnan-3-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol in ovarian venous plasma of androgen-sterilized rats treated with 25 IU of human chorionic gonadotropin (hCG) were assayed by gas chromatography. The compounds listed were essentially undetectable in polycystic ovaries of the androgen-sterilized rats. However, after injection of hCG, levels of these steroids were high. Levels of progesterone and 5 alpha-pregnane compounds reached a peak within 1 or 2 days after hCG treatment and then fell slowly. The level of 20 alpha-DHP reached a peak on day 4 after hCG treatment and remained high thereafter. Injection of 2 micrograms of luteinizing hormone (LH) before sample collection increased the secretion of progesterone at all times tested except when it was already at a peak. The secretion of 5 alpha-DHP and 3 alpha-OH was also increased by LH after hCG treatment, but the ability of the ovary to produce these steroids was not, suggesting that there was low 5 alpha-reductase activity in the cystic ovary before hCG treatment. The results suggest that ovulation and luteinization in cystic follicles may cause the low activities of 5 alpha-reductase and 20 alpha-hydroxysteroid dehydrogenase in polycystic ovaries of androgen-sterilized rats to increase.     

Progesterone metabolism in the ovaries and testes of the echinoid Lytechinus variegatus Lamarck (Echinodermata)

In the present study we examined the metabolic fate of progesterone (P4) in homogenate and tissue minces of the ovaries and testes of Lytechinus variegatus. P4 was metabolized primarily into 5alpha-reduced metabolites including, 5alpha-pregnane-3,20-dione (DHP), 3beta-hydroxy-5alpha-pregnan-20-one (3beta,20-one), 5alpha-pregnane-3beta,20alpha-diol (3beta,20alpha-diol), 5alpha-pregnane-3beta,20beta-diol (3beta,20beta-diol), and 5alpha-pregnane-3alpha,20alpha-diol (3alpha,20alpha-diol) by both the ovaries and testes. The capacity to metabolize P4 did not differ between the ovaries and testes. However, the relative quantity of Salpha-pregnane-3beta,20zeta-diol synthesized from ovary and testis tissue minces was about 3.3-fold higher than from homogenate preparations. Differences in the synthesis of 3beta,20-one and 3alpha,20alpha-diol in both ovary and testis minces were dependent on reproductive state. This study demonstrates the pathway of P4 conversion in the ovaries and testes of L. variegatus and indicates the rapid conversion of P4 into 5alpha-reduced metabolites in these tissues. Although P4 metabolism is not sex specific, sex-specific responses to P4 metabolites have been demonstrated previously. We hypothesize that the sex-specific responses of the ovaries and the testes to P4 may be associated with receptor-level regulatory processes.     

Stable isotope studies on steroid metabolism and kinetics: sulfates of 3 alpha-hydroxy-5 alpha-pregnane derivatives in human pregnancy

The metabolism and production rates of 3 alpha-hydroxy-5 alpha-pregnan-20-one sulfate and the 3-sulfate and 3,20-disulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol in pregnant women were studied. The steroid sulfates were labeled with deuterium in the 3 beta,11,11- or 3 beta,11,11,20 beta-positions and were injected intravenously. The deuterium content of steroids in the monosulfate and disulfate fraction of plasma collected at different times after the injection was determined by capillary column gas chromatography/mass spectrometry. The injected steroid sulfates underwent oxidoreduction at C-20 and 16 alpha-hydroxylation. In addition, the 3-sulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol became hydroxylated at C-21. The pregnanediol and pregnanetriol monosulfates were also converted to disulfates. No evidence was obtained for a metabolic sequence involving hydrolysis, oxidoreduction, and resulfation at the C-3 position. Production rates and rates of metabolic transformations were determined using different one- and two-pool models. The production rate of the pregnanolone/pregnanediol monosulfate couple was 0.08 to 0.5 mmol/24 h, the variability probably depending both on individual factors and stage of pregnancy. The half-life time for oxidation and reduction at C-20 was 0.1 to 0.4 hours, reduction being the faster process. The half-life time for the turnover of the steroid skeleton was 1.3 to 3.3 hours. The injected steroid monosulfates were 16 alpha-hydroxylated at a rate of 1 to 8 mumol/24 h. A significant fraction of these 16 alpha-hydroxylated steroid sulfates, 0.5 to 25 mumol/24 h, was formed from other, probably unconjugated, precursors. The 16 alpha-hydroxylated steroid monosulfates underwent rapid oxidoreduction at C-20. The 3-sulfate of 5 alpha-pregnane-3 alpha,20 alpha-diol was hydroxylated at C-21. The production rate of 5 alpha-pregnane-3 alpha,20 alpha,21-triol 3-sulfate was 8 to 36 mumol/24 h in four women and 180 mumol/24 h in one woman, and this steroid was not formed from other precursors to a significant extent. 5 alpha-Pregnane-3 alpha,20 alpha-diol disulfate was a metabolic end product accounting for a major part of the elimination of the steroids injected. Its half-life time was 1.4 to 2.8 hours. The results show that the formation of sulfated steroids with a 3 alpha-hydroxy-5 alpha configuration may account for 50% of the metabolism of progesterone in late pregnancy.     

Metabolism of progesterone by preimplantation mouse blastocysts in culture

This study examined the question whether or not preimplantation mouse blastocysts can metabolize progesterone (P). When young (Day 4) and implanting (Day 5) blastocysts were cultured in supplemented Eagle's minimum essential medium containing 0.4 microM [3H]P, metabolism of P and formation of metabolites were noticed at 10 h of culture. The metabolites accumulated in medium as the culture continued to 118 h. Three of the four metabolite fractions were identified, by crystallization to constant sp. act., to be 5 alpha-pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one (or allopregnanolone), accounting for 22 and 57% of radioactivity, respectively, and a small amount (1-10%) of 3 alpha-hydroxy-5 alpha-pregnan-20-one. This suggests that both delta 4-5 alpha-reductase and 3 alpha- and 3 beta-hydroxysteroid dehydrogenase are active. Day 5 blastocysts were much more active than Day 4 blastocysts in P metabolism. It is suggested that the ability of blastocysts to metabolize P could produce the following effects in the adjacent endometrium: a lessening of P effects; and consequently a change in P-estrogen interaction; and possible effects from the metabolites. These local effects of embryos on the endometrium may be important for embryonic development and implantation.     

Androgenic modulation of progesterone metabolism by rat granulosa cells in culture

Effects of androgens on progesterone accumulation, utilization of exogenous progesterone and accumulation of [4-14C]progesterone metabolites by rat granulosa cells in culture were studied. Androgen increased progesterone accumulation in cultures without exogenous progesterone and slowed the overall decline of progesterone concentration in cultures supplemented with exogenous progesterone. Both aromatizable testosterone and nonaromatizable 5 alpha-dihydrotestosterone decreased [4-14C]progesterone utilization by granulosa cells by 12 to 30%. This effect was observed irrespective of whether the cells were continuously exposed to androgens or only pre-exposed. In he same experiments, androgens decreased conversion of radiolabeled progesterone to 20 alpha-hydroxy-4-pregnen-3-one by 11 to 50% and to 5 alpha-pregnane-3 alpha, 20 alpha-diol by 26 to 49%. Accumulation of 3 alpha-hydroxy-5 alpha-pregnan-20-one was not altered in 3 h incubations and was increased by up to 43% in 24 h incubations by androgen treatment. It is suggested that androgens alter progesterone catabolism by granulosa cells by decreasing 20 alpha-hydroxysteroid dehydrogenase activity and that this effect may contribute to overall stimulatory action of androgens on progesterone accumulation.     

Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems.

Progesterone and several progesterone metabolites are capable of inhibiting uterine contractility. Some progesterone metabolites have shown little or no affinity for the progesterone receptor but have been found to be potent modulators of the GABAA receptor system. This study examined whether the inhibition of uterine contraction by progesterone and its metabolites was progesterone receptor-mediated or gamma amino butyric acidA (GABAA) receptor-mediated. Uterine contractions were measured in annular rings of uterine tissue, 5 mm in length, from diestrous II rats, under a fixed tension of 1 gram. The steroids tested were 3 beta-hydroxy-5 beta-pregnan-20-one (6 micrograms/ml), 5 beta-pregnane-3,20-dione (10 micrograms/ml), 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha,5 alpha-THP, 27.5 micrograms/ml), and progesterone (40 micrograms/ml). All compounds significantly inhibited spontaneous uterine contractions when compared to controls. No effect was seen by either 16 micrograms/ml of the progesterone antagonist, RU486, or 32 micrograms/ml of the GABAA antagonist, pictrotoxin, when administered alone. However, when uterine tissues were exposed to a combination of the steroid and the antagonist, the effect of 3 beta-hydroxy-5 beta-pregnan-20-one and 3 alpha,5 alpha-THP was blocked by picrotoxin but not by RU486, indicating that the action of these steroids was mediated through the GABAA system. The effect of 5 beta-pregnane-3,20-dione and progesterone was effectively blocked by RU486 but not by picrotoxin, suggesting that their actions were mediated through the progesterone receptor system. These results indicate that multiple mechanisms exist in the uterus for inhibiting uterine contractility by progesterone and its metabolites.     

Steroid metabolism by rat nasal mucosa: studies on progesterone and testosterone

The metabolism of [4-14C]progesterone and [4-14C]testosterone by slices of the nasal mucosa from rats was studied. As shown by gas chromatography-mass spectrometry there was a preferential formation of reduced progesterone-metabolites (5 alpha-pregnane-3,20-dione, 3 alpha- and 3 beta-hydroxy-5 alpha-pregnane-20-one, 20 alpha- and 20 beta-hydroxypregn-4-en-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha-pregnane-20-one, 3 alpha,16 alpha-dihydroxy-5 alpha-pregnane-20-one) and reduced testosterone-metabolites (4-androstene-3,17-dione, 5 alpha-dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstane-17-one, and 5 alpha-androstane-3 alpha, 17 beta-diol, 2 alpha-hydroxy-5 alpha-dihydrotestosterone, 5 alpha-androstane-2 alpha,3 alpha, 17 beta-triol) indicating the presence of 5 alpha-reductase, 3 alpha-, 3 beta-, 17 beta-, 20 alpha- and 20 beta-hydroxysteroid oxidoreductase activities in this tissue. Progesterone-metabolites hydroxylated at positions 2 alpha, 6 alpha, 6 beta, 15 alpha and 16 alpha and testosterone-metabolites hydroxylated at positions 1 beta, 2 alpha, 6 beta, 15 beta and 16 alpha were also identified, indicating the presence of several steroid hydroxylases in the nasal mucosa. Autoradiography of the nasal region of rats injected with [4-14C]progesterone or [4-14C]testosterone showed a selective localization of radioactivity in the mucosa covering the olfactory region of the nasal cavity.     

Metabolism of progesterone by avian granulosa cells in culture

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

Ovarian secretion of pregnane compounds at first ovulation in rats treated with pregnant mare's serum gonadotropin

Progesterone, 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH), 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-DHP), 20 alpha-hydroxy-5 alpha-pregnan-3-one (20 alpha, 5 alpha), and 5 alpha-pregnane-3 alpha, 20 alpha-diol (DIOL) in ovarian venous plasma at first ovulation in female rats treated on day 30 with 10 IU of pregnant mare's serum gonadotropin (PMSG) were assayed using gas chromatography. Progesterone peaked at late proestrus before ovulation (day 32) and at early diestrus after ovulation (day 34). With the first peak, 5 alpha-DHP and 3 alpha-OH increased. The 20 alpha-DHP level peaked at early diestrus after ovulation (day 34) and remained high thereafter. The 20 alpha, 5 alpha and DIOL peaked at estrus after ovulation (day 33) and then fell slowly. Injection of 2 micrograms of luteinizing hormone (LH) before sample collection increased secretion of 20-keto-pregnane compounds, except when they were at peak levels. The secretion of 20 alpha-hydroxy-pregnane compounds was unaffected by LH at all times tested. These results suggest that rat ovaries without corpora lutea secrete 20-keto-pregnane compounds in response to LH, but that 20 alpha-hydroxy-pregnane compounds are secreted only from ovaries with corpora lutea.     

Uterine progesterone metabolism during early pseudopregnancy in the rat

We measured uptake and metabolism of progesterone (P4) during the estrous cycle and Days 2-6 of pseudopregnancy (PSP) to determine uterine P4 dynamics during preimplantation. Rats were infused with [3H]P4 for 60 min, blood was obtained, the uterus was removed, and endometrium and myometrium were isolated. Tissue radiolabeled P4 and P4 metabolites (5 alpha-pregnane-3,20-dione, DHP; 20 alpha-hydroxy P4; 17 alpha-hydroxy P4, and hydroxylated DHP derivatives) were extracted and separated by thin-layer chromatography (TLC). Serum P4 was measured by radioimmunoassay (RIA) in another group of rats. Endometrial and myometrial concentrations of [3H]P4 were greater (p less than 0.05) than plasma values. In contrast, [3H]DHP levels in the endometrium were higher (p less than 0.01) than values in myometrium or plasma. Compared to values in the estrous cycle, endometrial ratios of [3H]DHP/[3H]P4 and [3H]metabolites/[3H]P4 decreased (p less than 0.02) on Days 3-5 of PSP. Serum P4 levels during the estrous cycle (13-25 ng/ml) increased (p less than 0.01) to 120 ng/ml on Days 3-5 of PSP. Estimated concentration of P4 in the endometrium during the estrous cycle (90 ng/g) increased (p less than 0.05) to 580 ng/g by Day 5 of PSP. Similar observations were noted for the estimated endometrial concentrations of DHP and all P4 metabolites. We suggest that both endometrium and myometrium take up and metabolize P4 during the estrous cycle and early PSP. However, endometrial P4 metabolism during PSP is greater than during the estrous cycle, in part because of increased ovarian secretion and endometrial concentration of P4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurosteroids: a new function in the brain

"Neurosteroids" accumulate in the central nervous system independently of supply by peripheral endocrine glands. Dehydroepiandrosterone (DHA) and pregnenolone (delta 5P) were first found in the rat brain. Then, a steroid biosynthetic pathway was demonstrated in oligodendrocytes, mostly by enzyme immunocytochemistry and biochemical studies in primary cultures of glial cells, where the formation, from appropriate radioactive precursors, of delta 5P, delta 5-pregn-3 beta, 20 alpha-diol (20 alpha-DH delta 5P), progesterone (P), 5 alpha-pregnane-3,20-dione (5 alpha-DHP) and 3 alpha-hydroxy-5 alpha-pregnane-20-one (3 alpha, 5 alpha-THP), as well as estrogen-induced nuclear progesterone receptor (PR) was observed. Several biological effects of neurosteroids have been observed, such as electrical stimulation of neurones, involvement in behaviorial activities, modulation of GABAA-receptor (GABAA-R) function (potentiated by 3 alpha, 5 alpha-THP and its 21-hydroxyderivative, antagonized by delta 5P- and DHA-sulfates) and growth/differentiation of glial cells in vitro. Preliminary findings suggest that the neurosteroid concept applies to all mammalian species, including man. Further investigations should assess the pathophysiological significance of the synthesis of neurosteroids and decipher their mechanisms of action via nuclear and membrane receptors.     

Steroidogenic correlates of pregnancy in the rock hyrax (Procavia capensis)

In pregnant rock hyraxes isolated leucocytes metabolise both [3H]pregnenolone and [3H]progesterone while whole blood, erythrocytes and an erythrocyte/leucocyte mixture only metabolised [3H]progesterone. Plasma displayed no tendency to metabolically convert any one of these two steroids. In whole blood [3H]progesterone appears to be converted to 5alpha-pregnane-3,20-dione and a compound with chromatographic properties similar to that of 5alpha-pregnan-3alpha-ol-20-one. 5Alpha-pregnane-3,20-dione exhibited a high relative binding affinity for the uterine progesterone eceptor (94%), but 5alpha-pregnan-3alpha-ol-20-one displayed very little affinity for the same receptor (0.4%). 5Alpha-pregnane-3,20-dione may therefore aid in the maintenance of pregnancy. Corpora lutea metabolised progesterone to 17alpha-hydroxyprogesterone, a compound exhibiting no progestational function because of its low relative binding affinity for the uterine progesterone receptor (2%). Progesterone appears to be the main product of the corpus luteum. However, 5alpha-pregnane-3,20-dione circulated at concentrations approximately 8.5 times higher than progesterone, probably due to the metabolic conversion of progesterone to 5alpha-pregnane-3,20-dione by the blood. We conclude that in the hyrax progesterone, produced by the corpora lutea, enters the circulation, where it is reduced to 5alpha-pregnanes. 5Alpha-pregane-3,20-dione may then be transported to the uterus where it binds to the progesterone receptor to assist in the maintenance of pregnancy. This mechanism appears to be analogous to that of the African elephant which is phylogenetically related to the hyrax, except that in the elephant the 5alpha-reduced metabolites are produced by luteal tissue and not the blood.     

Synthesis and GABA(A) receptor activity of 6-oxa-analogs of neurosteroids

The 6-oxasteroids 3alpha-hydroxy-6-oxa-5alpha-pregnan-20-one (3) and 3alpha-hydroxy-6-oxa-5beta-pregnan-20-one (4) were obtained from pregnenolone acetate via the corresponding (5alpha or 5beta) 3beta, 20beta-diacetoxy-6-oxa-pregnane. Both steroids showed ca. 100-fold reduced potency for modulating [(3)H]flunitrazepam, [(3)H]muscimol or [(35)S]TBPS binding to the GABA(A) receptor when compared to their natural carbon analogs 3alpha-hydroxy-5alpha-pregnan-20-one (1) and 3alpha-hydroxy-5beta-pregnan-20-one (2).     

Conversion of 17 alpha-hydroxyprogesterone to 5 alpha, 3 alpha, and 20 alpha-reduced metabolites by female rat anterior pituitary and hypothalamus

The metabolism of 17 alpha-[3H]hydroxyprogesterone was examined in female rat anterior pituitary and hypothalamic tissues. After reverse isotopic dilution analysis and purification to constant specific activity, the following 5 alpha-, 3 alpha- and 20 alpha-reduced products were detected in both tissues: 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione; 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one and 5 alpha-pregnane-3 alpha,17 alpha,20 alpha-triol. While the metabolites formed were qualitatively the same, there were quantitative differences between the two tissues. The 3 alpha,5 alpha-reduced metabolite, 3 alpha,17 alpha-dihydroxy-5 alpha-pregnan-20-one, was the principal product in the anterior pituitary while the 5 alpha-reduced metabolite, 17 alpha-hydroxy-5 alpha-pregnane-3,20-dione, was produced in largest amount by the hypothalamus. With both tissues, the aforementioned four products plus starting substrate accounted for nearly all of the starting radioactivity. There was no evidence for the formation of C19 steroids (androgens) despite the presence of the 17 alpha-hydroxy group.     

Steroid profiles of brown adipose tissue

In brown adipose tissue of alp-marmot (Marmota marmota), badger (Meles meles) and Wistar rats steroids of C21- and C19-type are identified and quantified. The detection of 3 alpha-hydroxy-5 alpha-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one, 3 alpha,21-dihydroxy-5 beta-pregnan-20-one and 3 beta,21-dihydroxy-5 alpha-pregnan-20-one is of special interest since sleep-inducing properties have been described with these steroids.