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.     

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.     

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.     

Secretion of pregnane compounds from follicular and luteal ovaries in rats

Differences during the follicular and luteal phases in the secretion of pregnane compounds by rat ovaries were studied. Daily s.c. injections of 2 mg of progesterone that began at early diestrus prolonged the diestrus stage for the duration of the treatment. In the follicular phase, normal proestrus and proestrus delayed by progesterone treatment were examined. In the luteal phase, day 6 of pseudopregnancy was examined. 20 alpha-hydroxy-4-pregnen-3-one accounted for most of total amount of pregnane compounds secreted from ovaries in the follicular phase, and progesterone and 20 alpha-hydroxy-4-pregnen-3-one for most at the luteal phase. The levels of 5 alpha-pregnane compounds were low in both phases. Injection of 2 micrograms of luteinizing hormone (LH) before sample collection increased the secretion of progesterone in the follicular phase, but not in the luteal phase. The secretion of 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one was increased by the injection of LH in either phase, but the ability of the ovaries to produce these steroids was low, suggesting that there was low 5 alpha-reductase activity in the ovaries of rats with delayed proestrus and pseudopregnant rats.     

Synthesis of the allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one and 3 alpha-hydroxy-4-androsten-17-one, and of 3 alpha-hydroxy-5 alpha-pregnan-20-one

A method for the convenient synthesis of the recently isolated allylic gonadal steroids, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha-dihydroprogesterone; 3 alpha-DHP) and 3 alpha-hydroxy-4-androsten-17-one (3 alpha-HA), was developed using 4-pregnene-3,20-dione (progesterone) and 4-androstene-3,17-dione as substrates and potassium trisiamylborohydride (KS-Selectride) as reducing agent. Similar reactions were also used for the reduction of 5 alpha-pregnane-3,20-dione to 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-HP). The yields were about 15%, 50%, and greater than 90% for 3 alpha-DHP, 3 alpha-HA and 3 alpha-HP, respectively. Structures of the products, including the 3 beta-isomers and the 17 alpha-epimer, formed in these reactions were determined by NMR and mass spectroscopic methods.     

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.     

Selective release of follicle-stimulating hormone by 5 alpha-dihydroprogesterone in immature ovariectomized estrogen-primed rats

The effect of 5 alpha-dihydroprogesterone (5 alpha-DHP) on gonadotropin release was examined in the immature acutely ovariectomized (OVX) rat primed with a low dose of estradiol (E2). Treatment with various doses of 5 alpha-DHP given in combination with E2 increased levels of follicle-stimulating hormone (FSH) but had no effect on serum luteinizing hormone (LH). A single injection of a maximally stimulating dose of 5 alpha-DHP (0.4 mg/kg) stimulated increases in serum FSH at 1200 h and, 6 h later, at 1800 h. Pituitary LH and FSH content was dramatically enhanced by 1600 h and levels remained elevated at 1800 h. The administration of pentobarbital at 1200 h, versus 1400 h or 1600 h, prevented the increase in basal serum FSH levels at 1800 h, implying that the release of hypothalamic LH releasing hormone (LHRH) is modulated by 5 alpha-DHP. In addition, changes in pituitary sensitivity to LHRH as a result of 5 alpha-DHP were measured and a significant increase in the magnitude of FSH release was observed at 1200 h and 1800 h. Although the LH response to LHRH in 5 alpha-DHP-treated rats was not different from controls, the duration of LH release was lengthened. These results suggest that 5 alpha-DHP may stimulate FSH release by a direct action at the pituitary level. Together, these observations support the theory that 5 alpha-DHP mediates the facilitative effect of progesterone on FSH secretion and further suggests an action of 5 alpha-DHP in this phenomenon at both pituitary and hypothalamic sites.     

The effects of neurotransmitters and other cellular modulators and factors on hypothalamic and anterior pituitary delta 4-steroid (progesterone) 5 alpha-reductase activity

A number of diverse biological compounds involved in the regulation of the hypothalamo-hypophyseal-ovarian axis have been examined for effects on the conversion of 3H-progesterone to 3H-5 alpha-dihydro-progesterone and 3H-3 alpha-hydroxy-5 alpha-pregnan-20-one by female rat hypothalamus and/or anterior pituitary. Broken cell preparations were incubated with 3H-progesterone and NADPH, and product 5 alpha-reduced progestins were quantitated by reverse isotopic dilution analysis. Progesterone 5 alpha-reductase activity was reduced up to 50% in the presence of 10(-2) to 10(-3) M serotonin in both preparations. At 10(-3) M, various indoles including n-acetylserotonin, melatonin, 5-methoxytryptamine, 5-methoxytryptophol, and 5-hydroxyindole acetic acid decreased by 10 to 30% 5 alpha-reduced product formation. At 10(-2) M, carbamylcholine and norepinephrine were without effect, while 10(-2) M dopamine reduced by 20% the 5 alpha-reduction of progesterone only in pituitary homogenates. The LHRH protease inhibitor bacitracin (2 X 10(-3) M) decreased by 10 to 40% progesterone 5 alpha-reductase activity in both tissues. By itself, LHRH did not affect the 5 alpha-reduction of progesterone nor did it potentiate the bacitracin effect. In the presence of 1 mM ATP, 100 micronM cAMP and 100 micronM cGMP increased 5 alpha-reduced product formation in the hypothalamus by 19 and 14%. The gonadotropins LH and FSH and the prostaglandins E1, E2, F1 alpha, and F2 alpha were without effect. Thus, these results and others indicate that a number of cellular components and other factors can affect the in vitro 5 alpha-reduction of progesterone in broken cell preparations.     

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.     

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.     

Role of 5 alpha-reduction in progesterone's ability to release FSH in estrogen-primed ovariectomized rats.

In ovariectomized estrogen-primed rats, progesterone as well as 5 alpha-dihydroprogesterone (5 alpha-DHP) are capable of inducing the release of gonadotropins. This study examined the need of 5 alpha-reduction as a prerequisite for the action of progesterone. The 5 alpha-reductase inhibitor, N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide was injected at a 1 or 2 mg dose/rat 2 h prior to an injection of 0.4 or 0.8 mg progesterone/kg body weight at 0900 h to immature ovariectomized, estrogen-primed rats and serum was analyzed for LH and FSH at 1500 h. Pituitary and hypothalamic 5 alpha-reductase activity was measured at the time of progesterone administration and at the time of the surge by incubating tissue homogenates with [3H]progesterone. Substrate, ([3H]progesterone) and product ([3H]5 alpha-DHP), were separated by reverse phase HPLC. The pituitary 5 alpha-reductase activity was not blocked at 1500 h. However, both pituitary and hypothalamic 5 alpha-reductase was blocked at the time of progesterone administration. No effect was seen by acute administration of the 5 alpha-reductase inhibitor upon either the 0.4 or 0.8 mg progesterone/kg-induced release of LH and FSH. There was, however, a specific, significant inhibition of progesterone-induced FSH but not LH release when the 5 alpha-reductase inhibition was sustained throughout the afternoon of the gonadotropin surge. These results indicate a biologically significant role for the irreversible 5 alpha-reduction of progesterone in the modulation of the release of FSH.     

Hypothalamic 5 alpha-reductase and 3 alpha-oxidoreductase activity in the male rat

The aim of the present study was to assess the progesterone (Pr) transforming 5 alpha-reductase (5 alpha-R) and 3 alpha-oxidoreductase (3 alpha-OR) activities in the hypothalamus of the male rat as a function of age and following castration and/or adrenalectomy performed at the sixth day of life. The hypothalamic activity of these enzymes was estimated from the sum of the 5 alpha- or 3 alpha-reduced metabolites produced from 14C-labeled Pr incubated "in vitro" with hypothalamic tissue. Plasma levels of testosterone (T), progesterone (Pr), estrone (E1), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured simultaneously. Special attention was paid to the GC/MS analysis of the endogenous content of the hypothalamic Pr-metabolites 3 alpha-hydroxy-pregn-4-en-20-one (3 alpha-Pr), 5 alpha-pregnane-3,20-dione (5 alpha-Pr) and 3 alpha-hydroxy-5 alpha-pregnan-20-one (5 alpha,3 alpha-Pr).The high 5 alpha-R and 3 alpha-OR activities estimated in the hypothalamus of prepubertal rats are not related to the action of gonadal or adrenal steroids. Substantial and comparable endogenous 3 alpha- and/or 5 alpha-Pr-metabolites were found in hypothalami from both prepubertal and mature rats. The results of the present study do not provide evidence for a contributory role of the 3 alpha-hydroxylated Pr derivative to the regulation of gonadotropin secretion in the male rat.     

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.     

Substrate-binding ability of Escherichia coli ribonucleic acid polymerase in relation to its protein composition.

The metabolism of [3H]progesterone in the rabbit endometrium and myometrium was studied in vitro. The major metabolities identified were 5alpha-pregnane-3,20-dione, 20alpha-hydroxypregn-4-en-3-one, 3beta-hydroxy-5alpha-preganan-20-one and 5alpha-pregnane-3beta,20alpha-diol. Other minor metabolites tentatively identified were 3alpha-hydroxy-5beta-pregnan-20-one,20alpha-hydroxy-5beta-pregnan-3-one and 5beta-pregnane-3alpha,20alpha-diol. The ability of the endometrium to metabolize progesterone on a unit weight bais was about 2.7 times that of the myometrium. The metabolism of [3H]progesterone in the rabbit uterus under the influnce of oestradiol-17beta and progesterone was studied. The ability of the oestradiol-treated rabbit uterus to metabolize progesterone was increased to 3.47 times that of the overiectomized control uterus, whereas the oestradiol-progesterone-treated rabbit uterus metabolized only 1.86 times that of the control. Study of the metabolism of progesterone with uterine subcellular preparations revealed that the 5alpha-reductase enzyme was present mainly in the nuclear fraction; 20alpha-hydroxysteroid dehydrogenase was found in the cytosol fraction and 3beta-hydroxysteroid dehydrogenase in the particulate fraction of the uterus. The metabolic pathways of progesterone in the rabbit uterine tissue are discussed.     

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.     

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.     

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.     

Selective release of luteinizing hormone by 3 alpha-hydroxy-5 alpha-pregnan-20-one in immature ovariectomized estrogen-primed rats

This study investigated the role of 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-THP) in the modulation of gonadotropin secretion using the immature ovariectomized (OVX) rat primed with a low dose of estradiol. A treatment regimen of either 0.2 or 0.4 mg/kg of 3 alpha,5 alpha-THP given in conjunction with estradiol for 4 days significantly increased levels of serum luteinizing hormone (LH) but had no effect on serum follicle-stimulating hormone (FSH). Estrogen-primed rats receiving a single injection of 3 alpha,5 alpha-THP at 0930 h showed an increase in serum and pituitary LH levels at 1200 h and 1500 h. At 1800 h, only pituitary levels of LH remained significantly higher than controls. An injection of 3 alpha,5 alpha-THP at 1230 h in estrogen-primed rats resulted in enhanced levels of pituitary LH at 1500 h and elevated levels of both serum and pituitary LH at 1800 h. When 3 alpha,5 alpha-THP was given at 0930 h and 1230 h, elevated serum levels of LH were maintained for over 6 h. The administration of pentobarbital (Pb) 30 min after an injection of 3 alpha,5 alpha-THP at 0930 h or 1230 h prevented the increases in serum LH at 1200 h, 1500 h or 1800 h. This suggests that LH-releasing hormone (LHRH) is involved in mediating the LH response by 3 alpha,5 alpha-THP. There was no change in the sensitivity of the pituitary to LHRH following 3 alpha,5 alpha-THP treatment, indicating the absence of a pituitary effect of this steroid.     

Purification of the NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase from female rat pituitary cytosol

The NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) [EC 1.1.1.50] which catalyzes the reversible conversion of 5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone; 5 alpha-DHP) to 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha-,5 alpha-tetrahydroprogesterone; 3 alpha,5 alpha-THP) was purified to apparent homogeneity from female rat anterior pituitary cytosol by a three step micro-purification procedure. Specific activity of purified 3 alpha-HSOR was enriched 438-fold from that in pituitary cytosol using successive ion exchange, chromatofocusing and affinity column chromatography purification steps. 3 alpha-HSOR appears to be a monomer with an approximate molecular weight of 36 kDa and an isoelectric point of about 5.75. The purified enzyme appears as a single protein staining band (36 kDa) when examined by polyacrylamide gel electrophoresis and with both silver or Coomassie blue staining. Under non-dissociating electrophoretic conditions, all of the 3 alpha-HSOR activity co-migrated with the 36 kDa protein staining band. The purified enzyme in the presence of the preferred cofactor, NADPH, has an apparent Km for 5 alpha-DHP of 82 nM and a Vmax of 1.2 mumol of 3 alpha,5 alpha-THP formed per mg protein/30 min. The Km for NADPH was 0.71 microM. In the oxidative direction, the enzyme in the presence of NADP+ has a Km for 3 alpha,5 alpha-THP of 1.4 microM and a Vmax of 9.7 mumol of 5 alpha-DHP formed per mg protein/30 min. The Km for NADP+ was 1.6 microM.     

Radioimmunoassay of 3 alpha-hydroxy-5 alpha-pregnan-20-one in rat and human plasma

A radioimmunoassay for measuring 3 alpha-hydroxy-5 alpha-pregnan-20-one in plasma has been developed. Polyclonal antibodies were raised in rabbits against 3 alpha-hydroxy-20-oxo-5 alpha-pregnan-11 alpha-yl carboxymethyl ether coupled to bovine serum albumin. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was purified from either extracts of plasma by high-performance liquid chromatography. These antibodies were then used for the radioimmunoassay of this centrally active progesterone metabolite in rat and human plasma. 3 alpha-Hydroxy-5 alpha-pregnan-20-one was detected in plasma from female rats on the day of estrus (2.0 to 9.3 ng/ml) and in the plasma of women during the luteal phase of the menstrual cycle at levels ranging from 0.25 to 2.5 ng/ml. The latter was highly correlated with plasma progesterone levels.