Circulating neuroactive C21- and C19-steroids in young men before and after ejaculation

Twelve neuroactive and neuroprotective steroids, androgens and androgen precursors i.e. 3alpha,17beta-dihydroxy-5alpha-androstane, 3alpha-hydroxy-5alpha-androstan-17-one, 3alpha-hydroxy-5beta-androstan-17-one, androst-5-ene-3beta,17beta-diol, 3beta,17alpha-dihydroxy-pregn-5-en-20-one (17alpha-hydroxy-pregnenolone), 3beta-hydroxy-androst-5-en-17-one (dehydroepiandrosterone, DHEA), testosterone, androst-4-ene-3,17-dione (androstenedione), 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone), 3beta-hydroxy-pregn-5-en-20-one (pregnenolone), 7alpha-hydroxy-DHEA, and 7beta-hydroxy-DHEA were measured using the GC-MS system in young men before and after ejaculation provoked by masturbation. The circulating level of 17alpha-hydroxypregnenolone increased significantly, whereas the other circulating steroids were not changed at all. This fact speaks against the hypothesis that a drop in the level of neuroactive steroids, e.g. allopregnanolone may trigger the orgasm-related increase of oxytocin, reported by other authors.     

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.     

Neuroactive steroids, their precursors and polar conjugates during parturition and postpartum in maternal blood: 2. Time profiles of pregnanolone isomers

Time profiles of the pregnanolone isomers epipregnanolone (3 beta-hydroxy-5 beta-pregnan-20-one), allopregnanolone (3 alpha-hydroxy-5 alpha-pregnan-20-one), pregnanolone (3 alpha-hydroxy-5 beta-pregnan-20-one), and isopregnanolone (3 beta-hydroxy-5 alpha-pregnan-20-one) were measured around parturition and in the postpartum period in the serum of 13 and three women with subarachnoidal and epidural analgesia, respectively. In addition, the levels of polar conjugates of all pregnanolone isomers were followed during parturition. GC/MS analysis was used for the measurement of steroid levels. Changes in concentrations of free steroids exhibited a similar pattern, with a fall primarily within the first hour after delivery. The decrease in conjugated steroids was shifted to the interval within the first hour and first day after delivery, and the changes were more pronounced. The time profile of the conjugated/free steroid ratio exhibited a significant decrease within the first hour and the first day after delivery in all of the isomers investigated. A decrease was also observed in the ratio of 3 alpha/3 beta-isomers and 5 alpha/5 beta-isomers around parturition. The possible physiological consequences of the findings are indicated.     

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).     

Neuropsychopharmacological properties of neuroactive steroids.

In addition to the well-known genomic effects of steroid molecules via intracellular steroid receptors, certain steroids rapidly alter neuronal excitability through interaction with neurotransmitter-gated ion channels. Several of these steroids accumulate in the brain after local synthesis or after metabolism of adrenal steroids. The 3alpha-hydroxy ring A-reduced pregnane steroids allopregnanolone and tetrahydrodeoxycorticosterone have been thought not to interact with intracellular receptors, but enhance gamma-aminobutyric acid (GABA)-mediated chloride currents, whereas pregnenolone sulfate and dehydroepiandrosterone (DHEA) sulfate display functional antagonistic properties at GABA(A) receptors. We demonstrated that these neuroactive steroids can regulate also gene expression via the progesterone receptor after intracellular oxidation. Thus, in physiological concentrations these neuroactive steroids regulate neuronal function through their concurrent influence on transmitter-gated ion channels and gene expression. When administered in animal studies, memory-enhancing effects have been shown for pregnenolone sulfate and DHEA. The 3alpha-hydroxy ring A-reduced neuroactive steroids predominantly display anxiolytic, anticonvulsant, and hypnotic activities. Sleep studies evaluating the effects of progesterone as a precursor molecule for these neuroactive steroids revealed a sleep electroencephalogram pattern similar to that obtained by the administration of benzodiazepines. These findings extend the concept of a "cross-talk" between membrane and nuclear hormone effects and provide a new role for the therapeutic application of these steroids in neurology and psychiatry.     

Conversion of 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one to delta 16-C19 steroids by the reconstituted delta 16-C19-steroid synthetase system

The substrate specificity of the reconstituted delta 16-C19-steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3 beta-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and NADH-cytochrome b5 reductase all from pig testicular microsomes. It was found that 5 alpha-reduced C21 steroids such as 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 alpha-ol and 5 alpha-androst-16-en-3 beta-ol, respectively. The results suggest that 5 alpha-reduced delta 16-C19 steroids might be synthesized from pregnenolone and progesterone via 5 alpha-reduced C21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5 alpha-reduced delta 16-C19 steroids from pregnenolone and progesterone.     

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 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.     

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.     

The neurosteroid allopregnanolone modulates oxytocin expression in the hypothalamic paraventricular nucleus

Virgin, ovariectomized rats exposed to 2 wk of sequential estradiol (E(2)) and progesterone (P) followed by P withdrawal have increased hypothalamic oxytocin (OT) mRNA and peptide levels relative to sham-treated animals. This increase is prevented if P is sustained. In the central nervous system, P is metabolized to the neurosteroid allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), which exerts effects by acting as a positive allosteric modulator of GABA(A) receptor/Cl(-)-channel complexes. In the present study, ovariectomized rats that received sequential E(2) and P for 2 wk followed by P withdrawal were administered allopregnanolone at the time of P withdrawal. Hypothalamic and plasma allopregnanolone concentrations, serum E(2) and P concentrations, and hypothalamic OT mRNA levels were measured at death. Steroid-induced increases in OT mRNA were attenuated in animals treated with allopregnanolone at the time of P withdrawal. The results suggest that allopregnanolone plays an important modulatory role in steroid-mediated increases in hypothalamic OT.     

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.     

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.     

Neurosteroids act on recombinant human GABAA receptors

The endogenous steroid metabolites 3 alpha,21dihydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 alpha-pregnan-20-one potentiate GABA-activated Cl- currents recorded from a human cell line transfected with the beta 1, alpha 1 beta 1, and alpha 1 beta 1 gamma 2 combinations of human GABAA receptor subunits. These steroids are active at nanomolar concentrations in potentiating GABA-activated Cl- currents and directly elicit bicuculline-sensitive Cl- currents when applied at micromolar concentrations. The potentiating and direct actions of both steroids were expressed with every combination of subunits tested. However, an examination of single-channel currents recorded from outside-out patches excised from these transfected cells suggests that despite the common minimal structural requirements for expressing steroid and barbiturate actions, the mechanism of GABAA receptor modulation by these pregnane steroids may differ from that of barbiturates.     

5alpha-reduced C21 steroids are substrates for human cytochrome P450c17

The 5alpha-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5alpha-reduced C(19) steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate. We studied the metabolism of 5alpha-reduced C(21) steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C(21) steroids to C(19) steroids via its 17alpha-hydroxylase and 17,20-lyase activities. hCYP17 17alpha-hydroxylates 5alpha-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity. hCYP17 also 17alpha-hydroxylates 5alpha-pregnan-3alpha-ol-20-one and the 5alpha-pregnan-3alpha,17alpha-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity. Furthermore, 5alpha-pregnan-3alpha,17alpha-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17alpha-hydroxypregnenolone and cytochrome b(5) stimulates androsterone formation only 3-fold. Both 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3alpha,17alpha-diol-20-one bind to hCYP17 with higher affinity than does progesterone. We conclude that 5alpha-reduced, 3alpha-hydroxy-C(21) steroids are excellent, high-affinity substrates for hCYP17. The brisk metabolism of 5alpha-pregnan-3alpha,17alpha-diol-20-one to androsterone by CYP17 explains how, when 5alpha-reductases are present, the testis can produce C(19) steroids androsterone and androstanediol from 17alpha-hydroxyprogesterone without the intermediacy of androstenedione and testosterone.     

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.     

Synthesis of a [1,2-3H]-labeled pregnanolone.

A method is described for the synthesis and purification of 3 alpha-hydroxy-5 alpha-[1,2-3H]pregnan-20-one. [1,2-3H]progesterone (55 Ci/mmol) was incubated with a homogenate of rat brain tissue. The product was purified by Sephadex chromatography and thin-layer chromatography. The identity and purity of the product were established by successive recrystallizations and high-performance liquid chromatography. A 34% portion of the starting material was converted to 3 alpha-hydroxy-5 alpha-[1,2-3H]pregnan-20-one. The final radiopurity of 3 alpha-hydroxy-5 alpha-pregnan-20-one obtained from four independent preparations was 94% to 99%.     

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.     

Synthesis and GABA(A) receptor activity of oxygen-bridged neurosteroid analogs

Three analogs of neuroactive steroids were prepared (4-6) in which 1,11- or 11,19-oxygen bridges give a constrained conformation. Their 3D structures were obtained by ab initio calculations and in the case of 3alpha-hydroxy-11,19-epoxypregn-4-ene-20-one (4), confirmed by X-ray analysis. Biological activity of the synthetic steroids was assayed in vitro using t-[(3)H]butylbicycloorthobenzoate as radiolabeled ligand for the GABA(A) receptor. The activity of compound 4 was similar to that of allopregnanolone (1). 1alpha,11alpha-Epoxypregnanolone (6) was more active than pregnanolone (2).     

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.