A high-performance liquid chromatography-tandem mass spectrometry assay of the androgenic neurosteroid 3alpha-androstanediol (5alpha-androstane-3alpha,17beta-diol) in plasma

The testosterone metabolite 3alpha-androstanediol (5alpha-androstane-3alpha,17-diol) is a potential GABA(A) receptor-modulating neurosteroid with anticonvulsant properties and hence could act as a key neuromodulator in the central nervous system. However, there is no specific and sensitive assay for quantitative determination of the androgenic neurosteroid 3alpha-androstanediol in biological samples. We have established a liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay to measure 3alpha-androstanediol in rat plasma. Standard 3alpha-androstanediol added to rat plasma has been successfully analysed with excellent linearity, specificity, sensitivity, and reproducibility. The sensitivity of the method was < 10 ng/ml with a detection limit of 2 ng/ml (6.8 nmol/l) and a linear range of 10-2000 ng/ml. The method was used for the analysis of testosterone-induced increase in plasma 3alpha-androstanediol levels in rats. Testosterone produced a dose-dependent elevation in plasma 3alpha-androstanediol, which was almost completely prevented by pretreatment with the 5alpha-reductase inhibitor finasteride, indicating that 3alpha-androstanediol is synthesized from testosterone via a 5alpha-reductase pathway. This LC-MS-MS method allows accurate, high-throughput analysis of 3alpha-androstanediol in small amounts (200 microl) of plasma and possibly other biological samples.     

Modulation of neurosteroid production in human neuroblastoma cells by Alzheimer's disease key proteins

Studies performed with animals suggest neurosteroid involvement in neuroprotection. However in humans, the role of neurosteroidogenesis in the regulation of degenerative processes is unknown. To determine whether cellular factors intervening in degenerative mechanisms may interfere with the process of neurosteroidogenesis in humans, we combined pulse-chase experiments with HPLC and continuous flow scintillation detection to compare neurosteroid production in normal and transfected SH-SY5Y cells with key proteins involved in Alzheimer's disease (AD). Microscope analyses revealed that cell morphology was unchanged in stably transfected SH-SY5Y cells overexpressing human native tau (hTau40), mutant tau (P301L), and wild-type amyloid precursor protein (APPwt) compared to controls. Biochemical investigations showed that hTau40 enhanced progesterone (PROG), 17OHPROG, testosterone, and 3alpha-androstanediol neosynthesis from pregnenolone. In contrast, tau with the pathogenic P301L mutation was devoid of action on neurosteroidogenesis. Overexpression of APPwt inhibited PROG formation, did not affect 17OHPROG and testosterone, but increased 3alpha-androstanediol and estradiol synthesis. Extracellular treatment of control cells with aggregated amyloid peptide mimicked the action of APPwt expression on PROG but not on 3alpha-androstanediol and estradiol production. Moreover, PROG biosynthesis in APPwt cells was up-regulated in the presence of a gamma-secretase inhibitor. Our results provide the first evidence for the regulation of neurosteroid biosynthesis by key proteins involved in the etiology of AD. The data suggest that pathogenic factors may induce neurodegeneration in humans through the reduction of the synthesis of endogenous neuroprotective neurosteroids in nerve cells.     

Epoxy Fatty Acids and Inhibition of the Soluble Epoxide Hydrolase Selectively Modulate GABA Mediated Neurotransmission to Delay Onset of Seizures

In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA). ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs). The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH), the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders.     

Neurosteroid biosynthesis in the quail brain: a review

The brain traditionally has been considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have shown that the brain itself also has the capability of forming steroids de novo, the so-called "neurosteroids". De novo neurosteroidogenesis in the brain from cholesterol is a conserved property of vertebrates. Our studies using the quail, as an excellent animal model, have demonstrated that the avian brain possesses cytochrome P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/c17,20-lyase (P450(17alpha,lyase)), 17beta-HSD, etc., and produces pregnenolone, progesterone, 3beta, 5beta-tetrahydroprogesterone, androstenedione, testosterone and estradiol from cholesterol. However, the biosynthetic pathway of neurosteroids in the avian brain from cholesterol may be still incomplete, because we recently found that the quail brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed avian neurosteroid. This paper summarize the advances made in our understanding of biosynthesis of neurosteroids in the avian brain.     

Testosterone inhibits osteoclast formation stimulated by parathyroid hormone through androgen receptor

Androgens play an important role in the regulation of bone metabolism in animals and humans. The present study was performed to investigate whether androgens would affect osteoclast formation stimulated by parathyroid hormone (PTH) in mouse bone cell cultures and its mechanism. Testosterone as well as alpha-dihydrotestosterone (DHT) concentration-dependently inhibited osteoclast formation induced by PTH-(1-34). 10(-8) M ICI 182780, an estrogen receptor inhibitor, did not affect PTH-induced osteoclast formation antagonized by 10(-8) M testosterone, although it completely antagonized the effects of 10(-8) M 17beta-estradiol. Moreover, 3 microM 4-androsten-4-ol-3,17-dione, an aromatase inhibitor, did not affect PTH-induced osteoclast formation antagonized by testosterone. Hydroxyflutamide, an androgen receptor antagonist, concentration-dependently antagonized the inhibitory effects of testosterone as well as DHT on PTH-stimulated osteoclast formation. In conclusion, the present study first demonstrated that testosterone inhibited osteoclast formation stimulated by PTH through the androgen receptor, but not through the production of intrinsic estrogen in mouse bone cell cultures.     

Effects of hydroxyflutamide on rats treated with a superovulatory dose of pregnant mare serum gonadotropin

Immature female rats treated with superovulatory doses of pregnant mare serum gonadotropin (PMSG) were used to study the effects of the antiandrogen hydroxyflutamide on steroid production, particularly the biologically active androgens, in two experiments. In the first experiment, animals were given either 5 mg hydroxyflutamide or vehicle alone at 30 and 36 h following 40 IU PMSG. Compared with the vehicle group, hydroxyflutamide treatment significantly reduced the percentage of degenerate oocytes recovered from oviducts (p less than 0.05). Serum levels of testosterone and androstenedione, and their aromatized product 17 beta-estradiol, significantly decreased (p less than 0.05) in the hydroxyflutamide-treated group; however, nonaromatizable androgen, 5 alpha-dihydrotestosterone, was not affected. In the second experiment, ovaries obtained 48 h after stimulation with 4 or 40 IU PMSG were incubated with and without hydroxyflutamide (10(-5) M) and (or) testosterone (10(-7) M) to study [4-14C]pregnenolone metabolism to major steroids. In 40 IU stimulated ovaries, hydroxyflutamide significantly decreased the metabolism of pregnenolone to progesterone (p less than 0.01) and androstenedione (p less than 0.01), while the production of 17 beta-estradiol increased significantly (p less than 0.05); however, pregnenolone conversions to testosterone and 5 alpha-dihydrotestosterone were not affected. Testosterone completely reversed the hydroxyflutamide-induced alteration of pregnenolone metabolism. In contrast, there was no difference in the pregnenolone conversion patterns between untreated and hydroxyflutamide or hydroxyflutamide plus testosterone groups in 4 IU stimulated ovaries. Present results confirm our previous finding that hydroxyflutamide decreases the percentage of abnormal oocytes recovered from superovulating rats and indicates that this hydroxyflutamide effect may be partly mediated by altered ovarian steroidogenesis following inhibition of androgen binding in the ovary.     

Interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol in rat kidney: heterogeneity of 3alpha-hydroxysteroid oxidoreductases.

3alpha-Hydroxysteroid oxidoreductases catalyzing the interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol (3alpha-androstanediol) have been studied in rat kidney. Three enzymes can be distinguished: a soluble NADPH-dependent oxidoreductase, a microsomal NADPH-dependent enzyme and a microsomal NADH-linked enzyme. Traces of the microsomal enzymes are consistently observed in the 108 000 X g supernatant. Studies on crude preparations reveal that these enzymes differ not only in subcellular localization and co-factor requirement, but also in optimum pH, kinetic characteristics, sensitivity to potential steroidal inhibitors and sensitivity to detergents, ionic strength and temperature. Moreover, salient sex differences exist in the activity of all three kidney enzymes. The soluble NADPH-dependent enzyme is more active in female rats whereas both microsomal enzymes are considerably more active in male animals. The microsomal NADH-dependent oxidoreductase displays favorable characteristics to catalyze the 3alpha-dehydrogenation of 3alpha-androstanediol. Evidence is presented that it is mainly this enzyme that enables the kidney to use 3alpha-androstanediol as an efficient precursor for the local formation of 5alpha-dihydrotestosterone.     

Neurosteroid modulation of GABAA receptors: molecular determinants and significance in health and disease

Over the past 20 years it has become apparent that certain steroids, synthesised de novo in the brain, hence named neurosteroids, produce immediate changes (within seconds) in neuronal excitability, a time scale that precludes a genomic locus of action. Identified molecular targets underlying modulation of brain excitability include both the inhibitory GABA(A) and the excitatory NMDA receptor. Of particular interest is the interaction of certain neurosteroids with the GABA(A) receptor, the major inhibitory receptor in mammalian brain. During the last decade, compelling evidence has accrued to reveal that locally produced neurosteroids may selectively "fine tune" neuronal inhibition. A range of molecular mechanisms including the subunit composition of the receptor(s), phosphorylation and local steroid metabolism, underpin the region- and neuronal selectivity of action of neurosteroids at synaptic and extrasynaptic GABA(A) receptors. The relative contribution played by each of these mechanisms in a variety of physiological and pathophysiological scenarios is currently being scrutinised at a cellular and molecular level. However, it is not known how such mechanisms may act in concert to influence behavioural profiles in health and disease. An important question concerns the identification of the anatomical substrates mediating the repertoire of behaviours produced by neurosteroids. "Knock-in" mice expressing mutant GABA(A) subunits engineered to be insensitive to benzodiazepines or general anaesthetics have proved invaluable in evaluating the role of GABA(A) receptor subtypes in complex behaviours such as sedation, cognition and anxiety [Rudolph, U., Mohler, H., 2006. GABA-based therapeutic approaches: GABA(A) receptor subtype functions. Curr. Opin. Pharmacol. 6, 18-23]. However, the development of a similar approach for neurosteroids has been hampered by the limited knowledge that, until recently, has surrounded the identity of the amino acid residues contributing to the neurosteroid binding pocket. Here, we will review recent progress in identifying the neurosteroid binding site on the GABA(A) receptor, and discuss how these discoveries will impact on our understanding of the role of neurosteroids in health and disease.     

Endocrine modulating actions of a phytosterol mixture and its oxidation products in zebrafish (Danio rerio)

In this study, a phytosterol preparation ("ultrasitosterol"; 80% beta-sitosterol) and an oxidized ultrasitosterol preparation were evaluated for reproductive effects in zebrafish. Adults were exposed in a continuous flow to 10 microg/L and 100 microg/L ultrasitosterol and oxidized ultrasitosterol, and to 0.27 microg/L 17beta-estradiol and 0.28 microg/L testosterone for 3 weeks. Biomarkers analysed included plasma vitellogenin, testosterone, 11-ketotestosterone, 17beta-estradiol, and gonadal histopathology. Ovarian steroid production of testosterone and 17beta-estradiol was examined in isolated zebrafish follicles exposed in vitro to the compounds at the same concentrations as in vivo. Vtg was induced in males exposed to ultrasitosterol, and in males and females exposed to 17beta-estradiol. Males exposed to oxidized phytosterols showed increased levels of testosterone and 11-ketotestosterone, and accelerated spermatogenesis. Increased follicular atresia was observed in females exposed to oxidized phytosterols and 17beta-estradiol. Correlation analyses between biomarkers revealed more intercorrelated values for females than for males, and the strongest associations were found in females exposed to oxidized phytosterols. Testosterone production was significantly increased in follicles exposed to the oxidized phytosterol preparations. These findings indicate that the phytosterol mixture is weakly estrogenic in male fish, and that the oxidized phytosterol mixture contains substances that may interfere with spermatogenesis, oogenesis and gonadal steroidogenesis in zebrafish.     

Enzyme induction in Streptomyces hydrogenans. V. Characterization of testosterone-17 beta-dehydrogenase and its induction by steroids]

Testosterone 17beta-dehydrogenase can be enriched from Streptomyces hydrogenans. The enzyme dehydrogenizes testosterone with Km=13muM and estradiol-17beta with Km=21muM to the corresponding 17-ketoderivatives. NAD forms NADH with Km=125muM. The enzyme is strongly inhibited by androstandione and 17alpha-methyltestosterone. The Ki for 17alpha-methyltestosterone is 18muM. The enzyme activity increases with increasing pH up to alkali-mediated denaturation at about pH 10. The optimum temperature is at 45 degrees C. If Streptomyces hydrogenans is cultivated in the absence of steroids, the specific activity of testosterone 17beta-dehydrogenase in the cytosol of the microorganisms amounts to 10 mU/mg protein, and increases up to 10-fold if the cells are cultivated in the presence of certain steroids. Testosterone, alpha-dihydrotestosterone, beta-dihydrotestosterone, estradiol-17beta, and 17alpha-methyltestosterone are very effective inducers. Thus, for the first time, the ability of estradiol-17beta to induce an enzyme synthesis in a microorganism is shown. The steroid-dependent induction is inhibited by testosterone acetate and rifamycin SV. Cyproterone, however, does not decrease the testosterone-dependent enzyme induction of testosterone 17beta-dehydrogenase.     

The determination of five steroids in avian plasma by radioimmunoassay and competitive protein-binding.

A method has been developed for the simultaneous determination of testosterone, 5alpha-dihydrotestosterone and corticosterone, or of estrone, estradiol-17beta and corticosterone, after separation on a Celite:propylene glycol:ethylene glycol column (6:1.5:1.5 w/v/v). The lower quarter of the column was packed with a Celite: water mixture (3:1 w/v) as a stationary phase (glycol) 'trap'. This effectively prevented leaching of the glycols into the eluate as the concentration of ethyl acetate in the mobile phase was increased to elute the more polar steroids. In addition, a second system utilizing a Celite: ethylene glycol column (2:1 w/v) for the separation of estrone and estradiol-17beta is described. Testosterone, 5alpha-dihydrotestosterone, estrone and estradiol-17beta were measured by radioimmunoassay and corticosterone by a competitive protein-binding technique. Reliability criteria are presented showing that the assay systems used are accurate and reproducible. Plasma-steroid levels of eight avian species are also presented and compared with those found by other investigators.     

Inhibition of Acid Sensing Ion Channel 3 Aggravates Seizures by Regulating NMDAR Function

The existing data about whether acid sensing ion channels (ASICs) are proconvulsant or anticonvulsant are controversial. Particularly, acid sensing ion channel 3 (ASIC3) is the most sensitive to extracellular pH and has the characteristic ability to generate a biphasic current, but few studies have focused on the role of ASIC3 in seizure. Here we found ASIC3 expression was increased in the hippocampus of pilocarpine induced seizure rats, as well as in hippocampal neuronal cultures undergoing epileptiform discharge elicited by Mg²⁺-free media. Furthermore, ASIC3 blockade by the selective inhibitor APETx2 shortened seizure onset latency and increased seizure severity compared with the control in the pilocarpine induced seizure model. Incubation with APETx2 enhanced the excitability of primary cultured hippocampal neurons in Mg²⁺-free media. Notably, the aggravated seizure was associated with upregulation of the N-methyl-d-aspartate subtype of glutamate receptors (NMDARs), increased NMDAR mediated excitatory neurotransmission and subsequent activation of the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cAMP-response element binding protein (CREB) signaling pathway. Moreover, co-immunoprecipitation confirmed the interaction between ASIC3 and NMDAR subunits, and NMDARs blockade prevented the aggravated seizure caused by ASIC3 inhibition. Taken together, our findings suggest that ASIC3 inhibition aggravates seizure and potentiates seizure induced hyperexcitability at least partly by the NMDAR/CaMKII/CREB signaling pathway, which implies that ASIC3 agonists may be a promising approach for seizure treatment.     

Steroid control of gonadotrophin secretion in the orchidectomized dog

The effects of s.c. administration of oil, testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and oestradiol-17 beta on plasma concentrations of LH and FSH were determined in 5 orchidectomized dogs. The dosages for the androgens and oestradiol-17 beta were 500 and 50 micrograms/kg body weight, respectively. Testosterone and oestradiol-17 beta significantly reduced plasma gonadotrophin concentrations, although the onset and duration of their suppressive effects differed. Dihydrotestosterone and oil had no effect on either gonadotrophin. Administration of androstanediol had no effect on plasma concentrations of LH but did cause a temporary and significant reduction in FSH. It is concluded that testosterone and oestradiol-17 beta are major regulators of gonadotrophin secretion in the male dog, but the 5 alpha-reduction of testosterone seems to play only a minor role in this control.     

The effect of naloxone administration on pregnancy-associated seizures

Pregnant mice are more susceptible to flurothyl-induced seizures than are non-pregnant controls. The possibility that the well-known increase in beta-endorphin concentration which accompanies pregnancy was involved in this effect was examined by testing whether naloxone administration could block the increased seizure susceptibility. Pregnant female, control female and male C3H mice were treated with 5-50 mg/kg naloxone 5 min before flurothyl seizure testing. Naloxone markedly increased clonic seizure susceptibility in all three groups at a dose of 50 mg/kg, but had little effect at lower doses. In contrast, naloxone had differential effects on myoclonic seizures in pregnant and control female mice, being anticonvulsant in the controls, but proconvulsant in the pregnant mice. A role for endogenous opiates is unlikely in mediating clonic seizures in pregnant mice, but may be involved in myoclonic seizures.     

Testosterone rapidly reduces anxiety in male house mice (Mus musculus)

Eight experiments supported the hypotheses that reflexive testosterone release by male mice during sexual encounters reduces male anxiety (operationally defined in terms of behavior on an elevated plus-maze) and that this anxiolysis is mediated by the conversion of testosterone to neurosteroids that interact with GABA(A) receptors. In Experiment 1, a 10-min exposure to opposite-sex conspecifics significantly reduced both male and female anxiety 20 min later (as indexed by increased open-arm time on an elevated plus-maze) compared to control mice not receiving this exposure. In contrast, locomotor activity (as indexed by enclosed-arm entries on the elevated plus-maze) was not significantly affected. The remaining experiments examined only male behavior. In Experiment 2, exposure to female urine alone was anxiolytic while locomotor activity was not significantly affected. Thus, urinary pheromones of female mice likely initiated the events leading to the male anxiolysis. In phase 1 of Experiment 3, sc injections of 500 microg of testosterone significantly reduced anxiety 30 min later while locomotor activity was not significantly affected. Thus, testosterone elevations were associated with reduced male anxiety and the time course consistent with a nongenomic, or very rapid genomic, mechanism of testosterone action. In phase 2 of Experiment 3, the anxiolytic effect of testosterone was dose dependent with a 250 microg sc injection required. Thus, testosterone levels likely must be well above baseline levels (i.e., in the range induced by pulsatile release) in order to induce anxiolysis. In Experiment 4, a high dosage of 5alpha-dihydrotestosterone was more anxiolytic than a high dosage of estradiol benzoate, suggesting that testosterone action may require 5alpha-reduction. In Experiments 5 and 6, 3alpha,5alpha-reduced neurosteroid metabolites of testosterone (androsterone and 3alpha-androstandione) were both anxiolytic at a lower dosage (100 microg/sc injection) than testosterone, supporting the notion that testosterone is converted into neurosteroid metabolites for anxiolytic activity. Experiments 7 and 8 found that either picrotoxin or bicucculine, noncompetitive and competitive antagonists of the GABA(A) receptor, respectively, blocked the anxiolytic effects of testosterone. However, conclusions from these 2 experiments must be tempered by the reduction in locomotor activity that was also seen. The possible brain locations of testosterone action as well as the possible adaptive significance of this anxiolytic response are discussed.     

Pharmacological and neurochemical characterization of the involvement of hippocampal adrenoreceptor subtypes in the modulation of acute limbic seizures

Noradrenaline exerts inhibitory effects on seizure susceptibility. Subtype selective agonists and antagonists were used to identify the anticonvulsant hippocampal adrenoreceptors. Intrahippocampal dialysis was used for administration of all compounds, including pilocarpine for limbic seizure induction, and as the neurotransmitter sampling tool. The noradrenaline reuptake inhibitor maprotiline mediated anticonvulsant effects, associated with dose-dependent increases in extracellular hippocampal noradrenaline, dopamine and GABA levels. At high concentrations, maprotiline produced proconvulsant effects associated with high levels of noradrenaline, dopamine and glutamate. Maprotiline's anticonvulsant effect was blocked by administration of either a selective α(2) - and β(2) -antagonist. α(2) -Antagonist administration with maprotiline was associated with a further increase in noradrenaline and dopamine from maprotiline alone; whereas β(2) -antagonist administered with maprotiline inhibited the dopamine increases produced by maprotiline. α(1A) -Antagonism blocked the GABA-ergic but not the anticonvulsive effect of maprotiline. These results were confirmed as combined but not separate α(2) - and β(2) -adrenoreceptor stimulation, using selective agonists, inhibited limbic seizures. Interestingly, α(1A) -receptor stimulation and α(1D) -antagonism alone also inhibited seizures associated with respectively significant hippocampal GABA increases and glutamate decreases. The main findings of this study are that (i) increased hippocampal noradrenergic neurotransmission inhibits limbic seizures via combined α(2) - and β(2) -receptor activation and (ii) α(1A) - and α(1D) -adrenoreceptors mediate opposite effects on hippocampal excitability.     

Pituitary-gonadal axis before puberty: evaluation of testicular steroids in the male rat

Orchidectomy was performed on 26-day-old rats via a single midscrotal incision following which 1 of 6 steroids was administered subcutaneously twice daily for 7 days. Each hormone treatment set had its own controls both castrate and intact. Levels of serum LH and FSH were measured by radioimmunoassay. It was found that LH was suppressed to intact levels by testosterone or its active metabolites at doses within the "physiologic dosage range" (equivalent in biological activity to endogenously secreted androgens). FSH suppression with androgens occurred at considerably higher doses; only testosterone could maintain FSH at intact levels with a physiologic dosage. Both 5alpha-dihydrotestosterone, and 3alpha-androstanediol suppressed LH well and FSH partially; 3beta-androstanediol and fluoxymesterone were ineffective over the same dosage range. Estradiol suppressed both LH and FSH. It was concluded that LH is more easily suppressed than FSH by androgens, that there is poor correlation between biologic potency and their gonadotropin-suppression ability, and that testosterone is almost certainly not the final active intracellular androgenic hormone. It was suggested that while a small amount of testicular androgen can maintain the low levels of LH, complete control of FSH secretion may require conversion of testosterone to estrogens.     

Immediate postnatal rise in whole body androgen content in male rats: correlation with increased testicular content and reduced body clearance of testosterone

Whole body content of androgen (testosterone + 5 alpha-dihydrotestosterone) was invariably higher in male than in female rat pups killed 1 or 3 h after natural delivery, whereas androgen content was equivalent in males and females killed immediately or 6, 12, and 24 h after birth. Testicular content of androgen was significantly elevated in males killed 1 and 24 h after birth, compared with levels in males killed immediately, or 3, 6, and 12 h after birth. Thus, heightened testicular androgen content was only initially associated with increased systemic levels of androgen in males during the immediate postpartum period. A second study assessed the possibility that the body's clearance (i.e., metabolism plus excretion) of testosterone is lower in newborn rats upon separation from the placental circulation than in slightly older pups. Rats of both sexes killed 1 and 3 h after s.c. injection of [3H] testosterone had significantly higher plasma concentrations of [3H] testosterone as well as several 5 alpha-reduced androgens (5 alpha-dihydrotestosterone, 3 alpha-androstanediol, and androsterone) when injections were given within minutes as opposed to 24 h after birth. This suggests that in both sexes the clearance of testosterone is slower immediately after birth than at later ages. This phenomenon together with a brief postnatal elevation in the testicular synthesis and secretion of testosterone may explain the temporary rise in circulating androgen concentrations that occurs in the newborn male rat.     

Determination of endogenous testosterone in rat tissues following fetal alcohol exposure using HPLC with UV detection

A novel method for quantitation of brain neurosteroid levels using HPLC with UV detection is described. In this simple and reliable method, testosterone from the brain and whole blood, and the internal standard, 17alpha-methyl testosterone, were extracted in 20% acetonitrile-phosphate buffer (pH 2.8), followed by solid phase extraction (SPE). The calibration curve was linear in concentration ranges from 0.1 to 10 ng from 0.2 g of tissue. We successfully applied this method to the analysis of endogenous testosterone in the male offspring of rats exposed to alcohol in utero. The concentration of testosterone at 21 post delivery in fetal alcohol exposure (FAE) group was significantly greater than the concentrations in either pair-fed or the ad libitum controls. These results support the usefulness of this method as a means of quantitating neurosteroids, and illustrate its applicability to fetal alcohol exposure.     

Crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase/bile acid binding protein complexed with NADP(+) and ursodeoxycholate

The crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase (HSD)/bile acid binding protein (AKR1C2) complexed with NADP(+) and 3alpha,7beta-dihydroxy-5beta-cholanic acid (ursodeoxycholate) at 3.0 A resolution is presented. Thus, the three-dimensional structure has now been solved for a human HSD member of the aldo-keto reductase superfamily. AKR1C2 is implicated in the prostatic production of the potent androgen 5alpha-dihydrotestosterone and the hepatic transport of bile acids. It also catalyzes the formation of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in the central nervous system, and its allosteric modulation by fluoxetine has been linked to the use of this drug for premenstrual dsyphoria. Like other members of the superfamily, AKR1C2 folds into an alpha/beta-barrel and binds NADP(+) in an extended conformation. The carboxylate of ursodeoxycholate binds to AKR1C2 in the oxyanion hole at the active site. More interestingly, the orientation of ursodeoxycholate is essentially "backwards" and "upside-down" from that observed for testosterone in the related rat 3alpha-HSD.NADP(+).testosterone ternary complex, where testosterone assumes the position of a 3-ketosteroid substrate. The orientation of ursodeoxycholate is thus similar to that expected of a 17beta-HSD substrate. The ternary structure explains the ability of AKR1C2 to catalyze 3alpha-, 17beta-, and 20alpha-HSD reactions. Comparison of the steroid binding pocket of AKR1C2 with that of rat 3alpha-HSD reveals significant differences in the positions of conserved and nonconserved loop residues, providing insights into the structural basis for the functional flexibility that is observed in all the human 3alpha-HSD isoforms but not in the rat isoform.