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NMDARs are ligand-gated ion channels that cause an influx of Na + and Ca 2+ into postsynaptic neurons. The resulting intracellular Ca 2+ transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca 2+ (Ca 2+ challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5β-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5β-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 μm PAhPim had virtually no effect on NMDAR responses; however, following the Ca 2+ challenge, it inhibited the responses by 62%; similarly, the Ca 2+ challenge induced a 3.7-fold decrease in the steroid IC 50 on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca 2+ challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results of in silico modeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity. SIGNIFICANCE STATEMENT There is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate or de novo gain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca 2+ increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions., (Copyright © 2021 the authors.)

Context: Oral once-daily dual-release hydrocortisone (DR-HC) replacement therapy has demonstrated an improved metabolic profile compared to conventional 3-times-daily (TID-HC) therapy among patients with primary adrenal insufficiency. This effect might be related to a more physiological cortisol profile, but also to a modified pattern of cortisol metabolism., Objective: This work aimed to study cortisol metabolism during DR-HC and TID-HC., Design: A randomized, 12-week, crossover study was conducted., Intervention and Participants: DC-HC and same daily dose of TID-HC were administered to patients with primary adrenal insufficiency (n = 50) vs healthy individuals (n = 124) as controls., Main Outcome Measures: Urinary corticosteroid metabolites were measured by gas chromatography/mass spectrometry at 24-hour urinary collections., Results: Total cortisol metabolites decreased during DR-HC compared to TID-HC (P < .001) and reached control values (P = .089). During DR-HC, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activity measured by tetrahydrocortisol + 5α-tetrahydrocortisol/tetrahydrocortisone ratio was reduced compared to TID-HC (P < .05), but remained increased vs controls (P < .001). 11β-HSD2 activity measured by urinary free cortisone/free cortisol ratio was decreased with TID-HC vs controls (P < .01) but normalized with DR-HC (P = .358). 5α- and 5β-reduced metabolites were decreased with DR-HC compared to TID-HC. Tetrahydrocortisol/5α-tetrahydrocortisol ratio was increased during both treatments, suggesting increased 5β-reductase activity., Conclusions: The urinary cortisol metabolome shows striking abnormalities in patients receiving conventional TID-HC replacement therapy, with increased 11β-HSD1 activity that may account for the unfavorable metabolic phenotype in primary adrenal insufficiency. Its change toward normalization with DR-HC may mediate beneficial metabolic effects. The urinary cortisol metabolome may serve as a tool to assess optimal cortisol replacement therapy., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society.)

Steroidal C19-hydroxylation is pivotal to the synthesis of naturally occurring bioactive C19-OH steroids and 19-norsteroidal pharmaceuticals. However, realizing this transformation is proved to be challenging through either chemical or biological synthesis. Herein, we report a highly efficient method to synthesize 19-OH-cortexolone in 80% efficiency at the multi-gram scale. The obtained C 19 -OH-cortexolone can be readily transformed to various synthetically useful intermediates including the industrially valuable 19-OH-androstenedione, which can serve as a basis for synthesis of C19-functionalized steroids as well as 19-nor steroidal drugs. Using this biocatalytic C19-hydroxylation method, the unified synthesis of six C19-hydroxylated pregnanes is achieved in just 4 to 9 steps. In addition, the structure of sclerosteroid B is revised on the basis of our synthesis.

Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3β-hydroxysteroid dehydrogenase (3βHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3βHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3βHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3βHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.

Furanosteroids, represented by wortmannin, viridin, and demethoxyviridin, are a special group of fungal-derived, highly oxygenated steroids featured by an extra furan ring. They are well-known nanomolar-potency inhibitors of phosphatidylinositol 3-kinase and widely used in biological studies. Despite their importance, the biosyntheses of these molecules are poorly understood. Here, we report the identification of the biosynthetic gene cluster for demethoxyviridin, consisting of 19 genes, and among them 15 biosynthetic genes, including six cytochrome P450 monooxygenase genes, are deleted. As a result, 14 biosynthetic intermediates are isolated, and the biosynthetic pathway for demethoxyviridin is elucidated. Notably, the pregnane side-chain cleavage requires three enzymes: flavin-dependent Baeyer-Villiger monooxygenase, esterase, and dehydrogenase, in sharp contrast to the single cytochrome P450-mediated process in mammalian cells. Structure-activity analyses of these obtained biosynthetic intermediates reveal that the 3-keto group, the C1β-OH, and the aromatic ring C are important for the inhibition of phosphatidylinositol 3-kinase.

In the latter half of gestation in the mare, progesterone concentrations decline to near undetectable levels while other 5α-reduced pregnanes are elevated. Of these, 5α-dihydroprogesterone and allopregnanolone have been reported to have important roles in either pregnancy maintenance or fetal quiescence. During this time, the placenta is necessary for pregnane metabolism, with the enzyme 5α-reductase being required for the conversion of progesterone to 5α-dihydroprogesterone. The objectives of this study were to assess the effects of a 5α-reductase inhibitor, dutasteride on pregnane metabolism (pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3β,20α-diol and allopregnanolone), to determine circulating dutasteride concentrations and to assess effects of dutasteride treatment on gestational parameters. Pregnant mares ( n  = 5) received dutasteride (0.01 mg/kg/day, IM) and control mares ( n  = 4) received vehicle alone from 300 to 320 days of gestation or until parturition. Concentrations of dutasteride, pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3β,20α-diol, and allopregnanolone were evaluated via liquid chromatography-tandem mass spectrometry. Samples were analyzed as both days post treatment and as days prepartum. No significant treatment effects were detected in pregnenolone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3β,20α-diol or allopregnanolone for either analysis; however, progesterone concentrations were increased ( P  < 0.05) sixfold in dutasteride-treated mares compared to control mares. Dutasteride concentrations increased in the treated mares, with a significant correlation ( P  < 0.05) between dutasteride concentrations and pregnenolone or progesterone concentrations. Gestational length and neonatal outcomes were not significantly altered in dutasteride-treated mares. Although 5α-reduced metabolites were unchanged, these data suggest an accumulation of precursor progesterone with inhibition of 5α-reductase, indicating the ability of dutasteride to alter progesterone metabolism., (© 2018 Society for Reproduction and Fertility.)

Neurosteroids are endogenous sterols that potentiate or inhibit pentameric ligand-gated ion channels (pLGICs) and can be effective anesthetics, analgesics, or anti-epileptic drugs. The complex effects of neurosteroids on pLGICs suggest the presence of multiple binding sites in these receptors. Here, using a series of novel neurosteroid-photolabeling reagents combined with top-down and middle-down mass spectrometry, we have determined the stoichiometry, sites, and orientation of binding for 3α,5α-pregnane neurosteroids in the Gloeobacter ligand-gated ion channel (GLIC), a prototypic pLGIC. The neurosteroid-based reagents photolabeled two sites per GLIC subunit, both within the transmembrane domain; one site was an intrasubunit site, and the other was located in the interface between subunits. By using reagents with photoreactive groups positioned throughout the neurosteroid backbone, we precisely map the orientation of neurosteroid binding within each site. Amino acid substitutions introduced at either site altered neurosteroid modulation of GLIC channel activity, demonstrating the functional role of both sites. These results provide a detailed molecular model of multisite neurosteroid modulation of GLIC, which may be applicable to other mammalian pLGICs., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Human Pregnane X Receptor (hPXR) is a regulator of drug metabolising enzymes (DME) and efflux transporters (ET). The prediction of hPXR activators and non-activators has pharmaceutical importance to predict the multiple drug resistance (MDR) and drug-drug interactions (DDI). In this study, we developed and validated the computational prediction models to classify hPXR activators and non-activators. We employed four machine learning methods support vector machine (SVM), k-nearest neighbour (k-NN), random forest (RF) and naïve bayesian (NB). These methods were used to develop molecular and fingerprint based descriptors for the prediction of hPXR activators and non-activators. Total 529 molecules consitsting of 317 activators and 212 non-activators were used for model development. The overall prediction accuracy of models was 69% to 99% to classify hPXR activators and nonactivators using RDkit descriptors. In case of 5 and 10-fold cross validation the prediction accuracy for training set is 74% to 82% and 79% to 83% for hPXR activators respectively and 50% to 62% and 49% to 65% non-activators, respectively. The external test prediction is between 59% to 73% for hPXR activators and 55% to 68% for hPXR non-activators. In addition, consensus models were developed in which the best model shows overall 75% to 83% accuracy for fingerprint and RDkit descriptors, respectively. The best developed model will be utilized for the prediction of hPXR activators and non-activators.

This study examined the reproductive physiology of female European mink (Mustela lutreola) to augment the available information on estrus, ovulation, and pregnancy with the long-term goal of supporting ex situ breeding management of this highly endangered species. Fecal reproductive hormone metabolites were measured using EIAs for estrogen and 20-oxo-pregnane metabolites. Seasonal hormone profiles were established. A comparison of hormone fluctuations in pregnant and nonpregnant females showed that both estrogen and 20-oxo-pregnane metabolites were significantly elevated during gestation, which is 42 days in length. Delayed implantation or embryonic diapause does not occur in this species. Litter size was correlated with 20-oxo-pregnane levels but not with estrogen concentrations. During lactation, 20-oxo-pregnane metabolite levels remained higher than in nonpregnant females. The breeding season was characterized by peaks in vaginal cornified cells and fecal estrogen metabolite levels. Up to four peaks in estrogen levels were identified and confirmed that European mink are seasonally polyestrous. The results of 20-oxo-pregnane measurements indicated that hCG can be applied to induce ovulation. With the establishment of this noninvasive method, we present a new tool to support population management of this species., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To screen the effective acupoints for anti-depression in depression rats and to explore the mechanisms of acupuncture for relieving depression., Methods: Fifty rats were randomly divided into normal control, model, "Yin-tang" (EX-HN 3)-"Baihui" (GV 20, 2-acupoints), EX-HN 3-GV 20-"Fengchi" (GB 20)-"Shenshu" (BL 23, 4-acupoints) and medication groups, with 10 rats in each group. The depression model was established by unpredictable chronic mild stress (UCMS) for 28 days. For rats of the 2-acupoints and 4-acupoints groups, EX-HN 3 and GV 20, and EX-HN 3, GV 20, GB 20 and BL 23 were punctured with filiform needles respectively before performing mild stress every time. The acupuncture needles were retained for 30 min during each intervention and the treatment was conducted once daily for 28 days. The rats of the medication group were treated by intragastric administration of Fluoxetine (0.18 mg/kg) once a day for 28 days. The rats' anxiety-like behavior (rearing and crossing times) was detected by open-field test. The contents of adrenocorticotropic hormone (ACTH) in the pituitary, 5-hydroxytryptamine (5-HT) in the hippocampus, the cortisol (CORT) in the adrenal gland, and the brain derived neurotrophic factor (BDNF) in the serum were examined by ELISA., Results: Compared to the normal control group, the numbers of both rearing and crossing motions in the model group were significantly decreased (P<0.01, P<0.05), while in comparison with the model group, the rearing and crossing numbers of rats in the 2-acupoints and 4-acupoints and medication groups were significantly increased (P<0.01). ELISA showed that after modeling, the content of adrenal CORT was significantly increased (P<0.01), and those of hippocampal 5-HT and serum BDNF were obviously down-regulated in the model group (P<0.01). After the treatment, the adrenal CORT levels in the three intervention groups were notably down-regulated, and hippocampal 5-HT and serum BONE evidently up-regulated in these 3 intervention groups (P<0.05, P<0.01). No marked changes were found in the pituitary ACTH contents of the model and 3 intervention groups (P>0.05), and no significant differences were shown among the three intervention groups in the levels of the aforementioned 6 indexes (P>0.05)., Conclusion: Acupuncture intervention can effectively improve the unprompted activates of the depression rats, which may be related to its effects in up-regulating hippocampal 5-HT and serum BONE levels, and in down-regulating adrenal CORT content.

In plants androstanes, estranes, pregnanes and corticoids have been described. Sometimes 17β-estradiol, androsterone, testosterone or progesterone were summarized as sex hormones. These steroids influence plant development: cell divisions, root and shoot growth, embryo growth, flowering, pollen tube growth and callus proliferation. First reports on the effect of applicated substances and of their endogenous occurrence date from the early twenties of the last century. This caused later on doubts on the identity of the compounds. Best investigated is the effect of progesterone. Main steps of the progesterone biosynthetic pathway have been analyzed in Digitalis. Cholesterol-side-chain-cleavage, pregnenolone and progesterone formation as well as the stereospecific reduction of progesterone are described and the corresponding enzymes are presented. Biosynthesis of androstanes, estranes and corticoids is discussed. Possible progesterone receptors and physiological reactions on progesterone application are reviewed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Endometrial cancer is the most frequently diagnosed gynecological malignancy. It is associated with prolonged exposure to estrogens that is unopposed by progesterone, whereby enhanced metabolism of progesterone may decrease its protective effects, as it can deprive progesterone receptors of their active ligand. Furthermore, the 5α-pregnane metabolites formed can stimulate proliferation and may thus contribute to carcinogenesis. The aims of our study were to: (1) identify and quantify progesterone metabolites formed in the HEC-1A and Ishikawa model cell lines of endometrial cancer; and (2) pinpoint the enzymes involved in progesterone metabolism, and delineate their roles. Progesterone metabolism studies combined with liquid chromatography-tandem mass spectrometry enabled identification and quantification of the metabolites formed in these cells. Further quantitative PCR analysis and small-interfering-RNA-mediated gene silencing identified individual progesterone metabolizing enzymes and their relevant roles. In Ishikawa and HEC-1A cells, progesterone was metabolized mainly to 20α-hydroxy-pregn-4-ene-3-one, 20α-hydroxy-5α-pregnane-3-one, and 5α-pregnane-3α/β,20α-diol. The major difference between these cell lines was rate of progesterone metabolism, which was faster in HEC-1A cells. In the Ishikawa and HEC-1A cells, expression of AKR1C2 was 110-fold and 6800-fold greater, respectively, than expression of AKR1C1, which suggests that 20-ketosteroid reduction of 5α-pregnanes and 4-pregnenes is catalyzed mainly by AKR1C2. AKR1C1/AKR1C2 gene silencing showed decreased progesterone metabolism in both cell lines, thus further supporting the significant role of AKR1C2. SRD5A1 was also expressed in these cells, and its silencing confirmed that 5α-reduction is catalyzed by 5α-reductase type 1. Silencing of SRD5A1 also had the most pronounced effects, with decreased rate of progesterone metabolism, and consequently higher concentrations of unmetabolized progesterone. Our data confirm that in model cell lines of endometrial cancer, AKR1C2 and SRD5A1 have crucial roles in progesterone metabolism, and may represent novel targets for treatment., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

New oxazolinyl derivatives of [17(20)E]-pregna-5,17(20)-diene: 2'-{[(E)-3β-hydroxyandrost-5-en-17-ylidene]methyl}-4',5'-dihydro-1',3'-oxazole 1 and 2'-{[(E)-3β-hydroxyandrost-5-en-17-ylidene]methyl}-4',4'-dimethyl-4',5'-dihydro-1',3'-oxazole 2 were evaluated as potential CYP17A1 inhibitors in comparison with 17-(pyridin-3-yl)androsta-5,16-dien-3β-ol 3 (abiraterone). Differential absorption spectra of human recombinant CYP17A1 in the presence of compound 1 (λmax=422 nm, λmin=386 nm) and compound 2 (λmax=416 nm) indicated significant differences in enzyme/inhibitors complexes. CYP17A1 activity was measured using electrochemical methods. Inhibitory activity of compound 1 was comparable with abiraterone 3 (IC50=0.9±0.1 μM, and IC50=1.3±0.1 μM, for compounds 1 and 3, respectively), while compound 2 was found to be weaker inhibitor (IC50=13±1 μM). Docking of aforementioned compounds to CYP17A1 revealed that steroid fragments of compound 1 and abiraterone 3 occupied close positions; oxazoline cycle of compound 1 was coordinated with heme iron similarly to pyridine cycle of abiraterone 3. Configuration of substituents at 17(20) double bond in preferred docked position corresponded to Z-isomers of compounds 1 and 2. Presence of 4'-substituents in oxazoline ring of compound 2 prevents coordination of oxazoline nitrogen with heme iron and worsens its docking score in comparison with compound 1. These data indicate that oxazolinyl derivative of [17(20)E]-pregna-5,17(20)-diene 1 (rather than 4',4'-dimethyl derivative 2) may be considered as potential CYP17A1 inhibitor and template for development of new compounds affecting growth and proliferation of prostate cancer cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Interpretation of plasma cortisol levels in wild-caught fish is confounded by the stress of capture. Measurement of cortisol metabolites in fish bile could provide a method for assessing the stress level of wild fish because the time-lag for metabolism, conjugation and excretion into bile avoids the effects of sampling stress. To determine which biliary metabolite(s) to target, four Atlantic cod, Gadus morhua L., were injected with radioactive cortisol. After 22 h, the bile was collected and found to contain 30% of the injected activity. Cortisol metabolites were extracted from diluted bile samples using solid phase extraction and the radioactive metabolites separated by several different chromatographic procedures. The metabolites were predominantly present as sulfates (95%) with the remainder being glucuronidated. Chromatography split the sulfates into at least seven peaks, and acid solvolysis (which removes sulfate groups from steroids) generated four major radioactive steroids. These were identified, using microchemical reactions and re-crystallization to constant specific activity, as: 11β,17,21-trihydroxypregn-4-ene-3,20-dione (cortisol), 3α,11β,17,21-tetrahydroxy-5β-pregnan-20-one (tetrahydrocortisol; THF), 3α,17,21-trihydroxy-5β-pregnane-11,20-dione (tetrahydrocortisone; THE) and 3α,17,20β,21-tetrahydroxy-5β-pregnan-11-one (β-cortolone). The last of these was the most abundant, and thus a likely target for a biliary stress assay. Studies were also carried out to determine the best method for extraction and solvolysis of sulfates. Solid phase extraction (i.e. using octadecylsilane) was found to be too unreliable for routine use. Even though the extraction efficiency could be improved by acidifying the bile, this caused premature solvolysis of sulfated steroids. Acid solvolysis of unextracted bile worked best (c. 90% converted to free steroids) on volumes that were 1 μL or lower. Aryl sulfatase digestion of unextracted bile did not work well (only 20% of radioactivity was converted to free steroids)., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

To help save the Sumatran rhino from extinction, the captive breeding program must capitalize on each rhino's reproductive lifespan. Doing so requires knowing when calves are sexually mature. The goal of this study was to monitor physiological changes associated with sexual maturation in two captive born calves (one male and one female) to determine the approximate age of maturity for both sexes of this species. Fecal testosterone metabolites were monitored in the male calf from 6 months to 7 years of age, and fecal pregnane metabolites were measured in the female calf from 6 months to 5.5 years of age. In addition, rectal ultrasonography was employed to monitor changes in ovarian activity from 2 to 5.5 years of age. The male calf's fecal testosterone concentrations reached levels comparable to those detected in samples from adult males when he was 6-6.5 years of age. The first pre-ovulatory sized follicle was observed on the ovaries of the female calf when she was 4.75 years old, but fecal pregnane metabolite concentrations only reached maximum mean concentrations and variability when she was 5-5.5 years of age. Results from this study indicate that male and female Sumatran rhino calves are sexually mature at 6-6.5 and 5-5.5 years of age, respectively., (© 2013 Wiley Periodicals, Inc.)

The main and side products of hydroxylation by the C. lunata VKPM F-981 mycelium of fourteen delta(4)-3-ketosteroids of the estrane, androstane, and pregnane series and six of their delta(5)-3beta-hydroxy analogues were identified by H1 PMR spectroscopy and comparison with standard samples. The obtained experimental data are considered in terms of the triangular model of the enzyme-substrate interaction. The dependence of the direction of hydroxylation of steroid molecules and the orientation of hydroxy groups on the structure of the initial substrate was revealed.

Citrus juice intake has been highlighted because of its health-promoting effects. LC-MS based metabolomics approaches are applied to obtain a better knowledge on changes in the concentration of metabolites due to its dietary intake and allow a better understanding of involved metabolic pathways. Eight volunteers daily consumed 400 mL of juice for four consecutive days and urine samples were collected before intake and 24h after each citrus juice intake. Urine samples were analysed by nanoHPLC-q-TOF, followed by principal component analysis (PCA) and Student's t-test (p<0.05). PCA showed a separation between two groups (before and after citrus juice consumption). This approach allowed the identification of four endocrine compounds (tetrahydroaldosterone-3-glucuronide, cortolone-3-glucuronide, testosterone-glucuronide and 17-hydroxyprogesterone), which belonged to the steroid biosynthesis pathway as significant metabolites upregulated by citrus juice intake. Additionally, these results confirmed the importance of using the non-targeted metabolomics technique to identify new endogenous metabolites, up- or down-regulated as a consequence of food intake., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Because of overpopulation of African elephants in South Africa and the consequent threat to biodiversity, the need for a method of population control has become evident. In this regard, the potential use of the porcine zona pellucida (pZP) vaccine as an effective means for population control is explored. While potential effects of pZP treatment on social behavior of African elephants have been investigated, no examination of the influence of pZP vaccination on the endocrine correlates in treated females has been undertaken. In this study, ovarian activity of free-ranging, pZP-treated African elephant females was monitored noninvasively for 1 yr at Thornybush Private Nature Reserve, South Africa, by measuring fecal 5α-pregnan-3β-ol-20-on concentrations via enzyme immunoassay. A total of 719 fecal samples from 19 individuals were collected over the study period, averaging 38 samples collected per individual (minimum, maximum: 16, 52). Simultaneously, behavioral observations were made to record the occurrence of estrous behavior for comparison. Each elephant under study showed 5α-pregnan-3β-ol-20-on concentrations rising above baseline at some period during the study indicating luteal activity. Average 5α-pregnan-3β-ol-20-on concentrations were 1.61 ± 0.46 μg/g (mean ± SD). Within sampled females, 42.9% exhibited estrous cycles within the range reported for captive African elephants, 14.3% had irregular cycles, and 42.9% did not appear to be cycling. Average estrous cycle duration was 14.72 ± 0.85 wk. Estrous behavior coincided with the onset of the luteal phase and a subsequent rise in 5α-pregnan-3β-ol-20-on concentrations. Average 5α-pregnan-3β-ol-20-on levels positively correlated with rainfall. No association between average individual 5α-pregnan-3β-ol-20-on concentrations or cyclicity status with age or parity were detected. Earlier determination of efficacy was established via fecal hormone analysis with no pregnancies determined 22 mo post-treatment and onward. Results indicate the presence of ovarian activity amongst pZP-treated female African elephants in 2 yr after initial immunization. Further study should now be aimed toward investigating the long-term effects of pZP vaccination on the reproductive function of female African elephants., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Urine from neonates with 21-hydroxylase deficiency contains a large range of metabolites of 17-hydroxyprogesterone, 21-deoxycortisol and androgens but few have been previously described. We present the second part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of the A- and B-rings containing an oxo group. Fragmentations indicating presence of 3-, 6-, and 7-oxo groups and also 1β-, 2α-, 4β-, and 6β-hydroxyls are presented and discussed for the first time. Interpretation was aided by comparison with spectra of available relevant standards, of oxidation products of standards and urinary metabolites and of deuterated derivatives. Endogenous 1-enes and 2(3)-ene artifacts of non-hydrolyzed 3α-sulfates are also reported. D-ring and side chain structure was determined according to our previously published criteria. Likely metabolic relationships were also explored. We conclude that GC-MS combined with GC-MS/MS allows identification of the A- and B-ring structure of pregnane and pregnenes in the presence of an oxo group on one of these rings. Major oxygenations are 1β, 15β, 16α and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2α, 4β and 6β. Three major metabolic streams exist in affected neonates in addition to the classical 3α-hydroxy-5β-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3α- and 3β-hydroxy-5α-anes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Regulative endocrine mechanisms influence the reproductive behaviour and success of mammals, but they have been studied predominantly in domestic and captive animals. The study aims at describing the pattern of faecal 20-oxopregnane and oestrogen concentrations during pregnancy in wild plains zebra Equus quagga chapmani. Data were collected during wet and dry seasons 2007-2009. Enzyme Immunoassays were used to determine 20-oxopregnane and oestrogen concentrations in faecal samples (n=74) collected from individual mares (n=32) whose dates of foaling were known through long-term monitoring. Hormonal profiles were described with a General Additive Model (GAM: Hormone ∼ Days to Foaling). Faecal 20-oxopregnanes have a complex cycle during pregnancy (GAM, n=70, R(2)=0.616, p<0.001). From -250 days to foaling, faecal 20-oxopregnane concentrations were above the baseline levels found in non-pregnant mares, peaking in the last 50 days. Faecal oestrogen levels showed a clear peak in mid-pregnancy (GAM, n=62, R(2)=0.539, p<0.001). The sex of the foetus and season had no detectable effect on hormone concentrations during pregnancy. High levels (>200 ng/g DW) of faecal 20-oxopregnanes associated with high (>160 ng/g DW) faecal oestrogen levels indicate mid-pregnancy in c.90% of cases (16/17). High faecal 20-oxopregnanes (>200 ng/g DW) and low faecal oestrogen levels (<160 ng/g DW) indicate late pregnancy, again in c.90% of cases. Two faecal samples would allow the stage of pregnancy to be determined with confidence., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The biological activity of two seven-membered A-ring (A-homo) analogues of progesterone was evaluated by transactivation assays in Cos-1 cells and by determination of Bcl-x(L) expression levels in T47D cells. The results show that both compounds act as selective progesterone receptor (PR) agonists but lack mineralocorticoid receptor (MR) activity. Molecular modelling using semiempirical AM1 and ab initio HF/6-31G** calculations, showed that the A-ring of the A-homo steroids may adopt five different conformations, although only three correspond to low energy conformers. The low energy conformers of each analogue were introduced into the ligand binding pocket of the PR ligand binding domain (LBD) obtained from the PR LBD-progesterone crystal structure. The steroid binding mode was then analyzed using 10 ns of molecular dynamics (MD) simulation. The PR LBD-progesterone complex was also simulated as a control system. The MD results showed that both A-homo steroids have one conformer that may be properly recognized by the PR, in agreement with the observed progestagen activity. Moreover, the simulation revealed the importance of a water molecule in the formation of a hydrogen bonding network among specific receptor residues and the steroid A-ring carbonyl., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Regio- and stereospecificity of microbial hydroxylation was studied at the transformation of 3-keto-4-ene steroids of androstane and pregnane series by the filamentous fungus of Curvularia lunata VKMF-644. The products of the transformations were isolated by column chromatography and identified using HPLC, mass-spectrometry (MS) and proton nuclear magnetic resonance (1H NMR) analyses. Androst-4-ene-3,17-dione (AD) and its 1(2)-dehydro- and 9alpha-hydroxylated (9-OH-AD) derivatives were hydroxylated by the fungus mainly in position 14alpha, while 6alpha-, 6beta- and 7alpha-hydroxylated products were revealed in minor amounts. At the transformation of C21-steroids (cortexolone and its acetylated derivatives) the presence of 17-acetyl group was shown to facilitate further selectivity of 11beta-hydroxylation. Original procedures for protoplasts obtaining, mutagenesis and mutant strain selection have been developed. A stable mutant (M4) of C. lunata with high 11beta-hydroxylase activity towards 21-acetate and 17alpha,21-diacetate of cortexolone was obtained. Yield of 11beta-hydroxylated products reached about 90% at the transformation of 17alpha, 21-diacetate of cortexolone using mutant strain M4.

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring. All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized. GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed. We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.

The present study is addressed to ascertain the inhibitory effect of several progesterone derivatives having a chlorine substituent at C-6 (12a-12d), 15 with a bromine substituent at C-6 and 14a-14d, without any halogen atom at C-6 all having an ester side chain at C-17 (benzoate ester bearing a Cl, F and a Br atom at C-4 position of the phenyl ring) on the 5alpha-reductase enzyme activity present in human prostate. In addition, it was also of interest to investigate the pharmacological effect on hamster flank organs diameter size. In order to study the structure-activity relationships of steroids 12a-12d, 14a-14d and 15 we determined the concentration of these steroids that inhibited 50% of the activity of human prostate 5alpha-reductase enzyme (IC(50)), as well as the in vivo effect of these compounds in the hamster flank organs diameter size. We also ascertained, the capacity of these steroids to bind to the androgen receptors present in the rat prostate cytosol using labeled mibolerone (MIB) for monitoring the binding to the androgen receptor. The results from this study indicated that compounds 12a-12d (having a chlorine substituent at C-6), 14a-14d (lacking a halogen atom at C-6), 13 and 15 (having a bromine atom at C-6) showed an increased antiandrogenic effect (lower value for the diameter of the flank organs) as compared to the flank organs from testosterone-treated hamsters. On the other hand, the series of compounds containing a chlorine substituent at C-6 compounds (12a-12d) showed a higher antiandrogenic activity as compared to the compounds lacking a halogen atom at C-6 (14a, 14b and 14d). Although compounds 13 and 15 decreased the flank organs diameter size, however, this increase was not statistically significant as compared to that of the commercially available product finasteride. The steroidal derivatives 13, 14a-14d (lacking the chlorine substituent at C-6) and 15 (having a bromine atom at C-6) exhibited a higher 5alpha-reductase inhibitory activity (lower IC(50) values) as compared to the series of compounds 12a-12d having the halogen substituent at C-6. Finasteride reduced the diameter size of the flank organs. The effect of this steroid and compounds 12a-12d, 13, 14a-14d and 15 on hamster flank organs can be explained by the fact that these steroids did not bind to the androgens receptor, which indicates that its mechanism of action is an inhibiting for the 5alpha-reductase activity. This enzyme is present in the hamster flank organs and was inhibited by the novel steroids in the human prostate homogenates.

The biotransformation of the synthetic hormone dydrogesterone (1) by a number of fungal strains - including Cephalosporium aphidicola, Rhizopus stolonifer, Cunninghamella elegans, and Fusarium lini - afforded ten different metabolites, compounds 2-11. From a structural point of view, these transformations involved a combination of hydroxylation, oxidation, reduction, and/or epoxidation. The pregnane-based metabolites 10 and 11 are new compounds. All the known compounds were obtained for the first time from 1 by fungal transformation. The metabolites 3, 5, and 8 showed more-potent respiratory-burst inhibitory activity than the substrate 1 in a neutrophil-based cellular assay (Table 3).

It is almost 70 years since the discovery by Huggins et al. that androgens are essential for prostate cancer (PC) growth and progression, and there has been about 30 years experience using ketoconazole for PC therapy. Since then we have come a long way in learning about the disease and developing new strategies to approach it, among which is cytochrome 17alpha-hydroxylase-C(17,20)-lyase (CYP17) inhibition. This review focuses on the efforts to find prospective CYP17 inhibitors, both steroidal and nonsteroidal, in the absence of a 3D structure of the enzyme. It covers almost 4 decades of literature with highlights on the most significant achievements in this area, providing insight into PC pathophysiology, management and treatment options.

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG) with a 3' single-stranded extension (the G-overhang). The stabilization of G-quadruplexes in the human telomeric sequence by small-molecule ligands inhibits the activity of telomerase and results in telomere uncapping, leading to senescence or apoptosis of tumor cells. Therefore, the search for new and selective G-quadruplex ligands is of considerable interest because a selective ligand might provide a telomere-targeted therapeutic approach to treatment of cancer. We have screened a bank of derivatives from natural and synthetic origin using a temperature fluorescence assay and have identified two related compounds that induce G-quadruplex stabilization: malouetine and steroid FG. These steroid derivatives have nonplanar and nonaromatic structures, different from currently known G-quadruplex ligands. Malouetine is a natural product isolated from the leaves of Malouetia bequaaertiana E. Woodson and is known for its curarizing and DNA-binding properties. Steroid FG, a funtumine derivative substituted with a guanylhydrazone moiety, interacted selectively with the telomeric G-quadruplex in vitro. This derivative induced senescence and telomere shortening of HT1080 tumor cells at submicromolar concentrations, corresponding to the phenotypic inactivation of telomerase activity. In addition, steroid FG induced a rapid degradation of the telomeric G-overhang and the formation of anaphase bridges, characteristics of telomere uncapping. Finally, the expression of protection of telomere 1 (POT1) induced resistance to the growth effect of steroid FG. These results indicate that these steroid ligands represent a new class of telomere-targeted agents with potential as antitumor drugs.

tert-Butyl hydroperoxide catalyzed by (5,10,15,20-tetramesitylporphyrinate) osmium(II) carbonyl [Os(TMP)CO] complex was found to be a highly efficient versatile oxidant for C-H carbons in steroid substrates. When reacted with representative steroids with an estrane, pregnane, 5beta-cholane, or 5alpha-cholestane structure, regioselective oxyfunctionalization and/or oxidative degradation occurred to give a variety of novel and uncommon derivatives in one step.

Recent evidence indicates that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that breast carcinoma and tumorigenic breast cell lines have higher 5alpha-reductase and lower 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO activities and mRNA expression levels than normal tissue and non-tumorigenic cell lines. The 5alpha-reduced progesterone metabolites such as 5alpha-dihydroprogesterone (5alphaP) promote both mitogenic and metastatic activity in breast cell lines in culture, whereas the 4-pregnene metabolites, 4-pregnen-3alpha-ol-20-one (3alphaHP) and 4-pregnen-20alpha-ol-3-one (20alphaHP) have the opposite (anti-cancer-like) effects. The 5alpha-reductase inhibitor dutasteride has been shown to inhibit 5alpha-reduction of testosterone to 5alpha-dihydrotestosterone in prostate tissue, resulting in decreased prostate volume. The aim of this study was to determine if dutasteride is an effective inhibitor of progesterone 5alpha-reduction in human breast cell lines and if such inhibition reduces mammary cell proliferation and detachment. The effect of dutasteride on progesterone metabolizing enzyme activities and mRNA expression were examined in tumorigenic MCF-7 and non-tumorigenic MCF-10A human breast cell lines. Dutasteride (10(-6)M) inhibited progesterone conversion to 5alpha-pregnanes by >95% and increased 4-pregnene production. The results indicated that effects of dutasteride on the progesterone metabolizing enzymes are due to direct inhibition of 5alpha-reductase activity and to altered levels of expression of 5alpha-reductase and HSO mRNAs. Treatment of cells with progesterone without medium change for 72 h resulted in significant conversion to 5alpha-pregnanes and increases in cell proliferation and detachment. The increases in proliferation and detachment were blocked by dutasteride and were reinstated by concomitant treatment with 5alphaP, providing proof-of-principle that the effects were due not to progesterone but to the 5alpha-reduced metabolites. This study provides the first evidence that dutasteride is a potent progesterone 5alpha-reductase inhibitor and that such inhibition may be beneficial in breast cancer.

Neuroactive steroids are a large group of substances having effect in the brain and on brain function. The steroids most studied are allopregnanolone (ALLO), tetrahydrodesoxycorticosterone (THDOC), pregnenolone sulfate (PS) dihydroepiandrosteronesulfate (DHEAS), and estradiol (E2). ALLO and THDOC are called gamma-aminobutyric acid (GABA) steroids as they are positive modulators of the GABAA receptor in a similar way as benzodiazepines, barbiturates, and alcohol. GABA steroids not only have similar behavioral effects as benzodiazepines and barbiturates but, possibly, also similar adverse effects as well. This review aims to elucidate the possible role that neuroactive steroids play in the development of mood disorders in women. One of the most clear-cut examples of the interaction between mood, neuroactive steroids, and the GABA system is premenstrual dysphoric disorder (PMDD), which is a cluster of negative mood symptoms occurring during the luteal phase of the menstrual cycle in 2-6% of reproductive women. Furthermore, certain women also experience adverse mood effects during sequential progestin addition to postmenopausal estrogen treatment, which is why the role of neuroactive steroids in postmenopausal women is also addressed in this review.

It has recently become more clearly understood that in human brain pathophysiology, neurosteroids play a role in anxiety disorders, premenstrual syndrome, postpartum depression, posttraumatic stress disorder, and depression. In the treatment of major depression, recent clinical studies indicate that the pharmacological profiles of fluoxetine and fluvoxamine are correlated with the ability of these drugs to increase the brain and cerebrospinal fluid content of allopregnanolone (Allo), a potent positive allosteric modulator of gamma-aminobutyric acid (GABA) action at GABAA receptors. Thus, the neurosteroid-induced positive allosteric modulation of GABA action at GABAA receptors is facilitated by fluoxetine or its congeners (i.e., paroxetine, fluvoxamine, sertraline), which may not block 5-HT reuptake at the doses currently prescribed in the clinic. However, these doses are effective in the treatment of premenstrual dysphoria, anxiety, and depression. In socially isolated mice, we tested the hypothesis that fluoxetine, norfluoxetine, and other specific serotonin reuptake inhibitor (SSRI) congeners stereoselectively upregulate neurosteroid content at doses insufficient to inhibit 5-HT reuptake; although they potentiate pentobarbital-induced sedation and exert antiaggressive action. Very importantly, the inhibition of 5-HT reuptake lacks stereospecificity and requires fluoxetine and norfluoxetine doses that are 50-fold greater than those required to increase brain Allo content, potentiate the action of pentobarbital, or antagonize isolation-induced aggression. Based on these findings, it could be inferred that the increase of brain Allo content elicited by fluoxetine and norfluoxetine, rather than the inhibition selective of 5-HT reuptake, may be operative in the fluoxetine-induced remission of the behavioral abnormalities associated with mood disorders. Therefore, the term "SSRI" may be misleading in defining the pharmacological profile of fluoxetine and its congeners. To this extent, the term "selective brain steroidogenic stimulants" (SBSSs) could be proposed.

Velutinol A is a pregnane compound isolated from the rhizomes of the Brazilian plant Mandevilla velutina that interferes with kinin actions and possesses anti-inflammatory action. Here, we investigate the effect produced by velutinol A in different models of inflammatory nociception. The nociceptive effect caused by the intraplantar injection of phorbol myristate acetate (PMA, 50 pmol/paw) in mice was practically abolished by coadministration of velutinol A (1-10 nmol/paw). In contrast, the coadministration of velutinol A (10 nmol/paw) failed to affect the nociceptive response elicited by either bradykinin (BK, 10 nmol/paw) or prostaglandin E(2) (PGE(2), 10 nmol/paw). Of note, velutinol A (10 nmol/paw) partially inhibited the nociceptive response caused by capsaicin (1 nmol/paw). However, velutinol A (10 microM) did not significantly interfere with the specific binding sites of [(3)H]resiniferatoxin or [(3)H]BK in vitro. Our data also suggest that these effects are related with its ability to interact with kinin B(1) receptor-mediated mechanisms, as the cotreatment of mice with velutinol A (10 nmol/paw) consistently blocked the nociceptive response induced by the selective B(1) receptor agonist des-Arg(9)-BK. Finally, the persistent hyperalgesia produced by intraplantar injection of carrageenan (300 microg/paw) was completely reversed by the coadministration of velutinol A (10 nmol/paw). Collectively, the present results show that the pregnane compound velutinol A produces peripheral antinociceptive action in some models of acute and persistent inflammatory pain by interacting with kinin B(1)-receptor mediated effects. Thus, velutinol A or its derivatives could constitute an attractive molecule of interest for the development of new analgesic drugs. Additional studies are now in progress in order to further explore its precise mechanism of action on B(1) receptor pathways.

GABA(A) (gamma-aminobutyric acid type A) receptors mediate most of the 'fast' synaptic inhibition in the mammalian brain and are targeted by many clinically important drugs. Certain naturally occurring pregnane steroids can potently and specifically enhance GABA(A) receptor function in a nongenomic (direct) manner, and consequently have anxiolytic, analgesic, anticonvulsant, sedative, hypnotic and anaesthetic properties. These steroids not only act as remote endocrine messengers, but also can be synthesized in the brain, where they modify neuronal activity locally by modulating GABA(A) receptor function. Such 'neurosteroids' can influence mood and behaviour in various physiological and pathophysiological situations, and might contribute to the behavioural effects of psychoactive drugs.

In the rat brain, gamma-hydroxybutyric-acid (GHB) increases the concentrations of 3alpha-hydroxy,5alpha-pregnan-20-one (allopregnanolone, 3alpha,5alpha-THP) and 3alpha,21-dihydroxy,5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone/3alpha,5alphaTHDOC), two neurosteroids acting as positive allosteric modulators of gamma-aminobutyric acid (GABA)(A) receptors. This study was aimed at assessing whether neurosteroids play a role in GHB-induced loss of righting reflex (LORR). Basal and GHB-stimulated brain concentrations of endogenous 3alpha,5alpha-THP and 3alpha,5alpha-THDOC were analyzed in two rat lines, GHB-sensitive (GHB-S) and GHB-resistant (GHB-R), selectively bred for opposite sensitivity to GHB-induced sedation/hypnosis. Basal neurosteroid concentrations were similar in brain cortex of the two rat lines. However, in male GHB-S rats, administration of GHB (1000 mg/kg, i.p., 30 min) increased brain cortical concentrations of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC 7- and 2.5-fold, respectively, whilst male GHB-R animals displayed only a 4- and 2-fold increase, respectively. In GHB-S rats this increase lasted up to 90 min and declined 180 min following GHB administration, a time course that matches LORR onset and duration. In contrast, in GHB-R rats, which failed to show GHB-induced LORR, brain cortical 3alpha,5alpha-THP and 3alpha,5alpha-THDOC had returned to control values within 90 min. At onset of LORR, a similar increase in brain cortical levels of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC (2-3-fold) was observed in GHB-S female rats and in the few female GHB-R rats that lost the righting reflex after GHB administration, but not in female GHB-R rats failing to show LORR. Sub-hypnotic doses (7.5 and 12.5 mg/kg, i.p.) of pregnanolone, administered 10 min before GHB, dose-dependently facilitated the expression of GHB-induced LORR in GHB-R male rats. These results suggest that the GHB-induced increases of brain 3alpha,5alpha-THP and 3alpha,5alpha-THDOC concentrations are implicated in the eliciting of the sedative/hypnotic action of GHB.

Gestation and lactation can impose substantial energetic costs on female mammals. We developed a non-invasive means to determine reproductive condition in female giraffe using fecal steroid analysis. Giraffe may be especially challenged during their reproductive cycle because of two characteristics: they are impregnated while lactating and they do not breed seasonally. We studied the social behavior and endocrinology of seven female giraffe in a large naturalistic outdoor enclosure in order to chart connections between maternal physiology and behavior across the reproductive cycle. We found that giraffe gestation averages 448 days among females producing a calf that survived, with fecal pregnane concentrations reaching a zenith during the last trimester of pregnancy. Resumption of ovarian cyclicity following parturition was accelerated after neonatal calf mortality, but ovarian cycles resumed as early as 39 days postparturition while nursing. Although time spent feeding was unaffected by reproductive state, pregnant females significantly reduced time allocated to social behavior and had a tendency to locomote less than when cycling or acyclic. We suggest that modifications in foraging strategies as a function of reproductive state among wild giraffe derive from antipredator activity rather than from metabolic demands. Female giraffe probably cope with simultaneous lactation and gestation by producing high quality milk for neonatal calves commensurate with slow fetal growth and accelerating fetal growth simultaneous with weaning of nursing calves.

Proliferative changes in the normal breast are known to be controlled by female sex steroids. However, only a portion of all breast cancer patients respond to current estrogen based endocrine therapy, and with continued treatment nearly all will become unresponsive and experience relapse. Therefore, ultimately for the majority of breast carcinomas, explanations and treatments based on estrogen are inadequate. Recent observations indicate that 5alpha-pregnane and 4-pregnene progesterone metabolites may serve as regulators of estrogen-responsive as well as unresponsive human breast cancers. The conversion of progesterone to the 5alpha-pregnanes is increased while conversion to the 4-pregnenes is decreased in breast carcinoma tissue, as a result of changes in progesterone metabolizing 5alpha-reductase, 3alpha-hydroxysteroid oxidoreductase (3alpha-HSO) and 20alpha-HSO activities and gene expression. The 5alpha-pregnane, 5alpha-pregnane-3,20-dione (5alphaP) stimulates, whereas the 4-pregnene, 3alpha-hydroxy-4-pregnen-20-one (3alphaHP), inhibits cell proliferation and detachment, by modulation of cytoskeletal and adhesion plaque molecules via the MAP kinase pathway and involving separate and specific plasma membrane-based receptors. The promotion of breast cancer appears to be related to changes in in situ concentrations of cancer-inhibiting and cancer-promoting progesterone metabolites. New diagnostic and therapeutic possibilities for breast cancer are suggested.

That the brain is a target for hormones is a well established fact. Today we also know that brains can secrete the whole gamut of peptides and steroid hormones, i.e., pregnanes and pregnenes. Considering that the ancestral neuron was a neurosecreting cell, this is not surprising. As CRH and cortisol secretion occur in situ in nervous systems, the intriguing possibility that anxio-depressive syndromes may be associated with paracrine effects of these compounds on the brain should now be considered. The concept of stress as a specific reaction of the nueroendocrine system to nocuous stimuli is re-examined in light of new evidence. The fundamental importance of historical, social, and psychological contexts in evaluating hormonal actions is emphasized. Problems emerging from the attempt to search for specific biological markers in different psychiatric disorders are discussed. The suggestion is made that, rather than to categorize nosological entities, biological dysfunctions should be elated with psychological abnormalities.

When female rats pace their coital interaction, a reward state evaluated by conditioned place preference is induced. Progesterone (P) is essential for the expression of proceptive behavior and for the induction of CPP. However, the functional significance of ring A reduction of P for the induction of this state during estrous is unsettled. In the present study, we evaluated whether ring A-reduced metabolites of P are involved in the reward state induced when the females are allowed to pace their sexual contacts. Ovariectomized (ovx) female rats treated with estradiol benzoate (EB, 5 microg) and P (13 microg), Megestrol acetate (MA; 13 microg ), 5 alpha-pregnan-20 dione (5 alphaDHP; 3 microg), or 5 beta-pregnan-3 alpha-ol-20-one (5 beta,3 alpha-Pgl; 3 microg) were used. Progestins were dissolved in propylene glycol and intravenously (iv) injected through an indwelling jugular catheter before females were tested for pacing behavior. After 15 intromissions or one ejaculation, females were gently placed in the nonpreferred compartment of a CPP box. Paced mating in all groups treated with progestins induced a clear change of preference. The administration of progestins alone did not induce CPP. These results suggest that P and ring A-reduced metabolites facilitate the reward state following pacing.

Within the last 20 years, rapid nongenomic actions of steroid hormones have been demonstrated to occur via an interaction with ligand-gated ion channels. For example, the pregnane neurosteroid allopregnanolone (ALLOP) is a potent positive modulator of gamma-aminobutyric acid(A) (GABA(A)) receptors. The physiological significance of fluctuations in endogenous ALLOP levels has been investigated with regard to disease states and the effect of therapeutic agents on ALLOP levels. Because the pharmacological profile of ALLOP is similar to that of ethanol (EtOH), the modulatory effect of pregnane neurosteroids on EtOH dependence and withdrawal will be the focus of this review. Data on the effects of chronic EtOH exposure and withdrawal on pregnane neurosteroid levels, biosynthetic enzymes, and changes in neurosteroid sensitivity will be summarized. Results from genetic animal models indicate that seizure-prone animals have a persistent decrease in endogenous ALLOP levels during EtOH withdrawal in conjunction with tolerance to ALLOP's anticonvulsant effect. Manipulation of endogenous ALLOP levels with finasteride also markedly reduced the severity of chronic EtOH withdrawal. Gene mapping studies provide a hint for an interaction between genes for GABA(A) receptor subunits and the biosynthetic enzyme 5alpha-reductase. Overall, the results are suggestive of a relationship between endogenous pregnane neurosteroid levels and behavioral changes in excitability during EtOH withdrawal, consistent with recent findings in humans. While the findings with ALLOP emphasize the therapeutic potential of neurosteroid treatment during EtOH withdrawal, the gene mapping studies suggest that pregnane neurosteroid biosynthesis may represent a target for therapeutic intervention in the treatment of alcohol dependence.

The aim of this study was to correlate mineralocorticoid action and steroid structure. Inasmuch as Na(+) retention follows a parabolic dose-response curve for most pregnanesteroids, the second-order coefficient of the function was used as a representative factor for this bipartite biological effect. The C(3)=O/D angle of the ligands was correlated with both Na(+)-retaining activity and binding affinity for the mineralocorticoid receptor. Because some steroids exhibit identical functional groups and different conformational structure, we also postulate that the flat conformation of a pregnanesteroid determines its Na(+)-retaining capacity in vivo. No correlations were found in vitro, which demonstrates the multifactorial nature of the second-order coefficient determined in vivo under more complex and interactive conditions that include various pre-receptor variables. These findings may allow the estimation of the putative biological activity of a given steroid simply by knowing its conformational structure, which may be important for designing compounds in a pharmaceutical setting.

The metabolism in vitro of [(3)H]17-hydroxyprogesterone by gonadal tissues of the grouper (Epinephelus coioides) during 17alpha-methyltestosterone (MT)-induced female-to-male sex inversion was examined. In the female phase, C(17,20) lyase, 5beta-reductase, 3alpha/beta-HSD, 20beta-HSD, and 17beta-HSD activities resulted in the biosynthesis of 5beta-pregnans and 5beta-androstanes (including 5beta-androstane-3alpha/beta, 17beta-diol, 3alpha/beta, 17alpha-dihydroxy-5beta-pregnen-20-one, and 5beta-androstane-3,17-dione). In the MT-induced male phase, however, the abrogation of C(17,20) lyase activity and the concomitant activation of 21alpha-hydroxylase/11beta-hydroxylase resulted in the preferential synthesis of polar 21alpha-hydroxlyated 5beta-pregnans (5beta-pregnan-3beta,17alpha,20beta,21alpha-tetrol and 3beta,20beta,21alpha-trihydroxy-5beta-pregnan-3-one) and corticosteroids (11-deoxycortisol and cortisol). Interestingly, synthesis of these 21alpha-hydroxylated 5beta-pregnans and corticosteroids was uniquely compartmentalized in only testicular tissues of the MT-induced males. This study shows that there is selective activation of specific steroidogenic enzymes in the different sexual phases leading to the synthesis of metabolites that may be involved in regulating sex inversion of the grouper., ((c) 2002 Elsevier Science (USA).)

Non-invasive pregnancy diagnosis in mares by measuring faecal oestrogens has been performed over years with great accuracy. However, results have indicated breed-related differences in the amount of excreted steroids during late pregnancy. Therefore faecal samples were collected during the last 4 months of pregnancy of Thoroughbred (n = 10), New Forest pony (n = 9), Shetland pony (n = 10) and Iceland pony mares (n = 11). Concentrations of oestrogens, 20alpha-hydroxy- and 20-oxopregnanes were measured using enzyme immunoassays. Breed differences concerning both levels (though significant only in case of oestrogens) and time course of measured steroids were observed. There was a highly significant time effect (p < 0.00001) and an interaction between time and breeds (p < 0.02) for all steroids measured, suggesting that the time effect differs for different breeds. Oestrogen concentrations showed a decrease towards parturition, whereas in 20alpha-hydroxy- and 20-oxopregnane levels a pronounced increase was found 2 and I months, respectively, before parturition. A breed effect was only significant (p = 0.001) when comparing oestrogen concentrations and was mainly due to Iceland ponies, which had the lowest concentrations especially during the last 2 months of pregnancy. An almost significant (p = 0.06) breed effect was found for 20-oxopregnanes. In Iceland mares an additional increase in faecal pregnane content was already observed earlier, reaching maximum levels before the 60th day ante-partum (a.p.), followed by a decrease until the 30th day a.p. The ratio of 20-oxopregnanes to oestrogens in the samples was significantly higher (p < 0.006) in Iceland ponies in comparison with any other breed throughout all months before parturition. The breed differences observed in the amounts of oestrogens and/or progestagens present during late pregnancy may demonstrate micro-evolutionary changes in the endocrine system of a species.

This study examines the effect of inhibiting the synthesis of gamma-aminobutyric acid(A) (GABA(A)) agonist steroids on behavioural activity and somatosensory evoked potentials (SEP) in late gestation fetuses. Pregnane steroid production was suppressed by infusion of the 5alpha-reductase inhibitor, finasteride in chronically catheterised fetal sheep, 130-135 days gestation. Finasteride treatment (160 mg in 10 ml of vehicle over 2 h) significantly increased the incidence of fetal arousal during the period 4-10 h after commencing the infusion (P<0.05, n=6), whereas other behavioural parameters were not effected. In three of four animals, finasteride produced an increase in the amplitude of the N22 peak of the SEP during high voltage electrocortical activity. We conclude that suppression of pregnane steroid synthesis, by inhibition of the 5alpha-reductase enzyme, increases arousal activity in the fetus which is consistent with a reduction in GABA(A) receptor mediated inhibition.

Certain pregnane steroids are now established as potent, positive allosteric modulators of the gamma-aminobutyric acid type A (GABAA) receptor. These compounds are known to be synthesized in the periphery by endocrine glands, such as the ovaries and the adrenal glands, and can rapidly cross the blood-brain barrier. Therefore, such steroids could act as endogeneous modulators of the major inhibitory receptor in the mammalian central nervous system. However, the demonstration that certain neurons and glia can synthesize the pregnane steroids (i.e., neurosteroids) additionally suggests that they may serve a paracrine role by influencing GABAA-receptor function through their local release in the brain itself. Here, we demonstrate that these neurosteroids are highly selective and extremely potent modulators of the GABAA receptor. The subunit composition of the GABAA receptor may influence the actions of the neurosteroids, particularly when considering concentrations of these agents thought to occur physiologically, which may underlie their reported differential effects at certain inhibitory synapses. However, recent work suggests that the phosphorylation status of either the synaptic GABAA receptor or its associated proteins may also influence neurosteroid sensitivity; these findings are discussed. Upon administration, the neurosteroids exhibit clear behavioral effects, including sedation, anticonvulsant actions, and behaviors predictive of anxiolysis; when given at high doses, they induce general anesthesia. Numerous synthetic steroids have been synthesized in an attempt to therapeutically exploit these properties, and these data are reviewed in this chapter. However, targeting the brain enzymes that synthesize and metabolize the neurosteroids may offer a new approach to exploit this novel endocrine-paracrine neurotransmitter interaction.

Two pregnane steroids, pregnane (1) and 3β-hydroxypregnane (2), were oxidized by fermentation with the fungus Cephalosporium aphidicola. The fermentation of pregnane (1) yielded 3β-hydroxypregnane (2) and 3β, 6β,11α-trihydroxypregnane (3), while that of 3β-hydroxypregnane (2) afforded 6β,11α-dihydroxypregn-3,20-dione (4), 3β,6β,15α-trihydroxypregn-20-one (5) and 3β,5α,11α-trihydroxypregn-20-one (6). The metabolites are characterized by detailed physical and spectroscopic studies.

The in vitro metabolism of [3H]17-hydroxyprogesterone by spermatozoa of primary and 17alpha-methyltestosterone (MT)-induced secondary male grouper (Epinephelus coioides) has been examined. With only the labeled precursor, the four predominant 5beta-pregnanes (17, 20alpha-dihydroxy-5beta-pregnan-3-one; 5beta-pregnan-3alpha,17, 20alpha-triol; 3alpha,17-dihydroxy-5beta-pregnan-20-one; and 17hydroxy-5beta-pregnan-3,20-dione) accounted for 75% of the total product yield, with the remainder comprising 17, 20alpha-dihydroxy-4-pregnen-3-one (17,20alphaP) and traces (3%) of 17,20beta-dihydroxy-4-pregnen-3-one. This is the first report of the synthesis of these 5beta-pregnanes by teleost spermatozoa. The addition of excess unlabeled precursor caused a marked shift to the synthesis of only 17,20alphaP. The steroidogenic profile in spermatozoa of the primary and secondary males appears similar in both cases. The shift to 17,20alphaP synthesis at high substrate concentration may suggest a gonadotropin (GtH) control of 17, 20alphaP synthesis, involving the competition for 17P between the high-activity, low-capacity 5beta-reductases and the low-activity, high-capacity 20alpha-HSD. Poor spermiation in MT-induced males may thus be due to the lack of 17P arising from low GtH secretion in vivo in the secondary males., (Copyright 1998 Academic Press.)

Steroid binding domains of Na,K-ATPase and the nuclear hormone receptors share amino acid sequence homologies. In a ouabain radioligand assay, the potencies of series of planar or bent steroid moieties suggest that the domain in Na,K-ATPase can accommodate compounds with a planar configuration. The A/B -cis, C/D-cis steroid configuration in the cardenolides, in conjunction with appropriate substituents at the 3 and 17 positions, may represent a fortuitous "fit" for the exogenous compounds. It remains to be determined if the putative physiological digitalis-like substance is a member of a hormonal steroid family, an endogenous ouabain-like compound or both.