Divergent mechanisms of steroid inhibition in the human ρ1 GABAA receptor.

ρ-type γ-aminobutyric acid-A (GABAA) receptors are widely distributed in the retina and brain, and are potential drug targets for the treatment of visual, sleep and cognitive disorders. Endogenous neuroactive steroids including ß-estradiol and pregnenolone sulfate negatively modulate the function of ρ1 GABAA receptors, but their inhibitory mechanisms are not clear. By combining five cryo-EM structures with electrophysiology and molecular dynamics simulations, we characterize binding sites and negative modulation mechanisms of ß-estradiol and pregnenolone sulfate at the human ρ1 GABAA receptor. ß-estradiol binds in a pocket at the interface between extracellular and transmembrane domains, apparently specific to the ρ subfamily, and disturbs allosteric conformational transitions linking GABA binding to pore opening. In contrast, pregnenolone sulfate binds inside the pore to block ion permeation, with a preference for activated structures. These results illuminate contrasting mechanisms of ρ1 inhibition by two different neuroactive steroids, with potential implications for subtype-specific gating and pharmacological design.

Optical annealing of peroxo-ferric intermediates in CYP17A1 and product formation.

Human cytochrome P450 CYP17A1 catalyzes the hydroxylation of pregnenolone and progesterone at the C17 position, with subsequent C17-C20 bond scission, to form dehydroepiandrosterone and androstenedione respectively. The first hydroxylation reaction is faster in H2O than in D2O, while the second carbon­carbon bond scission event demonstrates an inverse solvent isotope effect, which is more pronounced for 17-hydroxy pregnenolone. In order to better understand the cause of this difference, we compared the optical absorption spectra of oxygenated CYP17A1 with the four substrates (pregnenolone, progesterone, 17-hydroxy pregnenolone and 17-hydroxy progesterone) in both H2O and D2O. We also studied the temperature-dependent decay of the peroxo-ferric and hydroperoxo-ferric intermediates generated by cryoradiolysis of the corresponding oxygenated heme proteins at 77 K. For both pregnenolone and 17-hydroxypregnenolone, annealing of the peroxo-intermediates was observed at lower temperatures in H2O than in D2O. In contrast, no solvent isotope effect was detected when progesterone or 17-hydroxyprogesterone were used as substrates. These differences are attributed to their different positioning in the P450 active site with respect to the heme bound peroxo (Fe-OO-) moiety, which is in agreement with earlier structural and spectroscopic investigations. Analysis of the samples run in both H2O and in D2O, where 17-hydroxyprogesterone is the substrate, demonstrated significant (∼25%) yield of androstenedione product relative to the oxygenated starting material.

Steroidomics in Men with Schizophrenia.

Schizophrenia is associated with numerous abnormalities, including imbalances in all hormonal axes, among which steroids play a major role. Steroidomic studies therefore represent a promising tool for early diagnosis and appropriate treatment of schizophrenia. A total of 51 adult male schizophrenics aged 27 (22, 34) years (shown as median with quartiles) and 16 healthy controls (HCs) aged 28 (25, 32) years were enrolled into this study. Our results showed the effective differentiation of men with schizophrenia from controls based on steroidomic profiles. We also found an altered metabolic pathway from pregnenolone and its sulfate (PREG/S) to cortisol in schizophrenics with several metabolic bottlenecks such as lower PREG levels due to increased PREG sulfation and/or suppressed PREGS desulfation and attenuated conversion of 17-hydroxy-PREG to 17-hydroxy-progesterone, as well as the results suggestive of suppressed CYP11B1 activity. In contrast, steroid molar ratios suggested two counterregulatory steps involving increased conversion of PREG/S to 17-hydroxy-PREG/S and decreased conversion of cortisol to cortisone, which may maintain unchanged basal cortisol levels but may not ensure a sufficient cortisol response to stress. Our data also indicated a trend to higher 7α-, 7ß-, and 16α-hydroxylation that may counteract the autoimmune complications and proinflammatory processes accompanying schizophrenia. Finally, a possible suppression of HSD17B3 activity was suggested, resulting in decreased circulating testosterone levels with increased androstenedione levels.

Neuroactive steroids fluctuate with regional specificity in the central and peripheral nervous system across the rat estrous cycle.

Neuroactive steroids (i.e., sex steroid hormones and neurosteroids) are important physiological regulators of nervous function and potential neuroprotective agents for neurodegenerative and psychiatric disorders. Sex is an important component of such effects. However, even if fluctuations in sex steroid hormone level during the menstrual cycle are associated with neuropathological events in some women, the neuroactive steroid pattern in the brain across the ovarian cycle has been poorly explored. Therefore, we assessed the levels of pregnenolone, progesterone, and its metabolites (i.e., dihydroprogesterone, allopregnanolone and isoallopregnanolone), dehydroepiandrosterone, testosterone and its metabolites (i.e., dihydrotestosterone, 3α-diol and 17ß-estradiol) across the rat ovarian cycle to determine whether their plasma fluctuations are similar to those occurring in the central (i.e., hippocampus and cerebral cortex) and peripheral (i.e., sciatic nerve) nervous system. Data obtained indicate that the plasma pattern of these molecules generally does not fully reflect the events occurring in the nervous system. In addition, for some neuroactive steroid levels, the pattern is not identical between the two brain regions and between the brain and peripheral nerves. Indeed, with the exception of progesterone, all other neuroactive steroids assessed here showed peculiar regional differences in their pattern of fluctuation in the nervous system during the estrous cycle. These observations may have important diagnostic and therapeutic consequences for neuropathological events influenced by the menstrual cycle.

Rescuing tri-heteromeric NMDA receptor function: the potential of pregnenolone-sulfate in loss-of-function GRIN2B variants.

N-methyl-D-aspartate receptors (NMDARs emerging from GRIN genes) are tetrameric receptors that form diverse channel compositions in neurons, typically consisting of two GluN1 subunits combined with two GluN2(A-D) subunits. During prenatal stages, the predominant channels are di-heteromers with two GluN1 and two GluN2B subunits due to the high abundance of GluN2B subunits. Postnatally, the expression of GluN2A subunits increases, giving rise to additional subtypes, including GluN2A-containing di-heteromers and tri-heteromers with GluN1, GluN2A, and GluN2B subunits. The latter  emerge as the major receptor subtype at mature synapses in the hippocampus. Despite extensive research on purely di-heteromeric receptors containing two identical GRIN variants, the impact of a single variant on the function of other channel forms, notably tri-heteromers, is lagging. In this study, we systematically investigated the effects of two de novo GRIN2B variants (G689C and G689S) in pure, mixed di- and tri-heteromers. Our findings reveal that incorporating a single variant in mixed di-heteromers or tri-heteromers exerts a dominant negative effect on glutamate potency, although 'mixed' channels show improved potency compared to pure variant-containing di-heteromers. We show that a single variant within a receptor complex does not impair the response of all receptor subtypes to the positive allosteric modulator pregnenolone-sulfate (PS), whereas spermine completely fails to potentiate tri-heteromers containing GluN2A and -2B-subunits. We examined PS on primary cultured hippocampal neurons transfected with the variants, and observed a positive impact over current amplitudes and synaptic activity. Together, our study supports previous observations showing that mixed di-heteromers exhibit improved glutamate potency and extend these findings towards the exploration of the effect of Loss-of-Function variants over tri-heteromers. Notably, we provide an initial and crucial demonstration of the beneficial effects of GRIN2B-relevant potentiators on tri-heteromers. Our results underscore the significance of studying how different variants affect distinct receptor subtypes, as these effects cannot be inferred solely from observations made on pure di-heteromers. Overall, this study contributes to ongoing efforts to understand the pathophysiology of GRINopathies and provides insights into potential treatment strategies.

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system.

BACKGROUND: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. RESULTS: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. CONCLUSION: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Pregnenolone sulfate induces transcriptional and immunoregulatory effects on T cells.

Pregnenolone sulfate is a steroid metabolite of the steroidogenesis precursor, pregnenolone, with similar functional properties, including immunosuppression. We recently reported an elevation in serum levels of pregnenolone sulfate in children with malaria, contributing to an immunosuppressed state. Yet, the molecular mechanisms in which this steroid exerts its immunoregulatory functions are lacking. In this study, we examined the effects of pregnenolone sulfate on T cell viability, proliferation and transcriptome. We observed a pregnenolone sulfate dose-dependent induction of T cell death and reduction in proliferation. RNA sequencing analysis of pregnenolone sulfate-treated T cells for 2 and 24 h revealed the downregulation of pro-inflammatory genes and the upregulation of the steroid nuclear receptor superfamily, NR4A, as early-response genes. We also report a strong activation of the integrated stress response mediated by the upregulation of EIF2AK3. These results contribute to the knowledge on transcriptional regulation driving the immunoregulatory effects of pregnenolone sulfate on T cells.

De novo steroidogenesis in tumor cells drives bone metastasis and osteoclastogenesis.

Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.

[Cloning and functional analysis of 3ß-hydroxysteroid dehydrogenase gene involved in biosynthesis of digitoxin].

Digitoxin, an important secondary metabolite of Digitalis purpurea, is a commonly used cardiotonic in clinical practice. 3ß-Hydroxysteroid dehydrogenase(3ßHSD) is a key enzyme involved in the biosynthesis of digitoxin. It belongs to the short-chain dehydrogenase/reductase(SDR) family, playing a role in the biosynthesis of cardiac glycosides by oxidizing and isomerizing the precursor sterol. In this study, two 3ßHSD genes were cloned from D. purpurea. The results showed that the open reading frame(ORF) of Dp3ßHSD1 was 780 bp, encoding 259 amino acid residues. The ORF of Dp3ßHSD2 was 774 bp and encoded 257 residues. Dp3ßHSD1/2 had the cofactor binding site TGxxxA/GxG and the catalytic site YxxxK. In vitro experiments confirmed that Dp3ßHSD1/2 catalyzed the generation of progesterone from pregnenolone, and Dp3ßHSD1 had stronger catalytic capacity than Dp3ßHSD2. The expression level of Dp3ßHSD1 was much higher than that of Dp3ßHSD2 in leaves, and digitoxin was only accumulated in leaves. The results implied that Dp3ßHSD1 played a role in the dehydrogenation of pregnenolone to produce progesterone in the biosynthesis of digitoxin. This study provides a reference for further exploring the biosynthetic pathway of cardiac glycosides in D. purpurea.

Advances in steroid research from the pioneering neurosteroid concept to metabolomics: New insights into pregnenolone function.

Advances in neuroendocrinology have led to major discoveries since the 19th century, identifying adaptive loops for maintaining homeostasis. One of the most remarkable discoveries was the concept of neurosteroids, according to which the brain is not only a target but also a source of steroid production. The identification of new membrane steroid targets now underpins the neuromodulatory effects of neurosteroids such as pregnenolone, which is involved in functions mediated by the GPCR CB1 receptor. Structural analysis of steroids is a key feature of their interactions with the phospholipid membrane, receptors and resulting activity. Therefore, mass spectrometry-based methods have been developed to elucidate the metabolic pathways of steroids, the ultimate approach being metabolomics, which allows the identification of a large number of metabolites in a single sample. This approach should enable us to make progress in understanding the role of neurosteroids in the functioning of physiological and pathological processes.

The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive.

Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and koff rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH)2 cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d3-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.

The hypothalamic steroidogenic pathway mediates susceptibility to inflammation-evoked depression in female mice.

BACKGROUND: Depression is two-to-three times more frequent among women. The hypothalamus, a sexually dimorphic area, has been implicated in the pathophysiology of depression. Neuroinflammation-induced hypothalamic dysfunction underlies behaviors associated with depression. The lipopolysaccharide (LPS)-induced mouse model of depression has been well-validated in numerous laboratories, including our own, and is widely used to investigate the relationship between neuroinflammation and depression. However, the sex-specific differences in metabolic alterations underlying depression-associated hypothalamic neuroinflammation remain unknown. METHODS: Here, we employed the LPS-induced mouse model of depression to investigate hypothalamic metabolic changes in both male and female mice using a metabolomics approach. Through bioinformatics analysis, we confirmed the molecular pathways and biological processes associated with the identified metabolites. Furthermore, we employed quantitative real-time PCR, enzyme-linked immunosorbent assay, western blotting, and pharmacological interventions to further elucidate the underlying mechanisms. RESULTS: A total of 124 and 61 differential metabolites (DMs) were detected in male and female mice with depressive-like behavior, respectively, compared to their respective sex-matched control groups. Moreover, a comparison between female and male model mice identified 37 DMs. We capitalized on biochemical clustering and functional enrichment analyses to define the major metabolic changes in these DMs. More than 55% of the DMs clustered into lipids and lipid-like molecules, and an imbalance in lipids metabolism was presented in the hypothalamus. Furthermore, steroidogenic pathway was confirmed as a potential sex-specific pathway in the hypothalamus of female mice with depression. Pregnenolone, an upstream component of the steroid hormone biosynthesis pathway, was downregulated in female mice with depressive-like phenotypes but not in males and had considerable relevance to depressive-like behaviors in females. Moreover, exogenous pregnenolone infusion reversed depressive-like behaviors in female mice with depression. The 5α-reductase type I (SRD5A1), a steroidogenic hub enzyme involved in pregnenolone metabolism, was increased in the hypothalamus of female mice with depression. Its inhibition increased hypothalamic pregnenolone levels and ameliorated depressive-like behaviors in female mice with depression. CONCLUSIONS: Our study findings demonstrate a marked sexual dimorphism at the metabolic level in depression, particularly in hypothalamic steroidogenic metabolism, identifying a potential sex-specific pathway in female mice with depressive-like behaviors.

CLICK-chemoproteomics and molecular dynamics simulation reveals pregnenolone targets and their binding conformations in Th2 cells.

Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 de novo; P5, in turn, play important role in immune homeostasis and regulation. However, P5's biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5's mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5's biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5-protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.

Microwave-assisted synthesis of novel steroidal heterocyclic analogs as potent inhibitors of RANKL-induced osteoclastogenesis.

Osteoporosis is a significant public health issue in our aging population. It is an excessive bone resorption condition brought on by osteoclastogenesis, which makes bones more brittle. In the present work, a series of novel heterosteroidal derivatives have been synthesized using the microwave technique and were evaluated as antiosteoclastogenic agents. The structures of the newly synthesized compounds have been confirmed using analytical and spectral data. The antiosteoclastogenic activity of the newly synthesized compounds was estimated in vitro against osteoclast-differentiated cells from the RAW 264.7 cell line. The pregnenolone dimer 10, the pyridinotestosterone derivative 2, and the phenylnicotinonitrile pregnenolone derivative 8a attained the most promising antiosteoclastogenic activity displaying IC50 (the half maximal inhibitory concentration) values of 5.45 ± 5.30, 11.88 ± 2.09, and 13.40 ± 3.00 µM, respectively, in comparison with dimethyl itaconate (IC50 = 17.76 ± 3.20 µM) and alendronate (IC50 = 4.48 ± 1.89 µM) as reference compounds. Finally, an in silico ADME (Absorption, Distribution, Metabolism, and Excretion) study was conducted to evaluate the synthesized compounds' pharmacokinetic and drug-likeness properties. The results manifested that almost all the investigated compounds' properties were compatible with the specified optimal range, which indicates their reassuring pharmacokinetic properties.

Pregnenolone sulfate analogues differentially modulate GABAA receptor closed/desensitised states.

BACKGROUND AND PURPOSE: GABAA receptors are regulated by numerous classes of allosteric modulators. However, regulation of receptor macroscopic desensitisation remains largely unexplored and may offer new therapeutic opportunities. Here, we report the emerging potential for modulating desensitisation with analogues of the endogenous inhibitory neurosteroid, pregnenolone sulfate. EXPERIMENTAL APPROACH: New pregnenolone sulfate analogues were synthesised incorporating various heterocyclic substitutions located at the C-21 position on ring D. The pharmacological profiles of these compounds were assessed using electrophysiology and recombinant GABAA receptors together with mutagenesis, molecular dynamics simulations, structural modelling and kinetic simulations. KEY RESULTS: All seven analogues retained a negative allosteric modulatory capability whilst exhibiting diverse potencies. Interestingly, we observed differential effects on GABA current decay by compounds incorporating either a six- (compound 5) or five-membered heterocyclic ring (compound 6) on C-21, which was independent of their potencies as inhibitors. We propose that differences in molecular charges, and the targeted binding of analogues to specific states of the GABAA receptor, are the most likely cause of the distinctive functional profiles. CONCLUSIONS AND IMPLICATIONS: Our findings reveal that heterocyclic addition to inhibitory neurosteroids not only affected their potency and macroscopic efficacy but also affected innate receptor mechanisms that underlie desensitisation. Acute modulation of macroscopic desensitisation will determine the degree and duration of GABA inhibition, which are vital for the integration of neural circuit activity. Discovery of this form of modulation could present an opportunity for next-generation GABAA receptor drug design and development.

The analysis of pesticides and fungicides in the inhibition of human and rat placental 3ß-hydroxysteroid dehydrogenase activity: Mode of inhibition and mechanism.

3ß-Hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase 1 (3ß-HSD1) plays a critical role in the biosynthesis of progesterone from pregnenolone in the human placenta to maintain normal pregnancy. Whether they inhibit placental 3ß-HSD1 and mode of inhibition remains unclear. In this study, we screened 21 pesticides and fungicides in five classes to inhibit human 3ß-HSD1 and compared them to rat homolog 3ß-HSD4. 3ß-HSD activity was measured by catalyzing pregnenolone to progesterone in the presence of NAD+. Of the 21 chemicals, azoles (difenoconazole), thiocarbamates (thiram and ferbam) and organochlorine (hexachlorophene) significantly inhibited human 3ß-HSD1 with half maximal inhibitory concentration (IC50) values of 2.77, 0.24, 0.68, and 17.96 µM, respectively. We also found that difenoconazole, ferbam and hexachlorophene are mixed/competitive inhibitors of 3ß-HSD1 while thiram is a mixed/noncompetitive inhibitor. Docking analysis showed that difenoconazole and hexachlorophene bound steroid-binding site. Difenoconazole and hexachlorophene except thiram and ferbam also significantly inhibited rat 3ß-HSD4 activity with IC50 of 1.12 and 2.28 µM, respectively. Thiram and ferbam significantly inhibited human 3ß-HSD1 possibly by interfering with cysteine residues, while they had no effects on rat 3ß-HSD4. In conclusion, some pesticides potently inhibit placental 3ß-HSD, leading to the reduction of progesterone formation.

Oxytocin modulates steroidogenesis-associated genes and estradiol levels in the placenta.

Oxytocin (OXT) plays a significant role during pregnancy, especially toward the end of pregnancy. Some studies have reported that OXT is involved in the stimulation of steroidogenesis in several organs. However, the effects of OXT on placental steroidogenesis have not yet been established. In this study, we investigated the regulation of steroid hormones and steroidogenic enzymes by OXT-associated signaling in vitro and in vivo. OXT increased the gene expression of steroidogenic enzymes, which convert pregnenolone to progesterone and dehydroepiandrosterone (DHEA) in vitro. In OXT-administered pregnant rats, pregnenolone and DHEA levels were significantly enhanced in the plasma and the expression of the enzymes synthesizing DHEA, testosterone, and estradiol (E2) was increased in placental tissues. Furthermore, OXT was found to affect placental cell differentiation, which is closely related to steroid hormone synthesis. After treatment of the pregnant rats with atosiban, an antagonist of the OXT receptor, the concentration of E2 in the plasma and the expression of E2-synthesizing enzyme were reduced. This regulation may be due to OXT-mediated differentiation, because OXT increases the expression of corticotropin-releasing hormone, which is a biomarker of placental cell differentiation. Our findings suggest that OXT contributes to maintaining pregnancy by regulating the differentiation of placental cells and steroidogenesis during pregnancy.

Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1).

Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (kcat/Km) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b5 produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that -as in human CYP17A1-these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b5 protein in the lyase reactions.

Inhibition of human and rat placental 3ß-hydroxysteroid dehydrogenase/Δ5,4-isomerase activities by insecticides and fungicides: Mode action by docking analysis.

Many insecticides and fungicides are endocrine-disrupting compounds, which possibly interfere with the placental endocrine system. In the placenta, 3ß-hydroxysteroid dehydrogenase/Δ5,4-isomerase type 1 (HSD3B1) is the major steroidogenic enzyme, which makes progesterone from pregnenolone to support the placental stability. In this study, we screened 12 classes of insecticides and fungicides to inhibit placental HSD3B1 activity and compared them to the rat homolog type 4 (HSD3B4) isoform. Human HSD3B1 activity and rat HSD3B4 activity were measured in the presence of 200 nM pregnenolone and 0.2 mM NAD+ and 100 µM of test chemical. Triclosan, triflumizole, dichlone, and oxine at 100 µM significantly inhibited human HSD3B1 activity with the residual activity being less than 50% of the control. Further study showed that the half-maximal inhibitory concentration (IC50) values of triclosan, triflumizole, dichlone, and oxine were 85.53 ± 9.14, 73.75 ± 3.42, 2.54 ± 0.40, and 102.93 ± 6.10 µM, respectively. In the presence of pregnenolone, triclosan, triflumizole, and dichlone were mixed inhibitors of HSD3B1, while oxine was a noncompetitive inhibitor. In the presence of NAD+, triclosan exhibited competitive inhibition while triflumizole possessed uncompetitive inhibition. Docking analysis showed that triclosan bound NAD+-binding site, while triflumizole, dichlone, and oxine mostly bound steroid-binding site. When the effect of these insecticides on rat placental HSD3B4 activity was screened in the presence of 200 nM pregnenolone, atrazine, triclosan, triflumizole, oxine, cyprodinil, and diphenyltin at 100 µM significantly inhibited rat HSD3B4 activity, with IC50 values of triclosan, triflumizole, oxine, and cyprodinil were 82.99 ± 6.48, 35.45 ± 2.73, 105.59 ± 12.04, and 43.37 ± 3.00 µM, respectively. The mode action analysis showed that triflumizole and cyprodinil were almost competitive inhibitors, while triclosan and oxine were almost noncompetitive inhibitors of rat HSD3B4. Docking analysis showed that triclosan and oxine bound cofactor NAD+ binding residues more than steroid-binding residues of rat HSD3B4 while triflumizole and cyprodinil bound most pregnenolone-interactive residues. In conclusion, some insecticides such as triclosan, triflumizole, and oxine can effectively inhibit both human and rat placental HSD3B activity and they have unique mode action due to the structure difference.

Evaluation of two gene ablation methods for the analysis of pregnenolone function in zebrafish embryonic development.

Pregnenolone (P5) is a steroid that functions in the brain and in zebrafish embryogenesis. It is synthesized from cholesterol via the enzymatic activity of P450scc, encoded by CYP11A1. P5 exerts its function by activating CLIP1, which in turn promotes microtubule assembly necessary for many biological processes including embryogenesis. To examine the functional relatedness of CYP11A1 and CLIP1, we ablated the embryonic expression of both genes in zebrafish, i.e. cyp11a1 and clip1a. Two cyp11a1 knockout fish lines were generated. Both homozygous cyp11a1 knockout lines appeared normal. But the development of fish embryos was delayed and embryonic cell migration was reduced when cyp11a1 function was depleted of by morpholinos. This discrepancy in phenotypes by two different gene depletion methods was also observed for clip1a. While clip1a morphants are defective in embryogenesis, clip1a knockout fish appeared normal. The phenotypes depend on the methods that create gene depletion. While knockout fish lines do not have expected phenotypic defects, clip1a and cyp11a1 morpholinos both reduce embryonic cell migration. We have evaluated the usefulness of both methods of gene ablation, and conclude that CYP11A1 and CLIP1 function in the same pathway to promote embryogenesis.