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Background: Biosynthesis of 17β-estradiol (E2) is a crucial ovarian function in mammals, which is essential for follicular development and pregnancy outcome. Exploring the epigenetic regulation of E2 synthesis is beneficial for maintaining ovary health and the optimal reproductive traits. NORFA is the first validated sow fertility-associated long non-coding RNA (lncRNA). However, its role on steroidogenesis is elusive. The aim of this study is to investigate the regulation and underlying mechanism of NORFA to E2 synthesis in sow granulosa cells (GCs)., Results: Through Pearson correlation analysis and comparative detection, we found that NORFA expression was positively correlated with the levels of pregnenolone (PREG) and E2 in follicles, which also exhibited similar alteration patterns during follicular atresia. ELISA was conducted and indicated for the first time that NORFA induced the synthesis of PREG and E2 in sow GCs in a dose- and time-dependent manner. RNA-seq, GSEA and quantitative analyses results validated that CYP11A1, the coding gene of P450SCC which is the first step rate-limiting enzyme of E2 synthesis, was a positive functional target of NORFA. Mechanistically, NORFA promotes SF-1 expression by stabilizing NR5A1 mRNA through directly interacting with its 3'-UTR, and also tethers SF-1 to shuttle into nucleus. Additionally, SF-1 in the nucleus activates CYP11A1 transcription by directly binding to its promoter, which ultimately induces E2 synthesis and inhibits GC apoptosis., Conclusion: Our findings highlight that NORFA, a multifunctional lncRNA, induces E2 synthesis and inhibits GC apoptosis through the SF-1/CYP11A1 axis in a ceRNA-independent manner, which provide valuable clues and potential targets for follicular atresia inhibition and female fertility improvement., Competing Interests: Declarations Ethics approval and consent to participate All the animal-involved experiments in this study were conducted in accordance with the Chinese guidelines for administration of affairs concerning experimental animals and were reviewed, approved, and supervised by the Institutional Animal Ethics Committee of Nanjing Agricultural University (NJAU.No20220324059). Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The development of ovarian follicles in poultry is a key factor affecting the performance of egg production. Ovarian follicle development is regulated via the Wnt/β-catenin signaling pathway, and β-catenin, encoded by CTNNB1, is a core component of this pathway. In this study, using ovary GCs from laying hens, we investigated the regulatory role of CTNNB1 in steroid synthesis. We found that CTNNB1 significantly regulates the expression of StAR and CYP11A1 (key genes related to progesterone synthesis) and the secretion of progesterone (P4). Furthermore, simultaneous overexpression of CTNNB1 and SF1 resulted in significantly higher levels of CYP11A1 and secretion of P4 than in cells overexpressing CTNNB1 or SF1 alone. We also found that in GCs overexpressing SF1 , levels of CYP11A1 and secreted P4 were significantly greater than in controls. Silencing of CYP11A1 resulted in the inhibition of P4 secretion while overexpression of SF1 in CYP11A1- silenced cells restored P4 secretion to normal levels. Together, these results indicate that synergistic cooperation between the β-catenin and SF1 regulates progesterone synthesis in laying hen ovarian hierarchical granulosa cells to promote CYP11A1 expression.

Steroidogenesis occurs locally in peripheral tissues and via adrenal and gonadal glands' biosynthesis. The C2C12 mouse myoblast cell line and rat skeletal muscles harbor a local steroidogenesis pathway for glucocorticoids, and corticosterone is biosynthesized from skeletal muscle cells. However, Cyp11a1 and StAR protein expressions are not observed in C2C12 cells or rat muscular tissues. In this context, this study investigated the relationship between DNA methylation and key steroidogenic genes. Bioinformatics analysis of methylated DNA immune precipitation showed that C2C12 myoblasts and myotubes did not have remarkable DNA methylated regions in the gene-body of Cyp11a1. However, a highly methylated region in the CpG island was detected in the intronic enhancer of Ad4BP/SF-1, known as the transcriptional factor for steroidogenic genes. After C2C12 myoblasts treatment with 5-aza-2-deoxycytidine, the gene expressions of Ad4BP/SF-1, Cyp11a1, and StAR were significantly time- and concentration-dependent upregulated. To clarify the contribution of Ad4BP/SF-1 on Cyp11a1 and StAR transcripts, we silenced Ad4BP/SF-1 during the 5-aza-2-deoxycytidine treatment in C2C12 myoblasts, resulting in significant suppression of both Cyp11a1 and StAR. Additionally, pregnenolone levels in the supernatants of C2C12 cells were enhanced by 5-aza-2-deoxycytidine treatment, whereas pregnenolone production by C2C12 myoblasts was significantly suppressed by Ad4BP/SF-1 knockdown. These results indicate that DNA methylation of Ad4BP/SF-1 might be involved in the downregulation of steroidogenic genes, such as Cyp11a1 and StAR in C2C12 myoblasts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alterations in steroid hormone regulation have been implicated in the etiology and progression of autism spectrum disorders (ASD), with the enzyme cytochrome P450 family 11 subfamily A member 1 (CYP11A1)-a key catalyst in cholesterol side-chain cleavage, prominently expressed in the adrenal glands, ovaries, testes, and placenta-standing at the forefront of these investigations. The potential link between aberrations in placental Cyp11a1 expression and the resultant neurodevelopmental disorders, along with the mechanisms underpinning such associations, remains inadequately delineated. In this study, we employed a placental trophoblast-specific Cyp11a1 Hipp11 (H11) knock-in murine model to dissect the phenotypic manifestations within the placenta and progeny, thereby elucidating the underlying mechanistic pathways. Behavioral analyses revealed a diminution in social interaction capabilities alongside an augmented anxiety phenotype, as evidenced by open field and elevated plus maze assessments; both phenotypes were ameliorated after vitamin D3 supplementation. Electrophysiological assays underscored the augmented inhibition of paired-pulse facilitation, indicating impaired neuroplasticity in Cyp11a1 H11-modified mice. An elevation in progesterone concentrations was noted, alongside a significant upregulation of Th1-related cytokines (IL-6 and TNFα) across the plasma, placental, and frontal cortex-a pathological state mitigable through vitamin D3 intervention. Western blotting revealed a vitamin D-mediated rectification of vitamin D receptor and PGC-1α expression dysregulations. Immunofluorescence assays revealed microglial activation in the knock-in model, which was reversible upon vitamin D3 treatment. In conclusion, Cyp11a1 overexpression in the placenta recapitulated an autism-like phenotype in murine models, and vitamin D3 administration effectively ameliorated the resultant neurobehavioral and neuroinflammatory derangements. This study substantiates the application of Cyp11a1 as a biomarker in prenatal diagnostics and posits that prenatal vitamin D3 supplementation is a viable prophylactic measure against perturbations in steroid hormone metabolism associated with ASD pathogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The first and rate-limiting step of steroid hormone biosynthesis is catalysed by mitochondrial cytochrome P450 side-chain cleavage enzyme (CYP11A1). CYP11A1 deficiency is commonly associated with adrenal insufficiency and, in 46 XY individuals, with variable degrees of differences in sex development (DSD). Here, we present a case report of a preadolescent male who presented to our emergency outpatient department in a state of decompensated shock necessitating ionotropic support. Further evaluation confirmed primary adrenal insufficiency. Subsequent clinical exome sequencing uncovered a compound heterozygous mutation in exons one and five of the CYP11A1 gene. This case highlights the varied presentation of CYP11A1 deficiency, showing that it can present as adrenal insufficiency without DSD., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

The ovarian KGN granulosa-like tumour cell line is commonly used as a model for human granulosa cells, especially since it produces steroid hormones. To explore this further, we identified genes that were differentially expressed by KGN cells compared to primary human granulosa cells using three public RNA sequence datasets. Of significance, we identified that the expression of the antioxidant gene TXNRD1 (thioredoxin reductase 1) was extremely high in KGN cells. This is ominous since cytochrome P450 enzymes leak electrons and produce reactive oxygen species during the biosynthesis of steroid hormones. Gene Ontology (GO) analysis identified steroid biosynthetic and cholesterol metabolic processes were more active in primary granulosa cells, whilst in KGN cells, DNA processing, chromosome segregation and kinetochore pathways were more prominent. Expression of cytochrome P450 cholesterol side-chain cleavage (CYP11A1) and cytochrome P450 aromatase (CYP19A1), which are important for the biosynthesis of the steroid hormones progesterone and oestrogen, plus their electron transport chain members (FDXR, FDX1, POR) were measured in cultured KGN cells. KGN cells were treated with 1 mM dibutyryl cAMP (dbcAMP) or 10 μM forskolin, with or without siRNA knockdown of TXNRD1. We also examined expression of antioxidant genes, H2O2 production by Amplex Red assay and DNA damage by γH2Ax staining. Significant increases in CYP11A1 and CYP19A1 were observed by either dbcAMP or forskolin treatments. However, no significant changes in H2O2 levels or DNA damage were found. Knockdown of expression of TXNRD1 by siRNA blocked the stimulation of expression of CYP11A1 and CYP19A1 by dbcAMP. Thus, with TXNRD1 playing such a pivotal role in steroidogenesis in the KGN cells and it being so highly overexpressed, we conclude that KGN cells might not be the most appropriate model of primary granulosa cells for studying the interplay between ovarian steroidogenesis, reactive oxygen species and antioxidants., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: The objective of this study was to investigate the allele frequencies of polymorphisms in genes CYP11A1 rs4886595 and CYP11A1 rs4887139 that are responsible for the steroidogenesis mechanism in polycystic ovary syndrome patients and control females., Methods: Samples were obtained from the Department of Obstetrics and Gynecology in the Near East University Hospital from September 2019 to December 2019. Only the nonobese patients between the ages of 18-40 years were included in this study following informed consent. Obese patients and patients more than 40 years of age were excluded from the study. Nonobese women and normal ovulation were included in the control group. DNA was isolated from blood samples. Real-time polymerase chain reaction (PCR) was used to analyze single nucleotide polymorphisms (SNPs) in various genes linked to polycystic ovary syndrome. The studies were carried out using the samples obtained from 120 women, of whom 55 were nonobese and had normal ovulation, and 65 were polycystic ovary syndrome patients. The allelic frequencies of SNPs in genes linked to polycystic ovary syndrome were calculated using real-time PCR outcomes., Results: The variation of the CYP11A1 rs4887139 G>A did not show any significance, while the variation of CYP11A1 rs4886595 C>A showed significant differences between the patient and the control groups (p=0.01), respectively., Conclusion: Future research ought to focus on elucidating the susceptible causes of polycystic ovary syndrome with a wide range of SNPs and more sample size. The genome-wide association studies in polycystic ovary syndrome patients of different origin will be important to identify candidate genes as well as proteins that are implied in polycystic ovary syndrome risk.

Polycystic ovary syndrome (PCOS), a prevalent reproductive disorder in women of reproductive age, features androgen excess, ovulatory dysfunction, and polycystic ovaries. Despite its high prevalence, specific pharmacologic intervention for PCOS is challenging. In this study, we identified artemisinins as anti-PCOS agents. Our finding demonstrated the efficacy of artemisinin derivatives in alleviating PCOS symptoms in both rodent models and human patients, curbing hyperandrogenemia through suppression of ovarian androgen synthesis. Artemisinins promoted cytochrome P450 family 11 subfamily A member 1 (CYP11A1) protein degradation to block androgen overproduction. Mechanistically, artemisinins directly targeted lon peptidase 1 (LONP1), enhanced LONP1-CYP11A1 interaction, and facilitated LONP1-catalyzed CYP11A1 degradation. Overexpression of LONP1 replicated the androgen-lowering effect of artemisinins. Our data suggest that artemisinin application is a promising approach for treating PCOS and highlight the crucial role of the LONP1-CYP11A1 interaction in controlling hyperandrogenism and PCOS occurrence.

Steroid metabolism is a fundament to testicular development and function. The cytochrome P450, family 11, subfamily A, polypeptide 1 ( CYP11A1 ) is a key rate-limiting enzyme for catalyzing the conversion of cholesterol to pregnenolone. However, despite its importance, what expression and roles of CYP11A1 possesses and how it regulates the testicular development and spermatogenesis in Tibetan sheep remains largely unknown. Based on this, we evaluated the expression and localization patterns of CYP11A1 in testes and epididymides of Tibetan sheep at three developmental stages (three-month-old, pre-puberty; one-year-old, sexual maturity and three-year-old, adult) by quantitative real-time PCR (qPCR), western blot and immunofluorescence. The results showed that CYP11A1 mRNA and protein were expressed in testes and epididymides throughout the development stages and obviously more intense in one- and three-year-old groups than three-month-old group (except for the caput epididymidis). Immunofluorescence assay showed that the CYP11A1 protein was mainly located in Leydig cells and epididymal epithelial cells. In addition, positive signals of CYP11A1 protein were observed in germ cells, epididymal connective tissue and sperms stored in the epididymal lumen. Collectively, these results suggested that the CYP11A1 gene might be mainly involved in regulating spermatogenesis and androgen synthesis in developmental Tibetan sheep testis and epididymis.

Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Due to the ubiquitous presence of PFOA in the environment, the impacts of PFOA exposure not only affect human reproductive health but may also affect livestock reproductive health. The focus of this study was to determine the effects of PFOA on the physiological functions of bovine granulosa cells in vitro. Primary bovine granulosa cells were exposed to 0, 4, and 40 μM PFOA for 48 and 96 h followed by analysis of granulosa cell function including cell viability, steroidogenesis, and mitochondrial activity. Results revealed that PFOA inhibited steroid hormone secretion and altered the expression of key enzymes required for steroidogenesis. Gene expression analysis revealed decreases in mRNA transcripts for CYP11A1, HSD3B, and CYP19A1 and an increase in STAR expression after PFOA exposure. Similarly, PFOA decreased levels of CYP11A1 and CYP19A1 protein. PFOA did not impact live cell number, alter the cell cycle, or induce apoptosis, although it reduced metabolic activity, indicative of mitochondrial dysfunction. We observed that PFOA treatment caused a loss of mitochondrial membrane potential and increases in PINK protein expression, suggestive of mitophagy and mitochondrial damage. Further analysis revealed that these changes were associated with increased levels of reactive oxygen species. Expression of autophagy related proteins phosphoULK1 and LAMP2 were increased after PFOA exposure, in addition to an increased abundance of lysosomes, characteristic of increased autophagy. Taken together, these findings suggest that PFOA can negatively impact granulosa cell steroidogenesis via mitochondrial dysfunction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

The process of steroidogenesis plays a vital role in human physiology as it governs the biosynthesis of mineralocorticoids, glucocorticoids, and androgens. These three classes of steroid hormones are primarily produced in the adrenal and gonadal glands through steroidogenesis pathways. Initiated by the side chain cleavage of cholesterol (CLR), this process leads to the conversion of cholesterol into pregnenolone and isocaproic aldehyde. The enzyme CYP11A1, encoded by the CYP11A1 gene, plays a key role in catalyzing the side chain cleavage of CLR. Several single nucleotide polymorphisms (SNPs) have been identified in the CYP11A1 gene, which may predispose carriers to disorders associated with abnormal steroidogenesis. Specifically, missense SNPs in the CYP11A1 gene have the potential to negatively impact the interaction between CYP11A1 and CLR, thus affecting the overall metabolome of steroid hormones. In this computational study, we focused on a specific set of missense SNPs reported in the CYP11A1 gene, aiming to identify variants that directly impact the interaction between CYP11A1 and CLR. The three-dimensional structure of the CYP11A1-CLR complex was obtained from the RCSB Protein Data Bank, while missense SNPs in the CYP11A1 gene were retrieved from Ensembl. To predict the most deleterious variants, we utilized the ConSurf server, SIFT, and PolyPhen. Furthermore, we assessed the impact of induced amino acid (AA) substitutions on the CYP11A1-CLR interaction using the PRODIGY server, PyMol, and Ligplot programs. Additionally, molecular dynamics (MD) simulations were conducted to analyze the effects of deleterious variants on the structural dynamics of the CYP11A1-CLR complex. Among the 8096 retrieved variants, we identified ten missense SNPs (E91K, W147G, R151W, R151Q, S391C, V392M, Q395K, Q416E, R460W, and R460Q) as deleterious for the interaction between CYP11A1 and CLR. MD simulations of the CYP11A1-CLR complexes carrying these deleterious AA substitutions revealed that Q416E, W147G, R460Q, and R460W had the most pronounced impacts on the structural dynamics of the complex. Consequently, these missense SNPs were considered the most deleterious ones. Further functional tests are recommended to assess the impact of these four missense SNPs on the enzymatic activity of CYP11A1. Moreover, Genome-Wide Association Studies (GWAS) should be conducted to determine the significance of their association with abnormal steroidogenesis diseases in various patient groups., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Neurosteroids, which are steroids synthesized by the nervous system, can exert neuromodulatory and neuroprotective effects via genomic and nongenomic pathways. The neurosteroid and major steroid precursor pregnenolone has therapeutical potential in various diseases, such as psychiatric and pain disorders, and may play important roles in myelination, neuroinflammation, neurotransmission, and neuroplasticity. Although pregnenolone is synthesized by CYP11A1 in peripheral steroidogenic organs, our recent study showed that pregnenolone must be synthesized by another mitochondrial cytochrome P450 (CYP450) enzyme other than CYP11A1 in human glial cells. Therefore, we sought to identify the CYP450 responsible for pregnenolone production in the human brain. Upon screening for CYP450s expressed in the human brain that have mitochondrial localization, we identified three enzyme candidates: CYP27A1, CYP1A1, and CYP1B1. We found that inhibition of CYP27A1 through inhibitors and siRNA knockdown did not negatively affect pregnenolone synthesis in human glial cells. Meanwhile, treatment of human glial cells with CYP1A1/CYP1B1 inhibitors significantly reduced pregnenolone production in the presence of 22(R)-hydroxycholesterol. We performed siRNA knockdown of CYP1A1 or CYP1B1 in human glial cells and found that only CYP1B1 knockdown significantly decreased pregnenolone production. Furthermore, overexpression of mitochondria-targeted CYP1B1 significantly increased pregnenolone production under basal conditions and in the presence of hydroxycholesterols and low-density lipoprotein. Inhibition of CYP1A1 and/or CYP1B1 via inhibitors or siRNA knockdown did not significantly reduce pregnenolone synthesis in human adrenal cortical cells, implying that CYP1B1 is not a major pregnenolone-producing enzyme in the periphery. These data suggest that mitochondrial CYP1B1 is involved in pregnenolone synthesis in human glial cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In the present study, granulosa cells (GCs) from domestic cats and Persian leopard were cultured and characterized from selected days. The culture period was divided into two phases: maintenance, which lasted for 7 days, and luteinization, which followed for up to 11 days. Luteinization was performed on ultra-low attachment plates, supporting the formation of spheroids in a medium supplemented with insulin, forskolin, and luteinizing hormone (LH). GCs of domestic cat produced estradiol (E2) and progesterone (P4) during the maintenance phase. The gene expressions of some proteins involved in steroidogenesis were stable (STAR, HSD3B1) or decreased over time (CYP11A1, HSD17B1, CYP17A1, and CYP19A1), which was similar to the expressions of gonatropin receptors (LHCGR and FSHR). During the luteinization phase, P4 concentration significantly increased (P < 0.05), and E2, in contrast to the proliferation phase, was below detection range. The expression of genes of proteins involved in steroidogenesis (STAR, CYP11A1, HSD3B1, HSD17B1, CYP17A1, and CYP19A1) and of gonadotropin receptors (LHCGR and FSHR) significantly increased during the luteinization period, but some expressions exhibited a decrease at the end of the phase (LHCGR, FSHR, HSD17B1, CYP19A1). The morphology of the luteinized GCs of domestic cat resembled large luteal cells and had numerous vacuole-like structures. Also, the GCs of Persian leopard underwent luteinization, shown by increasing P4 production and HSD3B1 expression. This study confirms that GCs from felids can be luteinized in a 3D spheroid system which can be a basis for further studies on luteal cell function of felids. Additionally, we could show that the domestic cat can serve as a model species for establishing cell culture methods which can be transferred to other felids., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that can be perceived as prejudicing the impartiality of the research reported., (Copyright © 2023 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Steroid drugs, the second largest class of pharmaceuticals after antibiotics, have shown significant anti-inflammatory, anti-allergic, and endocrine-regulating effects. A group of cytochrome P450 enzymes, namely, CYP11A1 isoenzymes from different organisms are capable of converting cholesterol into pregnenolone, which is a pivotal reaction in both steroid metabolism and (bio)synthetic network of steroid products. However, the low activity of CYP11A1s greatly restricts the industrial application of these cholesterol side-chain cleavage enzymes. Herein, we investigate ten CYP11A1 enzymes of different origins and in vitro characterize two CYP11A1s with a relatively higher expression level from Capra hircus and Sus scrofa, together with the CYP11A1s from Homo sapiens and Bos taurus as references. Towards five selected sterol substrates with different side chain structures, S. scrofa CYP11A1 displays relatively higher activities. Through redox partners combination screening, we reveal the optimal redox partner pair of S. scrofa adrenodoxin and C. hircus adrenodoxin reductase. Moreover, the semi-rational mutagenesis for the active sites and substrate entrance channels of human and bovine CYP11A1s is performed based on comparative analysis of their crystal structures. The mutant mBtCYP11A1-Q377A derived from mature B. taurus CYP11A1 shows a 1.46 times higher activity than the wild type enzyme. These results not only demonstrate the tunability of the highly conserved CYP11A1 isoenzymes, but also lay a foundation for the following engineering efforts on these industrially relevant P450 enzymes., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Context: Bushen Yiyuan recipe (BYR) is an effective Chinese prescription with antifatigue and antioxidation effects., Objective: The effects of BYR on testosterone synthesis in rat Leydig cells with exercise-induced low serum testosterone levels (EILST) are assessed., Materials and Methods: Thirty-two Sprague-Dawley rats were chronically trained for 6 weeks to establish an EILST model. EILST rats were divided into model (physiological saline), EFE (700 mg/kg ethanol extract of Epimedii folium , the dried leaves of Epimedium brevicornu Maxim [Berberidaceae]), and BYR groups (350 and 700 mg/kg) for 6 weeks. Expression of HMG-CoA, LDL-R, SR-BI, STAR and CYP11A1 were quantified by RT qPCR and Western blots., Results: Compared with the model group (115.52 ± 13.05 μg/dL; 67.83 ± 14.29; 0.32 ± 0.04; 0.33 ± 0.02; 0.38 ± 0.01), serum testosterone, testosterone/cortisol ratio, HMG-CoA, STAR and CYP11A1 relative protein expression significantly increased in low-dose BYR (210.60 ± 5.08 μg/dL; 119.38 ± 13.02; 0.47 ± 0.01; 0.46 ± 0.03; 0.46 ± 0.02), high-dose BYR (220.57 ± 14.71 μg/dL; 124.26 ± 14.79; 0.49 ± 0.02; 0.42 ± 0.03; 0.51 ± 0.02), and EFE groups (206.83 ± 5.54 μg/dL; 119.53 ± 25.04; 0.45 ± 0.02; 0.42 ± 0.02; 0.41 ± 0.02) (all p  < 0.01, except for CYP11A1 in EFE group). HMG-CoA, STAR and CYP11A1 mRNA relative expression significantly increased in low-dose and high-dose BYR group compared to model group (all p  < 0.01)., Conclusions: BYR affects endogenous cholesterol synthesis and testosterone synthesis to prevent and treat EILST levels in rats. It can improve the body's sports ability.

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: CYP11A1 is a protein located in the inner membrane of mitochondria catalyzing the first step of steroid synthesis. As a marker gene for steroid-producing cells, the abundance of CYP11A1 characterizes the extent of steroidogenic cell differentiation. Besides, the mitochondria of fully differentiated steroidogenic cells are specialized with tubulovesicular cristae. The participation of CYP11A1 in the change of mitochondrial structure and the differentiation of steroid-producing cells, however, has not been investigated., Methods: We engineered nonsteroidogenic monkey kidney COS1 cells to express CYP11A1 upon doxycycline induction and examined the mitochondrial structure of these cells. We also mapped the CYP11A1 domains that confer structural changes of mitochondria. We searched for CYP11A1-interacting proteins and investigated the role of this interacting protein in shaping mitochondrial structure. Finally, we examined the effect of CYP11A1 overexpression on the amount of mitochondrial contact site and cristae organizing system., Results: We found that CYP11A1 overexpression led to the formation of tubulovesicular cristae in mitochondria. We also identified the A'-helix located at amino acid #57-68 to be sufficient for membrane insertion and crista remodeling. We identified heat shock protein 60 (Hsp60) as the CYP11A1-interacting protein and showed that Hsp60 is required for CYP11A1 accumulation and crista remodeling. Finally, we found that the small MIC10 subcomplex of the mitochondrial contact site and cristae organizing system was reduced when CYP11A1 was overexpressed., Conclusions: CYP11A1 participates in the formation of tubulovesicular cristae in the mitochondria of steroidogenic cells. Its A'-helix is sufficient for the formation of tubulovesicular cristae and for protein integration into the membrane. CYP11A1 interacts with Hsp60, which is required for CYP11A1 accumulation. The accumulation of CYP11A1 leads to the reduction of MIC10 complex and changes mitochondrial structure., (© 2022. The Author(s).)

An eight-year old South Asian boy presenting with progressive hyperpigmentation was found to have primary adrenal insufficiency (PAI) in the form of isolated glucocorticoid deficiency. Follow up of this boy for nine years, until the age of 17 years showed normal pubertal onset and progression. Molecular evaluation, by targeted next generation sequencing of candidate genes linked to PAI revealed changes in two genes that are intricately linked in the early stages of steroid biosynthesis: compound heterozygous variants in STAR , c.465+1G>A and p.(E99K), plus a heterozygous rs6161 change in CYP11A1 . No variants in other known causal genes were detected. The proband's mother was heterozygous for the c.465+1G>A STAR and rs6161 CYP11A1 variants, while the father was homozygous for the p.(E99K) alteration in STAR but wild-type for CYP11A1 . Both parents had normal adrenal cortical function as revealed by short Synacthen tests. The STAR variant c.465+1G>A will lead to abnormal splicing of exon 4 in mRNA and the addition of the p.(E99K) variant, predicted damaging by SIFT and CADD, may be sufficient to cause PAI but this is by no means certain given that the unaffected father is homozygous for the latter change. The rs6161 CYP11A1 variant [c.940G>A, p.(E314K)] has recently been demonstrated to cause PAI in conjunction with a severe rare disruptive change on the other allele, however sequencing of the coding region of CYP11A1 revealed no further changes in this subject. We wondered whether the phenotype of isolated glucocorticoid deficiency had arisen in this child due to tri-allelic inheritance of a heterozygous CYP11A1 change along with the two STAR variants each of which contribute a partial loss-of-function burden that, when combined, is sufficient to cause PAI or if the loss-of-function c.465+1G>A combined with the presumed partial loss-of-function p.(E99K) in STAR could be causative., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ali, Maharaj, Buonocore, Achermann and Metherell.)

Cytochrome P45011A1, encoded by Cyp11a1, converts cholesterol to pregnenolone (P5), the first and rate-limiting step in steroidogenesis. In zebrafish, cyp11a1 is maternally expressed and cyp11a2 is considered the ortholog of Cyp11a1 in mammals. A recent study has shown that depletion of cyp11a2 resulted in steroidogenic deficiencies and the mutants developed into males with feminized secondary sexual characteristics. Here, we independently generated cyp11a2 mutants in zebrafish and showed that the mutants can develop into males and females in the juvenile stage, but finally into infertile males with defective mating behavior in the adult stage. In the developing ovaries, the cyp11a2 mutation led to stage I oocyte apoptosis and final sex reversal, which could be partially rescued by treatment with P5 but not estradiol. In the developing testes, depletion of cyp11a2 resulted in dysfunction of Sertoli cells and lack of functional Leydig cells. Spermatogonial stem cells (SSCs) in the mutant testes underwent active self-renewal but no differentiation, resulting in a high abundance of SSCs in the testis, as revealed by immunofluorescence staining with Nanos2 antibody. The high abundance and differentiation competence of SSCs in the mutant testes were verified by a novel testicular cell transplantation method developed in this study, by transplanting mutant testicular cells into germline-depleted wild-type (WT) fish. The transplanted mutant SSCs efficiently differentiated into functional spermatids in WT hosts. Overall, our study demonstrates the functional importance of cyp11a2 in early oogenesis and differentiation of SSCs., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glucose is a major energy source for growth. At birth, neonates must change their energy source from maternal supply to its own glucose production. The mechanism of this transition has not been clearly elucidated. To evaluate the possible roles of steroids in this transition, here we examine the defects associated with energy production of a mouse line that cannot synthesize steroids de novo due to the disruption of its Cyp11a1 (cytochrome P450 family 11 subfamily A member 1) gene. The Cyp11a1 null embryos had insufficient blood insulin and failed to store glycogen in the liver since embryonic day 16.5. Their blood glucose dropped soon after maternal deprivation, and the expression of hepatic gluconeogenic and glycogenic genes were reduced. Insulin was synthesized in the mutant fetal pancreas but failed to be secreted. Maternal glucocorticoid supply rescued the amounts of blood glucose, insulin, and liver glycogen in the fetus but did not restore expression of genes for glycogen synthesis, indicating the requirement of de novo glucocorticoid synthesis for glycogen storage. Thus, our investigation of Cyp11a1 null embryos reveals that the energy homeostasis is established before birth, and fetal steroids are required for the regulation of glycogen synthesis, hepatic gluconeogenesis, and insulin secretion at the fetal stage., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mitochondrial cytochromes P450 presumably originated from a common microsomal P450 ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation to the mitochondrial matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used the approach of ancestral sequence reconstruction (ASR) to resurrect ancient CYP11A1 enzymes and characterize their unique biochemical properties. Two ancestral CYP11A1 variants, CYP11A&#95;Mammal&#95;N101 and CYP11A&#95;N1, as well as an extant bovine form were recombinantly expressed and purified to homogeneity. All enzymes showed characteristic P450 spectral properties and were able to convert cholesterol as well as other sterol substrates to pregnenolone, yet with different specificities. The vertebrate CYP11A&#95;N1 ancestor preferred the cholesterol precursor, desmosterol, as substrate suggesting a convergent evolution of early cholesterol metabolism and CYP11A1 enzymes. Both ancestors were able to withstand increased levels of hydrogen peroxide but only the ancestor CYP11A&#95;N1 showed increased thermostability (˜ 25 °C increase in T 50 ) compared with the extant CYP11A1. The extraordinary robustness of ancient mitochondrial P450s, as demonstrated for CYP11A&#95;N1, may have allowed them to stay active when presented with poorly compatible electron transfer proteins and resulting harmful ROS in the new environment of the mitochondrial matrix. To the best of our knowledge, this work represents the first study that describes the resurrection of ancient mitochondrial P450 enzymes. The results will help to understand and gain fundamental functional insights into the evolutionary origins of steroid hormone biosynthesis in animals., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The most common multifactorial endocrine disorder in females of reproductive age is polycystic ovary syndrome (PCOS), affecting about 5-10% of females worldwide and 9.3% of females in India. Androgen excess in PCOS is caused as a result of defects in steroidogenesis genes. CYP11A1 is an imperative marker in the steroid synthesis pathway, and the altered expression of CYP11A1 has been reported to disrupt the synthesis of steroids and hence conferring risk for the development of PCOS. The present study aimed to analyze genetic variants (rs11632698, rs4077582, rs4887139) of CYP11A1 with PCOS from North India. The study included 270 PCOS females diagnosed according to Rotterdam 2003 criteria and 270 age-matched healthy non-PCOS females. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for the genotypic analysis of the selected genetic variants. Association analysis of biochemical parameters (cholesterol, triglyceride, high-density lipoprotein) and anthropometric measurements with PCOS cases was done. The genetic variants of CYP11A1 (rs11632698, rs4077582, and rs4887139) demonstrated significant association with PCOS cases (p=1.0E-12, p=3.0E-3, p=1.0E-2, respectively). Binary logistic regression revealed that the dominant model of rs11632698 conferred 2.0 risk, and dominant as well as the co-dominant model of rs4887139 conferred risk of 2.2 and 2.4 fold, respectively, towards the progression of PCOS. The overall mean triglyceride levels were elevated, and mean HDL levels were lower in PCOS cases as compared to threshold values. The significant association of studied genetic variants suggested the important role of CYP11A1 in susceptibility to PCOS. The study was the first of its kind from our region and provided baseline data of genetics of PCOS., (© 2021. Society for Reproductive Investigation.)

Background: One of the causes of congenital adrenal insufficiency, a genetically heterogeneous disorder is a mutation in the CYP11A1 gene, which is responsible for the initiation of steriodogenesis by converting cholesterol to pregnenolone. Case: In a now 3 years and 3 months-old girl, adrenal insufficiency was diagnosed in the neonatal period. Clinical exome sequencing for primary adrenal insufficiency revealed a homozygous p.Thr330Met (c.989C>T) variant in the CYP11A1 (NM&#95;000781) gene. Conclusion: Different types of inheritance patterns have been observed in CYP11A1 -related adrenal insufficiency cases. We consider our case is an due to an autosomal recessive inheritance.

Musk secreted by Chinese forest musk deer (FMD; Moschus berezovskii) is a highly valuable ingredient in the fields of perfumery and medicine, and the main factor affecting the production of musk is the androgen level of male FMD. To clarify whether the musk gland of FMD can synthesize androgen, we compared and analyzed the expression patterns of steroid hormone biosynthesis-related genes in the musk gland and testis of FMD by RNA-seq and RT-qPCR. We obtained 33,308 and 38,602 unigenes from the musk gland and testis, respectively, and 26,780 co-expressed unigenes. Analysis of co-expressed genes revealed that 12,647 genes were annotated to 11,484 Gene Ontology terms and 10,941 genes were annotated to 6120 pathways, including several pathways important in metabolic and synthetic activity. Next, 21 steroid hormone synthesis-related genes were screened from the transcriptome of the musk gland of 4-month-old FMD. The expression levels of three key genes of steroid hormone biosynthesis (CYP11A1, CYP17A1, and HSD3B) in the musk gland differed from their expression levels in the testis based on RT-qPCR. Furthermore, immunohistochemistry indicated that CYP11A1, CYP17A1, and HSD3B were localized in the glandular tubular columnar cells of the musk gland. These results suggested that the musk gland of male FMD has the potential to locally synthesize steroid hormone and thus plays a critically important role in musk secretion., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Our present knowledge on interrelation between morphology/ultrastructure of mitochondria of the Leydig cell and its steroidogenic function is far from satisfactory and needs additional studies. Here, we analyzed the effects of blockade of androgen receptor, triggered by exposure to flutamide, on the expression of steroidogenic proteins (1) and ultrastructure of Leydig cells' constituents (2). We demonstrated that increase in the expression level of steroidogenic (StAR, CYP11A1, 3β-HSD, and CYP19A1) proteins (and respective mRNAs) in rat testicular tissue as well as elevation of intratesticular sex steroid hormone (testosterone and estradiol) levels observed in treated animals correspond well to morphological alterations of the Leydig cell ultrastructure. Most importantly, up-regulation of steroidogenic proteins' expression apparently correlates with considerable multiplication of Leydig cell mitochondria and subsequent formation of local mitochondrial networks. Interestingly, we showed also that the above-mentioned processes were associated with elevated transcription of Drp1 and Mfn2 genes, encoding proteins implicated in mitochondrial dynamics. Collectively, our studies emphasize the importance of mitochondrial homeostasis to the steroidogenic function of Leydig cells.

Obese men have lower circulating testosterone than men with an optimal body mass index. Elevated fatty acids (FAs) caused by obesity have been reported to suppress the steroidogenesis of Leydig cells. Recent studies have demonstrated that autophagy regulates steroidogenesis in endocrine cells; however, few studies have investigated the molecular mechanisms of FA-impaired steroidogenesis. To study FA regulation in the steroidogenesis of Leydig cells, MA-10 cells were treated with an FA mixture and co-treated with 8-Br-cAMP to stimulate the steroidogenesis capacity. We showed that FAs led to cellular lipid accumulation and decreased steroidogenesis of MA-10 cells, and FA-suppressed steroidogenesis was largely recovered by P5 treatment but not by 22R-OHC treatment, suggesting the primary defect was the deficiency of CYP11A1. To examine the involvement of autophagy in the steroidogenesis of Leydig cells, we treated MA-10 cells with autophagy regulators, including rapamycin, bafilomycin, and chloroquine. Inhibition of late-stage autophagy including FA-upregulated Rubicon suppressed the steroidogenesis of MA-10 cells. More interestingly, Rubicon played a novel regulatory role in the steroidogenesis of MA-10 cells, independent of inhibitors of late-stage autophagy. Collectively, this study provides novel targets to investigate the interaction between FAs and steroidogenesis in steroidogenic cells.

CYP11A1, a member of the cytochrome P450 family, plays several key roles in the human body. It catalyzes the first and rate-limiting step in steroidogenesis, converting cholesterol to pregnenolone. Aside from the classical steroidogenic tissues such as the adrenals, gonads and placenta, CYP11A1 has also been found in the brain, gastrointestinal tract, immune systems, and finally the skin. CYP11A1 activity in the skin is regulated predominately by StAR protein and hence cholesterol levels in the mitochondria. However, UVB, UVC, CRH, ACTH, cAMP, and cytokines IL-1, IL-6 and TNFα can also regulate its expression and activity. Indeed, CYP11A1 plays several critical roles in the skin through its initiation of local steroidogenesis and specific metabolism of vitamin D, lumisterol, and 7-dehydrocholesterol. Products of these pathways regulate the protective barrier and skin immune functions in a context-dependent fashion through interactions with a number of receptors. Disturbances in CYP11A1 activity can lead to skin pathology., (Published by Elsevier B.V.)

Steroid metabolism is a fundamental process in the porcine testis to provide testosterone but also estrogens and androstenone, which are essential for the physiology of the boar. This study concerns boars at an early stage of puberty. Using a RT-qPCR approach, we showed that the transcriptional activities of several genes providing key enzymes involved in this metabolism (such as CYP11A1 ) are correlated. Surprisingly, HSD17B3 , a key gene for testosterone production, was absent from this group. An additional weighted gene co-expression network analysis was performed on two large sets of mRNA-seq to identify co-expression modules. Of these modules, two containing either CYP11A1 or HSD17B3 were further analyzed. This comprehensive correlation meta-analysis identified a group of 85 genes with CYP11A1 as hub gene, but did not allow the characterization of a robust correlation network around HSD17B3 . As the CYP11A1-group includes most of the genes involved in steroid synthesis pathways (including LHCGR encoding for the LH receptor), it may control the synthesis of most of the testicular steroids. The independent expression of HSD17B3 probably allows part of the production of testosterone to escape this control. This CYP11A1-group contained also INSL3 and AGT genes encoding a peptide hormone and an angiotensin peptide precursor, respectively.

Mitochondrial cytochrome P450 side-chain cleavage (P450scc), encoded by the cyp11a1 gene, initiates the first step of steroid biosynthesis. In this study, a 1554-bp open reading frame (ORF) of black rockfish (Sebastes schlegelii) cyp11a1 was cloned. The cyp11a1 gene is located on chromosome 5 and has 9 exons. The ORF encodes a putative precursor protein of 517 amino acids, and the predicted cleavable mitochondrial targeting peptide is located at amino acids 1-39. P450scc shares homology with other teleosts and tetrapods, which have relatively conserved binding regions with heme, cholesterol and adrenodoxin. Tissue distribution analysis revealed that the highest expression levels of cyp11a1 were detected in mature gonads and head kidney but that low levels were detected in gestational/regressed ovaries, regressed testes and other tissues. Immunostaining of P450scc was observed in testicular Leydig cells, ovarian theca cells, interrenal glands of head kidney, pituitary and multiple regions of brain. Particularly, two kinds of fish-specific P450scc-positive cells, including coronet cells of brain saccus vasculosus and hypophyseal somatolactin cells, were identified in black rockfish. Our results provide novel evidence for the potential role played by P450scc in reproduction behavior by mediating steroidogenesis in viviparous teleost., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Peripherally derived steroids affect steroid production in the brain via the blood-brain barrier. However, steroid concentrations are lower in the cerebrospinal fluid than those in the blood, indicating restricted influx of steroids because of their metabolization by choroid plexus (CP) epithelial cells. Here, we analyzed the gene expression of steroidogenic enzymes [cholesterol side-chain cleavage enzyme (P450scc), 17α-hydroxylase/C17-C20 lyase (P450c17), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), aromatase (Cyp19a1), and 5α-reductase type 1 (5α-R1)]. These genes were expressed to a lesser extent in the CP than in the testis and to a similar extent in the cerebral cortex. However, P450scc levels were higher in the CP than in the cerebral cortex, whereas Cyp19a1 levels showed the opposite trend. We also evaluated the effects of orchiectomy and testosterone on the expression of these genes. P450c17 and 5α-R1 levels were unaffected by orchiectomy, whereas P450scc and 3β-HSD levels were increased and decreased, respectively. Cyp19a1 expression increased upon testosterone treatment, whereas that of 17β-HSD decreased upon orchiectomy or administration of testosterone. Immunohistochemistry analysis revealed that 17β-HSD was expressed in the cytoplasm of CP epithelial cells. These results indicate that CP epithelial cells synthesize and convert the certain types of steroids to contribute to the homeostasis of steroids in the brain. J. Med. Invest. 68 : 238-243, August, 2021.

The steroidogenesis occurs in specific cells and tissues in the mammals which begins with the transfer and intracellular processing of cholesterol converted to pregnenolone. This study investigated the gene and protein expression levels of steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR) and cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc) in the scent glands of the muskrats during the breeding and non-breeding seasons. The immunohistochemical localizations of StAR and P450scc were identified in the glandular cells and epithelial cells while SF-1 was only expressed in glandular cells during the breeding and non-breeding seasons. The gene and protein expression levels of SF-1, StAR and P450scc in the scent glands were remarkedly higher in the breeding season than those of the non-breeding season. The interaction of micro RNAs (miRNAs) and transcriptome results showed that miR-762 and miR-4454 might be the genes encoding (Nr5a1, Star and Cyp11a1) in key biological processes. Taken together, these results suggested that the scent glands of the muskrats potentially owned ability to synthesize steroid hormones de novo, and the steroid hormones might affect the scent glandular functions of the muskrats during the breeding and non-breeding seasons., (Copyright © 2020. Published by Elsevier Ltd.)

One of the variants of congenital dysfunction of the adrenal cortex is a deficiency of the enzyme P450scc, which catalyzes the first stage of steroidogenesis. This is a rare autosomal recessive disease, the classic manifestation of which is primary adrenal insufficiency with a deficiency of gluco-and mineralocorticoids and a violation of the synthesis of sex steroids, which usually leads to a complete lack of masculinization in patients with karyotype 46, XY and hypergonadotropic hypogonadism in both sexes. Previously, it was suggested That p450scc deficiency is incompatible with the normal course of pregnancy, since the enzyme is expressed in the placenta, where it is necessary for the synthesis of progesterone, the main pregnancy hormone, and, consequently, the birth of a child with A p450scc deficiency is impossible. However, the literature describes clinical cases of p450scc deficiency with partially preserved enzyme function, which explains the normal course of pregnancy. Whereas cases of confirmed p450scc deficiency with zero enzyme activity are unique, not being explained until now. We present a description of severe p450scc deficiency in a child born from a dizygotic twin pregnancy in which the second Sib was healthy. It is possible that the preserved hormonal function of the second placenta and (or) treatment with progesterone analogs during gestation contributed to gestation in this rare form of steroidogenesis disorder.

Activation of the steroidogenic enzyme CYP11A1 was shown to be necessary for the development of peanut-induced intestinal anaphylaxis and IL-13 production in allergic mice. We determined if levels of CYP11A1 in peripheral blood T cells from peanut-allergic (PA) children compared to non-allergic controls were increased and if levels correlated to IL-13 production and oral challenge outcomes to peanut. CYP11A1 mRNA and protein levels were significantly increased in activated CD4+ T cells from PA patients. In parallel, IL-13 production was significantly increased; IFNγ levels were not different between groups. There were significant correlations between expression levels of CYP11A1 mRNA and levels of IL13 mRNA and protein, levels of serum IgE anti-Ara h 2 and to outcomes of peanut challenge. The importance of CYP11A1 on cytokine production was tested using a CYP11A1 CRISPR/Cas9 KO plasmid or an inhibitor of enzymatic CYP11A1 activity. Inhibition of CYP11A1 activation in patient cells treated with the inhibitor, aminoglutethimide, or CD4+ T cell line transfected with the CYP11A1 KO plasmid resulted in reduced IL-13 production. These data suggest that the CYP11A1-CD4+Tcell-IL-13 axis in activated CD4+ T cells from PA children is associated with development of PA reactions. CYP11A1 may represent a novel target for therapeutic intervention in PA children., Competing Interests: The authors have no conflicts of interest to declare.

Exposure to arsenic (AS) causes abnormalities in the reproductive system; however, the precise cellular pathway of AS toxicity on steroidogenesis in developing F1-male mice has not been clearly defined. In this study, paternal mice were treated with arsenic trioxide (As 2 O 3 ; 0, 0.2, 2, and 20 ppm in drinking water) from 5 weeks before mating until weaning and continued for male offspring from weaning until maturity (in vivo). Additionally, Leydig cells (LCs) were isolated from the testes of sacrificed F1-intact mature male mice and incubated with As 2 O 3 (0, 1, 10, and 100 μM) for 48 h (in vitro). Biomarkers of mitochondrial impairment, oxidative stress, and several steroidogenic genes, including the steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleaving enzyme (P450scc; Cyp11a), 3β-hydroxysteroid dehydrogenase (3β-HSD), and 17β-hydroxysteroid dehydrogenase (17β-HSD), were evaluated. High doses of As 2 O 3 interrupted testosterone (T) biosynthesis and T-related gene expression in these experimental models. Altogether, overconsumption of As 2 O 3 can cause testicular and LC toxicity through mitochondrial-related pathways and oxidative stress indices as well as downregulation of androgenic-related genes in mice and isolated LCs. These results could lead to the development of preventive/therapeutic procedures against As 2 O 3 -induced reproductive toxicity. Graphical Abstract Mohammad Mehdi Ommati and Reza Heidari contributed equally to this study.

Bisphenol S (BPS) is a structural analog of the endocrine disruptor bisphenol A (BPA); it is the main BPA replacement in the plastics industry. Previous studies have shown that BPA and BPS exhibit similar effects on reproduction in fish and rodent species. BPS reportedly alters steroidogenesis in bovine granulosa cells. Luteinised granulosa cells collected from 59 women who were undergoing an in vitro fertilization procedure were cultured for 48 h in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). BPS exposure was investigated by assessing follicular fluids from these 59 women for their BPS content. Culture medium, cells, total messenger RNA (mRNA) and total protein extracted from the luteinised granulosa cells were examined for oestradiol and progesterone secretion, cellular proliferation, viability, gene expression, steroidogenic enzyme expression and cell signaling. BPS was measured in follicular fluids using mass spectrometry. Exposure of granulosa cells to 10 or 50 µM BPS for 48 h induced a 16% ( p = 0.0059) and 64% ( p < 0.0001) decrease, respectively, in progesterone secretion; 50 µM BPS decreased oestradiol secretion by 46% ( p < 0.0001). Ten µM BPS also tended to reduce CYP11A1 protein expression by 37% ( p = 0.0947) without affecting HSD3B1 and CYP19A1 expression. Fifty µM BPS increased ERRγ expression. Environmental levels of BPS (nanomolar range) did not induce changes in steroidogenesis in human granulosa cells. The effects of BPS were observed after only 48 h of BPS exposure. These acute effects might be similar to chronic effects of physiological BPS levels.

Background: This study aimed to investigate the roles of CYP3A4 and CYP11A1 variants in ischemic stroke (IS) susceptibility among the Han Chinese population., Methods: Four hundred seventy-seven patients with IS and 493 healthy controls were enrolled. Seven single-nucleotide polymorphisms (SNPs) of CYP3A4 and CYP11A1 were genotyped by Agena MassARRAY. Odds ratio (OR) and 95% confidence intervals (CI) were calculated by logistic regression adjusted for age and gender., Results: We found that CYP3A4 rs3735451 (OR = 0.81, p = 0.039) and rs4646440 (OR = 0.72, p = 0.021) polymorphisms decreased the risk of IS. CYP3A4 rs4646440 (OR = 0.74, p = 0.038) and CYP11A1 rs12912592 (OR = 1.58, p = 0.034) polymorphisms were correlated with IS risk in males. CYP3A4 rs3735451 (OR = 0.63, p = 0.031) and rs4646440 (OR = 0.57, p = 0.012) possibly weaken the IS susceptibility at age > 61 years. Besides, CYP3A4 rs4646437 (OR = 0.59, p = 0.029), CYP11A1 rs12912592 (OR = 1.84, p = 0.017) and rs28681535 (OR = 0.66, p = 0.038) were associated with IS risk at age ≤ 61 years. CYP11A1 rs28681535 TT genotype was higher high-density lipoprotein cholesterol level than the GT and GG genotype (p = 0.027)., Conclusions: Our findings indicated that rs3735451, rs4646440, rs4646437 in CYP3A4 and rs28681535 in CYP11A1 might be protective factors for IS, while CYP11A1 rs12912592 polymorphism be a risk factor for IS in Chinese Han population.

Dimethoate is an organophosphate pesticide. It is widely used in agriculture. However, whether it blocks pubertal development of Leydig cells remains unknown. In the current study, we exposed male Sprague Dawley rats with 7.5 and 15 mg kg -1 dimethoate from postnatal day 35-56. We also exposed Leydig cells isolated from 35-day-old rats for 3 h. Dimethoate reduced serum testosterone levels at 7.5 and 15 mg kg-1 but increased serum luteinizing hormone and follicle stimulating hormone levels at 15 mg kg-1. Dimethoate did not influence Leydig cell number but reduced Leydig cell size and down-regulated Star, Cyp11a1, and Hsd3b1 in Leydig cells as well as their protein expression. Dimethoate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.05 μM. It significantly inhibited luteinizing hormone and 8Br-cAMP stimulated androgen outputs at 50 μM. It significantly inhibited 22R-hydroxycholesterol and progesterone-mediated androgen outputs at 50 μM. Further study demonstrated that dimethoate also down-regulated the expression of Star, Cyp11a1, and Hsd3b1 at 5 or 50 μM in vitro. Dimethoate did not directly inhibit rat testicular steroidogenic enzyme activities at 50 μM. In conclusion, dimethoate targets Star, Cyp11a1, and Hsd3b1 transcription, thus blocking Leydig cell differentiation during puberty., (Copyright © 2019. Published by Elsevier Ltd.)

Cytochrome P450 side-chain cleavage enzyme, encoded by the CYP11A1 gene, catalyzes the first and rate-limiting step of steroid hormone biosynthesis. Previous morpholino-knockdown studies in zebrafish suggested cyp11a2 is a functional equivalent of human CYP11A1 and is essential for interrenal steroidogenesis in zebrafish larvae. The role of Cyp11a2 in adult zebrafish, particularly in gonadal steroidogenesis, remains elusive. To explore the role of Cyp11a2 in adults, we developed zebrafish mutant lines by creating deletions in cyp11a2 using the CRISPR/Cas9 genomic engineering approach. Homozygous cyp11a2 mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal axis. Furthermore, these Cyp11a2-deficient zebrafish demonstrated profound glucocorticoid and androgen deficiencies. Cyp11a2 homozygotes only developed into males with feminized secondary sex characteristics. Adult cyp11a2 -/- mutant fish showed a lack of natural breeding behaviors. Histological characterization revealed disorganized testicular structure and significantly decreased numbers of mature spermatozoa. These findings are further supported by the downregulation of the expression of several pro-male genes in the testes of cyp11a2 homozygous zebrafish, including sox9a, dmrt1 and amh. Moreover, the spermatogonia markers nanos2 and piwil1 were upregulated, while the spermatocytes marker sycp3 and spermatids marker odf3b were downregulated in the testes of cyp11a2 homozygous mutants. Our expression analysis is consistent with our histological studies, suggesting that spermatogonia are the predominant cell types in the testes of cyp11a2 homozygous mutants. Our work thus demonstrates the crucial role of Cyp11a2 in interrenal and gonadal steroidogenesis in zebrafish larvae and adults.

Orexin A (OXA) is primarily known for its involvement in the regulation of feeding behaviour, energy metabolism and sleep/wake cycle. Nevertheless, studies indicate its engagement in the regulation of the porcine reproductive system. Therefore, the aim of this study was to investigate OXA effect (1, 10, 100 nM), in the presence or absence of the selective orexin receptor type 1 antagonist (SB-3348667; 1 μM), on the gene expression of key steroidogenic enzymes: steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B1), as well as on progesterone (P 4 ) and androstenedione (A 4 ) secretion. Endometrial and myometrial tissue explants were collected from gilts on days 10 to 11, 12 to 13, 15 to 16 and 27 to 28 of pregnancy, and on days 10 to 11 of the oestrous cycle (n = 5 per studied period of pregnancy or mid-luteal phase of the oestrous cycle). Gene expression was evaluated by real-time PCR. The level of steroid hormones secreted into the culture medium was examined by radioimmunoassay (RIA). In the present study, in the endometrium, OXA significantly stimulated StAR expression on days 12 to 13, CYP11A1 expression on days 27 to 28 and HSD3B1 expression on days 15 to 16 of pregnancy. Further, in this tissue, OXA decreased StAR mRNA level on days 10 to 11, CYP11A1 mRNA level on days 15 to 16, as well as HSD3B1 mRNA level on days 10 to 11 and 12 to 13 of gestation. Regarding the myometrium, OXA stimulated CYP11A1 gene expression on days 15 to 16 of pregnancy. In this tissue, OXA decreased StAR transcript content on days 15 to 16 and CYP11A1 mRNA level on days 27 to 28. We also demonstrated that OXA alone enhanced P 4 secretion in the endometrium on days 10 to 11 and 12 to 13 of gestation. OXA alone has no significant effect on endometrial and myometrial A 4 secretion, whereas OXA in combination with OX1R antagonist increased this hormone secretion during all studied stages of pregnancy. Therefore, we can conclude that OXA may affect de novo synthesis and secretion of P 4 and A 4 in the porcine uterus via participating in the regulation of key steroidogenic enzymes gene expression, as well as modulating steroid hormones secretion during early pregnancy and mid-luteal phase of the oestrous cycle in pigs. However, further research is required to explain the exact role of OXA in the porcine uterus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We analyzed three cases of Complete Androgen Insensitivity Syndrome (CAIS) and report three hitherto undisclosed causes of the disease. RNA-Seq, Real-timePCR, Western immunoblotting, and immunohistochemistry were performed with the aim of characterizing the disease-causing variants. In case No.1, we have identified a novel androgen receptor (AR) mutation (c.840delT) within the first exon in the N-terminal transactivation domain. This thymine deletion resulted in a frameshift and thus introduced a premature stop codon at amino acid 282. In case No.2, we observed a nonsynonymous mutation in the ligand-binding domain (c.2491C>T). Case No.3 did not reveal AR mutation; however, we have found a heterozygous mutation in CYP11A1 gene, which has a role in steroid hormone biosynthesis. Comparative RNA-Seq analysis of CAIS and control revealed 4293 significantly deregulated genes. In patients with CAIS, we observed a significant increase in the expression levels of PLCXD3 , TM4SF18 , CFI , GPX8 , and SFRP4, and a significant decrease in the expression of SPATA16, TSACC, TCP10L, and DPY19L2 genes (more than 10-fold, p < 0.05). Our findings will be helpful in molecular diagnostics of patients with CAIS, as well as the identified genes could be also potential biomarkers for the germ cells differentiation process., Competing Interests: Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1.

Defects in the initial steps of steroidogenesis usually present with female external genitalia in both 46,XX and 46,XY. Hence, they are not often considered in the differential diagnosis of primary adrenal insufficiency children with normal male external genitalia. Here, we report a boy with normal male external genitalia who presented with hyperpigmentation since the age of 2 years but diagnosis was delayed till 11 years of age. Evaluation revealed low-serum cortisol with elevated adrenocorticotropic hormone and direct renin level confirming primary adrenal insufficiency. Clinical exome sequencing analysis revealed a homozygous c.1351C>T (p.R451W) mutation in exon 8 of the CYP11A1 gene which was confirmed on Sanger sequencing. Both parents were heterozygous for the variation. To conclude, we report the first case of CYP11A1 deficiency from India. The report reiterates the existence of non-classic CYP11A1 deficiency characterised by primary adrenal insufficiency and normal male external genitalia in 46,XY., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

In this investigation, the effects of genistein (GEN) on the expression of steroidogenic genes such as steroidogenic acute regulatory protein (StAR), side-chain cleavage enzymes (P450scc) and cytochrome P450 aromatase (CYP19) were assessed. For this study, forty young female Sprague Dawley (SD) rats at aged 2-3 months (200±20 g) and forty aged female SD rats aged 10-12 months (490±20 g) were selected. Also, based on weight they were divided into a negative control group (NC), three different GEN dose groups, which received GEN of 15, 30, 60 mg/kg, and a positive control group (PC). The experiment lasted 30 days. Concentrations of serum hormones were determined by Enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions of StAR, P450scc and CYP19 were determined by Real-Time PCR and western blot techniques. It was observed that 30-60 mg/kg GEN could increase the expression of androgen generating key enzymes in the young rat ovary. GEN also significantly increased progesterone and E2 levels in the serum of aged rats and reduced the levels of LH and FSH in the serum of both young and aged rats. Compared with young rats, the effect of GEN on the ovary of aged rats was stronger and a lower dose of GEN (15 mg/kg) showed an obvious effect on these indicators. GEN influenced both estrogen level and indicators associated with estrogen and androgen transformation processes, which indicates that GEN can impair the growth and maturation of the ovary., (Copyright© by the Polish Academy of Sciences.)

Emerging epidemiological studies indicate that hypercholesterolaemia is a risk factor for testosterone deficiency. However, the underlying mechanism is unclear. Testicular Leydig cells are the primary source of testosterone in males. To identify the effect and mechanism of cholesterol overload on Leydig cell function, rats were fed with a HC (HC) diet to induce hypercholesterolaemia. During the 16-week feeding period, serum testosterone levels were reduced in a time-dependent manner in rats fed the HC diet. Accordingly, these steroidogenic enzymes within the Leydig cells, including steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), were down-regulated. Notably, the HC-fed rats showed evident endoplasmic reticulum (ER) stress in the testis, including a dilated ER as an evident pathological change in the Leydig cell ultrastructure, up-regulated ER stress biomarker (binding immunoglobulin protein) levels and activation of the activating transcription factor 6 (ATF6)-related unfolded protein response pathway. Further analysis showed that when 4-phenyl butyric acid (4-PBA) was used to block ER stress in HC-fed rats for 8 weeks, the testosterone deficiency was significantly alleviated. Our findings suggested that high dietary cholesterol intake affected serum testosterone levels by down-regulating steroidogenic enzymes and that activated ER stress might serve as the underlying mechanism., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The cholesterol hydroxylase/lyase (CHL) system, located in the mitochondria of the mammalian adrenal cortex cells, consists of cytochrome P450scc (CYP11A1), adrenodoxin (Adx), and adrenodoxin reductase (AdR) and performs the first stage of the steroidogenesis: AdR and Adx enable the electron transfer between NADPH and cytochrome P450scc, and P450scc catalyzes the conversion of cholesterol into pregnenolone. CHL system was reconstructed in Escherichia coli using the polycistronic plasmid pTrc99A/CHL. In E. coli cells, the recombinant proteins form the catalytically active system. CHL activity towards 22R-hydroxycholesterol was 4.0 ± 1.3 nmol pregnenolone/h per 1 mg homogenate protein. The alteration of the order of heterologous cDNAs in the expression cassette from AdR-Adx-P450scc to P450scc-Adx-AdR results in alteration of stoichiometric ratio P450scc/Adx/AdR from 1:1.45:4.2 to 1:1.67:0.98; the former ratio is more optimal for the functioning of the cytochrome P450scc. The application of modified cDNA of Adx (AdxS112W) does not increase the CHL activity; however, the introduction of the second copy of AdxS112W gene into the expression cassette increases both the expression level of АdxS112W and the CHL activity in comparison with P450scc/АdxS112W/AdR system. In vivo activity of the CHL system in bacteria is limited by the substrate uptake by bacterial cells: it varied in the range of 0.05-0.62 mg pregnenolone/l resting cell suspension per 1-day cultivation, depending on the type and concentration of permeabilizing agents in the medium. The obtained results contribute to the knowledge of CHL system functioning in living bacteria.

The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot-and-mouth disease virus 2A-based method allows to express multiple proteins from a single transcript. We developed a 2A-based multicistronic system for the coexpression of three bovine CHL system proteins as the self-processing polyprotein pCoxIV-P450scc-2A-Adx-2A-AdR-GFP (pCoxIV-CHL-GFP), with a cleavable N-terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV-CHL-GFP, efficiently performed the expression of P450scc-2A, targeted to mitochondria, and Adx-2A, AdR-GFP and the fusion protein Adx-2A-AdR-GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of cells expressing all three CHL system components, and to that of nature steroidogenic cells. Thus, the P450scc activity detected in cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3β-hydroxysteroid dehydrogenase. Therefore, HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics., (© 2018 Wiley Periodicals, Inc.)