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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.

Backgruound: Phosphatidylinositol 3-kinase (PI3K) regulates cellular development and energy homeostasis. However, the roles of its subunits in organ development remain largely unknown., Methods: We explored the roles of PI3K catalytic subunits in steroidogenic factor 1 (SF1)-expressing cells through knockout (KO) of the p110α and p110β subunits., Results: We examined mice with a double KO of p110α and p110β in SF1-expressing cells (p110αβ KOSF1). Although these animals exhibited no significant changes in the development of the ventromedial hypothalamus, we noted pronounced hypotrophy in the adrenal cortex, testis, and ovary. Additionally, corticosterone and aldosterone levels were significantly reduced. The absence of these subunits also resulted in decreased body weight and survival rate, along with impaired glucose homeostasis, in p110αβ KOSF1 mice., Conclusion: The data demonstrate the specific roles of PI3K catalytic subunits in the development and function of SF1-expressing organs.

Purpose: We previously showed the clinical characteristics of acromegaly with a paradoxical growth hormone (GH) response to oral glucose or thyrotropin-releasing hormone. However, the clinical characteristics of acromegaly with an increased GH response to luteinizing hormone-releasing hormone (LHRH responders) remain unclear. The aim of the present study was to evaluate the clinical characteristics, especially gonadotroph-related characteristics of LHRH responders in acromegaly., Methods: The clinical characteristics of 33 LHRH responders and 81 LHRH nonresponders were compared., Results: No differences in age, sex or basal serum levels of GH, insulin-like growth factor-1 (IGF-1), and gonadotropin were observed between the two groups. Steroidogenic factor 1 (SF-1), gonadotropin-releasing hormone receptor (GnRHR), and LH expression was more frequently observed in LHRH responders (P < 0.05). In addition, a greater increased rate of GH after LHRH loading, and the proportion of GnRHR and gonadotropin expression was observed in pituitary tumor with SF-1 expression than that without the expression (P < 0.01). LHRH responders showed a greater GH decrease in the octreotide test and a greater IGF-1 decrease after first-generation somatostatin ligand than LHRH nonresponders (P < 0.05). Furthermore, the proportion of hypointense pituitary tumors on T2-weighted magnetic resonance imaging and tumors with densely granulated type was higher in LHRH responders than in LHRH nonresponders, respectively (P < 0.05). No difference between the two groups was observed in either somatostatin receptor 2 or 5 expression., Conclusions: The increased GH response to LHRH is associated with the gonadotroph-related characteristics. This response may reflect the biological characteristics of somatotroph tumors., (© 2024. The Author(s).)

Steroidogenic factor 1 (SF-1) is a nuclear receptor that regulates steroidogenesis and reproductive development. NR5A1 /SF-1 variants are associated with a broad spectrum of phenotypes across individuals with disorders of sex development (DSDs). Oligogenic inheritance has been suggested as an explanation. SF-1 interacts with numerous partners. Here, we investigated a constellation of gene variants identified in a 46,XY severely undervirilized individual carrying an ACMG-categorized 'pathogenic' NR5A1 /SF-1 variant in comparison to the healthy carrier father. Candidate genes were revealed by whole exome sequencing, and pathogenicity was predicted by different in silico tools. We found variants in NR1H2 and INHA associated with steroidogenesis, sex development, and reproduction. The identified variants were tested in cell models. Novel SF-1 and NR1H2 binding sites in the AR and INHA gene promoters were found. Transactivation studies showed that wild-type NR5A1 /SF-1 regulates INHA and AR gene expression, while the NR5A1 /SF-1 variant had decreased transcriptional activity. NR1H2 was found to regulate AR gene transcription; however, the NR1H2 variant showed normal activity. This study expands the NR5A1 /SF-1 network of interacting partners, while not solving the exact interplay of different variants that might be involved in revealing the observed DSD phenotype. It also illustrates that understanding complex genetics in DSDs is challenging.

Background: Sex steroid hormones, primarily synthesized by gonadal somatic cells, are pivotal for sexual development and reproduction. Mice studies have shown that two transcription factors, steroidogenic factor 1 (SF-1) and Wilms' tumor 1 (WT1), are involved in gonadal development. However, their role in human gonadal somatic differentiation remains unclear. We therefore aimed to investigate the roles of SF-1 and WT1 in human gonadal steroidogenic cell differentiation., Methods: Using a transient lentivirus-mediated gene expression system, we assessed the effects of SF-1 and WT1 expression on the steroidogenic potential of human amniotic membrane-derived mesenchymal stem cells (hAmMSCs)., Results: SF-1 and WT1-KTS, a splice variant of WT1, played distinct roles in human steroidogenic differentiation of hAmMSCs. SF-1 induced hAmMSC differentiation into progesterone- and androgen-producing cell lineages, whereas WT1-KTS promoted hAmMSC differentiation into estrogen-producing cell lineages., Conclusion: Our findings revealed that SF-1 and WT1-KTS play important roles in human gonadal steroidogenic cell differentiation, especially during ovarian development. These findings may pave the way for future studies on human ovarian differentiation and development., 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 © 2024 Miyazaki, Orisaka, Fujita, Mizutani, Yazawa and Yoshida.)

Background: The Axl gene is a receptor tyrosine kinase essential for male fertility. With other Tyro3 family members, it regulates cell apoptosis and preserves the organization of seminiferous tubules. However, the regulation of the expression of Axl in testicular Sertoli cells is not entirely understood. The transcription factors NR5A1 and JUNB are involved in several male fertility mechanisms such as sex development and steroidogenesis. We hypothesize that Axl promoter activity is regulated by cooperation between JUNB and NR5A1 in Sertoli cells., Methods and Results: Following transfections of TM4 Sertoli cells with DsiRNA interference against Axl, our results show that cell morphology may be regulated by AXL. Using transfections of expression plasmids and reporter plasmids containing the Axl promoter, we report that Axl expression is highly activated by cooperation between NR5A1 and JUNB in TM4 and 15P-1 Sertoli cells. Chromatin immunoprecipitation and luciferase reporter assays with 5' promoter deletions demonstrate that JUNB and NR5A1 are being recruited to DNA regulatory elements in the proximal region of the Axl promoter. The fourth intronic region of Axl also participates in the recruitment of JUNB., Conclusion: Thus, Axl expression is regulated by a cooperation between the transcription factors JUNB and NR5A1 and influences the morphology of TM4 Sertoli cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Directed differentiation of pluripotent stem cells into specialized cell types represents an invaluable tool for a wide range of applications. Here, we have exploited single-cell transcriptomic data to develop a stepwise in vitro differentiation system from mouse embryonic stem cells into adrenocortical cells. We show that during development, the adrenal primordium is embedded in an extracellular matrix containing tenascin and fibronectin. Culturing cells on fibronectin during differentiation increased the expression of the steroidogenic marker NR5A1. Furthermore, 3D cultures in the presence of protein kinase A (PKA)-pathway activators led to the formation of aggregates composed of different cell types expressing adrenal progenitor or steroidogenic markers, including the adrenocortical-specific enzyme CYP21A1. Importantly, in-vitro-differentiated cells responded to adrenocorticotropic hormone (ACTH) and angiotensin II with the production of glucocorticoids and mineralocorticoids, respectively, thus confirming the specificity of differentiation toward the adrenal lineage., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Endometriosis (EMS) is a common gynecological disease that causes dysmenorrhea, chronic pelvic pain and infertility. Luoshi Neiyi Prescription (LSNYP), a traditional Chinese medicine (TCM) formula, is used to relieve EMS in the clinic., Aims: This study aimed to examine the active components of LSNYP and the possible mechanism involved in its treatment of EMS., Materials and Methods: Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was used to identify the chemical components of LSNYP. Human primary ectopic endometrial stromal cells (ecESCs) and eutopic endometrial stromal cells (euESCs) were isolated, and the expression levels of hypoxia inducible factor 1A (HIF1A), enhancer of zeste homolog 2 (EZH2) and steroidogenic factor 1 (SF-1) were detected by immunofluorescence and qPCR. Cobalt chloride (CoCl 2 ) was utilized to construct an in vitro hypoxic environment, and lentiviruses were engineered to downregulate HIF1A and EZH2 and upregulate EZH2. Subsequently, the expression levels of HIF1A, EZH2, and SF-1 were measured using qPCR or western blotting. The binding of EZH2 to the SF-1 locus in ESCs was examined via ChIP. Furthermore, the effects of LSNYP on the HIF1A/EZH2/SF-1 pathway were evaluated both in vitro and in vivo., Results: A total of 185 components were identified in LSNYP. The protein and gene expression levels of HIF1A and SF-1 were increased, whereas those of EZH2 were decreased in ecESCs. After treating euESCs with 50 μmol L -1 CoCl 2 for 24 h, cell viability and estradiol (E 2 ) production were enhanced. Hypoxia decreased EZH2 protein expression, while si-HIF1A increased it. SF-1 was increased when EZH2 was downregulated in normal and hypoxic environments, whereas the overexpression of EZH2 led to a decrease in SF-1 expression. ChIP revealed that hypoxia reduced EZH2 binding to the SF-1 locus in euESCs. In vitro, LSNYP-containing serum decreased E 2 and prostaglandin E 2 (PGE 2 ) production, inhibited cell proliferation and invasion, and reduced the expression of HIF1A, SF-1, steroidogenic acute regulatory protein (StAR), and aromatase cytochrome P450 (P450arom). In vivo, LSNYP suppressed inflammation and adhesion and inhibited the HIF1A/EZH2/SF-1 pathway in endometriotic tissues., Conclusions: LSNYP may exert pharmacological effects on EMS by inhibiting E 2 synthesis and inflammation through regulation of the HIF1A/EZH2/SF-1 pathway. These results suggest that LSNYP may be a promising candidate for the treatment of EMS., 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.)

Cell therapy for adrenocortical insufficiency can potentially provide steroid replacement in response to physiological stimuli. Previously, we reported that adipose tissue-derived stromal cells (ADSCs) are transformed into steroid-producing cells by overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1). The steroidogenic cells are characterized by the production of both adrenal and gonadal steroids. Cytotherapy for adrenocortical insufficiency requires cells with more adrenocortical characteristics. Considering the highly developed vascular network within the adrenal cortex, all adrenocortical cells are adjacent to and interact with vascular endothelial cells (VECs). In this study, NR5A1-induced steroidogenic cells derived from mouse ADSCs (NR5A1-ADSCs) were co-cultured with mouse VECs. Testosterone secretion in NR5A1-ADSCs was not altered; however, corticosterone secretion significantly increased while levels of steroidogenic enzymes significantly increased in the corticosterone synthesis pathway. Co-culture with lymphatic endothelial cells (LECs) or ADSCs, or transwell culture with NR5A1-ADSCs and VECs did not alter corticosterone production. VECs expressed higher levels of collagen and laminin than LECs. Culture in type-IV collagen and laminin-coated dishes increased corticosterone secretion in NR5A1-ADSCs. These results suggest that VECs may characterize ADSC-derived steroidogenic cells into a more corticosterone-producing phenotype, and VECs may be useful for generating adrenal steroidogenic cells from stem cells., (© 2024. The Author(s).)

NR2F2 encodes COUP-TFII, an orphan nuclear receptor required for the development of the steroidogenic lineages of the murine fetal testes and ovaries. Pathogenic variants in human NR2F2 are associated with testis formation in 46,XX individuals, however, the function of COUP-TFII in the human testis is unknown. We report a de novo heterozygous variant in NR2F2 (c.737G > A, p.Arg246His) in a 46,XY under-masculinized boy with primary hypogonadism. The variant, located within the ligand-binding domain, is predicted to be highly damaging. In vitro studies indicated that the mutation does not impact the stability or subcellular localization of the protein. NR5A1, a related nuclear receptor that is a key factor in gonad formation and function, is known to physically interact with COUP-TFII to regulate gene expression. The mutant protein did not affect the physical interaction with NR5A1. However, in-vitro assays demonstrated that the mutant protein significantly loses the inhibitory effect on NR5A1-mediated activation of both the LHB and INSL3 promoters. The data support a role for COUP-TFII in human testis formation. Although mutually antagonistic sets of genes are known to regulate testis and ovarian pathways, we extend the list of genes, that together with NR5A1 and WT1, are associated with both 46,XX and 46,XY DSD., (© 2024. The Author(s).)

Allostatic adaptations to a perceived threat are crucial for survival and may tap into mechanisms serving the homeostatic control of energy balance. We previously established that exposure to predator odor (PO) in rats significantly increases skeletal muscle thermogenesis and energy expenditure (EE). Evidence highlights steroidogenic factor 1 (SF1) cells within the central and dorsomedial ventromedial hypothalamus (c/dmVMH) as a modulator of both energy homeostasis and defensive behavior. However, the brain mechanism driving elevated EE and muscle thermogenesis during PO exposure has yet to be elucidated. To assess the ability of SF1 neurons of the c/dmVMH to induce muscle thermogenesis, we used the combined technology of chemogenetics, transgenic mice, temperature transponders, and indirect calorimetry. Here, we evaluate EE and muscle thermogenesis in SF1-Cre mice exposed to PO (ferret odor) compared to transgenic and viral controls. We detected significant increases in muscle temperature, EE, and oxygen consumption following the chemogenetic stimulation of SF1 cells. However, there were no detectable changes in muscle temperature in response to PO in either the presence or absence of chemogenetic stimulation. While the specific role of the VMH SF1 cells in PO-induced thermogenesis remains uncertain, these data establish a supporting role for SF1 neurons in the induction of muscle thermogenesis and EE similar to what is seen after predator threats.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17β (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels., 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.)

Objective: Hypothalamic signals potently stimulate energy expenditure by engaging peripheral mechanisms to restore energy homeostasis. Previous studies have identified several critical hypothalamic sites (e.g. preoptic area (POA) and ventromedial hypothalamic nucleus (VMN)) that could be part of an interconnected neurocircuit that controls tissue thermogenesis and essential for body weight control. However, the key neurocircuit that can stimulate energy expenditure has not yet been established., Methods: Here, we investigated the downstream mechanisms by which VMN neurons stimulate adipose tissue thermogenesis. We manipulated subsets of VMN neurons acutely as well as chronically and studied its effect on tissue thermogenesis and body weight control, using Sf1 Cre and Adcyap1 Cre mice and measured physiological parameters under both high-fat diet and standard chow diet conditions. To determine the node efferent to these VMN neurons, that is involved in modulating energy expenditure, we employed electrophysiology and optogenetics experiments combined with measurements using tissue-implantable temperature microchips., Results: Activation of the VMN neurons that express the steroidogenic factor 1 (Sf1; VMN Sf1 neurons) reduced body weight, adiposity and increased energy expenditure in diet-induced obese mice. This function is likely mediated, at least in part, by the release of the pituitary adenylate cyclase-activating polypeptide (PACAP; encoded by the Adcyap1 gene) by the VMN neurons, since we previously demonstrated that PACAP, at the VMN, plays a key role in energy expenditure control. Thus, we then shifted focus to the subpopulation of VMN Sf1 neurons that contain the neuropeptide PACAP (VMN PACAP neurons). Since the VMN neurons do not directly project to the peripheral tissues, we traced the location of the VMN PACAP neurons' efferents. We identified that VMN PACAP neurons project to and activate neurons in the caudal regions of the POA whereby these projections stimulate tissue thermogenesis in brown and beige adipose tissue. We demonstrated that selective activation of caudal POA projections from VMN PACAP neurons induces tissue thermogenesis, most potently in negative energy balance and activating these projections lead to some similar, but mostly unique, patterns of gene expression in brown and beige tissue. Finally, we demonstrated that the activation of the VMN PACAP neurons' efferents that lie at the caudal POA are necessary for inducing tissue thermogenesis in brown and beige adipose tissue., Conclusions: These data indicate that VMN PACAP connections with the caudal POA neurons impact adipose tissue function and are important for induction of tissue thermogenesis. Our data suggests that the VMN PACAP → caudal POA neurocircuit and its components are critical for controlling energy balance by activating energy expenditure and body weight control., Competing Interests: Declaration of competing interest Jonathan N. Flak and Travis S. Johnson have received research support from Eli Lilly and Company, United States for separate projects in their respective laboratories., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The current study aimed to investigate the effect of Sox9-Cre-directed Nr5a1-conditional knockout (Sox9-Cre;Nr5a1 flox/flox ) on adrenal development. We showed that SOX9 is expressed by adrenocortical cells at E10.5-E11.5 but is extinguished no later than E12.5. The number of adrenocortical cells significantly reduced in Sox9-Cre;Nr5a1 flox/flox mice while the number of cleaved caspase 3-positive cells increased compared to that in the controls at E11.5-E12.5, when the adrenal primordium (AP) is about to expand. This indicated that fetal adrenocortical cells are lost via apoptosis due to Nr5a1 ablation by E12.5. Both medulla formation and encapsulation were perturbed, accompanied by a smaller AP size, in Sox9-Cre;Nr5a1 flox/flox mice during embryonic development. Adult Sox9-Cre;Nr5a1 flox/flox adrenals were hypoplastic and exhibited irregular organization of the medulla with aberrant sex differentiation in the X zone. Additionally, there were histologically eosin-negative vacuolated cells, which were negative for both the X-zone marker 20αHSD and the steroidogenesis marker 3βHSD at the innermost cortex of Sox9-Cre;Nr5a1 flox/flox adrenals. Although Nr5a1 +/- adrenals were hypoplastic, a small number of chromaffin cells were properly located in the center, having normal sex differences in the X-zone. The results collectively provided in-vivo evidence that Nr5a1 plays a critical role in AP expansion and subsequent adrenal development., (© 2024. The Author(s).)

Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad., (© 2024. The Author(s).)

Chicken ovarian follicle development is regulated by complex and dynamic gene expression. Nuclear receptor 5A1 and 5A2 (NR5A1 and NR5A2, respectively) are key genes that regulate steroid hormone production and gonadal development in mammals; however, studies on follicular development in the chicken ovary are scarce. In this study, we investigated the functions of NR5A1 and NR5A2 on follicle development in chickens. The results showed that the expression of NR5A1 and NR5A2 was significantly higher in small yellow follicles and F5. Furthermore, the expression of NR5A1 and NR5A2 was significantly higher in follicular tissues of peak-laying hens (30 wk) than in follicular tissues of late-laying hens (60 wk), with high expression abundance in granulosa cells (GC). The overexpression of NR5A1 and NR5A2 significantly promoted proliferation and inhibited apoptosis of cultured GC; upregulated STAR, CYP11A1, and CYP19A1 expression and estradiol (E2) and progesterone (P4) synthesis in GC from preovulatory follicles (po-GC); and increased STAR, CYP11A1, and CYP19A1 promoter activities. In addition, follicle-stimulating hormone treatment significantly upregulated NR5A1 and NR5A2 expression in po-GC and significantly promoted FSHR, CYP11A1, and HSD3B1 expression in GC from pre-hierarchical follicles and po-GC. The core promoter region of NR5A1 was identified at the -1,095- to -483-bp and -2,054- to -1,536-bp regions from the translation start site (+1), and the core promoter region of NR5A2 was at -998 to -489 bp. Two single nucleotide polymorphisms (SNP) were identified in the core promoter region of the NR5A1 gene, which differed between high- and low-yielding chicken groups. Our study suggested that NR5A1 and NR5A2 promoted chicken follicle development by promoting GC proliferation and E2 and P4 hormone synthesis and inhibiting apoptosis. Moreover, we identified the promoter core region or functional site that regulates NR5A1 and NR5A2 expression., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human granulosa cells in different stages are essential for maintaining normal ovarian function, and granulosa cell defect is the main cause of ovarian dysfunction. To address this problem, it is necessary to induce functional granulosa cells at different stages in vitro. In this study, we established a reprogramming method to induce early- and late-stage granulosa cells with different steroidogenic abilities. We used an AMH-fluorescence-reporter system to screen candidate factors for cellular reprogramming and generated human induced granulosa-like cells (hiGC) by overexpressing FOXL2 and NR5A1. AMH-EGFP + hiGC resembled human cumulus cells in transcriptome profiling and secreted high levels of oestrogen and progesterone, similar to late-stage granulosa cells at antral or preovulatory stage. Moreover, we identified CD55 as a cell surface marker that can be used to isolate early-stage granulosa cells. CD55 + AMH-EGFP - hiGC secreted high levels of oestrogen but low levels of progesterone, and their transcriptome profiles were more similar to early-stage granulosa cells. More importantly, CD55 + hiGC transplantation alleviated polycystic ovary syndrome (PCOS) in a mouse model. Therefore, hiGC provides a cellular model to study the developmental program of human granulosa cells and has potential to treat PCOS., (© 2024 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5' regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process., (© 2024. The Author(s).)

Pituitary neuroendocrine tumors (PitNETs) are classified according to cell lineage, which requires immunohistochemistry for adenohypophyseal hormones and the transcription factors (TFs) PIT1, SF1, and TPIT. According to the current WHO 2022 classification, PitNETs with co-expression of multiple TFs are termed "plurihormonal". Previously, PIT1/SF1 co-expression was prevailingly reported in PitNETs, which otherwise correspond to the somatotroph lineage. However, little is known about such tumors and the WHO classification has not recognized their significance. We compiled an in-house case series of 100 tumors, previously diagnosed as somatotroph PitNETs. Following TF staining, histopathological features associated with PIT1/SF1 co-expression were assessed. Integration of in-house and publicly available sample data allowed for a meta-analysis of SF1-associated clinicopathological and molecular features across a total of 270 somatotroph PitNETs. The majority (74%, 52/70) of our densely granulated somatotroph PitNETs (DGST) unequivocally co-expressed PIT1 and SF1 (DGST-PIT1/SF1). None (0%, 0/30) of our sparsely granulated somatotroph PitNETs (SGST) stained positive for SF1 (SGST-PIT1). Among DGST, PIT1/SF1 co-expression was significantly associated with scarce FSH/LH expression and fewer fibrous bodies compared to DGST-PIT1. Integrated molecular analyses including publicly available samples confirmed that DGST-PIT1/SF1, DGST-PIT1 and SGST-PIT1 represent distinct tumor subtypes. Clinicopathological meta-analyses indicated that DGST-PIT1 respond more favorably towards treatment with somatostatin analogs compared to DGST-PIT1/SF1, while both these subtypes show an overall less aggressive clinical course than SGST-PIT1. In this study, we spotlight that DGST with co-expression of PIT1 and SF1 represent a common, yet underrecognized, distinct PitNET subtype. Our study questions the rationale of generally classifying such tumors as "plurihormonal", and calls for a refinement of the WHO classification. We propose the term "somatogonadotroph PitNET"., (© 2024. The Author(s).)

The prospect that the ventromedial hypothalamic nucleus (VMN) transcription factor steroidogenic factor-1/NR5A1 (SF-1) may exert sex-dimorphic control of glucose counterregulation is unresolved. Recent studies in male rats show that SF-1 regulates transcription of co-expressed hypoglycemia-sensitive neurochemicals in dorsomedial VMN growth hormone-releasing hormone (Ghrh) neurons. Gene knockdown and laser-catapult-microdissection/single-cell multiplex qPCR techniques were used here in a female rat model to determine if SF-1 control of Ghrh neuron transmitter marker, energy sensor, and estrogen receptor (ER) variant mRNAs varies according to sex. Data show that in females, hypoglycemia elicits a gain of SF-1 inhibitory control of VMNdm Ghrh neuron Ghrh and Ghrh-receptor gene profiles and loss of augmentation of glutaminase transcription; SF-1 gene silencing diminished eu- and hypoglycemic patterns of neuronal nitric oxide gene transcription. SF-1 imposes divergent control of baseline and hypoglycemic glutamate decarboxylase 65 (GAD)-1 (stimulatory) versus GAD2 (inhibitory) mRNAs in that sex. SF-1 stimulates baseline VMNdm Ghrh neuron PRKAA1/AMPKα1 and PRKAA2/AMPKα2 gene expression, yet causes opposite changes in these gene profiles during hypoglycemia. SF-1 exerts glucose-dependent control of ER-alpha and G-protein-coupled ER-1 transcription, but blunts ER-beta gene profiles during eu- and hypoglycemia. In females, SF-1 knockdown did not affect hypercorticosteronemia or hyperglucagonemia, but blunted hypoglycemic suppression of growth hormone secretion. Results show that SF-1 expression is critical for female rat VMNdm Ghrh neuron counterregulatory neurochemical, AMPK catalytic subunit, and ER gene transcription responses to hypoglycemia. Sex differences in direction of SF-1 control of distinctive gene profiles may result in observed disparities in SF-1 regulation of counterregulatory hormone secretion between sexes.

Ventromedial hypothalamic nucleus (VMN) growth hormone-releasing hormone (Ghrh) neurotransmission shapes counterregulatory hormone secretion. Dorsomedial VMN Ghrh neurons express the metabolic-sensitive transcription factor steroidogenic factor-1/NR5A1 (SF-1). In vivo SF-1 gene knockdown tools were used here to address the premise that in male rats, SF-1 may regulate basal and/or hypoglycemic patterns of Ghrh, co-transmitter biosynthetic enzyme, and estrogen receptor (ER) gene expression in these neurons. Single-cell multiplex qPCR analyses showed that SF-1 regulates basal profiles of mRNAs that encode Ghrh and protein markers for neurochemicals that suppress (γ-aminobutyric acid) or enhance (nitric oxide; glutamate) counterregulation. SF-1 siRNA pretreatment respectively exacerbated or blunted hypoglycemia-associated inhibition of glutamate decarboxylase 67 (GAD 67 /GAD1) and - 65 (GAD 65 /GAD2) transcripts. Hypoglycemia augmented or reduced nitric oxide synthase and glutaminase mRNAs, responses that were attenuated by SF-1 gene silencing. Ghrh and Ghrh receptor transcripts were correspondingly refractory to or increased by hypoglycemia, yet SF-1 knockdown decreased both gene profiles. Hypoglycemic inhibition of ER-alpha and G protein-coupled-ER gene expression was amplified by SF-1 siRNA pretreatment, whereas as ER-beta mRNA was amplified. SF-1 knockdown decreased (corticosterone) or elevated [glucagon, growth hormone (GH)] basal counterregulatory hormone profiles, but amplified hypoglycemic hypercorticosteronemia and -glucagonemia or prevented elevated GH release. Outcomes document SF-1 control of VMN Ghrh neuron counterregulatory neurotransmitter and ER gene transcription. SF-1 likely regulates Ghrh nerve cell receptivity to estradiol and release of distinctive neurochemicals during glucose homeostasis and systemic imbalance. VMN Ghrh neurons emerge as a likely substrate for SF-1 control of glucose counterregulation in the male rat.

Steroidogenic Factor-1 (SF-1, NR5A1) is a member of the nuclear receptor superfamily of ligand-regulated transcription factors, consisting of a DNA-binding domain (DBD) connected to a transcriptional regulatory ligand binding domain (LBD) via an unstructured hinge domain. SF-1 is a master regulator of development and adult function along the hypothalamic pituitary adrenal and gonadal axes, with strong pathophysiological association with endometriosis and adrenocortical carcinoma. SF-1 was shown to bind and be regulated by phospholipids, one of the most interesting aspects of SF-1 regulation is the manner in which SF-1 interacts with phospholipids: SF-1 buries the phospholipid acyl chains deep in the hydrophobic core of the SF-1 protein, while the lipid headgroups remain solvent-exposed on the exterior of the SF-1 protein surface. Here, we have reviewed several aspects of SF-1 structure, function and physiology, touching on other transcription factors that help regulate SF-1 target genes, non-canonical functions of SF-1, the DNA-binding properties of SF-1, the use of mass spectrometry to identify lipids that associate with SF-1, how protein phosphorylation regulates SF-1 and the structural biology of the phospholipid-ligand binding domain. Together this review summarizes the form and function of Steroidogenic Factor-1 in physiology and in human disease, with particular emphasis on adrenal cancer., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster., Methods: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 μmol/L) or pretreated with a p38 inhibitor (50 μmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 μmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 μmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR)., Results: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 μmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression significantly increased (P < 0.05) in the 40 μmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 μmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 μmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 μmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3β-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3β-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3β-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05)., Conclusion: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3β-HSD, and P450c17 expression in primary rooster Leydig cells., (Copyright © 2023. Published by Elsevier Inc.)

Dexamethasone is widely used to treat pregnancy disorders related to premature delivery. However, lots of researches have confirmed that prenatal dexamethasone exposure (PDE) could increase the risk of offspring multiple diseases. This study was designed to elucidate the epigenetic mechanism of adrenal developmental programming and explore its early warning marker in peripheral blood mononuclear cells (PBMC). We found the adrenal morphological and functional changes of PDE male offspring rats before and after birth, which were mainly performed as the decreased serum corticosterone concentration, steroidogenic acute regulatory (StAR) protein expression, and histone 3 lysine 27 acetylation (H3K27ac) level of steroidogenic factor 1 (SF1) promoter region and its expression. Simultaneously, the expressions of glucocorticoid receptor (GR) and histone acetylation enzyme 5 (HDAC5) in the PDE male fetal rats were increased. In vitro, dexamethasone reduced the expression of SF1, StAR, and cortisol production and still increased the expression of GR and HDAC5, the binding between GR and SF1 promoter region, and protein interaction between GR and HDAC5. GR siRNA or HDAC5 siRNA was able to reverse the above roles of dexamethasone. Furthermore, in vivo, we confirmed that H3K27ac levels of SF1 promoter region and its expression in PBMC of the PDE group were decreased before and after birth, showing a positive correlation with the same indexes in adrenal. Meanwhile, in clinical trials, we confirmed that prenatal dexamethasone application decreased H3K27ac of SF1 promoter region and its expression in neonatal PBMC. In conclusion, PDE-caused adrenal insufficiency of male offspring rats was related to adrenal GR activated by dexamethasone in uterus. The activated GR, on the one hand, increased its direct binding to SF1 promoter region to inhibit its expression, on the other hand, upregulated and recruited HDAC5 to decrease H3K27ac level of SF1 promoter region, and strengthened the inhibition of SF1 and subsequent StAR expression., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The ovarian follicle reserve, formed pre- or perinatally, comprises all oocytes for lifetime reproduction. Depletion of this reserve results in infertility. Steroidogenic factor 1 (SF-1; Nr5a1 ) and liver receptor homolog 1 (LRH-1; Nr5a2 ) are two orphan nuclear receptors that regulate adult endocrine function, but their role in follicle formation is unknown. We developed models of conditional depletion of SF-1 or LRH-1 from prenatal ovaries. Depletion of SF-1, but not LRH-1, resulted in dramatically smaller ovaries and fewer primordial follicles. This was mediated by increased oocyte death, resulting from increased ovarian inflammation and increased Notch signaling. Major dysregulated genes were Iroquois homeobox 3 and 5 and their downstream targets involved in the establishment of the ovarian laminin matrix and oocyte-granulosa cell gap junctions. Disruptions of these pathways resulted in follicles with impaired basement membrane formation and compromised oocyte-granulosa communication networks, believed to render them more prone to atresia. This study identifies SF-1 as a key regulator of the formation of the ovarian reserve.

Endometriosis (EMS) is an estrogen-dependent disease. However, little is known about the regulation of estrogen, a potential therapeutic target, in EMS, which remains very poorly managed in the clinic. We hypothesized that microRNAs (miRNAs) can be exploited therapeutically to regulate transcription factor 21 (TCF21) and steroidogenic factor-1 (SF-1) gene expression. In our study, paired eutopic and ectopic endometrial samples were obtained from women with EMS and processed by a standard protocol to obtain human endometrial stromal cells (EMs) for in vitro studies. We found that miR-92a-3p levels were decreased in ectopic endometrium and ectopic stromal cells (ESCs) compared with paired eutopic lesions. miR-92a-3p overexpression significantly suppressed the proliferation and migration of ESCs, whereas a decreased level of miR-92a-3p generated the opposite results. Next, we identified TCF21 as a candidate target gene of miR-92a-3p. In vitro cell experiments showed that miR-92a-3p negatively regulated the expression of TCF21 and its downstream target gene SF-1. Moreover, cell proliferation and invasion ability decreased after the silencing of SF-1 and increased after SF-1 overexpression. We also observed that silencing SF-1 while inhibiting miR-92a-3p partially blocked the increase in cell proliferation and invasion ability caused by miR-92a-3p knockdown while overexpressing both SF-1 and miR-92a-3p mitigated the impairment in cell proliferation and invasion ability caused by miR-92a-3p overexpression. Our results may provide a novel potential therapeutic target for the treatment of EMS., (© 2023. The Author(s).)

Inflammation and thermogenesis in white adipose tissue (WAT) at different sites influence the overall effects of obesity on metabolic health. In mice fed a high-fat diet (HFD), inflammatory responses are less pronounced in inguinal WAT (ingWAT) than in epididymal WAT (epiWAT). Here we show that ablation and activation of steroidogenic factor 1 (SF1)-expressing neurons in the ventromedial hypothalamus (VMH) oppositely affect the expression of inflammation-related genes and the formation of crown-like structures by infiltrating macrophages in ingWAT, but not in epiWAT, of HFD-fed mice, with these effects being mediated by sympathetic nerves innervating ingWAT. In contrast, SF1 neurons of the VMH preferentially regulated the expression of thermogenesis-related genes in interscapular brown adipose tissue (BAT) of HFD-fed mice. These results suggest that SF1 neurons of the VMH differentially regulate inflammatory responses and thermogenesis among various adipose tissue depots and restrain inflammation associated with diet-induced obesity specifically in ingWAT., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Steroidogenic factor-1 (SF-1, also termed Ad4BP; NR5A1 in the official nomenclature) is a nuclear receptor transcription factor that plays a crucial role in the regulation of adrenal and gonadal development, function and maintenance. In addition to its classical role in regulating the expression of P450 steroid hydroxylases and other steroidogenic genes, involvement in other key processes such as cell survival/proliferation and cytoskeleton dynamics have also been highlighted for SF-1. SF-1 has a restricted pattern of expression, being expressed along the hypothalamic-pituitary axis and in steroidogenic organs since the time of their establishment. Reduced SF-1 expression affects proper gonadal and adrenal organogenesis and function. On the other hand, SF-1 overexpression is found in adrenocortical carcinoma and represents a prognostic marker for patients' survival. This review is focused on the current knowledge about SF-1 and the crucial importance of its dosage for adrenal gland development and function, from its involvement in adrenal cortex formation to tumorigenesis. Overall, data converge towards SF-1 being a key player in the complex network of transcriptional regulation within the adrenal gland in a dosage-dependent manner.

Introduction: Testis differentiation is initiated by the SRY gene on the Y chromosome in mammalian species. However, the Amami spiny rat, Tokudaia osimensis, lacks both the Y chromosome and the Sry gene and acquired a unique Sox9 regulatory mechanism via a male-specific duplication upstream of Sox9, without Sry. In general mammalian species, the SRY protein binds to a testis-specific enhancer to promote SOX9 gene expression. Several enhancers located upstream of Sox9/SOX9 have been reported in mice and humans. In particular, the binding of SRY to the highly conserved enhancer Enh13 is thought to be a common mechanism underlying testis differentiation and sex determination in mammals., Methods: Sequences of T. osimensis homologues of three Sox9 enhancers that were previously reported in mice, Enh8, Enh14, and Enh13, were determined. We performed in vitro assays to confirm enhancer activity involved in Sox9 regulation in T. osimensis., Results: T. osimensis Enh13 showed enhancer activity when co-transfected with NR5A1 and SOX9. Mouse Enh13 was activated by NR5A1 and SRY; however, T. osimensis Enh13 did not respond to SRY, even though the binding sites of SRY and NR5A1 were conserved. To identify the key sequence that is present in mouse but absent from T. osimensis, we performed reporter gene assays using vectors in which partial sequences of T. osimensis Enh13 were replaced with mouse sequences. For T. osimensis Enh13 in which the second half (approximately 430 bp) was replaced with the corresponding mouse sequence, activity in response to NR5A1 and SRY was recovered. Further, reporter assays revealed that multiple regions in the second half of the mouse Enh13 sequence are required for the response to NR5A1 and SRY. The latter 49 bp was particularly important and contained four binding sites for three transcription factors, POU2F1, HOXA3, and GATA1., Conclusion: We showed that there are unknown sequences responsible for the interaction between NR5A1 and SRY and mEnh13 based on comparative analyses of Sry-dependent and Sry-independent species. Our comparative analyses revealed new molecular mechanisms underlying mammalian sex determination., (© 2024 S. Karger AG, Basel.)

In Brief: The nuclear receptor steroidogenic factor 1 (SF-1) is essential for mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and depletion of SF-1 in steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility., Abstract: Steroidogenic factor 1 (SF-1 or NR5A1) plays an essential role in the development of fetal gonads and regulates genes involved in steroid biosynthesis. Since SF-1 is expressed in multiple cell types in mouse gonads, we developed three novel conditional knockout (cKO) mouse models employing Cre-recombinase and floxed alleles of SF-1 (Nr5a1f/f) to identify its role in testes and ovaries of mature mice: Cytochrome P450 17α-hydroxylase (Cyp17Cre/+;Nr5a1f/f, Leydig and theca cell-specific), aromatase (Cyp19Cre/+;Nr5a1f/f, Sertoli and granulosa cell-specific), as well as a combination of both (Cyp17+Cyp19-Cre;Nr5a1f/f). Compared to control animals, Cyp19-Cre;Nr5a1f/f cKO males showed normal fertility and testicular function. The Cyp17Cre/+;Nr5a1f/f cKO males had smaller testis, with drastically reduced Leydig cell volumes and impaired steroidogenesis, though their reproductive performance remained comparable to controls. Some 50% of Cyp17Cre/++Cyp19Cre/+;Nr5a1f/f double-cKO (dKO) males were infertile, while the remaining 50% showed significantly reduced fertility. These dKO males also had smaller testis with degenerative seminiferous tubules, abnormal Leydig cell morphology and lower levels of intra-testicular testosterone. Abnormal Sertoli cell localization was noted in dKO testes, with increased Sox9, p27 and inhibin subunit ßb and decreased androgen receptor expression. Female mice from all genotypes showed normal reproductive capacity, though steroidogenic gene expression levels were significantly decreased in both Cyp17Cre/+;Nr5a1f/f cKO and dKO females. These results show the essential role of SF-1 in mature mouse gonad steroidogenic gene expression, for Leydig and Sertoli cell function, and that depletion SF-1 in all steroidogenic cells of the testis compromises steroidogenesis, spermatogenesis and male fertility.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans., Competing Interests: Declaration of competing interest none., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: Steroidogenic factor 1 (SF1) expressing neurons in the ventromedial hypothalamus (VMH) have been directly implicated in whole-body metabolism and in the onset of obesity. The co-chaperone FKBP51 is abundantly expressed in the VMH and was recently linked to type 2 diabetes, insulin resistance, adipogenesis, browning of white adipose tissue (WAT) and bodyweight regulation., Methods: We investigated the role of FKBP51 in the VMH by conditional deletion and virus-mediated overexpression of FKBP51 in SF1-positive neurons. Baseline and high fat diet (HFD)-induced metabolic- and stress-related phenotypes in male and female mice were obtained., Results: In contrast to previously reported robust phenotypes of FKBP51 manipulation in the entire mediobasal hypothalamus (MBH), selective deletion or overexpression of FKBP51 in the VMH resulted in only a moderate alteration of HFD-induced bodyweight gain and body composition, independent of sex., Conclusions: Overall, this study shows that animals lacking and overexpressing Fkbp5 in Sf1-expressing cells within the VMH display only a mild metabolic phenotype compared to an MBH-wide manipulation of this gene, suggesting that FKBP51 in SF1 neurons within this hypothalamic nucleus plays a subsidiary role in controlling whole-body metabolism., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The inhibins control reproduction by suppressing follicle-stimulating hormone synthesis in pituitary gonadotrope cells. The newly discovered inhibin B coreceptor, TGFBR3L, is selectively and highly expressed in gonadotropes in both mice and humans. Here, we describe our initial characterization of mechanisms controlling cell-specific Tgfbr3l/TGFBR3L transcription. We identified two steroidogenic factor 1 (SF-1 or NR5A1) cis-elements in the proximal Tgfbr3l promoter in mice. SF-1 induction of murine Tgfbr3l promoter-reporter activity was inhibited by mutations in one or both sites in heterologous cells. In homologous cells, mutation of these cis-elements or depletion of endogenous SF-1 similarly decreased reporter activity. We observed nearly identical results when using a human TGFBR3L promoter-reporter. The Tgfbr3l gene was tightly compacted and Tgfbr3l mRNA expression was essentially absent in gonadotropes of SF-1 (Nr5a1) conditional knockout mice. During murine embryonic development, Tgfbr3l precedes Nr5a1 expression, though the two transcripts are fully colocalized by embryonic day 18.5 and thereafter. Collectively, these data indicate that SF-1 directly regulates Tgfbr3l/TGFBR3L transcription and is required for postnatal expression of the gene in gonadotropes., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phthalates may interfere with the biosynthesis of steroid hormones in the adrenal cortex. Bis (2-butoxyethyl) phthalate (BBOP) is a phthalate containing oxygen atoms in the alcohol moiety. In this study, 35-day-old male Sprague-Dawley rats were daily gavaged with BBOP (0, 10, 100, 250, and 500 mg/kg body weight) for 21 days. BBOP did not affect the weight of body and adrenal glands. BBOP significantly reduced serum corticosterone levels at 250 and 500 mg/kg, and lowered aldosterone level at 500 mg/kg without affecting adrenocorticotropic hormone. BBOP did not alter the thickness of the adrenal cortex. BBOP significantly down-regulated the expression of steroidogenesis-related genes (Scarb1, Star, Cyp11a1, Cyp21, Cyp11b1, Cyp11b2, Nr5a1, Nr4a1, and Nr4a2) and proteins, and antioxidant enzymes (Sod1, Sod2, Gpx1, and Cat) and their proteins, while up-regulating the expression of Mc2r and Agtr1a at various doses. BBOP reduced the phosphorylation of AKT1, AKT2, and ERK1/2, as well as the levels of SIRT1 and PGC1α without affecting the phosphorylation of AMPK. BBOP significantly induced the production of reactive oxygen species and apoptosis rate in H295R cells at 100 μM and higher after 24 h of treatment. In conclusion, male rats exposed to BBOP in puberty have significant reduction of steroid biosynthesis with a potential mechanism that is involved in the decrease in the phosphorylation of AKT1, AKT2, ERK1/2, as well as SIRT1 and PGC1α and increase in ROS., (© 2022 Wiley Periodicals LLC.)

In Brief: The insulin-like 3 (INSL3) hormone produced by Leydig cells is essential for proper male sex differentiation, but the regulation of Insl3 expression remains poorly understood. This study describes a new physical and functional cooperation between the nuclear receptors SF1 and COUP-TFII in Insl3 expression., Abstract: INSL3, a hormone abundantly produced by Leydig cells, is essential for testis descent during fetal life and bone metabolism in adults. The mechanisms regulating Insl3 expression in Leydig cells have been studied in several species but remain poorly understood. To date, only a handful of transcription factors are known to activate the Insl3 promoter and include the nuclear receptors AR, NUR77, COUP-TFII, and SF1, as well as the Krüppel-like factor KLF6. Some of these transcription factors are known to transcriptionally cooperate on the Insl3 promoter, but the mechanisms at play remain unknown. Here, we report that COUP-TFII and SF1 functionally cooperate on the Insl3 promoter from various species but not on the Inha, Akr1c14, Cyp17a1, Hsd3b1, Star, Gsta3, and Amhr2 promoters that are known to be regulated by COUP-TFII and/or SF1. The Insl3 promoter contains species-conserved binding sites for COUP-TFII (-91 bp) and SF1 (-134 bp). Mutation of either the COUP-TFII or the SF1 sequence had no impact on the COUP-TFII/SF1 cooperation, but the mutation of both binding sites abolished the cooperation. In agreement with this, we found that COUP-TFII and SF1 physically interact in Leydig cells. Finally, we report that the transcriptional cooperation is not limited to COUP-TFII and SF1 as it also occurred between all NR2F and NR5A family members. Our data provide new mechanistic insights into the cooperation between the orphan nuclear receptors COUP-TFII and SF1 in the regulation of Insl3 gene expression in Leydig cells.

The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable regulator of adrenal and gonadal formation, playing roles in sex determination, hypothalamic development, and pituitary function. This study aimed to identify the roles of SF-1 in postnatal female reproductive function. Using a progesterone receptor-driven Cre recombinase, we developed a novel murine model, characterized by conditional depletion of SF-1 [PR-Cre;Nr5a1f/f; conditional knockout (cKO)] in the hypothalamic-pituitary-gonadal axis. Mature female cKO were infertile due to the absence of ovulation. Reduced gonadotropin concentrations in the pituitary gland that were nevertheless sufficient to maintain regular estrous cycles were observed in mature cKO females. The cKO ovaries showed abnormal lipid accumulation in the stroma, associated with an irregular expression of cholesterol homeostatic genes such as Star, Scp2, and Acat1. The depletion of SF-1 in granulosa cells prevented appropriate cumulus oöphorus expansion, characterized by reduced expression of Areg, Ereg, and Ptgs2. Exogenous delivery of gonadotropins to cKO females to induce ovulation did not restore fertility and was associated with impaired formation and function of corpora lutea accompanied by reduced expression of the steroidogenic genes Cyp11a1 and Cyp19a1 and attenuated progesterone production. Surgical transplantation of cKO ovaries to ovariectomized control animals (Nr5a1f/f) resulted in 2 separate phenotypes, either sterility or apparently normal fertility. The deletion of SF-1 in the pituitary and in granulosa cells near the moment of ovulation demonstrated that this nuclear receptor functions across the pituitary-gonadal axis and plays essential roles in gonadotropin synthesis, cumulus expansion, and luteinization., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Information on the effects of copper on reproduction is limited. Our previous study indicated that copper induces abnormal steroidogenesis in human ovarian granulosa cells, but the underlying mechanism remains unclear. In this study, human ovarian granulosa cells were treated with multiple concentrations of copper for 24 h. After treatment, the 17-estradiol levels were significantly increased (29.83 % and 45.12 %, respectively) in the 1.0 and 2.0 μg/mL groups but decreased (23.06 % and 31.56 %, respectively) in the 20.0 and 40.0 μg/mL groups (P < 0.05). Similar changes in the levels of FSHR, StAR, CYP11A1, CYP19A1, HSD3β1, and SF-1 were observed. The protein levels of FSHR were increased in the 2.0 μg/mL group but decreased in the 20.0 and 40.0 μg/mL groups (P < 0.05). Moreover, copper partially reversed the FSH-induced increase in FSHR, CYP19A1 and 17-estradiol levels, and the decreased effect of the FSH receptor binding inhibitor fragment on FSHR, CYP19A1, and 17-estradiol became more apparent after adding copper. Additionally, the total methylation levels of the SF-1 promoter and DNMTs expression were significantly decreased following copper treatment. Overall, our results indicate that copper exposure induces steroidogenesis disorders via the FSHR/CYP19A1 pathway and changes DNA methylation on the SF-1 promoter in human ovarian granulosa cells., 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 © 2021 Elsevier B.V. All rights reserved.)

The vomeronasal system plays an essential role in sensing various environmental chemical cues. Here we show that mice exposed to blood and, consequently, hemoglobin results in the activation of vomeronasal sensory neurons expressing a specific vomeronasal G protein-coupled receptor, Vmn2r88, which is mediated by the interaction site, Gly17, on hemoglobin. The hemoglobin signal reaches the medial amygdala (MeA) in both male and female mice. However, it activates the dorsal part of ventromedial hypothalamus (VMHd) only in lactating female mice. As a result, in lactating mothers, hemoglobin enhances digging and rearing behavior. Manipulation of steroidogenic factor 1 (SF1)-expressing neurons in the VMHd is sufficient to induce the hemoglobin-mediated behaviors. Our results suggest that the oxygen-carrier hemoglobin plays a role as a chemosensory signal, eliciting behavioral responses in mice in a state-dependent fashion., (© 2022. The Author(s).)

Steroidogenic factor 1 (sf1) (officially designated as nuclear receptor subfamily 5 group A member 1 [NR5A1]) is an important regulator of gonad development. Previous studies on sf1 in fish have been limited to cloning and in vitro expression experiments. In this study, we used antisense RNA to down-regulate sf1 transcription and sf1 protein expression. Down-regulation of sf1 resulted in an increase in body weight and inhibition of gonadal development in both males and females with the consequent lower gonadosomatic index compared to fish in the control group. Hematoxylin-eosin staining of the gonads of fish with down-regulated sf1 revealed fewer seminiferous tubules and sperm in the testis of males. In addition, the oocytes were mainly stage II and many of them were atretic follicle. We conducted comparative transcriptome and proteome analyses between the sf1-down-regulated group and the control group. These analyses revealed multiple gene-protein pairs and pathways involved in regulating the observed changes, including 44 and 74 differently expressed genes and proteins in males and females, respectively. The results indicated that dysfunctional retinal metabolism and fatty acid metabolism could be causes of the observed weight gain and gonad abnormalities in sf1-down-regulated fish. These findings demonstrate the feasibility of using antisense RNA for gene editing in fish. This methodology allows the study gene function in species less amenable to gene editing as for example aquaculture species with long life cycles., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: NR5A1 is an essential transcription factor that regulates several target genes involved in reproduction and endocrine function. Pathogenic variants in this gene are responsible for a wide spectrum of disorders/differences of sex development (DSD)., Methods: The molecular study involved Sanger sequencing, in vitro assays, and whole exome sequencing (WES)., Results: Four variants were identified within the NR5A1 non-coding region in 3 patients with 46,XY DSD. In vitro analyses showed that promoter activity was affected in all cases. WES revealed variants in SRA1, WWOX, and WDR11 genes., Discussion/conclusion: Evaluation of clinical and phenotypic significance of variants located in a non-coding region of a gene can be complex, and little is known regarding their association with DSD. Nevertheless, based on the important region for interaction with cofactors essential to promote appropriated sex development and on our in vitro results, it is feasible to say that an impact on gene expression can be expected and that this may be correlated with the DSD pathophysiology presented in our patients. Considering the number of cases that remain elusive after screening for the well-known DSD related genes, we emphasize the importance of a careful molecular analysis of NR5A1 non-coding region which is commonly neglected and might explain some idiopathic DSD cases., (© 2022 S. Karger AG, Basel.)

The functional role of the transcription factors NR5A1 and NR5A2 and their interaction with Clock gene and bone morphogenetic proteins (BMPs) were investigated in human granulosa KGN cells. Treatment with BMP-15 and GDF-9 suppressed forskolin (FSK)-induced steroidogenesis as shown by the mRNA expression levels of StAR and P450scc but not the mRNA expression level of P450arom. Of interest, treatment with BMP-15 and GDF-9 also suppressed FSK-induced NR5A2 mRNA expression. Treatment with BMP-15 suppressed NR5A2 mRNA and protein expression but increased Clock mRNA and protein expression levels by granulosa cells. The mRNA expression levels of NR5A1, but not those of NR5A2, were positively correlated with the levels of Clock mRNA, while the mRNA levels of Id-1, the target gene of BMP signaling, were positively correlated with those of NR5A1 but not with those of NR5A2. It was also demonstrated that the mRNA expression levels of NR5A1 were positively correlated with those of P450arom and 3βHSD, whereas the mRNA expression level of NR5A2 was correlated with those of StAR and P450scc. Furthermore, inhibition of Clock gene expression by siRNA attenuated the expression of NR5A1, and the mRNA levels of Clock gene were significantly correlated with those of NR5A1. Collectively, the results suggested a novel mechanism by which Clock gene expression induced by BMP-15 is functionally linked to the expression of NR5A1, whereas NR5A2 expression is suppressed by BMP-15 in granulosa cells. The interaction between Clock NR5A1/NR5A2 and BMP-15 is likely to be involved in the fine-tuning of steroidogenesis by ovarian granulosa cells.

The ventromedial nucleus of the hypothalamus (VMH) is a complex brain structure that is integral to many neuroendocrine functions, including glucose regulation, thermogenesis, and appetitive, social, and sexual behaviors. As such, it is of little surprise that the nucleus is under intensive investigation to decipher the mechanisms which underlie these diverse roles. Developments in genetic and investigative tools, for example the targeting of steroidogenic factor-1-expressing neurons, have allowed us to take a closer look at the VMH, its connections, and how it affects competing behaviors. In the current review, we aim to integrate recent findings into the literature and contemplate the conclusions that can be drawn., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Cyclic adenosine monophosphate responsive element-binding protein-1-regulated transcription coactivator-1 (CRTC1) is a cytoplasmic coactivator that translocates to the nucleus in response to cyclic adenosine monophosphate. Whole-body knockdown of Crtc1 causes obesity, resulting in increased food intake and reduced energy expenditure. CRTC1 is highly expressed in the brain; therefore, it might play an important role in energy metabolism via the neuronal pathway. However, the precise mechanism by which CRTC1 regulates energy metabolism remains unknown. Here, we showed that mice lacking CRTC1, specifically in steroidogenic factor-1 expressing cells (SF1 cells), were sensitive to high-fat diet (HFD)-induced obesity, exhibiting hyperphagia and increased body weight gain. The loss of CRTC1 in SF1 cells impaired glucose metabolism. Unlike whole-body CRTC1 knockout mice, SF1 cell-specific CRTC1 deletion did not affect body weight gain or food intake in normal chow feeding. Thus, CRTC1 in SF1 cells is required for normal appetite regulation in HFD-fed mice. CRTC1 is primarily expressed in the brain. Within the hypothalamus, which plays an important role for appetite regulation, SF1 cells are only found in ventromedial hypothalamus. RNA sequencing analysis of microdissected ventromedial hypothalamus samples revealed that the loss of CRTC1 significantly changed the expression levels of certain genes. Our results revealed the important protective role of CRTC1 in SF1 cells against dietary metabolic imbalance., (© 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.)

Aims: Increasing evidence has shown that hormone secretion is regulated by endocytosis. Eps15 homology domain-containing protein 3 (EHD3) is an endocytic-trafficking regulatory protein, but whether EHD3 is associated with testosterone secretion is not clear. This work aims to explore the role of EHD3 in testosterone synthesis., Main Methods: Testosterone concentration was determined by ELISA. The effects of EHD3 on endocytosis were assessed by exosomes tracing assay and Immunofluorescence. Targeting relationship between EHD3 and NR5A1 was verified by chromatin immunoprecipitation (ChIP) and dual luciferase reporter gene assay in Leydig cells. For in vivo assessments, conditional NR5A1 knockout mouse model was established with CRISPR/Cas9 gene targeting technology., Key Findings: EHD3 overexpression significantly increased the concentration of testosterone. EHD3 knockdown markedly decreased testosterone synthesis by reducing endocytosis. The activity of the EHD3 promoter was positively regulated by NR5A1, which occupied the conserved sequence "AGGTCA" in the EHD3 promoter. Furthermore, mice with a Leydig cell-specific conditional NR5A1 knockout displayed the blunted levels of EHD3 and clathrin (a key factor for endocytosis), and serum testosterone concentration compared with NR5A1 f/f mice., Significance: This study suggests a potential molecular mechanism of testosterone synthesis to fully understand male reproductive health., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Oocytes mature in a specialized fluid-filled sac, the ovarian follicle, which provides signals needed for meiosis and germ cell growth. Methods have been developed to generate functional oocytes from pluripotent stem cell-derived primordial germ cell-like cells (PGCLCs) when placed in culture with embryonic ovarian somatic cells. In this study, we developed culture conditions to recreate the stepwise differentiation process from pluripotent cells to fetal ovarian somatic cell-like cells (FOSLCs). When FOSLCs were aggregated with PGCLCs derived from mouse embryonic stem cells, the PGCLCs entered meiosis to generate functional oocytes capable of fertilization and development to live offspring. Generating functional mouse oocytes in a reconstituted ovarian environment provides a method for in vitro oocyte production and follicle generation for a better understanding of mammalian reproduction., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Despite the substantial role played by the hypothalamus in the regulation of energy balance and glucose homeostasis, the exact mechanisms and neuronal circuits underlying this regulation remain poorly understood. In the last 15 years, investigations using transgenic models, optogenetic, and chemogenetic approaches have revealed that SF1 neurons in the ventromedial hypothalamus are a specific lead in the brain's ability to sense glucose levels and conduct insulin and leptin signaling in energy expenditure and glucose homeostasis, with minor feeding control. Deletion of hormonal receptors, nutritional sensors, or synaptic receptors in SF1 neurons triggers metabolic alterations mostly appreciated under high-fat feeding, indicating that SF1 neurons are particularly important for metabolic adaptation in the early stages of obesity. Although these studies have provided exciting insight into the implications of hypothalamic SF1 neurons on whole-body energy homeostasis, new questions have arisen from these results. Particularly, the existence of neuronal sub-populations of SF1 neurons and the intricate neurocircuitry linking these neurons with other nuclei and with the periphery. In this review, we address the most relevant studies carried out in SF1 neurons to date, to provide a global view of the central role played by these neurons in the pathogenesis of obesity and diabetes.

The hypothalamus is a critical regulator of glucose metabolism and is capable of correcting diabetes conditions independently of an effect on energy balance. The small GTPase Rap1 in the forebrain is implicated in high-fat diet-induced (HFD-induced) obesity and glucose imbalance. Here, we report that increasing Rap1 activity selectively in the medial hypothalamus elevated blood glucose without increasing the body weight of HFD-fed mice. In contrast, decreasing hypothalamic Rap1 activity protected mice from diet-induced hyperglycemia but did not prevent weight gain. The remarkable glycemic effect of Rap1 was reproduced when Rap1 was specifically deleted in steroidogenic factor-1-positive (SF-1-positive) neurons in the ventromedial hypothalamic nucleus (VMH) known to regulate glucose metabolism. While having no effect on body weight regardless of sex, diet, and age, Rap1 deficiency in the VMH SF1 neurons markedly lowered blood glucose and insulin levels, improved glucose and insulin tolerance, and protected mice against HFD-induced neural leptin resistance and peripheral insulin resistance at the cellular and whole-body levels. Last, acute pharmacological inhibition of brain exchange protein directly activated by cAMP 2, a direct activator of Rap1, corrected glucose imbalance in obese mouse models. Our findings uncover the primary role of VMH Rap1 in glycemic control and implicate Rap1 signaling as a potential target for therapeutic intervention in diabetes.

In the mammalian testis, two distinct populations of Sertoli cells (SCs), the immature SCs (ISCs) and adult SCs (ASCs), play significant roles in regulating the development and function of Leydig cells. However, the effect of different SC types on the function of Leydig cells is poorly understood. Here, our study showed that miR-145-5p expression was significantly different in SCs at different stages, with the highest expression observed in ISCs. Exosomes mediate the transfer of miR-145-5p from ISCs to Leydig cells. Overexpression of miR-145-5p in Leydig cells significantly downregulated steroidogenic gene expression and inhibited testosterone synthesis. Additionally, miR-145-5p functioned by directly targeted steroidogenic factor-1 (Sf-1) and downregulated the expression of SF-1, which further downregulated the expression of steroidogenic genes, induced accumulation of lipid droplets, and eventually suppressed testosterone production. These findings demonstrate that SC-derived miR-145-5p plays a significant role in regulating the functions of Leydig cells and may therefore serve as a diagnostic biomarker for male hypogonadism developmental abnormalities during puberty., (© 2021 Federation of American Societies for Experimental Biology.)

In this study, the polycystic ovary syndrome (PCOS) mice model was randomly divided into 6 groups: blank control group, Clomiphene group, PCOS group, and Yulin mixture high-/medium-/low-dose group. Rats were killed after 5 weeks of administration. The expression levels of serum E 2 ,T,Insulin and LH were detected by ELISA. The localizations and quantities of Steroid-generating factor-1 (SF-1) and Cytochrome protein P450 a1 (Cyp19a1) were detected by immunohistochemistry and western blot. The quantities of miR-320 were detected by RT-PCR. The results showed that the mechanism of Yulin mixture inhibiting the growth of polycystic ovary on mouse PCOS model may be through the decreasing of serum T and LH levels and then reducing local estrogen content to make the polycystic ovary atrophy. Yulin mixture can decrease the level of miR-320 and increase the expression of SF-1 and Cyp19a1 in ovary, thereby regulating the ovarian granulosa cell proliferation and apoptosis.