Search Results (85)

Immortalized adrenal, placental and gonadal cell models are often termed steroidogenic based on steroid hormone production and steroidogenic enzymes. Profiling of ‘classic’ steroid metabolites is common; however, downstream untargeted metabolites remain unidentified. This study characterized steroidogenesis in human adrenal H295R and H295A; placental BeWo and JEG-3; mouse Leydig MA-10; and mouse adrenal Y-1 and OS-3 cells. Steroids were determined under basal, stimulated and serum-free conditions using liquid chromatography–mass spectrometry. This study identified distinct differences in mineralocorticoid and glucocorticoid production in the two human adrenal models and between the human and mouse adrenal models; unconventional hydroxylated progesterone steroid metabolites in all models which were most abundant in MA-10 cells; glucocorticoids and abundant classical androgens in MA-10 cells; 11-oxy androgens in H295R, H295A and MA-10 cells; comparable levels of the classical androgens in H295R and MA-10 cells, while 11-oxy androgen were more abundant in H295R and H295A cells; and high pregnenolone and progesterone in placental models with limited hydroxylated progesterone metabolites. Our detailed protocols and comprehensive steroid profiles provide an invaluable guide to researchers for in vitro investigations into steroidogenesis. Full article

Endometriosis-associated pain has debilitating effects on the quality of life of patients. Despite its high prevalence in reproductive-aged women, the pathophysiology is still unknown, impeding the development of targeted treatment approaches. The prospective ExPAND study proposes the neurosteroids pregnenolone sulphate (PS) and dehydroepiandrosterone sulphate (DHEAS) as potential contributors to endometriosis-associated pain, due to their agonistic action at the pain-related ion channel TRPM3. To this end, endometrium, deep endometriosis lesions, and peritoneal fluid were prospectively collected in four demarcated patient groups, which were characterised based on their pain symptoms, as scored via the WERF-EPHect questionnaire, i.e., (1) control (n = 44), (2) endometriosis patients with no pain symptoms (n = 24), (3) with only severe dysmenorrhea (n = 54), or (4) with both severe dysmenorrhea and non-cyclic pelvic pain (n = 78). Tissue mRNA expression levels of steroidogenic enzymes were investigated and showed significantly increased levels of CYP17A1 in the endometrium of patients with severe pain symptoms compared to control tissue. In addition, liquid chromatography with tandem mass spectrometry (LC-MS/MS) was performed to investigate neurosteroid concentrations in the peritoneal fluid. Both neurosteroids PS and DHEAS were present in the peritoneal fluid at concentrations that are known to stimulate TRPM3 activity in vitro. Finally, using microfluorimetric Ca²⁺ imaging, we demonstrate that both DHEAS and PS stimulate human stem-cell-derived sensory neurons in a TRPM3-dependent manner. Taken together, these data indicate a potential contribution of steroidogenesis and TRPM3 in endometriosis-associated pain. Full article

Thyroid hormones are fundamental regulators of cellular differentiation, development, and metabolism. Their receptors are expressed in reproductive tissues, including the ovary, and dysregulation of thyroid hormone homeostasis has been associated with menstrual disturbances, infertility, and adverse pregnancy outcomes. Bone morphogenetic protein (BMP) ligands and their receptors are functionally involved in gonadotropin-induced ovarian steroidogenesis in an autocrine or paracrine manner. In this study, we examined the effects of thyroid hormones on steroidogenesis and their interplay with BMP signaling by using human granulosa-like KGN cells and primary rat granulosa cells (GCs). In KGN cells, triiodothyronine (T3) enhanced forskolin-induced expression of key steroidogenic enzymes involved in both estradiol biosynthesis and progesterone synthesis/metabolism, whereas thyroxine (T4) exerted minimal effects. In rat GCs, T3 treatment increased follicle-stimulating hormone (FSH)-stimulated estradiol production without altering progesterone output. T3 pretreatment attenuated BMP-6-induced phosphorylation of Smad1/5/9 in KGN cells, accompanied by upregulation of inhibitory Smad6 and downregulation of the BMP type II receptor. Conversely, BMP-6 stimulation elevated thyroid hormone receptor β expression, indicating reciprocal regulatory interactions between thyroid hormone and BMP pathways. Collectively, these findings suggest that thyroid hormones modulate steroidogenesis, at least in part, through suppression of endogenous BMP-6 signaling in granulosa cells. Full article

(1) Objective: This study aimed to systematically elucidate the molecular mechanisms by which gypenosides (GP), a major active component of Gynostemma pentaphyllum, ameliorate hypercholesterolemia by modulating the hepatic steroidogenesis pathway, and to identify key therapeutic targets. (2) Methods: We established a high-fat diet (HFD)-induced hypercholesterolemia (HC) mouse model and performed GP intervention. An integrated multi-omics approach, combining transcriptomics and proteomics, was utilized to comprehensively analyze GP’s effects on the expression of genes and proteins associated with hepatic cholesterol synthesis, transport, and steroid hormone metabolism. (3) Results: HFD induced significant dysregulation, with 48 steroidogenesis pathway-related genes and 35 corresponding proteins exhibiting altered expression in HC mouse livers. GP treatment remarkably reversed these HFD-induced abnormalities, significantly restoring the expression levels of 42 genes and 14 proteins. Multi-omics integration identified seven critical genes/proteins—Cyp3a25, Fdft1, Tm7sf2, Hmgcs1, Fdps, Mvd, and Pmvk—that were consistently and significantly regulated by GP at both transcriptional and translational levels. Furthermore, correlation analyses demonstrated that Cyp3a25 was significantly negatively correlated with serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), whereas Fdft1, Tm7sf2, Hmgcs1, Fdps, Mvd, and Pmvk showed significant positive correlations. (4) Conclusions: GP effectively ameliorates cholesterol dyshomeostasis through a multi-targeted mechanism in the liver. It inhibits endogenous cholesterol synthesis by downregulating key enzymes (Hmgcs1, Fdft1, Pmvk, Mvd, Fdps, Tm7sf2), promotes cholesterol efflux and transport (upregulating Abca1, ApoB), and accelerates steroid hormone metabolism (upregulating Cyp3a11, Cyp3a25). These findings provide robust scientific evidence for the development of GP as a safe and effective novel therapeutic agent for hypercholesterolemia. Full article

This study elucidates the role of the steroidogenic acute regulatory protein (StAR) in sex steroid hormone dynamics and the gonadal development of the commercially important marine bivalve ark shell Scapharca broughtonii. The sequence of the StAR gene was obtained and verified from the transcriptome of ark shell, then the tissue localization and expression pattern during the gonad development of the StAR gene were detected by in situ hybridization and quantitative real-time PCR, respectively. Additionally, the concentrations of three critical sex steroid hormones (progesterone, testosterone, and estradiol) were measured throughout gonadal development using enzyme-linked immunosorbent assay (ELISA). The results showed that the length of the coding region of StAR was 1446 bp, encoding 481 amino acids. The results of qRT-PCR showed that the expression of the StAR gene varied with gonadal development, increased from the early active stage to the development stage, and decreased from the mature stage to the spent stage. Notably, the expression level in ovaries was higher than that in testes, suggesting the potential involvement of StAR in sex differentiation and gonadal development. Additionally, the results indicated that progesterone, testosterone, and estradiol accounted for 80%, 10%, and 10% of the total hormone content in the gonads, respectively. Correlation analysis revealed a highly significant strong positive correlation between progesterone/estradiol levels and StAR gene expression, demonstrating that StAR serves as a key regulator in sex steroid hormone biosynthesis. These findings provide crucial molecular evidence for StAR-mediated steroidogenesis in bivalve reproduction, offering fundamental insights into invertebrate endocrinology. Full article

Background: Prolactin (PRL) is a key reproductive hormone that regulates follicular development through endocrine and paracrine mechanisms. However, its specific role in porcine follicular development remains unclear. Methods: In the in vivo experiments, follicular fluid and tissue cells were obtained from small (1–2 mm), medium (3–4 mm), and large (5–6 mm) porcine follicles. PRL levels in follicular fluid were measured by ELISA. The expression levels of genes and proteins related to follicular development were assessed using quantitative real-time PCR (RT-qPCR) and Western blotting (WB). In the in vitro experiments, CCK-8, RT-qPCR, and WB were used to detect the effects of different concentrations (0, 30, and 300 ng/mL) of recombinant porcine prolactin (prPRL) on granulosa cell (GC) proliferation, steroid hormone synthesis, and angiogenesis, and transcriptome sequencing was performed. Results: The PRL concentration was significantly higher in large follicles compared to small and medium follicles. During follicular development, expression levels of PRL, PRL receptor (PRLR), proteolytic enzymes (CTSD, MMP2, MMP14, and BMP-1), and angiogenic factors (VEGFA and FGF-2) increased and then decreased. Moreover, prPRL promoted GC proliferation, increased the expression of PCNA and cyclin D1, upregulated steroidogenesis-related genes CYP11A1 and 3β-HSD, and significantly enhanced the expression of key angiogenic factors VEGFA and FGF-2. RNA-seq analysis identified 226 differentially expressed genes (DEGs), which were mainly enriched in signaling pathways such as the Hippo, JAK/STAT, and Rap1 pathways. Conclusions: PRL may regulate porcine follicle development by affecting cell proliferation and angiogenesis in GCs through the Hippo, JAK/STAT and Rap1 signaling pathways. Full article

Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system. The manifestation of MS is related to steroid changes during the menstrual cycle and pregnancy. As data focusing on the effect of anti-MS drug treatment on steroidome are scarce, we evaluated steroidomic changes (79 steroids) in 61 female MS patients of reproductive age 39 (29, 47) years (median with quartiles) after treatment with anti-MS drugs on the GC-MS/MS platform and immunoassays (cortisol and estradiol). The changes were assessed using steroid levels and steroid molar ratios (SMRs) that may reflect the activities of steroidogenic enzymes (SMRs). A repeated measures ANOVA, followed by multiple comparisons and OPLS models, were used for statistical analyses. The anti-MS treatment decreased steroid levels in the follicular phase. Anti-CD20 monoclonal antibodies (mAb), such as ofatumumab and ocrelizumab; inhibitors of the sphingosine-1-phosphate receptor (S1PRI); and IFNβ-1a decreased circulating 17-hydroxy-pregnanes and shifted the CYP17A1 functioning from the hydroxylase- toward the lyase step. Decreased conjugated/unconjugated steroid ratios were found after treatment with anti-MS drugs, especially for glatiramer acetate and anti-CD20 mAb. In the luteal phase, IFN-β1a treatment increased steroidogenesis; both IFN-β1a and ocrelizumab increased AKR1D1, and S1PRI increased SRD5A functioning. Anti-CD20 mAb reduced the functioning of enzymes catalyzing the synthesis of immunomodulatory 7α/β and 16α-hydroxy-androgens, which may affect the severity of MS. The above findings may be important concerning the alterations in bioactive steroids, such as cortisol; active androgens and estrogens; and neuroactive, neuroprotective, and immunomodulatory steroids in terms of optimization of anti-MS treatment. Full article

Background: Congenital adrenal hyperplasia (CAH) is a group of genetic disorders that lead to the dysfunction of the steroidogenesis pathway, resulting in steroid hormone deficiency of varied intensity. The cholesterol side-chain cleavage enzyme (P450scc), coded by the CYP11A1 gene, is vital to the first step in the biosynthesis of steroid hormones, which is the conversion of cholesterol to pregnenolone. Therefore, its deficiency causes a general steroid hormone shortage. Objective: We report a case of CAH caused by P450scc deficiency with complete 46, XY sex reversal, characteristic facial features (narrow middle section of the face, small ears with thick helix, fleshy upturned lobules), and dysmorphic macrocephaly along with shortened upper and lower extremities. Results: Our patient carries a compound heterozygotic pathogenic variant of the CYP11A1 gene, with two frameshift pathogenic variants NM_000781.3(CYP11A1):c.358del (p.Arg120Aspfs*18) in exon 2 and NM_000781.3(CYP11A1):c.835del (p.Ile279Tyrfs*10) in exon 5. To date, only around 50 cases with CYP11A1 pathogenic variants have been reported worldwide. We believe this is the first described case of a newborn with severe, classic P450scc deficiency in Poland. Conclusions: CYP11A1 (P450scc) deficiency is a rare and complex disorder that leads to primary adrenal insufficiency and may present with 46, XY disorders of sex development (DSD), phenotypic variations, and associated endocrinological abnormalities. This case, along with others cited, highlights the diverse presentations of DSD in individuals with pathogenic CYP11A1 variants. Optimal management necessitates a multidisciplinary approach by a specialized DSD team. Gonadectomy is a key consideration to decrease the teratogenic risk associated with intra-abdominal gonadal tissue. Full article

17α-Methyltestosterone (MT) is a widely used androgen for all-male fish production in aquaculture. However, the molecular mechanism underlying MT-induced masculinization remains unclear. In this study, we aim to identify the key gene responsible for MT-induced masculinization using the Nile tilapia (Oreochromis niloticus) amhy, dmrt1, and gsdf mutants, which exhibit male-to-female sex reversal. Nile tilapia fry from these three mutant lines were treated with 50 μg/g MT from 5 to 30 days after hatching (dah). The results showed that amhy and gsdf mutants, but not dmrt1 mutants, were masculinized by the MT treatment. Gonadal transcriptome analysis revealed that genes involved in steroidogenesis and germ cell development in MT-treated dmrt1 mutants exhibited a similar expression pattern to that of the wild type (WT) XX. In addition, the dmrt1 mutants cannot be masculinized by co-treatment with MT and the aromatase inhibitor fadrozole. The MT treatment completely blocked early steroidogenic enzyme (Star2, Cyp17a2, and Cyp19a1a) expression independent of amhy, gsdf, and dmrt1. A luciferase analysis showed that MT directly suppressed basal and Sf-1-activated cyp19a1a promoter activity through ara and arb in cultured HEK293 cells. Furthermore, MT treatment inhibited germ cell proliferation in amhy and gsdf mutants but not in dmrt1 mutants. Consistently, dmrt1 expression was induced in MT-treated WT XX, -amhy, and -gsdf mutants. Taken together, these results suggest that dmrt1 is indispensable for MT-induced masculinization in Nile tilapia and that MT functions by inhibiting early steroid synthesis and activating dmrt1 to promote testis development. Full article

D-aspartate (D-Asp) is an amino acid found in high concentrations in the testis and pituitary gland. Increasing evidence suggests that D-Asp promotes spermatogenesis by activating testosterone production in the Leydig cells via LH release from the pituitary gland. In vitro studies indicate that D-Asp may also influence steroidogenesis and spermatogenesis through autocrine and paracrine signals. D-Asp enhances StAR and steroidogenic enzyme expressions, facilitating testicular cell proliferation via the GluR/ERK1/2 pathway. Moreover, it supports spermatogenesis by enhancing the mitochondrial function in spermatocytes, aiding in the metabolic shift during meiosis. Enhanced mitochondrial function, along with improved MAM stability and reduced ER stress, has been observed in Leydig and Sertoli cells treated with D-Asp, indicating potential benefits in steroidogenesis and spermatogenesis efficiency. Conversely, D-Asp exerts a notable anti-apoptotic effect in the testis via the AMPAR/AKT pathway, potentially mediated by antioxidant enzyme modulation to mitigate testicular oxidative stress. This review lays the groundwork for future investigations into the molecules promoting spermatogenesis by stimulating endogenous testosterone biosynthesis, with D-amino acids emerging as promising candidates. Full article

Research question: Theca interna cells (TICs) are an indispensable cell source for ovarian follicle development and steroidogenesis. Recent studies have identified theca stem cells (TSCs) in both humans and animals. Interestingly, TSCs express mesenchymal stem cell (MSC)-related markers and can differentiate into mesenchymal lineages. MSCs are promising for tissue engineering and regenerative medicine due to their self-renewal and differentiation abilities. Therefore, this study investigated the potential origin of TICs from MSCs. Design: Whole ovaries from postmenopausal organ donors were obtained, and their cortex was cryopreserved prior to the isolation of stromal cells. These isolated cells were differentiated in vitro to TICs using cell media enriched with various growth factors and hormones. Immunocytochemistry, an enzyme-linked immunosorbent assay, flow cytometry, and reverse transcription–quantitative polymerase chain were employed at different timepoints. Data were analyzed using one-way ANOVA. Results: Immunocytochemistry showed an increase in TIC markers from day 0 to day 8 and a significant rise in MSC-like markers on day 2. This corresponds with rising androstenedione levels from day 2 to day 13. Flow cytometry identified a decreasing MSC-like cell population from day 2 onwards. The CD13+ cell population and its gene expression increased significantly over time. NGFR and PDGFRA expression was induced on days 0 and 2, respectively, compared to day 13. Conclusions: This study offers insights into MSC-like cells as the potential origin of TICs. Differentiating TICs from these widely accessible MSCs holds potential significance for toxicity studies and investigating TIC-related disorders like polycystic ovary syndrome (PCOS). Full article

Cellular metabolism, apoptosis, fertilization, and proliferation of granulosa cells belong to a battery of processes where microRNAs can be detected and associated with infertility. The aim of the present review is to focus on mammalian oocyte maturation events and the association between oocyte growth and miRNA expression. PubMed/Medline, Google Scholar and Scopus databases were searched, and 33 studies were included. Regarding the correlation among miRNA expression and the regulation of granulosa cells and cumulus cells, the most important miRNAs were let-7b, let-7c and miR-21. Additionally, the loss of Dicer, an enzyme involved in miRNA biogenesis, is probably a crucial factor in oogenesis, oocyte maturation and embryogenesis. Furthermore, miRNAs interfere with different cellular mechanisms like apoptosis, steroidogenesis, genome integrity, angiogenesis, antioxidative response and, consequently, oocyte maturation. Hence, it is of major importance to clarify the role and mechanism of each miRNA as understanding its action may develop new tools and establish new diagnostic and treatment approaches for infertility and ovarian disorders. Full article

Background and Objectives: The neuroendocrine system plays a crucial role in regulating various bodily functions, including reproduction, with evidence suggesting its significant involvement in male fertility and sperm development. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are expressed in both male and female reproductive tissues, influencing penile erection and regulating steroidogenesis in males. Therefore, our study aimed to compare the protein levels of VIP and PACAP in seminal plasma between healthy controls and sub-fertile patients. Additionally, we sought to correlate the levels of these biomarkers with clinical, functional, and laboratory findings in the participants. Materials and Methods: The study included a total of 163 male participants for analysis. The participants were further stratified into subgroups of fertile and sub-fertile men of four subgroups according to the 2021 WHO guidelines. Seminal plasma concentrations of the neuropeptides VIP and PACAP were measured using human enzyme-linked immunosorbent assay technique. Results: The findings showed statistically significant differences in total sperm count, sperm concentration, total motility, and vitality (p < 0.001) between the fertile group and the sub-fertile group. Specifically, significant differences found between healthy males and oligoasthenospermic patients (p = 0.002), and between asthenospermic and oligoasthenospermic patients (p = 0.039). An ROC analysis showed associated sensitivity and specificity values of 62.2% and 55.6%, respectively, to PACAP seminal levels differentiated between sub-fertile patients from fertile males (p = 0.028). No significant difference in seminal levels of VIP was found between the sub-fertile and fertile groups. Conclusions: Previous research leads to the point of PACAP active involvement in spermatogenesis. In accordance to our study, in human semen samples, we have seen a significance change in PACAP levels amongst patients with low sperm count or with both low sperm count and low motility, hinting at its contribution and acting as a possible factor in this complex process. Thus, alterations in the levels or actions of these neuropeptides have been associated with certain reproductive disorders in males. Full article

Mitochondria-Associated Endoplasmic Reticulum Membranes (MAMs) mediate the communication between the Endoplasmic Reticulum (ER) and the mitochondria, playing a fundamental role in steroidogenesis. This study aimed to understand how D-aspartate (D-Asp), a well-known stimulator of testosterone biosynthesis and spermatogenesis, affects the mechanism of steroidogenesis in rat testes. Our results suggested that D-Asp exerts this function through MAMs, affecting lipid trafficking, calcium signaling, ER stress, and mitochondrial dynamics. After 15 days of oral administration of D-Asp to rats, there was an increase in both antioxidant enzymes (SOD and Catalase) and in the protein expression levels of ATAD3A, FACL4, and SOAT1, which are markers of lipid transfer, as well as VDAC and GRP75, which are markers of calcium signaling. Additionally, there was a decrease in protein expression levels of GRP78, a marker of aging that counteracts ER stress. The effects of D-Asp on mitochondrial dynamics strongly suggested its active role as well. It induced the expression levels of proteins involved in fusion (MFN1, MFN2, and OPA1) and in biogenesis (NRF1 and TFAM), as well as in mitochondrial mass (TOMM20), and decreased the expression level of DRP1, a crucial mitochondrial fission marker. These findings suggested D-Asp involvement in the functional improvement of mitochondria during steroidogenesis. Immunofluorescent signals of ATAD3A, MFN1/2, TFAM, and TOMM20 confirmed their localization in Leydig cells showing an intensity upgrade in D-Asp-treated rat testes. Taken together, our results demonstrate the involvement of D-Asp in the steroidogenesis of rat testes, acting at multiple stages of both MAMs and mitochondrial dynamics, opening new opportunities for future investigation in other steroidogenic tissues. Full article

Day length is a critical environmental factor for regulating animal growth and development. This study aimed to investigate the effects of different day lengths on the developmental changes of growth parameters, testicular sizes, testosterone secretion in Meishan male pigs, and steroidogenesis proteins and melatonin receptors. Fourteen Meishan male pigs (10 weeks (wks) of age) with the same parity, paired in litter and body weight (BW), were evenly allocated into a short-day-length group (SDL, 10 light/14 dark) and long-day-length group (LDL, 14 light/10 dark). After 12 wks of the experiment, the LDL-treated boars had more lying time and less exploring time. The LDL treatment led to significant increases in body height, chest circumference, testicular length, testicular weight, crude protein digestibility, and fecal testosterone at the 10th and 12th wks of the experiment, and cortisol at the 10th wk, compared to the SDL treatment, with no differences in the final BW, testicular width, and epididymis weight. Furthermore, the LDL treatment significantly increased the protein levels of melatonin receptor 1b (MT2), aromatase (CYP19), and steroidogenic factor 1 (SF1) in the testis, with no differences in the protein levels of melatonin receptor 1a (MT1), steroidogenic acute regulatory (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cholesterol side-chain cleavage enzyme (P450scc). The present study suggests that day length has an effect on the growth and gonadal development in male pigs maybe via MT2 and influences steroid synthesis and secretion in the testis. Therefore, proper day length should be considered in male pig breeding. Full article