Lambda-cyhalothrin (LCT) is a pyrethroid widely employed in agriculture. However, their effects on adrenal function during puberty remain unclear. The objectives of the study are to demonstrate LCT-mediated impairments of adrenal steroidogenesis in rats. Male Sprague-Dawley rats were gavaged with LCT (0.25, 0.5, and 1 mg/kg) from postnatal days 28 to 58, and toxicity was analyzed. LCT exposure did not affect body weight but significantly reduced serum aldosterone at ≥0.25 mg/kg and corticosterone at ≥0.5 mg/kg, without altering ACTH levels. Histological assessment revealed no changes in zona glomerulosa thickness, but 1 mg/kg LCT significantly reduced the CYP11A1-positive zona fasciculata thickness. qPCR and Western blot analyses indicated selective downregulation of Agtr1a, Cyp11b2, and Igf1 transcripts, with corresponding decreases in AGTR1A, CYP11B2, and IGF1 protein expression, implicating disrupted hormone biosynthesis and adrenal growth signaling. In H295R cells, LCT at ≤1000 μM was noncytotoxic but suppressed aldosterone and cortisol secretion at 100 μM. This concentration also significantly inhibited AGTR1A and CYP11B2 expression, increased CASP3, and decreased BCL-2 levels, without changes in ROS generation or mitochondrial membrane potential, suggesting apoptosis induction through ROS- and MMP-independent pathways. Collectively, these findings reveal that LCT impairs adrenal endocrine development and function by downregulating key steroidogenic mediators and promoting apoptosis.

Polybrominated diphenyl ethers (PBDEs) and their metabolite 4-bromodiphenyl ether (BDE3) are pervasive environmental contaminants suspected of endocrine-disrupting effects. This study investigates their inhibitory potential and mechanism of action against human (h3β-HSD1) and rat (r3β-HSD4) placental 3β-hydroxysteroid dehydrogenase, a pivotal enzyme in progesterone biosynthesis. Comprehensive enzymatic analysis revealed that among eight tested PBDE congeners and the structural analog 4-bromobiphenyl (BDP), only BDE3 and BDP exhibited significant inhibition. BDE3 was a potent, mixed/competitive inhibitor with IC50 values of 5.86 μM (h3β-HSD1) and 12.35 μM (r3β-HSD4), significantly more potent than BDP (IC50 ∼ 100 μM). Mechanistic studies using pregnenolone as substrate confirmed mixed/competitive inhibition for both compounds, indicating binding at the steroid substrate site, while analysis with NAD⁺ suggested mixed/noncompetitive inhibition. Molecular docking and dynamics simulations corroborated these findings, indicating BDE3 and BDP binding at the steroid/NAD⁺ interface with favorable binding energies, stabilizing the active-site conformation. Network toxicology analysis identified HSD3B1 (encoding h3β-HSD1) as a central hub gene linking PBDE exposure to preeclampsia, enriched in steroidogenesis and estrogen signaling pathways. Single-cell RNA-seq analysis revealed predominant HSD3B1 expression in extravillous and syncytiotrophoblasts, with reduced expression in preeclampsia. In silico virtual knockout of HSD3B1 predicted downstream disruption of cell cycle regulation and steroidogenic pathways. Pharmacokinetic predictions indicated both compounds have high intestinal absorption but potential hepatotoxicity. These results suggest that BDE3 may act as a potent inhibitor of placental 3β-HSD, potentially linking its disruption of progesterone synthesis to adverse pregnancy outcomes.

Pentachlorophenol (PCP) is a pesticide and persistent endocrine disruptor. This study examined how PCP and its metabolite tetrachloro-1,4-benzoquinone (TCBQ) inhibit human (h3β-HSD2) and rat (r3β-HSD1) gonadal steroidogenic enzymes. In vitro assays using human KGN cells and rat testicular microsomes showed PCP moderately inhibited h3β-HSD2 (IC₅₀: 22.55 μM, mixed inhibitor), while TCBQ was more potent (IC₅₀: 6.42 μM). PCP (25-50 μM) significantly reduced progesterone in KGN cells, whereas TCBQ suppressed it at ≥ 1 μM. In rats, PCP weakly inhibited r3β-HSD1 (IC₅₀: 32.03 μM), but TCBQ was far stronger (IC₅₀: 41 nM, mixed/noncompetitive). Molecular docking revealed both compounds bind to the enzyme's steroid/NAD⁺ site, with TCBQ covalently attaching to Cys123 via Michael addition. Dithiothreitol (DTT) partially reversed TCBQ's inhibition but not PCP's, suggesting differing mechanisms. Findings indicate PCP's metabolic activation to TCBQ drives its stronger endocrine-disrupting effects, highlighting TCBQ's role as a potent 3β-HSD inhibitor in both species.

Pentachlorophenol (PCP) is a widely used pesticide. However, whether PCP and its metabolite chloranil have endocrine-disrupting effects by inhibiting placental 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) remains unclear. The study used in vitro assays with human and rat placental microsomes to measure 3β-HSD activity as well as human JAr cells to evaluate progesterone production. The results showed that PCP exhibited moderate inhibition of human 3β-HSD1, with an IC50 value of 29.83μM and displayed mixed inhibition in terms of mode of action. Conversely, chloranil proved to be a potent inhibitor, demonstrating an IC50 value of 147nM, and displaying a mixed mode of action. PCP significantly decreased progesterone production by JAr cells at 50μM, while chloranil markedly reduced progesterone production at ≥1μM. Interestingly, PCP and chloranil moderately inhibited rat placental homolog 3β-HSD4, with IC50 values of 27.94 and 23.42μM, respectively. Dithiothreitol (DTT) alone significantly increased human 3β-HSD1 activity. Chloranil not PCP mediated inhibition of human 3β-HSD1 activity was completely reversed by DTT and that of rat 3β-HSD4 was partially reversed by DTT. Docking analysis revealed that both PCP and chloranil can bind to the catalytic domain of 3β-HSDs. The difference in the amino acid residue Cys83 in human 3β-HSD1 may explain why chloranil is a potent inhibitor through its interaction with the cysteine residue of human 3β-HSD1. In conclusion, PCP is metabolically activated to chloranil as a potent inhibitor of human 3β-HSD1.

Bisphenol A (BPA) and its analogues are widely used industrial chemicals. Placental 3β-hydroxysteroid dehydrogenases (3β-HSDs) catalyse the conversion of pregnenolone to progesterone. However, the potency of BPA analogues in inhibiting 3β-HSDs activity remains unclear. We investigated the inhibitory effect of 10 BPA analogues on 3β-HSDs activity using an in vitro assay and performed the structure-activity relationship and in silico docking analysis. BPH was the most potent inhibitor of human 3β-HSD1, with an IC50 value of 0.95 μM. BPFL, BPG, DABPA, BPAP, BPZ, DMBPA, and BPB also inhibited human 3β-HSD1 activity, albeit with lower potency. BPG was the most potent inhibitor of rat 3β-HSD4, with an IC50 value of 1.14 μM. BPAP, BPFL, BPG, BPH, BPZ, DABPA, and DMBPA are mixed inhibitors of human 3β-HSD1 and they significantly inhibited human JAr cells to secrete progesterone. The LogP values were inversely correlated with the inhibitory effects. Docking analysis showed that most BPA analogues bind to steroid-binding site of both 3β-HSDs. A pharmacophore containing hydrogen bond donor and hydrophobic region was generated for predicting the inhibitory strength of BPA analogues. In conclusion, this study demonstrates that some BPA analogues are potent inhibitors of 3β-HSDs and lipophilicity determines the inhibitory potency.

Butorphanol is a synthetic opioid analgesic medication that is primarily used for the management of pain. Butorphanol may have an inhibitory effect on androgen biosynthesis and metabolism in rat immature Leydig cells. The objective of this study was to investigate the influence of butorphanol on androgen secretion by rat Leydig cells isolated from the 35 -day -old male rats. Rat Leydig cells were cultured with 0.5-50 mu M butorphanol for 3 h in vitro. Butorphanol at 5 and 50 mu M significantly inhibited androgen secretion in immature Leydig cells. At 50 mu M, butorphanol also blocked the effects of luteinizing hormone (LH) and 8bromo-cAMP-stimulated androgen secretion and 22R-hydroxycholesterol- and pregnenolone-mediated androgen production. Further analysis of the results showed that butorphanol downregulated the expression of genes involved in androgen production, including Lhcgr (LH receptor), Cyp11a1 (cholesterol side -chain cleavage enzyme), Srd5a1 (5 alpha -reductase 1), and Akr1c14 (3 alpha -hydroxysteroid dehydrogenase). Additionally, butorphanol directly inhibited HSD3B1 (3?-hydroxysteroid dehydrogenase 1) and SRD5A1 activity. In conclusion, butorphanol may have side effects of inhibiting androgen biosynthesis and metabolism in Leydig cells.

Bisphenol A (BPA) and its analogues are widely used industrial chemicals. Placental 3 beta-hydroxysteroid dehydrogenases (3 beta-HSDs) catalyse the conversion of pregnenolone to progesterone. However, the potency of BPA analogues in inhibiting 3 beta-HSDs activity remains unclear. We investigated the inhibitory effect of 10 BPA analogues on 3 beta-HSDs activity using an in vitro assay and performed the structure-activity relationship and in silico docking analysis. BPH was the most potent inhibitor of human 3 beta-HSD1, with an IC50 value of 0.95 mu M. BPFL, BPG, DABPA, BPAP, BPZ, DMBPA, and BPB also inhibited human 3 beta-HSD1 activity, albeit with lower potency. BPG was the most potent inhibitor of rat 3 beta-HSD4, with an IC50 value of 1.14 mu M. BPAP, BPFL, BPG, BPH, BPZ, DABPA, and DMBPA are mixed inhibitors of human 3 beta-HSD1 and they significantly inhibited human JAr cells to secrete progesterone. The LogP values were inversely correlated with the inhibitory effects. Docking analysis showed that most BPA analogues bind to steroid-binding site of both 3 beta-HSDs. A pharmacophore containing hydrogen bond donor and hydrophobic region was generated for predicting the inhibitory strength of BPA analogues. In conclusion, this study demonstrates that some BPA analogues are potent inhibitors of 3 beta-HSDs and lipophilicity determines the inhibitory potency.

Morphine is an analgesic in the opiate family, isolated from many plants. It can inhibit androgen biosynthesis by Leydig cells. Whether morphine directly inhibits androgen biosynthesis and underlying mechanism remains unclear. To investigate the influence of morphine on androgen secretion by rat immature Leydig cells (ILCs) and possible mechanism. Rat ILCs were treated with 0.5-50 mu M morphine for 3 h in vitro. Morphine at >= 0.5 mu M significantly reduced total androgen secretion. Morphine at 50 mu M also compromised luteinizing hormone (LH, 10 mg/kg), 8Br-cAMP (1 mM), and 22R-hydroxycholesterol (20 mu M) stimulated total androgen, androstanediol, and testosterone secretion, without affecting pregnenolone, progesterone, androstenedione mediated androgen secretion and testosterone and dihydrotestosterone mediated androstanediol secretion. Further analysis revealed that morphine at >= 0.5 mu M downregulated Star expression and at >= 5 mu M downregulated Cyp11a1 expression. Morphine also significantly reduced STAR (>= 0.5 mu M) and reduced CYP11A1 (>= 5 mu M) levels. 0.5 mu M naloxone significantly antagonized morphine-mediated action. In conclusion, morphine might cause side effects by suppressing androgen biosynthesis via u opioid receptor. Graphical Abstract

This research investigated the impact of cyclopiazonic acid (CPA), a mycotoxin, on the function of progenitor Leydig cells (PLCs) in prepubertal male rats, focusing on its potential disruption of mitochondrial integrity through mitofusin 1 (MFN1) modulation. In vivo, Sprague Dawley rats received CPA (0.2, 1, 5 mg/kg/day) via gavage from postnatal days 21-28 to evaluate PLC function and mitochondrial morphology using serum hormone levels, histology, qPCR, and Western blot analyses. In vitro, rat R2C cells were treated with CPA (0.1, 1, 10 μM) alone or in combination with 100 μM leflunomide to assess PLC development through testosterone measurements, Western blotting, flow cytometry, and Mito-Tracker Green Staining. The findings from in vivo experiments showed that CPA reduced serum testosterone and progesterone levels at 1 mg/kg/day. The qPCR and Western blotting analyses revealed significant alterations in the expression of genes and proteins pertinent to PLC function, such as Scarb1, Star, Cyp11a1, and Cyp17a1. Immunofluorescence staining further revealed a reduction in MFN1 expression following exposure to CPA. In vitro experiments corroborated these observations, demonstrating that CPA induced mitochondrial fragmentation by downregulating SIRT1, PGC1-α, MFN1, and OPA1, increase reactive oxygen species, and inhibit testosterone synthesis in R2C cells. The administration of leflunomide was shown to mitigate the detrimental effects of CPA on PLCs. In conclusion, this research sheds new light on the deleterious effects of CPA on the reproductive development of prepubertal males.

Pentachlorophenol (PCP) is a widely used pesticide. However, whether PCP and its metabolite chloranil have endocrine -disrupting effects by inhibiting placental 3 I3 -hydroxysteroid dehydrogenase 1 (3 I3 -HSD1) remains unclear. The study used in vitro assays with human and rat placental microsomes to measure 3 I3 -HSD activity as well as human JAr cells to evaluate progesterone production. The results showed that PCP exhibited moderate inhibition of human 3 I3 -HSD1, with an IC 50 value of 29.83 mu M and displayed mixed inhibition in terms of mode of action. Conversely, chloranil proved to be a potent inhibitor, demonstrating an IC 50 value of 147 nM, and displaying a mixed mode of action. PCP significantly decreased progesterone production by JAr cells at 50 mu M, while chloranil markedly reduced progesterone production at >= 1 mu M. Interestingly, PCP and chloranil moderately inhibited rat placental homolog 3 I3 -HSD4, with IC 50 values of 27.94 and 23.42 mu M, respectively. Dithiothreitol (DTT) alone significantly increased human 3 I3 -HSD1 activity. Chloranil not PCP mediated inhibition of human 3 I3 -HSD1 activity was completely reversed by DTT and that of rat 3 I3 -HSD4 was partially reversed by DTT. Docking analysis revealed that both PCP and chloranil can bind to the catalytic domain of 3 I3 -HSDs. The difference in the amino acid residue Cys83 in human 3 I3 -HSD1 may explain why chloranil is a potent inhibitor through its interaction with the cysteine residue of human 3 I3 -HSD1. In conclusion, PCP is metabolically activated to chloranil as a potent inhibitor of human 3 I3 -HSD1.

Bisphenol H (BPH) has emerged as a potential alternative to bisphenol A (BPA), which has been curtailed for use due to concerns over its reproductive and endocrine toxicity. This study investigates whether BPH exerts antiandrogenic effects by impairing Leydig cell function, a critical component in testosterone production. We administered orally BPH to adult male rats at doses of 0, 1, 10, and 100 mg/kg/day for 7 days. Notably, BPH treatment resulted in a dose-dependent reduction in testicular testosterone levels, with significant decreases observed at >= 1 mg/kg/day. Additionally, BPH affected the expression of key genes involved in steroidogenesis and cholesterol metabolism, including Nr5a1, Nr3c4, Lhcgr, Scarb1, and Star, at higher doses (10 and/or 100 mg/ kg/day). The study also revealed alterations in antioxidant gene expression (Sod2 and Cat) and modulation of m6A-related genes (Ythdf1-3 and Foxo3) and their proteins. Through MeRIP-qPCR analysis, we identified increased m6A modifications in Scarb1 and Star genes following BPH exposure. In vitro experiments with primary Leydig cells confirmed that BPH enhanced oxidative stress and diminished testosterone production, which were partially mitigated by antioxidant vitamin E supplementation and Ythdf3 knockdown. Meanwhile, simultaneous administration of BPH and vitamin E to primary Leydig cells partially counteracted BPH-induced alterations in the Ythdf3 expression. Our findings underscore a novel mechanism by which BPH disrupts Leydig cell function through the oxidative stress-m6A modification-autophagy pathway, raising concerns about its potential reproductive toxicity.

Deoxynivalenol (DON) is a common food contaminant that can impair male reproductive function. This study investigated the effects and mechanisms of DON exposure on progenitor Leydig cell (PLC) development in prepubertal male rats. Rats were orally administrated DON (0-4 mg/kg) from postnatal days 21-28. DON increased PLC proliferation but inhibited PLC maturation and function, including reducing testosterone levels and downregulating biomarkers like HSD11B1 and INSL3 at ≥2 mg/kg. DON also stimulated mitochondrial fission via upregulating DRP1 and FIS1 protein levels and increased oxidative stress by reducing antioxidant capacity (including NRF2, SOD1, SOD2, and CAT) in PLCs in vivo. In vitro, DON (2-4 μM) inhibited PLC androgen biosynthesis, increased reactive oxygen species production and protein levels of DRP1, FIS1, MFF, and pAMPK, decreased mitochondrial membrane potential and MFN1 protein levels, and caused mitochondrial fragmentation. The mitochondrial fission inhibitor mdivi-1 attenuated DON-induced impairments in PLCs. DON inhibited PLC steroidogenesis, increased oxidative stress, perturbed mitochondrial homeostasis, and impaired maturation. In conclusion, DON disrupts PLC development in prepubertal rats by stimulating mitochondrial fission.

Resveratrol and its analogs are phytochemicals. Human 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) synthesizes steroid hormones for normal pregnancy or promoting cancer metastasis. Whether they inhibit 3β-HSD1 remains unclear. In this study, the inhibitory potency, mode of action, structure-activity relationship, and docking parameters of resveratrol and its analogs on 3β-HSD1 and rat homolog 3β-HSD4 were analyzed. The inhibitory potency of these chemicals on human 3β-HSD1 was 4,4'-dihydroxystilbene (IC50, 3.68 μM) > pinostilbene (8.07 μM) > pinosylvin (10.60 μM) > lunularin (26.84 μM) > resveratrol (30.20 μM) > dihydroresveratrol (>100 μM) = oxyresveratrol (>100 μM) > dihydropinosylvin (ineffective at 100 μM). Resveratrol analogs and metabolites are mixed or competitive inhibitors of human 3β-HSD1. Resveratrol and 4,4'-dihydroxystilbene inhibited progesterone secretion by human JAr cells at ≥1 μM. Resveratrol (IC50, 32.09 μM) and pinosylvin (34.71 μM) significantly inhibited rat placental 3β-HSD4 activity. Docking analysis shows that resveratrol analogs and metabolites bind the steroid-binding sites of human 3β-HSD1 and rat 3β-HSD4 and interact with the catalytic residues Ser125/Thr125 and Tyr155. The negative correlation of LogP and IC50 values for human 3β-HSD1 indicates that lipophilicity of chemicals plays a critical role in the inhibitory effect of chemicals. In conclusion, 4,4'-dihydroxystilbene, pinostilbene, and pinosylvin effectively inhibit human 3β-HSD1 depending on their lipophilicity, thereby acting as potential therapeutic agents.

Chalcones from licorice and its related plants have many pharmacological effects. However, the effects of chalcones on the activity of human and rat 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), and associated side effects remain unclear. The inhibition of 11 chalcones on human and rat 11β-HSD2 were evaluated in microsomes and a 3D-quantitative structure-activity relationship (3D-QSAR) was analyzed. Screening revealed that bavachalcone, echinatin, isobavachalcone, isobavachromene, isoliquiritigenin, licochalcone A, and licochalcone B significantly inhibited human 11β-HSD2 with IC50 values ranging from 15.62 (licochalcone A) to 38.33 (echinatin) μM. Screening showed that the above chemicals and 4-hydroxychalcone significantly inhibited rat 11β-HSD2 with IC50 values ranging from 6.82 (isobavachalcone) to 72.26 (4-hydroxychalcone) μM. These chalcones acted as noncompetitive/mixed inhibitors for both enzymes. Comparative analysis revealed that inhibition of 11β-HSD2 depended on the species. Most chemicals bind to the NAD+ binding site or both the NAD+ and substrate binding sites. Bivariate correlation analysis showed that lipophilicity and molecular weight determine inhibitory strength. Through our 3D-QSAR models, we identified that the hydrophobic region, hydrophobic aliphatic groups, and hydrogen bond acceptors are pivotal factors in inhibiting 11β-HSD2. In conclusion, many chalcones inhibit human and rat 11β-HSD2, possibly causing side effects and there is structure-dependent and species-dependent inhibition on 11β-HSD2.

Patulin is a mycotoxin with potential reproductive toxicity. We explored the impact of patulin on Leydig cell (LC) development in male rats. Male Sprague Dawley rats (21 days postpartum) were gavaged patulin at doses of 0.5, 1, and 2 mg/kg/day for 7 days. Patulin markedly lowered serum testosterone at ≥0.5 mg/kg and progesterone at 1 and 2 mg/kg, while increasing LH levels at 2 mg/kg. Patulin increased the CYP11A1+ (cholesterol side-chain cleavage, a progenitor LC biomarker) cell number and their proliferation at 1 and 2 mg/kg. Additionally, patulin downregulated Lhcgr (luteinizing hormone receptor), Scarb1 (high-density lipoprotein receptor), and Cyp17a1 (17α-hydroxylase/17,20-lyase) at 1 and 2 mg/kg. It increased the activation of pAKT1 (protein kinase B), pERK1/2 (extracellular signal-related kinases 1 and 2), pCREB (cyclic AMP response binding protein), and CCND1 (cyclin D1), associated with cell cycle regulation, in vivo. Patulin increased EdU incorporation into R2C LC and stimulated cell cycle progression in vitro. Furthermore, patulin showed a direct inhibitory effect on 11β-HSD2 (11β-hydroxysteroid dehydrogenase 2) activity, which eliminates the adverse effects of glucocorticoids. This study provides insights into the potential mechanisms via which patulin affects progenitor LC development in young male rats.

Ethnopharmacological relevance: In traditional Chinese medicine, licorice (the roots of Glycyrrhiza glabra and G. inflata) has been used to treat inflammation and sexual debility for over 1000 years. Pharmacological studies have identified many biologically active chalcone derivatives from licorice.Aim of the study: Human 3ss-Hydroxysteroid dehydrogenase 2 (h3ss-HSD2) catalyzes the formation of precursors for sex hormones and corticosteroids, which play critical roles in reproduction and metabolism. We explored inhibition and mode action of chalcones of inhibiting h3ss-HSD2 and compared it with rat 3ss-HSD1.Materials and methods: We investigated the inhibition of 5 chalcones on h3ss-HSD2 and compared species-dependent difference with 3ss-HSD1.Results: The inhibitory strength on h3ss-HSD2 was isoliquiritigenin (IC50, 0.391 & mu;M) > licochalcone A (0.494 & mu;M) > licochalcone B (1.485 & mu;M) > echinatin (1.746 & mu;M) >chalcone (100.3 & mu;M). The inhibitory strength on r3ss-HSD1 was isoliquiritigenin (IC50, 0.829 & mu;M) > licochalcone A (1.165 & mu;M) > licochalcone B (1.866 & mu;M) > echinatin (2.593 & mu;M) > chalcone (101.2 & mu;M). Docking showed that all chemicals bind steroid and/or NAD+- binding site with the mixed mode. Structure-activity relationship analysis showed that strength was correlated with chemical's hydrogen bond acceptor.Conclusion: Some chalcones are potent h3ss-HSD2 and r3ss-HSD1 inhibitors, possibly being potential drugs to treat Cushing's syndrome or polycystic ovarian syndrome.

Trichlorfon is a widely used organophosphorus insecticide. It has been reported that it has reproductive toxicity to animal models. However, whether trichlorfon affects testosterone biosynthesis and metabolism remains unclear. In this study, we explored the effects of trichlorfon on the steroidogenesis and the expression of genes in androgen biosynthetic and metabolic cascades in immature Leydig cells isolated from pubertal male rats. Immature Leydig cells were treated with trichlorfon (0.5-50 µM) for 3 h. Trichlorfon significantly inhibited total androgen output under basal condition at 5 and 50 μM, and under LH- and cAMP-stimulated conditions at 50 μM. Trichlorfon also downregulated the expression of Star, Sod2, and Gpx1 and their proteins at 5 and 50 μM and the expression of Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a1 at 50 μM. Trichlorfon significantly inhibited total androgen output at 50 μM, which was partially reversed by 400 μg/ml vitamin E, which alone had no effects on androgen output. In conclusion, trichlorfon downregulates the expression of steroidogenesis-related genes and antioxidants, which leads to a decrease in androgen production in rat immature Leydig cells.

Fibroblast growth factor-4 (FGF4) is a heparin-binding growth factor that is a member of the fibroblast growth factor family. Proteins of this family play a central role during prenatal development, postnatal growth, and regeneration of a variety of tissues, by promoting cellular proliferation and differentiation. We setup an ethane-dimethane sulfonate (EDS) depleted Leydig cell regeneration model in adult male Sprague Dawley rats and treated them via intratesticular injection of FGF4 (0, 10, and 100 ng/testis/day) from post-EDS day 14 to 27. We measured serum hormone levels, Leydig cell number, gene, and protein expression in vivo. We explored the effects of FGF4 treatment on stem Leydig cell proliferation and differentiation and progenitor/adult Leydig cell steroidogenesis in vitro. FGF4 increased serum testosterone levels without affecting the levels of luteinizing hormone and follicle-stimulating hormone at 100 ng/testis on post-EDS day 28 in vivo. FGF4 increased Leydig cell number at 10 and 100 ng/mg without affecting Sertoli cell number and down-regulated the expression of Leydig cell genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b1, Insl3, and Nr5a1) per se and their proteins in vivo. FGF4 also lowered medium testosterone levels and down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, and Hsd17b3) but increased EdU incorporation into stem Leydig cells at 10 or 100 ng/ml in vitro. FGF4 inhibited steroidogenesis at ³ 1 ng/ml. In conclusion, FGF4 stimulated stem Leydig cell proliferation but blocked their differentiation. In brief: FGF 4 inhibits differentiation but stimulates proliferation of rat stem Leydig cells

Tetramethyl bisphenol A (TMBPA) is a widely used flame retardant. TMBPA has been a toxic to Leydig cells in puberty, but it remains unclear whether TMBPA has a similar inhibitor effect on fetal Leydig cells (FLCs). This study reported morphological and functional alterations of FLCs in the testes of male offspring at birth after in utero exposure to TMBPA. Pregnant Sprague Dawley rats were dosed via continuous gavage of TMBPA (0, 10, 50, and 200 mg/kg/day) from gestational day 14 to 21. TMBPA markedly raised serum total testosterone level, testicular volume, and FLC number of male offspring at 200 mg/kg dose. The up-regulation of Insl3, Star, and Cyp11a1 mRNAs was observed after 200 mg/kg TMBPA exposure. After normalization to the number of FLCs, TMBPA significantly reduced Lhcgr and Hsd3b1 expressions at 10 mg/kg, and Cyp17a1 at 200 mg/kg paralleling with their protein levels. TMBPA compromised the expression of Esr1, while increased the expression of Cdk2 and Cdk4 as well as their protein levels. TMBPA particularly increased the phosphorylation of AKT1 and AKT2 at 200 mg/kg. In conclusion, the present study suggests that TMBPA may promote FLC proliferation via ESR1-CDK2/4-AKT pathway, while inhibits the function of FLCs by reducing steroidogenic enzyme activity.

Fluornen-9-bisphenol (BPFL) is used as one of the alternatives for bisphenol A. However, whether BPFL has deleterious effects to the male reproductive system and the underlying mechanism remain unknown. Here, we report the effects of BPFL on Leydig cell development in male rats in puberty. Male Sprague-Dawley (28 days old) rats were dosed with 0, 10, 100, 200 mg/kg/day BPFL via gavage for 28 days. BPFL significantly decreased serum testosterone levels at 200 mg/kg while increasing serum luteinizing hormone and follicle-stimulating hormone levels at 200 mg/kg. BPFL markedly increased Leydig cell number but down-regulated the expression of Cyp17a1 and its protein level in Leydig cells at 200 mg/kg. Further study showed that BPFL significantly increased Pcna and Cdk2 expression and increased Leydig cell proliferation at 200 mg/kg. BPFL treatment to immature Leydig cells isolated from 28-day-old male rats for 24 h significantly inhibited testosterone biosynthesis at 50 μM, which was completely reversed by the androgen receptor agonist 7α-methyl-nortestosterone and estrogen receptor α antagonist ICI 182,780. In conclusion, BPFL increases Leydig cell proliferation but inhibits its maturation in male rats in puberty by blocking androgen receptor and activating estrogen receptor α.