Search

Perfluorotetradecanoic acid (PFTeDA) is a novel perfluoroalkyl substance that ubiquitously exists in the environment. However, whether PFTeDA affects adrenal cortex function remains unclear. Male Sprague–Dawley rats (age of 60 days) were daily administered with PFTeDA (0, 1, 5, and 10 mg/kg body weight) through gavage for 28 days. PFTeDA did not change body and adrenal gland weights. PFTeDA markedly elevated serum corticosterone level at 10 mg/kg but lowering serum aldosterone level at this dosage without influencing serum adrenocorticotropic hormone level. PFTeDA thickened zona fasciculata without affecting zona glomerulosa. PFTeDA remarkably upregulated the expression of corticosterone biosynthetic genes (Mc2r, Scarb1, Star, Cyp21, Cyp11b1, and Hsd11b1) and their proteins, whereas downregulating aldosterone biosynthetic enzyme Cyp11b2 and its protein, thereby distinctly altering their serum levels. PFTeDA markedly downregulated the expression of antioxidant genes (Sod1 and Sod2) and their proteins at 10 mg/kg. PFTeDA significantly decreased SIRT1/PGC1α and AMPK signaling while stimulating AKT1/mTOR signaling. Corticosterone significantly inhibited testosterone production by adult Leydig cells at >0.1 μM in vitro; however aldosterone significantly stimulated testosterone production at 0.1 nM. In conclusion, exposure to PFTeDA at male rat adulthood causes corticosterone excess and aldosterone deficiency via SIRT1/PGC1α, AMPK, and AKT1/mTOR signals, which in turn additively leads to testosterone deficiency. [ABSTRACT FROM AUTHOR]

Background: Ciliary neurotrophic factor is a member of the interleukin‐6 family of cytokines. Ciliary neurotrophic factor drives many cells for their development. However, its effects on Leydig cell development remain unclear. Methods: In the current study, we used three‐dimensional seminiferous tubule culture system to induce the proliferation and differentiation of tubule‐associated stem Leydig cells and primary progenitor Leydig cells culture to address the effects of ciliary neurotrophic factor. Results: We found that ciliary neurotrophic factor stimulated the proliferation of stem Leydig cells but inhibited their development into the Leydig cell lineage. The ciliary neurotrophic factor‐mediated effects can be reversed by signal transducer and activator 3 inhibitor S3I‐201 and phosphatidylinositol 3‐kinase inhibitor wortmannin, indicating that ciliary neurotrophic factor acts via signal transducer and activator 3–phosphatidylinositol 3‐kinase signaling pathways to increase stem/progenitor Leydig cell proliferation. Ciliary neurotrophic factor at 1 and 10 ng/mL significantly decreased androgen production by progenitor Leydig cells. Microarray analysis of ciliary neurotrophic factor‐treated progenitor Leydig cells showed that ciliary neurotrophic factor blocked steroidogenic pathways by downregulating Scarb1, Star, and Hsd3b1, possibly by downregulating the transcription factor Nr5a1 expression. Conclusion: Ciliary neurotrophic factor stimulates proliferation but blocks the differentiation of stem/progenitor Leydig cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cypermethrin is a broad‐spectrum pyrethroid insecticide that is widely used. It may induce adverse endocrine‐disrupting effects on the male reproductive system. Whether cypermethrin can disrupt Leydig cell development and function in the late puberty remains elusive. The objective of this study was to explore the effect of cypermethrin exposure to male rats on the development and function of Leydig cells in late puberty and explore the underlying mechanism. Thirty‐six male Sprague–Dawley rats (age of 35 days) were gavaged with cypermethrin (0, 12.5, 25, and 50 mg/kg/day) from postnatal day 35–49. Cypermethrin significantly lowered serum testosterone level while elevating serum luteinizing hormone level at a dose of 50 mg/kg, without altering serum follicle‐stimulating hormone level. Cypermethrin markedly decreased CYP11A1‐positive Leydig cell number at 50 mg/kg without affecting SOX9‐positive Sertoli cell number. It significantly down‐regulated the expression of Leydig cell genes, Lhcgr, Star, Cyp11a1, and Cyp17a1 and their proteins, while up‐regulating the expression of Sertoli cell genes, Dhh and Amh, and their proteins, at doses of 12.5–50 mg/kg. In addition, cypermethrin significantly increased malondialdehyde level while lowering the expression of Sod1 and Sod2 and their proteins at 50 mg/kg. Cypermethrin markedly induced reactive oxidative species at a concentration of 200 μM and reduced mitochondrial membrane potential at 25 μM and higher concentrations after 24 h of treatment to primary Leydig cells in vitro. In conclusion, cypermethrin inhibits the development and function of Leydig cells in male rats in late puberty. [ABSTRACT FROM AUTHOR]

The function of immature Leydig cells is regulated by hormones, such as androgen and luteinizing hormone (LH). However, the regulation of this process is still unclear. The objective of this study was to determine whether luteinizing hormone (LH) or androgens contribute to this process. Immature Leydig cells were purified from 35-day-old male Sprague Dawley rats and cultured with LH (1 ng/ml) or androgen (7α-methyl-19- nortestosterone, MENT, 100 nM) for 2 days. LH or MENT treatment significantly increased the androgens produced by immature Leydig cells in rats. Microarray and qPCR and enzymatic tests showed that LH up-regulated the expression of Scarb1 , Cyp11a1 , Cyp17a1 , and Srd5a1 while down-regulated the expression of Sult2a1 and Akr1c14. On the contrary, the expression of Cyp17a1 was up-regulated by MENT. LH and MENT regulate Leydig cell function through different sets of transcription factors. We conclude that LH and androgens participate in the regulation of rat immature Leydig cell function through different transcriptional pathways. [ABSTRACT FROM AUTHOR]

Epidermal growth factor (EGF) has many physiological roles. However, its effects on stem and progenitor Leydig cell development remain unclear. Rat stem and progenitor Leydig cells were cultured with different concentrations of EGF alone or in combination with EGF antagonist, erlotinib or cetuximab. EGF (1 and 10 ng/mL) stimulated the proliferation of stem Leydig cells on the surface of seminiferous tubules and isolated CD90+ stem Leydig cells and progenitor Leydig cells but it blocked their differentiation. EGF also exerted anti‐apoptotic effects of progenitor Leydig cells. Erlotinib and cetuximab are able to reverse EGF‐mediated action. Gene microarray and qPCR of EGF‐treated progenitor Leydig cells revealed that the down‐regulation of steroidogenesis‐related proteins (Star and Hsd3b1) and antioxidative genes. It was found that EGF acted as a proliferative agent via increasing phosphorylation of AKT1. In conclusion, EGF stimulates the proliferation of rat stem and progenitor Leydig cells but blocks their differentiation. [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl (PFAS) substances are enduring industrial materials. 17β-Hydroxysteroid dehydrogenase isoform 1 (17β-HSD1) is an estrogen metabolizing enzyme, which transforms estrone into estradiol in human placenta and rat ovary. Whether PFAS inhibit 17β-HSD1 and what the structure-activity relationship (SAR) remains unexplored. We screened 18 PFAS for inhibiting human and rat 17β-HSD1 in microsomes and studied their SAR and mode of action(MOA). Of the 11 perfluorocarboxylic acids (PFCAs), C8-C14 PFCAs at a concentration of 100 μM substantially inhibited human 17β-HSD1, with order of C11 (half-maximal inhibition concentration, IC 50 , 8.94 μM) > C10 (10.52 μM) > C12 (14.90 μM) > C13 (30.97 μM) > C9 (43.20 μM) > C14 (44.83 μM) > C8 (73.38 μM) > others. Of the 7 per- and poly-fluorosulfonic acids (PFSAs), the potency was C8S (IC 50 , 14.93 μM) > C7S (80.70 μM) > C6S (177.80 μM) > others. Of the PFCAs, C8-C14 PFCAs at 100 μM markedly reduced rat 17β-HSD1 activity, with order of C11 (IC 50 , 9.11 μM) > C12 (14.30 μM) > C10 (18.24 μM) > C13 (25.61 μM) > C9 (67.96 μM) > C8 (204.39 μM) > others. Of the PFSAs, the potency was C8S (IC 50 , 37.19 μM) > C7S (49.38 μM) > others. In contrast to PFOS (C6S), the partially fluorinated compound 6:2 FTS with an equivalent number of carbon atoms demonstrated no inhibition of human and rat 17β-HSD1 activity at a concentration of 100 μM. The inhibition of human and rat enzymes by PFAS followed a V-shaped trend from C4 to C14, with a nadir at C11. Moreover, human 17β-HSD1 was more sensitive than rat enzyme. PFAS inhibited human and rat 17β-HSD1 in a mixed mode. Docking analysis revealed that they bind to the NADPH and steroid binding site of both 17β-HSD1 enzymes. The 3D quantitative SAR (3D-QSAR) showed that hydrophobic region, hydrogen bond acceptor and donor are key factors in binding to 17β-HSD1 active sites. In conclusion, PFAS exhibit inhibitory effects on human and rat 17β-HSD1 depending on factors such as carbon chain length, degree of fluorination, and the presence of carboxylic acid or sulfonic acid groups, with a notable V-shaped shift observed at C11. [Display omitted] • Some PFAS are inhibitors of human and rat 17β-HSD1. • PFAS inhibit human and rat 17β-HSD1 depending on the carbon atom numbers. • PFAS inhibit human and rat 17β-HSD1 depending on fluorination and the presence of acid groups. • The potency of PFAS increases from C4 to C11 and then declines. • 3D QSAR showed hydrogen bond acceptor, hydrogen bond donor and hydrophobic region for human 17β-HSD1. [ABSTRACT FROM AUTHOR]

Background: Propofol is a widely used anesthetic. Whether propofol inhibits androgen production by rat Leydig cells and the underlying mechanism remains unclear. The objective of the current study was to examine the effects of propofol exposure to rat primary immature Leydig cells and to define propofol-induced inhibition of steroidogenic enzymes in both rat and human testes in vitro. Methods: Immature Leydig cells were purified from 35-day-old male Sprague–Dawley rats and were exposed to propofol for 3 h. The androgen production by Leydig cells under basal, luteinizing hormone, 8bromo-cAMP, and steroid-substrate stimulated conditions and gene expression of Leydig cells after exposure to propofol were measured. Immature Leydig cells were treated with propofol for 3 h and switched to propofol-free medium for additional 3 and 9 h to test whether propofol-induced inhibition is reversible. 3H-Steroids were used to evaluate the direct action of propofol on cytochrome P450 cholesterol side chain cleavage (CYP11A1), 3β-hydroxysteroid dehydrogenase (HSD3B), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1), and 17β-hydroxysteroid dehydrogenase 3 (HSD17B3) activities in rat and human testes in vitro. Results: Propofol significantly lowered luteinizing hormone and 8bromo-cAMP stimulated androgen production by Leydig cells after 3-h exposure. Further investigation showed that propofol down-regulated the expression of Cyp11a1 and Cyp17a1 and their proteins at 5 and 50 µM, although it up-regulated Lhcgr expression at 50 µM. Propofol significantly suppressed phosphorylation of ERK1/2 and induced ROS production in immature Leydig cells at 5 and 50 µM. Propofol significantly induced apoptosis of immature Leydig cells at 50 µM. Propofol specifically inhibited rat and human testis HSD3B activities in vitro. The half maximal inhibitory concentrations of propofol for rat and human HSD3B enzymes were 1.011 ± 0.065 and 3.498 ± 0.067 µM, respectively. The mode of action of propofol of inhibiting HSD3B was competitive when pregnenolone was added. At 50 µM, propofol did not directly inhibit rat and human testis CYP11A1, CYP17A1, and HSD17B3 activities in vitro. Conclusion: Propofol inhibits androgen production via both directly inhibiting HSD3B activity and down-regulating Cyp11a1 and Cyp17a1 expression in Leydig cells. Suppression of steroidogenic enzymes is presumably associated with the lower production of androgen by Leydig cells after propofol treatment. However, propofol-induced inhibition on androgen production is reversible. [ABSTRACT FROM AUTHOR]

Leukemia inhibitory factor (LIF) has many physiological roles. However, its effects on Leydig cell development are still unclear. Rat immature and adult Leydig cells were cultured with different concentrations of LIF alone or in combination with luteinizing hormone (LH) for 24 h. LIF (1 and 10 ng/ml) significantly increased androgen production in immature Leydig cells, but had no effects on testosterone production in adult Leydig cells. Further studies revealed that LIF dose-dependently increasedStarandHsd17b3expression levels in immature Leydig cells. Gene microarray revealed that the upregulation of anti-oxidative genes andStarmight contribute to LIF-induced androgen production. In conclusion, LIF has stimulatory effects on androgen production in rat immature Leydig cells. [ABSTRACT FROM PUBLISHER]

Butylated hydroxyanisole (BHA) is a synthetic antioxidant used for food preservation. Whether BHA affects testosterone biosynthesis is still unclear. The effects of BHA on the steroidogenesis in rat immature Leydig cells were investigated. Rat immature Leydig cells were isolated from 35-old-day rats and cultured with BHA (50 μM) for 3 h in combination with 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone or dihydrotestosterone, and the concentrations of 5α-androstanediol and testosterone in the media were measured. Leydig cells were cultured with BHA (0.05–50 μM) for 3 h. Q-PCR was used to measure the mRNA levels of following genes:Lhcgr,Scarb1,Star,Cyp11a1,Hsd3b1,Cyp17a1,Hsd17b3,Srd5a1andAkr1c14. The testis microsomes were prepared to detect the direct action of BHA on 3β-hydroxysteroid dehydrogenase 1 (HSD3B1), 17α-hydroxylase (CYP17A1) and 17β-hydroxysteroid dehydrogenase 3 activities. In Leydig cells, BHA (50 μM) significantly inhibited LH- and 8Br-cAMP-mediated androgen production. BHA directly inhibited rat testis CYP17A1 and HSD3B1 activities. At 50 μM, it also reduced the expression levels ofHsd17b3andSrd5a1and their protein levels. In conclusion, BHA directly inhibits the activities of CYP17A1 and HSD3B1, and the expression levels ofHsd17b3andSrd5a1, leading to the lower production of androgen in Leydig cells. [ABSTRACT FROM PUBLISHER]

Methyl tert-butyl ether (MTBE) is a widely used gasoline additive. It is considered an endocrine-disrupting chemical. Whether MTBE affects the development of Leydig cells in late puberty of males and its underlying mechanism remains unclear. Twenty-four male Sprague-Dawley rats (35 days old) were randomly allocated into four groups and were orally given MTBE (0, 300, 600, and 1200 mg/kg/day) from postnatal day (PND) 35–56. MTBE markedly reduced serum testosterone levels at 300 mg/kg and higher doses without altering the serum levels of luteinizing hormone and follicle-stimulating hormone. It mainly inhibited cell proliferation, induced mitochondrial autophagy and apoptosis, and indirectly stimulated Sertoli cells to secrete anti-Müllerian hormones, thereby significantly reducing the number of Leydig cells at 1200 mg/kg. MTBE also markedly down-regulated the expression of mature Leydig cell biomarker Cyp11a1 and Hsd3b1 and their proteins, while up-regulating the expression of immature Leydig cell biomarker Akr1c14 and its protein at 600 mg/kg and higher. MTBE significantly down-regulated the expression of cell cycle gene Ccnd1 , antioxidant gene Gpx1, and anti-apoptotic gene Bcl2, while increasing pro-apoptotic gene Bax level at 1200 mg/kg. In vitro study further confirmed that MTBE can inhibit testosterone synthesis by inducing reactive oxygen species (ROS) generation, mitophagy, and apoptosis at 200 and 300 mM. In conclusion, exposure to MTBE compromises the development of Leydig cells in late puberty in male rats. Illustration of action of MTBE exposure on Leydig cell (LC) development in the late puberty. MTBE induces oxidative stress by lowering the transcript of GPX1 to cause the following actions: 1) activating AMPK and inactivating mTOR to induce mitophagy (a kind of autophagy); 2) inhibiting BCL2 expression and increasing the expression of Bax, thus increasing the mitochondrial membrane permeability to low the MMP and then the activation of CASP3, thereby inducing apoptosis. Finally, exposure to MTBE compromises the development of Leydig cells. [Display omitted] • MTBE reduces serum testosterone level in male rats after late pubertal exposure. • MTBE inhibits Leydig cell proliferation and number. • MTBE down-regulates the expression of maturity biomarker of Leydig cells. • MTBE induces ROS, apoptosis, and mitophagy. • MTBE reduces phosphorylation of mTOR and induces phosphorylation of AMPK in the testis. [ABSTRACT FROM AUTHOR]

In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3β-hydroxysteroid dehydrogenase (3βHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5α-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells. [ABSTRACT FROM AUTHOR]

Abstract: Humans have significant exposures to phthalates, as these chemical plasticizers are ubiquitously present in flexible plastics. Recent epidemiological evidence indicates that boys born to women exposed to phthalates during pregnancy have an increased incidence of congenital genital malformations and spermatogenic dysfunction, signs of a condition referred to as testicular dysgenesis syndrome (TDS). TDS is thought to develop as a result of environmental factors that cause a testicular disturbance at an early fetal stage with a resultant spectrum of clinical testicular dysfunction, ranging from impaired spermatogenesis and genital malformations to increased risk for development of testicular cancer. Proposed environmental factors in the etiology of TDS include endocrine disrupting compounds such as the phthalates. Leydig cells have been classified as one of the main targets for phthalate ester toxicity in the body based on studies in rodents. In support of this hypothesis, two Leydig cell products – insulin-like growth factor 3 (INSL3) and testosterone (T) – are both suppressed after phthalate exposures. Both fetal and adult generations of Leydig cells are affected by phthalate esters, although their sensitivities may differ. In rodent models, when pregnant dams are exposed to phthalate esters, fetal Leydig cells form enlarged clusters that are retained in the testis even after birth, in contrast to untreated controls. Despite the retention of fetal Leydig cells, however, their numbers and average cell volume of total in exposed males are reduced, as are INSL3 production and steroidogenic competence. These alterations are directly associated with clinical features of TDS, including cryptorchidism and impaired spermatogenesis. [Copyright &y& Elsevier]

Triadimefon is a broad-spectrum fungicide widely applied in the agriculture. It is believed to be an endocrine disruptor. Whether triadimefon can inhibit the development of fetal Leydig cells and the underlying mechanisms are unknown. Thirty-two female pregnant Sprague-Dawley rats were randomly assigned into four groups and were dosed via gavage of triadimefon (0, 25, 50, and 100 mg/kg/day) for 9 days from gestational day (GD) 12–20. Triadimefon significantly reduced serum testosterone level in male fetuses at 100 mg/kg. The double immunofluorescence staining of proliferating cell nuclear antigen (PCNA) and cytochrome P450 cholesterol side-chain cleavage (a biomarker for fetal Leydig cells) was used to measure PCNA-labeling in fetal Leydig cells. It markedly increased fetal Leydig cell number primarily via increasing single cell population and elevated the PCNA-labeling of fetal Leydig cells in male fetuses at 100 mg/kg while it induced abnormal aggregation of fetal Leydig cells. The expression levels of fetal Leydig cell genes, Lhcgr , Scarb1 , Star , Cyp11a1 , Hsd3b1 , Cyp17a1 , Hsd17b3 , Insl3 and Nr5a1 , were determined to explore its effects on fetal Leydig cell development. We found that triadimefon markedly down-regulated the expression of Leydig cell genes, Hsd17b3 , Insl3 , and Nr5a1 as low as 25 mg/kg and Scarb1 and Cyp11a1 at 100 mg/kg. It did not affect Sertoli cell number but markedly down-regulated the expression of Sertoli cell gene Amh at 50 and 100 mg/kg. Triadimefon significantly down-regulated the expression of antioxidant genes Sod1 , Gpx1 , and Cat at 25–100 mg/kg, suggesting that it can induce oxidative stress in fetal testis, and it reduced the phosphorylation of ERK1/2 and AKT2 at 100 mg/kg, indicating that it can inhibit the development of fetal Leydig cells. In conclusion, gestational exposure to triadimefon inhibits the development of fetal Leydig cells in male fetuses by inhibiting its differentiation. Illustration of the mode of action of triadimefon (TDM) exposure on fetal Leydig cell (FLC) development. TDM induces oxidative stress by down-regulating the expression of SOD1, GPX1, and CAT to cause the following actions: 1) lowering the phosphorylation of AKT2 and ERK1/2; 2) down-regulating the expression of NR5A1, thereby inhibiting steroidogenesis and INSL3 synthesis on via down-regulating the expression of steroidogenesis-related genes and Insl3 ; 3) acting on Sertoli cells to reduce AMH expression, which blocks FLC proliferation. [Display omitted] • Triadimefon reduces serum testosterone level in male fetuses after in utero exposure. • Triadimefon increases fetal Leydig cell proliferation and number. • Triadimefon down-regulates the expression of steroidogenesis-related genes. • Triadimefon reduces antioxidant enzymes SOD1, GPX1, and CAT. • Triadimefon reduces AKT2 and ERK1/2 phosphorylation in the fetal testis. [ABSTRACT FROM AUTHOR]

Perfluorooctane sulfonate is related to male reproductive dysfunction in rats and humans. However, the underlying mechanism remains unknown. Here, we reported the effects of short-term exposure to perfluorooctane sulfonate on the regeneration of Leydig cells in vivo and investigated possible mechanisms in vitro. After adult male Sprague-Dawley rats were gavaged perfluorooctane sulfonate (0, 5 or 10 mg/kg/day) for 7 days and then injected intraperitoneally ethane dimethane sulfonate next day to eliminate Leydig cells, the Leydig cell regeneration process was monitored. Perfluorooctane sulfonate significantly lowered serum testosterone levels, reduced the number of regenerated Leydig cells, down-regulated the expression of Lhcgr , Scarb1 , Star , Cyp11a1 , Hsd3b1 , Cyp17a1 , and Dhh) and their proteins at doses of 5 and 10 mg/kg 35 and 56 days after ethane dimethane sulfonate. Using a 3D seminiferous tubule culture system to study the development of stem Leydig cells, we found that perfluorooctane sulfonate inhibited stem Leydig cell proliferation and differentiation and hedgehog signaling pathway. In conclusion, a short-term exposure to perfluorooctane sulfonate can inhibit the development of stem Leydig cells into the Leydig cell lineage via direct suppression of hedgehog signaling pathway and indirect inhibition of desert hedgehog section by Sertoli cells. [Display omitted] • PFOS reduces serum testosterone levels during Leydig cell regeneration. • PFOS reduces Leydig cell number during regeneration • PFOS down-regulates Leydig cell steroidogenesis-related gene expression. • PFOS down-regulates expression of DHH and antagonizes SMO action. • PFOS inhibits stem Leydig cell proliferation and differentiation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Triclosan affects sterol synthase in rats, which leads to dyssynthesis of testosterone in rats. • Triclosan causes m6A methylation of mRNA, leading to Leydig cell autophagy. • Leydig cell developed malignant autophagy and cell death. Triclosan is a potent antibacterial compound widely used in everyday products. Whether triclosan affects Leydig cell function in adult male rats remains unknown. In this study, 0, 50, 100, or 200 mg/kg/day triclosan was gavaged to Sprague-Dawley male rats from 56 to 63 days postpartum. Triclosan significantly reduced serum testosterone levels at ≥ 50 mg/kg/day via downregulating the expression of Leydig cell gene Lhcgr , Scarb1 , Star , Cyp11a1 , Hsd3b1 , Cyp17a1 , and Hsd17b3 and regulatory transcription factor Nr3c2 at 100–200 mg/kg. Further analysis showed that triclosan markedly increased autophagy as shown by increasing LC3II and BECN1 and decreasing SQSTM1. The mRNA m6A modification analysis revealed that triclosan significantly downregulated Fto expression at 200 mg/kg while upregulating Ythdf1 expression at 100 and 200 mg/kg, leading to methylation of Becn1 mRNA as shown by MeRIP assay. Triclosan significantly inhibited testosterone output in rat R2C Leydig cells at ≥ 5 μM via downregulating Fto and upregulating Ythdf1. SiRNA Ythdf1 knockdown can reverse triclosan-mediated mitophagy in R2C cells, thereby reversing the reduction of testosterone output. In summary, triclosan caused Becn1 m6A methylation by downregulating Fto and upregulating Ythdf1 , which accelerated Becn1 translation, thus leading to the occurrence of autophagy and the decrease of testosterone biosynthesis. [ABSTRACT FROM AUTHOR]

Paraquat, a widely used nonselective herbicide, is a serious hazard to human health. However, the effects of paraquat on the male reproductive system remain unclear. In this study, adult male Sprague Dawley rats were intraperitoneally injected ethane dimethane sulfonate (EDS, 75 mg/kg) to initiate a regeneration of Leydig cells. EDS-treated rats were orally exposed to paraquat (0.5, 2, 8 mg/kg/day) from post-EDS day 17 to day 28 and effects of paraquat on Leydig and Sertoli cell functions on post-EDS day 35 and day 56 were investigated. Paraquat significantly decreased serum testosterone levels at 2 and 8 mg/kg. Paraquat lowered Leydig cell Hsd17b3 , Srd5a1 , and Hsd11b1 mRNA levels but increased Hsd3b1 on post-EDS day 35. Paraquat lowered Cyp11a1 , Cyp17a1 , and Hsd11b1 but increased Srd5a1 on post-EDS day 56. However, paraquat did not alter Leydig cell number and PCNA labeling index. Epididymal staining showed that few sperms were observed in paraquat-treated rats. Primary culture of adult Leydig cells showed that paraquat diminished testosterone output and induced reactive oxygen species generation at 1 and 10 μM and apoptosis rate at 10 μM. In conclusion, a short-term exposure to paraquat delays Leydig cell regeneration from stem/progenitor Leydig cells, causing low production of testosterone and an arrest of spermatogenesis. Image 1 • Paraquat delays stem/progenitor Leydig cell regeneration. • Paraquat inhibits Leydig cell differentiation. • Paraquat blocks spermatogenesis. • Paraquat in vitro inhibits T secretion by primary Leydig cells. • Paraquat in vitro induces apoptosis and ROS generation of primary Leydig cells. [ABSTRACT FROM AUTHOR]

Abstract Testicular 3β-hydroxysteroid dehydrogenase (HSD3B) is a steroidogenic enzyme, catalyzing the conversion of 3β-hydroxysteroids into 3-keto-steroids. Two distinct isoforms in the human are cloned, HSD3B1 and HSD3B2, and HSD3B2 is located in the testis. HSD3B2 is a two-substrate enzyme, which binds to cofactor NAD+ and a 3β-steroid. Many endocrine disruptors, including industrial compounds (phthalates, bisphenols, and perfluoroalkyl substances), insecticides and biocides (organochlorine insecticides and organotins), food additives (butylated hydroxyanisole, resveratrol, gossypol, flavones, and isoflavones), and drugs (etomidate, troglitazone, medroxyprogesterone acetate, and ketoconazole) inhibit testicular HSD3B, possibly interfering with androgen synthesis. In this review, we discuss the distinct testicular isoform of HSD3B, its gene, chemistry, subcellular location, and the endocrine disruptors that directly inhibit testicular HSD3B and their inhibitory modes. Highlights • HSD3B catalyzes 3β-dehydrogenase of 3β-hydroxysteroids. • Testicular HSD3B is critical for testosterone synthesis. • There is a SAR difference for phthalate-mediated inhibition of HSD3B. • Some flavones and isoflavones are very potent HSD3B inhibitors. [ABSTRACT FROM AUTHOR]

Many flavonoids and isoflavonoids have anti-diabetic effects in animal models. However, the mechanisms that are involved are generally unclear. Since 11β-hydroxysteroid dehydrogenases (HSD11Bs) play important roles in diabetes, we hypothesize that flavonoids and isoflavonoids may affect diabetes by targeting two isoforms of HSD11B differently. The inhibitory effects of flavonoids (apigenin and quercetin) and isoflavonoids [genistein and (±) equol] on rat and human HSD11B1 and HSD11B2 were analyzed. The potencies of inhibition on human HSD11B1 reductase was in the order of apigenin > quercetin > genistein > (±) equol, with IC 50 values of 2.19, 5.36, 11.00, and over 100 μM, respectively. Genistein also inhibited rat HSD11B1 reductase with IC 50 value of 24.58 μM, while other three chemicals showed no effects on the enzyme activity with IC 50 values over 100 μM. However, apigenin and (±) equol did not inhibit human HSD11B2 at concentrations as high as 100 μM, while genistein and quercetin inhibited human HSD11B2 by 60% and 50% at 100 μM, respectively. The effective flavonoids and isoflavonoids are noncompetitive inhibitors of HSD11B1 when steroid substrates were used. Docking analysis showed that they bound to the steroid-binding site of the human HSD11B1. These data indicate that apigenin is a selective inhibitor of human HSD11B1 of two HSD11B isoforms, which may be useful in managing symptoms of the metabolic syndrome. [ABSTRACT FROM AUTHOR]

Taxifolin is a flavonoid. It has been used as a chemopreventive agent and supplement. It may have some beneficial effects to treat prostate cancer by suppressing androgen production in Leydig cells. The objective of the present study was to study the effects of taxifolin on androgen production of rat Leydig cells isolated from immature testis and some rat and human testosterone biosynthetic enzyme activities. Rat Leydig cells were incubated with 100 μM taxifolin without (basal) or with 10 ng/ml luteinizing hormone (LH), 10 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (8BR), and steroid enzyme substrates (20 μM): 22R-hydroxychloesterol, pregnenolone, progesterone, and androstenedione. The medium concentrations of 5α-androstane-3α, 17β-diol (DIOL) and testosterone were measured. Taxifolin significantly suppressed basal, LH-stimulated, 8BR-stimulated, pregnenolone-mediated, and progesterone-mediated androgen production by Leydig cells. Further study demonstrated that taxifolin inhibited rat 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase/17, 20-lyase with IC 50 values of 14.55 ± 0.013 and 16.75 ± 0.011 μM, respectively. Taxifolin also inhibited these two enzyme activities in human testis with IC 50 value of about 100 μM. Taxifolin was a competitive inhibitor for these two enzymes when steroid substrates were used. In conclusion, taxifolin may have benefits for the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Trichlorfon inhibits androgen biosynthesis in Leydig cells. • Trichlorfon inhibits androgen metabolism in Leydig cells. • Trichlorfon downregulates steroidogenesis-related gene expression. • Trichlorfon induces oxidative stress in Leydig cells. [ABSTRACT FROM AUTHOR]

Nicotine is consumed largely as a component of cigarettes and has a potential effect on pubertal development of Leydig cells in males. To investigate its effects, 49-day-old male Sprague Dawley rats received intraperitoneal injections of nicotine (0.5 or 1 mg/kg/day) for 2 weeks and immature Leydig cells were isolated from the testes of 35-day-old rats and treated with nicotine (0.05–50 μM). Serum hormones, Leydig cell number and related gene expression levels after in vivo treatment were determined and medium androgen levels were measured and cell cycle, apoptosis, mitochondrial membrane potential (△Ψm), and reactive oxygen species (ROS) of Leydig cells after in vitro treatment were measured. In vivo exposure to nicotine lowered serum luteinizing hormone, follicle stimulating hormone, and testosterone levels and reduced Leydig cell number and gene expression levels. Nicotine in vitro inhibited androgen production in Leydig cells by downregulating the expression levels of P450 cholesterol side cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1, and steroidogenic factor 1 at different concentration ranges. In conclusion, nicotine disrupts Leydig cell steroidogenesis during puberty possibly via down-regulating some key steroidogenic enzyme expressions. [ABSTRACT FROM AUTHOR]

The objective of the present study is to determine whether in utero exposure to methoxychlor (MXC) affects rat fetal Leydig cell number, cell size, or functions. Pregnant Sprague Dawley dams were gavaged with corn oil (control, 0 mg/kg/day MXC) or MXC at doses of 10, 50, or 100 mg/kg/day from gestational day (GD) 12 to 21. The results show that MXC increased fetal Leydig cell numbers dose-dependently from 95 ± 8 × 10 3 cells/testis (control, mean ± SEM) to 101 ± 6, 148 ± 22, and 168 ± 21 × 10 3 cells/testis, at the doses of 10, 50, and 100 mg/kg, respectively. The increase of Leydig cell number by MXC was contributed by the increase of single cell population of Leydig cells, which increased from 21 ± 2% of the control to 31 ± 4%, 39 ± 3%, or 40 ± 4% at the doses of 10, 50 or 100 mg/kg, respectively. Quantitative PCR results show that MXC increased Lhcgr expression at dose of 10 mg/kg and Scarb1 and Cyp11a1 mRNA levels at doses of 50 and 100 mg/kg. Immunohistochemical staining demonstrated the increase of CYP11A1 protein level from the dose of 10 mg/kg. However, at the highest dose (100 mg/kg) MXC reduced the testicular testosterone level and MXC (1 μM) in vitro treatment also inhibited androgen production from isolated fetal Leydig cells. In conclusion, our findings indicate that at low dose MXC may increase fetal Leydig cell numbers and the expressions of some steroidogenic enzymes, but at high dose it reduces the testicular testosterone level leading to reproductive tract malformations in the male offspring. [ABSTRACT FROM AUTHOR]

Apigenin is a natural flavone. It has recently been used as a chemopreventive agent. It may also have some beneficial effects to treat prostate cancer by inhibiting androgen production. The objective of the present study was to investigate the effects of apigenin on the steroidogenesis of rat immature Leydig cells and some human testosterone biosynthetic enzyme activities. Rat immature Leydig cells were incubated for 3 h with 100 μM apigenin without (basal) or with 1 ng/ml luteinizing hormone (LH), 10 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (8BR), and 20 μM of the following steroid substrates: 22R-hydroxychloesterol (22R), pregnenolone (P5), progesterone ( P 4), and androstenedione (D4). The medium levels of 5α-androstane-3α, 17β-diol (DIOL), the primary androgen produced by rat immature Leydig cells, were measured. Apigenin significantly inhibited basal, 8BR, 22R, PREG, P4, and D4 stimulated DIOL production in rat immature Leydig cells. Further study showed that apigenin inhibited rat 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/17, 20-lyase, and 17β-hydroxysteroid dehydrogenase 3 with IC 50 values of 11.41 ± 0.7, 8.98 ± 0.10, and 9.37 ± 0.07 μM, respectively. Apigenin inhibited human 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 with IC 50 values of 2.17 ± 0.04 and 1.31 ± 0.09 μM, respectively. Apigenin is a potent inhibitor of rat and human steroidogenic enzymes, being possible use for the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

Many insecticides and fungicides are endocrine-disrupting compounds, which possibly interfere with the placental endocrine system. In the placenta, 3β-hydroxysteroid dehydrogenase/Δ5,4-isomerase type 1 (HSD3B1) is the major steroidogenic enzyme, which makes progesterone from pregnenolone to support the placental stability. In this study, we screened 12 classes of insecticides and fungicides to inhibit placental HSD3B1 activity and compared them to the rat homolog type 4 (HSD3B4) isoform. Human HSD3B1 activity and rat HSD3B4 activity were measured in the presence of 200 nM pregnenolone and 0.2 mM NAD+ and 100 μM of test chemical. Triclosan, triflumizole, dichlone, and oxine at 100 μM significantly inhibited human HSD3B1 activity with the residual activity being less than 50% of the control. Further study showed that the half-maximal inhibitory concentration (IC 50) values of triclosan, triflumizole, dichlone, and oxine were 85.53 ± 9.14, 73.75 ± 3.42, 2.54 ± 0.40, and 102.93 ± 6.10 μM, respectively. In the presence of pregnenolone, triclosan, triflumizole, and dichlone were mixed inhibitors of HSD3B1, while oxine was a noncompetitive inhibitor. In the presence of NAD+, triclosan exhibited competitive inhibition while triflumizole possessed uncompetitive inhibition. Docking analysis showed that triclosan bound NAD+-binding site, while triflumizole, dichlone, and oxine mostly bound steroid-binding site. When the effect of these insecticides on rat placental HSD3B4 activity was screened in the presence of 200 nM pregnenolone, atrazine, triclosan, triflumizole, oxine, cyprodinil, and diphenyltin at 100 μM significantly inhibited rat HSD3B4 activity, with IC 50 values of triclosan, triflumizole, oxine, and cyprodinil were 82.99 ± 6.48, 35.45 ± 2.73, 105.59 ± 12.04, and 43.37 ± 3.00 μM, respectively. The mode action analysis showed that t riflumizole and cyprodinil were almost competitive inhibitors, while triclosan and oxine were almost noncompetitive inhibitors of rat HSD3B4. Docking analysis showed that triclosan and oxine bound cofactor NAD+ binding residues more than steroid-binding residues of rat HSD3B4 while triflumizole and cyprodinil bound most pregnenolone-interactive residues. In conclusion, some insecticides such as triclosan, triflumizole, and oxine can effectively inhibit both human and rat placental HSD3B activity and they have unique mode action due to the structure difference. [Display omitted] • Triclosan, triflumizole, and dichlone are mixed inhibitors of human HSD3B1. • Triclosan, triflumizole, cyprodinil are mixed inhibitors of rat HSD3B4. • Oxine is a non-competitive inhibitor of human HSD3B1 and rat HSD3B4. • Primary structure difference in catalytic residue S125T may explain the species-dependent difference of these chemicals. [ABSTRACT FROM AUTHOR]

Bisphenol F (BPF) is a new analog of bisphenol A (BPA). BPA has deleterious effects on the male reproductive system, but the effect of BPF has not been studied in detail. In this study we focus on the effect of BPF on Leydig cell maturation. Male Sprague-Dawley rats were gavaged with 0, 1, 10, or 100 mg/kg BPF from postnatal days 35–56. BPF significantly reduced serum testosterone levels and sperm count in cauda epididymis at dose ≥1 mg/kg. It significantly down-regulated the expression of steroidogenic enzymes, while increasing FSHR and SOX9 levels at 10 and 100 mg/kg. Further studies showed that BPF reduced NR3C4 expression in Leydig and Sertoli cells without affecting its levels in peritubular myoid cells. BPF markedly increased GPER1 in Leydig cells at 100 mg/kg, and it significantly reduced SIRT1 and PGC1α levels in the testes at 100 mg/kg. BPF significantly inhibited testosterone production by immature Leydig cells at 50 μM after 24 h of treatment, which was completely reversed by NR3C4 agonist 7α-methyl-19-nortestosterone and partially reversed by GPER1 antagonist G15 not by ESR1 antagonist ICI 182,780. In conclusion, BPF negatively affects Leydig cell maturation in pubertal male rats through NR3C4 antagonism and GPER1 agonism. [Display omitted] • BPF inhibits testosterone production in male rats in puberty. • BPF down-regulates the expression of Star, Cyp11a1, and Hsd3b1 in Leydig cells. • BPF reduces androgen receptor levels in Leydig and Sertoli cells. • BPF increases G-coupled estrogen receptor 1 in Leydig cells. • Androgen receptor agonist and GPER1 antagonist reverse BPF-mediated inhibition of testosterone by Leydig cells. [ABSTRACT FROM AUTHOR]

Highlights: [•] Resveratrol inhibits androgen production in rat Leydig cells. [•] Resveratrol inhibits rat and human 3β-HSD activities. [•] Resveratrol is a competitive inhibitor of 3β-HSD. [Copyright &y& Elsevier]

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. Illustration of the working mechanism of bisphenol FL (BPFL)-mediated stimulation of Leydig cell proliferation and inhibition of Leydig cell maturation and in pubertal male rats.The androgen 7α-methyl-nortestosterone (MENT) binds the androgen receptor (NR3C4) in the cytoplasm, resulting in the formation of a ligand-receptor dimer and its translocation to the nucleus to transactivate the expression of NR3C4 target genes (Cyp17a1) upon binding AREs in the target gene promoter region. BPFL binds NR3C4 but does not form a dimer, thereby blocking NR3C4 transactivation. BPFL also binds ESR1 to transactivate the ESR1 activity to suppress the expression of steroidogenesis-related genes, thereby inhibiting T biosynthesis or increasing the expression of proliferation-related genes (Pcna and Cdk2) to stimulate the proliferation of immature Leydig cells (ILC). [Display omitted] • BPFL inhibits testosterone production in male rats in puberty. • BPFL down-regulates the expression of Cyp17a1 in Leydig cells. • BPFL increases Leydig cell proliferation. • BPFL-mediated inhibition of testosterone is reversed by androgen receptor agonist and estrogen receptor 1 antagonist. [ABSTRACT FROM AUTHOR]

Illustration of the signaling pathway to regulate adrenal cortex development after triadimefon (TDM) exposure. TDM increases the generation of ROS to cause the following actions: 1) down-regulating IGF1, which can activate AKT1 signaling for the growth of the adrenal gland; 2) down-regulating AT1R and MC2R, which can activate ERK1/2 for the growth and survival of the adrenal gland; 3) down-regulating NR5A1, which can activate steroidogenesis via up-regulating the expression of steroidogenesis-related genes; 4) activating the phosphorylation of AMPK, which is a negative signal to control adrenal steroidogenesis mainly via down-regulating Star , Cyp11b1 , Cyp11b2 expression, eventually leading to reduced synthesis of aldosterone (ALDO) and corticosterone (CORT) [Display omitted] • Triadimefon reduces serum ACTH level in male fetuses after in utero exposure. • Triadimefon reduces serum corticosterone and aldosterone levels in male fetuses. • Triadimefon down-regulates adrenal gland steroidogenesis-related gene expression. • Triadimefon induces AMPK phosphorylation in adrenal gland of male fetuses. • Triadimefon reduces AKT1 and ERK1/2 phosphorylation in adrenal gland of male fetuses. Triadimefon is a broad-spectrum antifungal agent, which is widely used in agriculture to control mold and fungal infections. It is considered an endocrine disruptor. Whether triadimefon exposure can inhibit the development of fetal adrenal glands and the underlying mechanism remain unclear. Thirty-two pregnant female Sprague-Dawley rats were randomly divided into four groups. Dams were gavaged triadimefon (0, 25, 50, and 100 mg/kg/day) daily for 10 days from gestational day (GD) 12 to GD 21. Triadimefon significantly reduced the thickness of the zona fasciculata of male fetuses at 100 mg/kg, although it did not change the thickness of the zona glomerulosa. It significantly reduced the serum aldosterone levels of male fetuses at a dose of 100 mg/kg, and significantly reduced serum corticosterone and adrenocorticotropic hormone levels at doses of 50 and 100 mg/kg. Triadimefon significantly down-regulated the expression of Agtr1 , Mc2r , Star , Cyp11b1 , Cyp11b2 , Igf1 , Nr5a1 , Sod2 , Gpx1 , and Cat , but did not affect the mRNA levels of Scarb1 , Cyp11a1 , Cyp21 , Hsd3b1 , and Hsd11b2. Triadimefon markedly reduced AT1R, CYP11B2, IGF1, NR5A1, and MC2R protein levels. Triadimefon significantly reduced the phosphorylation of AKT1 and ERK1/2 at 100 mg/kg without affecting the phosphorylation of AKT2. In contrast, it significantly increased AMPK phosphorylation at 100 mg/kg. In conclusion, exposure to triadimefon during gestation inhibits the development of fetal adrenal cortex in male fetuses. This inhibition is possibly due to the reduction of several proteins required for the synthesis of steroid hormones, and may be involved in changes in antioxidant contents and the phosphorylation of AKT1, ERK1/2, and AMPK. [ABSTRACT FROM AUTHOR]

Perfluoroundecanoic acid (PFUnA) is one of long-chain perfluoroalkyl carboxylic acids. However, the effect of PFUnA on pubertal development of Leydig cells remains unclear. The goal of this study was to investigate the effect of PFUnA on Leydig cell development in pubertal male rats. We orally dosed male Sprague-Dawley rats (age 35 days) with PFUnA at doses of 0, 1, 5, and 10 mg/kg/day from postnatal day (PND) 35 to PND 56. Serum testosterone and luteinizing hormone levels were remarkably reduced by PFUnA at ≥1 mg/kg while serum follicle-stimulating hormone levels were lowered at 5 and 10 mg/kg. PFUnA down-regulated the expression of Lhcgr , Scarb1 , Star , Cyp11a1 , Hsd3b1 , Cyp17a1 , Hsd17b3 , Hsd11b1, Insl3 , Nr5a1 , Fshr , Dhh , Sod1 , and Sod2 and their proteins in the testis and the expression of Lhb and Fshb in the pituitary. PFUnA reduced Leydig cell number at 5 and 10 mg/kg. PFUnA induced oxidative stress and increased autophagy. These may result from the inhibition of phosphorylation of mTOR, AKT1, AKT2, and ERK1/2 in the testis. In conclusion, PFUnA exhibits inhibitory effects on pubertal Leydig cell development possibly via inducing oxidative stress and increasing autophagy. PFUnA exerts multiple mechanisms to prevent the proliferation and differentiation of Leydig cells (LC) during puberty in rats. It inhibits LH secretion, leading to low AKT1 phosphorylation, and induces ROS to lead to low AKT1 and ERK1/2 phosphorylation, which results in the inhibition of LC proliferation. PFUnA induces ROS, inducing autophagy by inhibiting phosphorylation of mTOR/AKT/ERK1/2, and also inhibits LH and FSH, thereby inhibiting the differentiation of LC. The combination of a decrease in testosterone (T) and a decrease in FSH results in a low spermatogenesis. [Display omitted] • PFUnA reduces serum testosterone levels during pubertal exposure. • PFUnA reduces Leydig cell number. • PFUnA down-regulates Leydig cell steroidogenesis-related gene expression. • PFUnA induces autophagy in the testis. • PFUnA inhibits pituitary section of LH and FSH. [ABSTRACT FROM AUTHOR]

Abstract: Non-steroidal compounds that inhibit 17β-hydroxysteroid dehydrogenase isoform 3 (17β-HSD3), an enzyme catalyzing the final step in testosterone biosynthesis in Leydig cells, are under development for male contraceptive or treatment of androgen dependent diseases including prostate cancer. A series of curcumin analogues with more stable chemical structures were compared to curcumin as inhibitors of 17β-HSD3 in rat intact Leydig cells as well as rat and human testis microsomes. [Copyright &y& Elsevier]

Acephate is an organophosphate pesticide. It is widely used. However, whether it inhibits androgen synthesis and metabolism remains unclear. In the current study, we investigated the effect of acephate on the inhibition of androgen synthetic and metabolic pathways in rat immature Leydig cells after 3-h culture. Acephate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.5 μM. It significantly inhibited luteinizing hormone and 8-Br-cAMP stimulated androgen output at 50 μM. It significantly inhibited progesterone-mediated androgen output at 50 μM. Further study demonstrated that acephate down-regulated the expression of Hsd3b1 and its protein at ≥ 0.5 μM, Lhcgr at 5 μM and Star at 50 μM. Acephate directly blocked rat testicular HSD3B1 activity at 50 μM. Acephate did not affect other androgen synthetic and metabolic enzyme activities as well as ROS production, proliferation, and apoptosis of immature Leydig cells. In conclusion, acephate targets LHCGR, STAR, and HSD3B1, thus blocking androgen synthesis in rat immature Leydig cells and HSD3B1 is being the most sensitive target of acephate. Image 1 • Acephate inhibits androgen synthesis. • Acephate down-regulates steroidogenic enzymes. • Acephate down-regulates cholesterol transport. • Acephate down-regulates LHCGR. [ABSTRACT FROM AUTHOR]

Triphenyltin is an organotin, which is widely used as a fungicide in agriculture. Here, we reported the effects of triphenyltin on adrenal function in adult male rats. Adult male Sprague Dawley rats were daily gavaged with triphenyltin (0, 0.5, 1, and 2 mg/kg body weight) from postnatal day 56–86. Triphenyltin significantly decreased serum corticosterone levels at 1 and 2 mg/kg without affecting serum levels of aldosterone and adrenocorticotropic hormone. Triphenyltin increased thickness of zona glomerulosa without affecting that of zona fasciculata. Triphenyltin did not affect cell number in zona fasciculata and zona glomerulosa. Triphenyltin down-regulated the expression of Scarb1 , Star , Cyp11a1 , Hsd3b1 , Cyp21, Cyp11b1, and Hsd11b1 at 1 and/or 2 mg/kg while it up-regulated the expression of At1, Nr4a2, and Hsd11b2 at 2 mg/kg. Triphenyltin activated the phosphorylation of AMPKα while suppressed the phosphorylation of AKT1 and SIRT1/PGC-1α in rat adrenals in vivo and H295R cells in vitro. In vitro, triphenyltin also induced ROS production in H295R cells at 100 nM, a concentration at which no apoptosis was induced. In conclusion, triphenyltin disrupts glucocorticoid synthesis in rat adrenal cortex via several mechanisms: 1) lowering AKT1 phosphorylation and SIRT1/PGC-1α levels; 2) activating AMPKα; and 3) possibly inducing ROS production. Image 1 • Triphenyltin inhibits corticosterone synthesis in rat adrenals. • Triphenyltin down-regulates steroidogenesis-related gene expression. • Triphenyltin increases the thickness of the zG in rat adrenals. [ABSTRACT FROM AUTHOR]

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

• Dextmedetomidine concentration-dependently inhibits androgen production in immature Leydig cells. • Dextmedetomidine blocks androgen biosynthetic pathway and metabolizing pathway in immature Leydig cells. • Dextmedetomidine increases ROS generation in immature Leydig cells. • Dextmedetomidine induces apoptosis of immature Leydig cells. • Dextmedetomidine decreases EKR1/2 phosphorylation that could lead to lower steroidogenesis in the immature Leydig cells. Dexmedetomidine (DEX), an imidazole compound, is an anesthetic drug used perioperatively. In the current study, we investigated the effects of DEX on androgen production in rat immature Leydig cells in vitro. Leydig cells isolated from pubertal Sprague Dawley rats were treated with various concentrations of DEX (0.015–1.5 µM) for 3 h and medium 5α-androstanediol and testosterone levels and the expression of Lhcgr , Scarb1 , Star , Cyp11a1 , Hsd3b1 , Cyp17a1 , Hsd17b3 , Srd5a1 and Akr1c14 in Leydig cells were determined. At 0.015–1.5 μM, DEX concentration-dependently inhibited androgen secretion and downregulated Cyp17a1 and Srd5a1 mRNA levels. DEX equally blocked the LH- and cAMP-stimulated secretion of androgens. Using the steroid substrates, 22R-hydroxycholesterol (for cytochrome P450 cholesterol side chain cleavage), pregnenolone (for 3β-hydroxysteroid dehydrogenase 1), progesterone (for cytochrome P450 17α-hydroxylase/C17,C20-lyase), androstenedione (for 17β-hydroxysteroid dehydrogenase 3), testosterone (for steroid 5α-reductase 1), and dihydrotestosterone (for 3α-hydroxysteroid dehydrogenase), it was demonstrated that DEX inhibited 22R-hydroxycholesterol, pregnenolone, progesterone, and testosterone-mediated 5α-androstanediol formation at 1.5 μM. Further study demonstrated that DEX also directly inhibited rat testis cholesterol side chain cleavage, 3β-hydroxysteroid dehydrogenation, and 17α-hydroxylation at 1.5 μM. DEX induced ROS production and increased apoptosis rate in immature Leydig cells after 24-h treatment at ≥0.15 μM. In conclusion, DEX directly inhibits the activities of some steroidogenic enzymes and downregulates the expression of Cyp17a1 and Srd5a1 , and increases ROS production, thus leading to lower production of androgens in immature Leydig cells. [ABSTRACT FROM AUTHOR]

• HSD3B1 catalyzes 3β-dehydrogenation and Δ5,4-isomeration of 3β-hydroxysteroids. • HSD3B1 is critical for progesterone synthesis. • HSD3B1 expression is controlled by transcription factors and epigenetics. • There is a SAR difference for phthalates in the inhibition of HSD3B1. Human placental 3β-hydroxysteroid dehydrogenase/steroid Δ5, 4-isomerase 1 (HSD3B1), a high-affinity type I enzyme, uses pregnenolone to make progesterone, which is critical for maintenance of pregnancy. HSD3B1 is located in the mitochondrion and the smooth endoplasmic reticulum of placental cells and is encoded by HSD3B1 gene. HSD3B1 contains GATA and TEF-5 regulatory elements. Many endocrine disruptors, including phthalates, methoxychlor and its metabolite, organotins, and gossypol directly inhibit placental HSD3B1 thus blocking progesterone production. In this review, we discuss the placental HSD3B1, its gene regulation, biochemistry, subcellular location, and inhibitors from the environment. [ABSTRACT FROM AUTHOR]