Short-term exposure to perfluorotetradecanoic acid affects the late-stage regeneration of Leydig cells in adult male rats.

Perfluorotetradecanoic acid (PFTeDA) is one of perfluoroalkyl substances widely found in the environment. PFTeDA may cause the dysfunction of male reproductive system. However, whether PFTeDA affects the regeneration of Leydig cells remains unclear. The objective of this study was to examine the effects of short-term exposure of PFTeDA on the late-stage maturation of Leydig cells. Fifty-four adult Sprague-Dawley male rats were daily gavaged with PFTeDA (0, 10, or 20 mg/kg body weight) for 10 days, and then were injected intraperitoneally with ethylene dimethane sulfonate (EDS, 75 mg/kg body weight/once) to ablate Leydig cells to induce their regeneration. On day 21 (early stage) and 56 (late stage) after EDS, hormone levels, gene expression, and protein levels were measured. PFTeDA did not affect the early stage of Leydig cell regeneration, because it had no effect on serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels, Leydig cell number, and its gene and protein expression. PFTeDA significantly reduced serum testosterone level and down-regulated the expression of Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, and Insl3) and their proteins (CYP11A1, HSD3B1, CYP17A1, HSD17B3, and INSL3), decreased the phosphorylation of AKT1 and ERK1/2, as well as lowered sperm count in the epididymis at 20 mg/kg. In conclusion, short-term exposure to PFTeDA blocks the late-stage maturation of Leydig cells.

Steroidogenesis during prenatal testicular development in Spix's cavy Galea spixii.

Spix's cavy is a potentially good experimental model for research on reproductive biology and sexual development. The aim of the present study was to evaluate the ontogeny of the steroidogenic enzymes involved in testicular androgen synthesis during prenatal development. Testes were investigated on Days 25, 30, 40 and >50 of gestation. Immunohistochemistry and immunoblotting were used to establish the site and relative amount of androgenic enzymes, including 5α-reductase, cytosolic 17ß-hydroxysteroid dehydrogenase (17ß-HSDI) and mitochondrial microsomal 3ß-hydroxysteroid dehydrogenase (3ß-HSDII), throughout prenatal development. The testicular parenchyma began to organise on Day 25 of gestation, with the development of recognisable testicular cords. The mesonephros was established after Day 25 of gestation and the ducts differentiated to form the epididymis, as testicular cords were beginning to proliferate and the interstitium to organise by Day 30 of gestation, continuing thereafter. The androgen-synthesising enzymes 5α-reductase, 17ß-HSDI and 3ß-HSDII were evident in Leydig cells as they differentiated at all subsequent gestational ages studied. In addition, immunoblotting showed an increase in immunoreactivity for the enzymes at Days 30 and 40 of gestation (P<0.05) and a decrease at Day 50 of gestation (P<0.05). It is concluded that the increase in androgenic enzymes in Leydig cells coincides with the functional differentiation of the testes, and with the stabilisation and differentiation of mesonephric ducts forming the epididymis.

Could COVID-19 have an impact on male fertility?

The pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to several hypotheses of functional alteration of different organs. The direct influence of this virus on the male urogenital organs is still to be evaluated. However some hypotheses can already be made, especially in the andrological field, for the biological similarity of the SARS-CoV and SARS-CoV2. As well as SARS-CoV, SARS CoV-2 uses the 'Angiotensin Converting Enzyme-2' (ACE2) as a receptor to enter human cells. It was found that ACE2, Angiotensin (1-7) and its MAS receptors are present, over in the lung, also in the testicles, in particular in Leydig and Sertoli cells. A first hypothesis is that the virus could enter the testicle and lead to alterations in testicular functionality. A second hypothesis is that the binding of the virus to the ACE2 receptor, could cause an excess of ACE2 and give rise to a typical inflammatory response. The inflammatory cells could interfere with the function of Leydig and Sertoli cells. Both hypotheses should be evaluated and confirmed, in order to possibly monitor fertility in patients COVID-19+.

Abnormal functions of Leydig cells in crossbred cattle-yak showing infertility.

In Mongolia, yak (Bos grunniens) are able to live in alpine areas and their products greatly influence the lives of the local people. Increased vigour in hybridized yak and cattle can offer benefits for livestock farmers. However, male hybrids show reproductive defects resulting from spermatogenesis arrest, affecting the conservation and maintenance of dominant traits in the next generation. The underlying mechanisms involved in hybrid cattle-yak infertility have recently been investigated; however, the genetic cause is still unclear. Androgens and androgen receptor (AR) signalling are required for spermatogenesis. We, therefore, evaluated the expression of AR, 3ß-hydroxysteroid dehydrogenase (3ßHSD) and 5α-reductase 2 (SRD5A2) in Leydig cells to investigate their function in cattle-yak spermatogenesis. Testicular tissues from yaks (1-3 years old) and hybrids (F1-F3, 2 years old) were collected and subjected to immunohistochemistry and image analyses to investigate the expression of each parameter in the Leydig cells. After maturation at 2 years, the expression levels of AR increased and the levels of 3ßHSD decreased, but the SRD5A2 levels remained constant in yak. However, the cattle-yak hybrid F2 showed immature testicular development and significantly different expression levels of AR and 3ßHSD compared with mature yak. These results suggest that the decreased expression of AR and increased expression of 3ßHSD in the Leydig cells of cattle-yak hybrid testes may represent one of the causes of infertility. Our study might help in solving the problem of infertility in crossbreeding.

Directing differentiation of human induced pluripotent stem cells toward androgen-producing Leydig cells rather than adrenal cells.

Reduced serum testosterone (T), or hypogonadism, affects millions of men and is associated with many pathologies, including infertility, cardiovascular diseases, metabolic syndrome, and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However, TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus, there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs), proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs, the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively, human induced pluripotent stem cells (hiPSCs), which are expandable in culture and have the potential to differentiate into all somatic cell types, have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis, synthesized T rather than cortisol, secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol, and displayed ultrastructural features resembling LCs. By contrast, hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration.

Pubertal exposure to tebuconazole increases testosterone production via inhibiting testicular aromatase activity in rats.

Tebuconazole is a triazole compound used agriculturally to treat plant pathogenic fungi. However, whether pubertal exposure to tebuconazole affects Leydig cell development remains unknown. Here, we exposed male Sprague-Dawley rats at 35 days of age to 0, 25, 50, or 100 mg kg-1 day-1 tebuconazole for 21 days. Tebuconazole exposure increased serum testosterone level but lowered estradiol level at a dose of 100 mg kg-1, without affecting serum luteinizing hormone and follicle-stimulating hormone concentrations. Tebuconazole up-regulated the expression of testicular Cyp11a1, Hsd11b1, and Fshr genes as well as their proteins at a dose of 100 mg kg-1. However, tebuconazole did not stimulate the proliferation of Leydig cells. Tebuconazole in vitro inhibits aromatase activity in primary rat Leydig cells with IC50 value of 40 µmol/L. In conclusion, tebuconazole exposure stimulates pubertal Leydig cell differentiation via inhibiting aromatase activity.

Cyanidin-3-O-glucoside promotes progesterone secretion by improving cells viability and mitochondrial function in cadmium-sulfate-damaged R2C cells.

Cadmium (Cd) is a poisonous metal that is toxic for male reproduction. Cyanidin-3-O-glucoside (C3G) as typical anthocyanin benefits many organs. In this study, we investigated the protective effects and associated underlying mechanisms of C3G against the toxicity of Cd on male reproduction in rat Leydig cell line R2C cells. Cells were pre-protected with C3G (5-160 µmol/L) for 2 h and then treated with cadmium sulfate (CdSO4) (10-160 µmol/L) for 24 h. The results showed that cytotoxicity, mitochondrial damage, superoxide dismutase 2 (SOD2), and overproduction of reactive oxygen species (ROS) in CdSO4-treated R2C cells were significantly reduced with C3G pre-treatment. Moreover, C3G pre-treatment led to upregulated expression of steroidogenic acute regulatory (StAR) protein and progesterone production. Our study suggests that C3G may be a potential therapeutic agent against Cd-induced reproductive toxicity.

Cadmium-induced apoptosis through reactive oxygen species-mediated mitochondrial oxidative stress and the JNK signaling pathway in TM3 cells, a model of mouse Leydig cells.

Cadmium (Cd) is a heavy metal that widely exists in the environment and industry, and which causes serious damages to reproductive system. Recent studies have reported that cadmium induces apoptosis of various germ cells in testes, resulting in male infertility. However, the exact mechanism of cadmium-induced apoptosis remains unclear. In this study, we hypothesized that reactive oxygen species (ROS)-mediated c-jun N-terminal kinase (JNK) signaling pathway was involved in cadmium-induced apoptosis in TM3 cells, a model of mouse Leydig cells. TM3 cells were exposed for various times to a range of cadmium concentrations. We found that cadmium reduced TM3 cell viability and increased apoptosis in a time- and dose- dependent manner. Moreover, the levels of ROS generation and the phosphorylation of JNK were elevated by cadmium treatment. In addition, the nuclear transcription factor c-jun was significantly activated, which led to increased expression of downstream c-jun targets and Bcl-2 was decreased, accompanied with downstream activation of apoptosis-related proteins such as Cleaved-Caspase3 and Cleaved-PARP. However, pretreatment with the ROS inhibitor N-acetyl-L-cysteine (NAC) and JNK inhibitor JNK-IN-8, ROS, JNK and cadmium-induced TM3 cell apoptosis were remarkably suppressed. Based on above-mentioned results, this study provides a mechanistic understanding of cadmium induced TM3 cell apoptosis through the ROS/JNK signaling pathways.

Beer consumption negatively regulates hormonal reproductive status and reduces apoptosis in Leydig cells in peripubertal rats.

Beer is one of the most popular alcoholic beverages consumed by young people. Ethanol intake is associated with harmful effects to the reproductive system. Bioactive compounds present in beer may diminish the toxics effect of ethanol. However, there is still little knowledge about the effect of beer consumption on hormonal regulation of male reproduction in organisms exposed to alcohol after the peripubertal age. Therefore, the aim of this study was to determine the influence of beer intake on plasma reproductive hormones, immunolocalization of cleaved caspase-3 (casp-3), and the level of the neuronal isoform of nitric oxide synthase (nNOS) in Leydig cells (LCs) in adolescent male Wistar rats. The animals, beginning at the age of 30 days, drank beer (10% ethanol; B2 group [2 weeks' exposure] and B4 group [4 weeks' exposure]), 10% ethanol solution (CE2 group [2 weeks' exposure] and CE4 group [4 weeks' exposure]), or water (C2 group [2 weeks' exposure] and C4 group [4 weeks' exposure]). Rats drinking beer for 4 weeks showed higher phenolic acid intake compared to rats drinking beer for 2 weeks. Rats exposed to beer for 4 weeks showed decreased plasma levels of follicle-stimulating hormone (FSH) and 17ß-estradiol (E2) (3.173 ng/mL and 11.49 pg/mL, respectively), compared to the CE4 (5.293 ng/mL and 43.912 pg/mL, respectively) and the C4 groups (5.002 ng/mL and 41.121 pg mL, respectively). Expression of cleaved caspase-3 in LCs was lower in the B4 group rats, compared to the CE4 group rats (ID score: 1.676 vs. 2.190). No changes in nNOS expression were observed. Beer consumption revealed a similar negative effect on hormonal regulation of male reproductive function, but lower apoptosis in LCs may be beneficial for steroidogenic activity.

Gossypol inhibits 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase: Its possible use for the treatment of prostate cancer.

Gossypol is a yellow polyphenol isolated from cotton seeds. It has the antitumor activity and it is being tested to treat prostate cancer. However, its underlying mechanisms are still not well understood. The present study investigated the inhibitory effects of gossypol acetate on rat 5α-reductase 1, 3α-hydroxysteroid dehydrogenase, and retinol dehydrogenase 2 for androgen metabolism. Rat 5α-reductase 1, 3α-hydroxysteroid dehydrogenase, and retinol dehydrogenase 2 were expressed in COS-1 cells. Immature Leydig cells that contain these enzymes were isolated from 35-day-old male Sprague Dawley rats. The potency and mode of action of gossypol acetate to inhibit these enzymes in both enzyme-expressed preparations and immature Leydig cells were examined. Molecular docking study of gossypol on the crystal structure of 3α-hydroxysteroid dehydrogenase was performed. Gossypol acetate inhibited 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase with IC50 values of 3.33 ±â€¯0.07 and 0.52 ±â€¯0.06 × 10-6 M in the expressed enzymes as well as 8.512 ±â€¯0.079 and 1.032 ±â€¯0.068 × 10-6 M in intact rat immature Leydig cells, respectively. Gossypol acetate inhibited rat 5α-reductase 1 in a noncompetitive mode and 3α-hydroxysteroid dehydrogenase in a mixed mode when steroid substrates were supplied. Gossypol acetate weakly inhibited retinol dehydrogenase 2 with IC50 value over 1 × 10-4 M. Molecular docking analysis showed that gossypol partially bound to the steroid-binding site of the crystal structure of rat 3α-hydroxysteroid dehydrogenase. Gossypol acetate is a potent inhibitor of rat 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase, possibly inhibiting the formation of androgen in the prostate cancer cells.

A novel role for zinc transporter 8 in the facilitation of zinc accumulation and regulation of testosterone synthesis in Leydig cells of human and mouse testicles.

OBJECTIVE: Zinc is intimately involved in testosterone production. Zinc transporter 8 (ZnT8) is found to be localized in insulin secretory granules as a ß-cell specific Zn transporter. The effect of ZnT8 and related zinc accumulation in steroidogenesis, however, is still unknown. The present study aimed to explore whether ZnT8 plays a role in the facilitation of zinc accumulation and regulation of testosterone synthesis in testicles. METHODS: Leydig cells were isolated from the testicles of human, CD-1 suckling and ZnT8-KO mice. Zn accumulation in mitochondria was induced by hCG stimulation. Transfection of hZnT8-EGFP and RNA interfere of mZnT8 were done in MLTC-1 cells. ZnT8 expression and its co-localization with steroidogenic acute regulatory (StAR) protein were analyzed with RT-PCR, Western blot and dual-fluorescent staining protocols. Serum testosterone levels in mice were determined with chemiluminescent enzyme immunoassay. RESULTS: ZnT8 was found to be presented in Leydig cells and up-regulated in suckling mouse Leydig cells and MLTC-1 cells after hCG administration, by which zinc accumulation occurred in mitochondria. ZnT8 gene silencing or knockout inhibited stimulated progesterone and testosterone production, reduced stimulated zinc accumulation and down-regulated phosphorylated steroidogenic acute regulatory (StAR) expression in Leydig cells. Furthermore, an inhibitor (H89) of PKA blocked hCG-stimulated progesterone caused by ZnT8 over-expression and zinc treatment. CONCLUSION: The present study provided the first evidence that ZnT8 transports Zn into Leydig cell mitochondria with gonadotropin stimulation and suggests that ZnT8 may play a role in testosterone production via the PKA signaling pathway.

l-arginine protects against oxidative damage induced by T-2 toxin in mouse Leydig cells.

To explore the protective mechanism of l-arginine against T-2 toxin-induced oxidative damage in mouse Leydig cells, Leydig cells were isolated and cultured with control, T-2 toxin (10 nM), l-arginine (0.25, 0.5, and 1.0 mM), and T-2 toxin (10 nM T-2 toxin) with l-arginine (0.25, 0.5, or 1.0 mM) for 24 hours. Cells and supernatants were harvested to examine cell viability, activities, and messenger RNA (mRNA) expression of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), malondialdehyde (MDA) content, and DNA damage. Results showed that T-2 toxin significantly reduced cell viability, improved MDA content and DNA damage, and decreased activities and mRNA expression of GSH-Px, SOD, and CAT. However, l-arginine reduced T-2 toxin-induced oxidative damage and tended to maintain normal levels. Furthermore, l-arginine upregulated mRNA expressions of GSH-Px, SOD, and CAT. Collectively, l-arginine, due to its antioxidative ability, could ameliorate T-2 toxin-induced cytotoxicities in mouse Leydig cells by regulating oxidative stress.

Taxifolin suppresses rat and human testicular androgen biosynthetic enzymes.

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 100µM taxifolin without (basal) or with 10ng/ml luteinizing hormone (LH), 10mM 8-bromoadenosine 3',5'-cyclic monophosphate (8BR), and steroid enzyme substrates (20µM): 22R-hydroxychloesterol, pregnenolone, progesterone, and androstenedione. The medium concentrations of 5α-androstane-3α, 17ß-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 3ß-hydroxysteroid dehydrogenase and 17α-hydroxylase/17, 20-lyase with IC50 values of 14.55±0.013 and 16.75±0.011µM, respectively. Taxifolin also inhibited these two enzyme activities in human testis with IC50 value of about 100µM. 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.

T-2 toxin induces apoptosis via the Bax-dependent caspase-3 activation in mouse primary Leydig cells.

To explore the toxic effect of T-2 toxin on mouse Leydig cells and its underlying molecular mechanisms, we isolated Leydig cells from mature mice, set-up Leydig cells culture, treated cells with T-2 toxin, evaluated cell proliferation, detected the caspase-3 activity, mitochondrial activity and apoptosis rate, and measured the mRNA levels of Bcl-2, Bax, PARP and caspase-3. T-2 toxin inhibited cell proliferation at concentrations higher than 10-9 M or time more than 12 h, T-2 toxin also decreased Bcl-2 expression at the mRNA levels and mitochondrial activity at concentrations higher than 10-9 M. While, T-2 toxin increased the mRNA expressions of Bax and PARP at concentrations higher than 10-8 M and 10-9 M, respectively, triggered mitochondria-mediated apoptosis, activated downstream caspase-3, and then increased caspase-3 at the activity and mRNA levels at concentrations higher than 10-9 M. These data showed that T-2 toxin appears to activate specific intracellular death-related pathways leading to Bax-dependent caspase-3 activation and the induction of apoptosis in Leydig cells.

Nicotine affects rat Leydig cell function in vivo and vitro via down-regulating some key steroidogenic enzyme expressions.

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, 3ß-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.

Arsenic activates the expression of 3ß-HSD in mouse Leydig cells through repression of histone H3K9 methylation.

Arsenic exposure has been associated with male reproductive dysfunction by disrupting steroidogenesis; however, the roles of epigenetic drivers, especially histone methylation in arsenic-induced steroidogenic toxicity remain not well documented. In this study, we investigated the role of histone H3 lysine 9 (H3K9) methylation in steroidogenesis disturbance in mouse Leydig cells (MLTC-1) due to arsenic exposure. Our results indicated that mRNA and protein expression levels of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were both significantly up-regulated while the rest of key genes involved in steroidogenesis were down-regulated. Moreover, arsenic exposure significantly decreased the histone H3K9 di- and tri-methylation (H3K9me2/3) levels in MLTC-1 cells. Since H3K9 demethylation leads to gene activation, we further investigated whether the induction of 3ß-HSD expression was ascribed to reduced H3K9 methylation. The results showed that H3K9me2/3 demethylase (JMJD2A) inhibitor, quercetin (Que) significantly attenuated the decrease of H3K9me2/3 and increase of 3ß-HSD expression induced by arsenic. To further elucidate the mechanism for the activation of 3ß-HSD, we determined the histone H3K9 methylation levels in Hsd3b gene promoter, which also showed significant decrease of H3K9me2/3 in the investigated region after arsenic exposure. Considering these results, we conclude that arsenic exposure induced 3ß-HSD up-regulation by suppressing H3K9me2/3 status, which is suggested as a compensatory mechanism for steroidogenic disturbance in MLTC-1 cells.

Rutin attenuates H2O2-induced oxidation damage and apoptosis in Leydig cells by activating PI3K/Akt signal pathways.

Oxidative stress is a primary factor in the pathology of male infertility. The strong antioxidative capacity of rutin has been proven by numerous studies, but a protective role in the context of male reproduction remains to be elucidated. To explore the biological role of rutin in protecting male reproductive function and the potential underlying mechanism, H2O2-induced Leydig cells were used as a cell model of oxidation damage. Our findings showed that rutin at concentrations of 10, 20, and 40µmol/L remarkably increased cell survival rate of H2O2-induced Leydig cells to 70.1%, 86.8%, and 80.3% respectively. Next, rutin with concentrations of 10, 20, and 40µmol/L decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels but increased the levels of glutathione (GSH) and testosterone in H2O2-induced Leydig cells. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were remarkably increased by rutin treatment with concentrations of 20 and 40µmol/L, but glutathione peroxidase (GSH-Px) activity was notably decreased. Moreover, rutin with concentrations of 10, 20, and 40µmol/L increased Bcl-2 protein levels but decreased protein levels of Bax and caspase-3. Furthermore, 20µmol/L rutin significantly abrogated the decrease in levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT) induced by H2O2. Pretreatment with LY294002, a PI3K inhibitor, antagonized protective action of 20µmol/L rutin against H2O2-induced cell activities, intracellular oxidant, testosterone, antioxidant enzyme activities, and the apoptosis related protein expression. Taken together, these results suggest that rutin attenuates H2O2-induced oxidation damage and apoptosis in Leydig cells by activating PI3K/Akt signal pathways, providing a promising strategy to decrease oxidative stress associated with male infertility.

Augmented expression of gamma-glutamyl transferase 5 (GGT5) impairs testicular steroidogenesis by deregulating local oxidative stress.

An increasingly pro-oxidant environment has been widely implicated in causing dysfunction of testicular steroidogenesis, but little progress has been made in understanding the underlying molecular mechanism. Here, we report that gamma-glutamyl transferase 5 (GGT5), a key metabolism component responsible for the catalysis of important anti-oxidant glutathione (GSH), is predominantly expressed in mammalian Leydig cells (LCs). Deregulated GGT5 expression negatively correlates with testosterone deficiency in the testes of type 2 diabetic mice. Consistently, overexpression of GGT5 potentiates the susceptibility of TM3 LCs to spontaneous oxidative stress during luteinizing hormone (LH)-stimulated steroidogenesis. From a mechanistic standpoint, the deleterious effect of GGT5 overexpression on testicular steroidogenesis may stem from an alteration of the local redox state because of GSH deficiency. The above-mentioned response might involve the impairment of extracellular signal-related kinase activation mediated directly by oxidative injury or indirectly by abnormal P38 activation, which in turn inhibits steroidogenic acute regulatory protein abundance in mitochondria and thus significantly sabotages the rate-limiting step during LH-induced steroidogenesis. Alternatively, GGT5 overexpression induces heme oxygenase 1 (HO-1) expression, which, as a key catalyst responsible for the oxidative degradation of heme, may inhibit the activities of the cytochrome P450 monooxygenases, thus substantially impairing testicular steroidogenesis. These results, coupled with the differential roles of mitogen-activated protein kinases and HO-1 signaling in spermatogenesis, lead us to propose a model in which a delicate balance between these two pathways modulated by the GGT5/oxidative stress cascade plays a central role during LH-stimulated steroidogenesis.

Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells.

Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5(flox/flox):Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings.

In utero-exposed di(n-butyl) phthalate induce dose dependent, age-related changes of morphology and testosterone-biosynthesis enzymes/associated proteins of Leydig cell mitochondria in rats.

Female pregnant Sprague-Dawley rats were intragastrically (ig) administered di(n-butyl) phthalate (DBP) at four doses (0, 10, 50 and 100 mg/kg) during gestation days (GD) 12-21 (n = 5 per group). The age-related morphological changes of Leydig cell mitochondrion (LC-Mt) and testosterone biosynthesis enzymes/associated genes/proteins expression levels were investigated. As compared to the control (no DBP), the 10 mg, and 50 mg DBP dose groups, the 100 mg DBP dose group at weeks 5 and 7 showed a significant amount of small LC-Mt. Thereafter, from weeks 9 to 17, the LC-Mt size and quantity in the 100 mg DBP dose group increased and became statistically similar to the other dose groups; hence, dose and time-dependent LC-Mt changes were observed. Throughout the study, the 100 mg DBP dose group had significantly lower testosterone levels. In addition, the 100 mg DBP dose group displayed lower StAR (StAR, steroidogenic acute regulatory protein) and P450scc (CYP11a1, cholesterol side-chain cleavage enzyme) levels at weeks 5 and 7, but they became statistically similar to all other dose groups at weeks 9 to 17; in contrast, the SR-B1 (Sarb1, scavenger receptor class B member 1) levels were similar for all DBP dose groups. The rats in utero 100 mg DBP /kg/day (GD 12-21) exposure results from this study indicate a dose-dependent, age-related morphological change in LC-Mt which are linked to reductions in testosterone biosynthesis genes / proteins expression, specifically StAR and P450scc.