Fei Ge

Perfluorooctane sulfonate impairs rat Leydig cell development during puberty

Perfluorooctane sulfonate (PFOS) possibly delays male sexual development. However, its effects on pubertal Leydig cell development are unclear. The objective of the present study was to investigate the effects of inxa0vivo PFOS exposure on rat Leydig cell development during puberty. Immature male Sprague Dawley rats were gavaged 5 or 10xa0mg/kg PFOS on postnatal day 35 for 21 days. Compared to the control (0xa0mg/kg), PFOS lowered serum testosterone levels without altering luteinizing hormone and follicle-stimulating hormone levels on postnatal day 56. PFOS inxa0vivo downregulated mRNA or protein levels of Leydig cells (Lhcgr, Cyp11a1, and Cyp17a1). PFOS inxa0vitro inhibited androgen secretion in immature Leydig cells at ≥ 50xa0nM, most possibly via downregulating Hsd17b3 mRNA level. At ≥ 500xa0nM, PFOS downregulated Lhcgr, inhibited BCL-2 and increased BAX levels to cause Leydig cell apoptosis. In conclusion, PFOS at a lower dose directly inhibited pubertal development of Leydig cells.

Ziram delays pubertal development of rat Leydig cells.

Ziram [zinc, bis (dimethyldithiocarbamate)] is an agricultural dithiocarbamate fungicide. By virtual screening, we have identified that ziram is a potential endocrine disruptor. To investigate its effects on pubertal development of Leydig cells, 35-day-old male Sprague Dawley rats orally received ziram (2 or 4u2009mg/kg/d) for 4u2009weeks and immature Leydig cells isolated from 35-day-old rat testes were treated with ziram (0.5-50u2009μM in vitro). Serum hormones, Leydig cell number and specific gene or protein expression levels after in vivo treatment were determined and medium androgen levels were measured as well as apoptosis of Leydig cells after in vitro treatment were determined. In vivo exposure to ziram lowered testosterone and follicle-stimulating hormone levels, and reduced Leydig cell number, and downregulated Leydig cell specific gene or protein expression levels. Ziram exposure in vitro inhibited androgen production and steroidogenic enzyme activities in Leydig cells by downregulating expression levels of P450 cholesterol side cleavage enzyme (Cyp11a1), 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1), 17α-hydroxylase/17,20-lyase (Cyp17a1), and 17β-hydroxysteroid dehydrogenase 3 (Hsd17b3) via downregulating the steroidogenic factor 1 (Nr5a1) at a concentration as low as 5u2009μM. In conclusion, ziram exposure disrupts Leydig cell development during puberty possibly via downregulating Nr5a1.

Diverged Effects of Piperine on Testicular Development: Stimulating Leydig Cell Development but Inhibiting Spermatogenesis in Rats

Background: Piperine is the primary pungent alkaloid isolated from the fruit of black peppercorns. Piperine is used frequently in dietary supplements and traditional medicines. The objective of the present study was to investigate the effects of piperine on the testis development in the pubertal rat. Methods: Piperine (0 or 5 or 10 mg/kg) was gavaged to 35-day-old male Sprague-Dawley rats for 30 days. Serum levels of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured. The development of adult Leydig cell population was also analyzed 65 days postpartum. For in vitro studies, immature Leydig cells were isolated from 35-day-old male rats and treated with 50 μM piperine in the presence of different steroidogenic stimulators/substrates for 24 h. Results: Thirty-day treatment of rats with piperine significantly increased serum T levels without affecting LH concentrations. However, piperine treatment reduced serum FSH levels. Consistent with increase in serum T, piperine increased Leydig cell number, cell size, and multiple steroidogenic pathway proteins, including steroidogenic acute regulatory protein, cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase 1, 17α-hydroxylase/20-lyase, and steroidogenic factor 1 expression levels. Piperine significantly increased the ratio of phospho-AKT1 (pAKT1)/AKT1, phosphos-AKT2 (pAKT2)/AKT2, and phospho-ERK1/2 (pERK1/2)/ERK1/2 in the testis. Interestingly, piperine inhibited spermatogenesis. Piperine in vitro also increased androgen production and stimulated cholesterol side-chain cleavage enzyme and 17α-hydroxylase/20-lyase activities in immature Leydig cells. Conclusion: Piperine stimulates pubertal Leydig cell development by increasing Leydig cell number and promoting its maturation while it inhibits spermatogenesis in the rat. ERK1/2 and AKT pathways may involve in the piperine-mediated stimulation of Leydig cell development.

In utero exposure to hexavalent chromium disrupts rat fetal testis development

Hexavalent chromium (Cr6+) acts as an endocrine disruptor. Herein, we investigated effects of Cr6+ on the development of rat fetal Leydig and Sertoli cells, which support differentiation of the male reproductive tract in late gestation. Female pregnant Sprague Dawley rats were gavaged with potassium dichromate (0, 3, 6, and 12u2009mg/kg) from gestational days (GD) 12 to GD 21. Leydig and Sertoli cell function was evaluated by investigating serum testosterone levels, cell number and distribution, and the expression levels of Leydig and Sertoli cell genes and proteins. Cr6+ increased serum testosterone level at dose of 3u2009mg/kg (1.170u2009±u20090.121u2009ng/ml vs. 0.720u2009±u20090.082u2009ng/ml in the control), while lowered it at dose of 12u2009mg/kg (0.400u2009±u20090.098u2009ng/ml). In addition, it showed that Cr6+ dose-dependently reduced Leydig cell size and cytoplasmic size and decreased the percentage of medium fetal Leydig cell cluster at dose of 12u2009mg/kg. Further study demonstrated that the expression of Leydig cell (Lhcgr, Scarb1, and Hsd3b1) and Sertoli cell (Fshr, Pdgfa, and Lif) genes in the testis was upregulated at dose of 3u2009mg/kg while the expression of Lhcgr, Hsd17b3 and Igf1 was downregulated by Cr6+ at dose of 12u2009mg/kg. In conclusion, Cr6+ had biphasic effects on fetal Leydig cell development with low dose to stimulate testosterone production and high dose to inhibit it, possibly via biphasically regulating growth factor gene expression in fetal Sertoli cells.

Zearalenone Delays Rat Leydig Cell Regeneration

Zearalenone (ZEA), a fungal mycotoxin, is present in a wide range of human foods. By virtual screening, we have identified that ZEA is a potential endocrine disruptor of Leydig cells. The effect of ZEA on Leydig cell development is still unclear. The objective of the present study was to explore whether ZEA affected Leydig cell developmental process and to clarify the underlying mechanism. Adult male Sprague-Dawley rats (60u2009days old) were randomly divided into three groups and these rats received a single intraperitoneal injection of 75u2009mg/kg ethane dimethane sulfonate (EDS) to eliminate all Leydig cells. Seven days after EDS treatment, rats intratesticularly received normal saline (control) or 150 or 300u2009ng/testis/day ZEA for 21u2009days. Immature Leydig cells isolated from 35-day-old rats were treated with ZEA (0.05-50u2009μM) for 24u2009h in vitro. In vivo ZEA exposure lowered serum testosterone levels, reduced Leydig cell number, and decreased Leydig cell-specific gene or protein expression levels possibly via downregulating the steroidogenic factor 1 (Nr5a1) expression. ZEA in vitro inhibited androgen production and steroidogenic enzyme activities in immature Leydig cells by downregulating expression levels of cholesterol side cleavage enzyme (Cyp11a1), 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1), and steroid 5α-reductase 1 (Srd5a1) at a concentration as low as 50u2009nM. In conclusion, ZEA exposure disrupts Leydig cell development and steroidogenesis possibly via downregulating Nr5a1.

Comparison of flavonoids and isoflavonoids to inhibit rat and human 11β-hydroxysteroid dehydrogenase 1 and 2

&NA; Many flavonoids and isoflavonoids have anti‐diabetic effects in animal models. However, the mechanisms that are involved are generally unclear. Since 11&bgr;‐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 IC50 values of 2.19, 5.36, 11.00, and over 100 &mgr;M, respectively. Genistein also inhibited rat HSD11B1 reductase with IC50 value of 24.58 &mgr;M, while other three chemicals showed no effects on the enzyme activity with IC50 values over 100 &mgr;M. However, apigenin and (±) equol did not inhibit human HSD11B2 at concentrations as high as 100 &mgr;M, while genistein and quercetin inhibited human HSD11B2 by 60% and 50% at 100 &mgr;M, 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.

Triphenyltin Chloride Delays Leydig Cell Maturation During Puberty in Rats

Triphenyltin chloride (TPT) is present in a wide range of human foods. TPT could disrupt testis function as a potential endocrine disruptor of Leydig cells. However, the effect of TPT on pubertal Leydig cell development is still unclear. The objective of the current study was to explore whether exposure to TPT affected Leydig cell developmental process and to clarify the underlying mechanisms. Male Sprague-Dawley rats at 35 days of age were randomly divided into four groups and received normal corn oil (control), 0.5, 1, or 2 mg/kg/day TPT for 18 days. Immature Leydig cells isolated from 35-day-old rat testes were treated with TPT (10 and 100 nM) for 24 h in vitro. In vivo exposure to ≥0.5 mg/kg TPT lowered serum testosterone levels and lowered Star mRNA. TPT at 2 mg/kg also lowered Lhcgr, Cyp11a1, Hsd3b1, Hsd17b3 as well as pAKT1/AKT1, pAKT2/AKT2, and pERK1/2/ERK1/2 ratios. In vitro exposure to TPT (100 nM) increased ROS production and induced cell apoptosis rate in rat immature Leydig cells. In conclusion, TPT exposure disrupts Leydig cell development possibly via interfering with the phosphorylation of AKT1, AKT2, and ERK1/2 kinases.

Dehydroepiandrosterone Antagonizes Pain Stress-Induced Suppression of Testosterone Production in Male Rats

Background: Leydig cells secrete the steroid hormone, testosterone, which is essential for male fertility and reproductive health. Stress increases the secretion of glucocorticoid [corticosterone, (CORT) in rats] that decreases circulating testosterone levels in part through a direct action on its receptors in Leydig cells. Intratesticular CORT level is dependent on oxidative inactivation of CORT by 11β-hydroxysteroid dehydrogenase 1 (HSD11B1) in rat Leydig cells. Pain may cause the stress, thus affecting testosterone production in Leydig cells. Methods: Adult male Sprague–Dawley rats orally received vehicle control or 5 or 10 mg/kg dehydroepiandrosterone (DHEA) 0.5 h before being subjected to pain stimulation for 1, 3, and 6 h. In the present study, we investigated the time-course changes of steroidogenic gene expression levels after acute pain-induced stress in rats and the possible mechanism of DHEA that prevented it. Plasma CORT, luteinizing hormone (LH), and testosterone (T) levels were measured, and Leydig cell gene expression levels were determined. The direct regulation of HSD11B1 catalytic direction by DHEA was detected in purified rat Leydig, liver, and rat Hsd11b1-transfected COS1 cells. Results: Plasma CORT levels were significantly increased at hour 1, 3, and 6 during the pain stimulation, while plasma T levels were significantly decreased starting at hour 3 and 6. Pain-induced stress also decreased Star, Hsd3b1, and Cyp17a1 expression levels at hour 3. When 5 and 10 mg/kg DHEA were orally administered to rats 0.5 h before starting pain stimulation, DHEA prevented pain-mediated decrease in plasma T levels and the expression of Star, Hsd3b1, and Cyp17a1 without affecting plasma CORT levels. DHEA was found to modulate HSD11B1 activities by increasing its oxidative activity and decreasing its reductive activity, thus decreasing the intracellular CORT levels in Leydig cells. Conclusion: Stress induced by acute pain can inhibit Leydig cell T production by upregulation of corticosterone. DHEA can prevent the negative effects of excessive corticosterone by modulating HSD11B1 activity.

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, 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 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.

In utero exposure to triphenyltin disrupts rat fetal testis development

Triphenyltin is an organotin that is widely used as an anti-fouling agent and may have endocrine-disrupting effects. The objective of the current study was to investigate effects of triphenyltin on the development of rat fetal testis. Female pregnant Sprague Dawley dams were gavaged daily with triphenyltin (0, 0.5, 1, and 2u202fmg/kg body weight/day) from gestational day 12 to day 21. Triphenyltin dose-dependently decreased serum testosterone levels (0.971u202f±u202f0.072 and 0.972u202f±u202f0.231u202fng/ml at 1 and 2u202fmg/kg, respectively) from control level (2.099u202f±u202f0.351u202fng/ml). Triphenyltin at 1 and 2u202fmg/kg doses also induced fetal Leydig cell aggregation, decreased fetal Leydig cell size and cytoplasmic size. Triphenyltin decreased the expression levels of Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, Insl3, Fshr, Pdgfa, and Sox9 by 0.5u202fmg/kg dose and above. However, triphenyltin did not affect Leydig and Sertoli cell numbers. In conclusion, the current study indicated that in utero exposure of triphenyltin disrupted fetal Leydig and Sertoli cell development.