Longitudinal assessment of Leydig cell function in male survivors of childhood cancer.

BACKGROUND: As the number and longevity of childhood cancer survivors increases, assessing treatment-associated late effects remains crucial. We longitudinally examined the incidence of and associated risk factors for Leydig cell dysfunction (LCD) and Leydig cell failure (LCF) in men treated for pediatric cancers at our institution. PROCEDURE: We performed a retrospective longitudinal cohort study of adult male survivors treated for various childhood cancers who are at risk for LCD. The outcomes of interest were serum testosterone and luteinizing hormone (LH) levels during childhood and adulthood. Risk factors assessed included treatment with stem cell transplant, total body irradiation (TBI), and exposure to alkylating agents. RESULTS: Out of 118 eligible subjects, 7.6% had LCF and 14.4% had LCD. Median age at last testosterone level was 20 years. Subjects with sufficient testosterone levels in adulthood (N = 105) remained sufficient for a mean of 11.1 years following completion of cancer treatment. We found significant associations between LCF and treatment with TBI (p < .003) and between LCF in adulthood and testosterone insufficiency in childhood (p < .001). No statistically significant association was found between LCF and cyclophosphamide equivalent dose greater than 20 g/m2 (p = .2). LCF/LCD occurred in a small number of nonirradiated patients treated with the highest doses of alkylators. CONCLUSIONS: Incidence of LCF and LCD are low in male survivors of childhood cancer. Longitudinally, there is an association between childhood testosterone insufficiency and LCF in adulthood. Alkylating agents and stem cell transplant without TBI were not associated with LCF in our study.

The Effect of Aqueous Lessertia frutescens Extract on TM3 Leydig Cells Exposed to TNF-α in vitro.

BACKGROUND: Extractions of Lessertia frutescens (Lf) are shown to have immune modulation, anti-inflammatory and antioxidant properties. However, Lf is also cytotoxic, antiproliferative, and pro-apoptotic in vitro. Furthermore, Lf extractions may influence steroidogenesis. Nevertheless, the impact on Leydig cell function has not previously been investigated. As tumor necrosis factor-alpha (TNF-α) is known to cause Leydig cell dysfunction under inflammatory conditions, it is further proposed that Lf extracts may protect against the negative impact of TNF-α on Leydig cells. The aim of the study was to investigate the effect of an aqueous Lessertia frutescens extract (LFE) on Leydig cells exposed to TNF-αin vitro. METHODS: Human chorionic gonadotrophin-stimulated TM3 Leydig cells were exposed for 24 h to (a) TNF-α (0.1, 1, 10, 100 ng/mL), (b) LFE (0.01, 0.1, 1, 10, 100 ng/mL), and (c) co-exposure to 10 ng/mL TNF-α and LFE (0.01, 0.1, 1, 10, 100 ng/mL). We analyzed cell viability, cytotoxicity, caspase 3/7 activation, testosterone concentration, and intracellular superoxide. RESULTS: TNF-α exposure decreased cell viability, increased cytotoxicity, and caspase 3/7, with no significant effect on intracellular superoxide in TM3 Leydig cells. When LFE concentrations of 0.01-10 ng/mL were tested, we observed improved vitality and reduced levels of caspase 3/7. At 100 ng/mL, LFE decreased viability and increased cytotoxicity and caspase 3/7. However, LFE did not affect intracellular superoxide. Furthermore, LFE protected against 10 ng/mL TNF-α-induced cytotoxicity and apoptosis, except at the highest concentration. LFE alone and in co-culture with 10 ng/mL TNF-α increased testosterone at high concentrations. CONCLUSIONS: In our TM3 Leydig cell model, LFE protected against TNF-α-induced cytotoxicity and early apoptosis, except at the highest experimental concentrations, where it was cytotoxic. These effects were not mediated through a change in intracellular superoxide. Although further investigations are warranted, aqueous LFE may protect against TNF-α-induced Leydig cell dysfunction.

Differentiation of Human Induced Pluripotent Stem Cells Into Testosterone-Producing Leydig-like Cells.

Late-onset hypogonadism (LOH) syndrome, due to a partial lack of testosterone, decreases the quality of life of older men. Testosterone is mainly secreted by Leydig cells in the testes. Leydig cell transplantation is expected to be a promising alternative to conventional testosterone replacement therapy for LOH syndrome. We herein report a simple and robust protocol for directed differentiation of human induced pluripotent stem cells (hiPSCs) into Leydig-like cells by doxycycline-inducible overexpression of NR5A1 and treatment with a combination of 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP) and forskolin. The differentiated cells expressed the steroidogenic enzyme genes STAR, CYP11A1, CYP17A1, and HSD3B2 and the specific markers of adult Leydig cells HSD17B3, INSL3, and LHCGR. Furthermore, we confirmed the secretion of functional testosterone from the cells into the culture supernatant by a testosterone-sensitive cell proliferation assay. These findings showed that the hiPSCs were able to be differentiated into Leydig-like cells, supporting the expectation that hiPSC-derived Leydig-like cells can be novel tools for treating LOH syndrome.

Comparative transcriptome analysis reveals potential testosterone function-related regulatory genes/pathways of Leydig cells in immature and mature buffalo (Bubalus bubalis) testes.

Leydig cells (LCs) are testosterone-generating endocrine cells that are located outside the seminiferous tubules in the testis, and testosterone is fundamental for retaining spermatogenesis and male fertility. In buffalo, adult Leydig cells (ALCs) are developed by immature Leydig cells (ILCs) in the postnatal testes. However, the genes/pathways associated to the regulation of testosterone secretion function during the development of postnatal LCs remains comprehensively unidentified. The present study comparatively analyzed the transcriptome profiles of ILC and ALC in buffalo with significant differences in testosterone secretion. Differentially expressed genes (DEGs) analysis identified 972 and 1,091 annotated genes that were significantly up- and down-regulated in buffalo ALC. Functional enrichment analysis showed that cAMP signaling being the most significantly enriched pathway, and testosterone synthesis and lipid transport-related genes/pathways were upregulated in ALC. Furthermore, gene set enrichment analysis (GSEA) shows that cAMP signaling and steroid hormone biosynthesis were activated in ALC, demonstrating that cAMP signaling may serve as a positive regulatory pathway in the maintenance of testosterone function during postnatal development of LCs. Protein-protein interaction (PPI) networks analysis highlighted that ADCY8, ADCY2, POMC, CHRM2, SST, PTGER3, SSTR2, SSTR1, NPY1R, and HTR1D as hub genes in the cAMP signaling pathway. In conclusion, this study identified key genes and pathways associated in the regulation of testosterone secretion function during the ILC-ALC transition in buffalo based on bioinformatics analysis, and these key genes might be deeply involved in cAMP generation to influencing testosterone levels in LCs. The results suggest that ALCs might increase testosterone levels by enhancing cAMP production than ILCs. Our data will enhance the understanding of developmental mechanism studies related to testosterone function and provide preliminary evidence for molecular mechanisms of LCs regulating spermatogenesis.

Di-n-butyl phthalate diminishes testicular steroidogenesis by blocking the hypothalamic-pituitary-testicular axis: relationship with germ cell apoptosis in Japanese quail.

Although di-n-butyl phthalate (DBP) induces germ cell apoptosis, the underlying mechanism is not yet clear in quail. In this study, prepubertal quails were given a single dose of 500mg kg-1 DBP by gavage and were then killed 3, 6 and 24h after treatment. There was a significant reduction in intratesticular testosterone (ITT) concentrations and testicular steroidogenic enzyme mRNA expression and a significant increase in germ cell apoptosis in DBP-treated compared with control quails at all time points. Maximum apoptosis was detected 6h after treatment and the maximum reduction in testosterone concentrations was at 3h. To investigate whether DBP suppressed testicular steroidogenesis by affecting the hypothalamic-pituitary-testicular axis, we analysed pituitary LH subunit ß (Lhb) mRNA expression and serum LH concentrations. At all time points, pituitary Lhb expression and serum LH concentrations were significantly decreased following DBP treatment. The present observations suggest the possibility that DBP blocked LH secretion from the hypothalamus and/or pituitary, thereby decreasing LH stimulation of Leydig cells and reducing ITT concentrations. DBP-induced decreases in ITT concentrations may cause changes to the physical structure of Sertoli cells, which, in turn, may induce germ cell apoptosis.

Low dose of fire retardant, 2,2',4,4'-tetrabromodiphenyl ether (BDE47), stimulates the proliferation and differentiation of progenitor Leydig cells of male rats during prepuberty.

2,2',4,4'-Tetrabromodiphenyl ether (BDE47), a flame retardant, is extensively distributed in the food chain. However, whether BDE47 affects Leydig cell development during prepuberty remains unclear. BDE47 was daily gavaged to 21-day-old Sprague-Dawley male rats with 0 (corn oil), 0.1, 0.2, and 0.4 mg/kg for 14 days. BDE47 did not affect the body weight or testis weight of rats. It significantly increased serum testosterone level at 0.4 mg/kg, but decreased luteinizing hormone (LH) level without affecting estradiol level. BDE47 induced Leydig cell hyperplasia (the number of CYP11A1-positive Leydig cells increased), and up-regulated the expression of Scarb1, Star, Hsd11b1, Pcna, and Ccnd1 in the testis. BDE47 significantly reduced p53 and p21 levels but increased CCND1 level. It also markedly increased the phosphorylation of AKT1, AKT2, ERK1/2, and CREB. BDE47 significantly up-regulated the expression of Scarb1, Star, and Hsd11b1 and stimulated androgen production by immature Leydig cells from rats under basal, LH, and 8Br-cAMP stimulated conditions at 100 nM in vitro. In conclusion, BDE47 increased Leydig cell number and up-regulated the expression of Scarb1 and Star, thereby leading to increased testosterone synthesis.

Immunization with oral KISS1 DNA vaccine inhibits testicular Leydig cell proliferation mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes in goats.

This study aimed to analyze the effect and mechanism of immunization of oral KISS1 DNA vaccine on the proliferation of goat testicular Leydig cells. Ten 8-week-old male goats were randomly divided into KISS1 DNA vaccine and control groups for immunization (five goats each group). These goats were sacrificed at 8 weeks after primary immunization, and the tissue samples of hypothalamus, pituitary, and testis and Leydig cell samples were collected for RT-PCR and CCK8 assay. Immunization with the oral KISS1 DNA vaccine effectively inhibited the proliferation of Leydig cells, the expression of hypothalamus KISS1, GPR54, and GnRH mRNA, pituitary GnRHR and LH mRNA, testicular LHR mRNA, and apoptosis-inhibitory gene Bcl-2 mRNA in Leydig cells. By contrast, the immunization enhanced the mRNA expression of apoptosis-promoting gene Bax and Clusterin in Leydig cells. These findings indicate that immunization with the oral KISS1 DNA vaccine can inhibit the proliferation of goat testicular Leydig cells mainly via the hypothalamic-pituitary-testicular axis and apoptosis-related genes.

Leydig stem cells and future therapies for hypogonadism.

PURPOSE OF REVIEW: In this review, we outline the most recent advances in the development of Leydig stem cells (LSCs) and summarize the current and upcoming treatments for hypogonadism. RECENT FINDINGS: In-vitro and in-vivo studies show that inducing stem cells to differentiate into testosterone-producing adult Leydig cells is possible. In addition, LSCs can be grafted with Sertoli cells to increase testosterone levels in vivo. This therapy causes minimal effects on luteinizing hormone and follicle stimulating hormone levels. Novel therapies for hypogonadism include varying methods of testosterone delivery such as intranasal and oral agents, as well as novel selective estrogen and androgen receptor modulators. SUMMARY: LSC therapies provide an effective way of increasing testosterone levels without detrimentally affecting gonadotropin levels. Next steps in developing viable Leydig cell grafting options for the treatment of hypogonadism should include the assessment of efficacy and potency of current animal models in human trials. Recently, both intranasal and oral testosterone have been made available and shown promising results in treating hypogonadism while maintaining fertility. Enclomiphene citrate and selective androgen receptor modulators have been suggested as future therapies for hypogonadism; however, further studies assessing efficacy and adverse effects are needed.

Luteolin modulates gene expression related to steroidogenesis, apoptosis, and stress response in rat LC540 tumor Leydig cells.

In males, androgens are mainly produced by Leydig cells from the testis. A critical and highly regulated step of steroidogenesis involves the importation of cholesterol within the mitochondria by the steroidogenic acute regulatory (STAR) protein. During aging, STAR protein levels in Leydig cells gradually decrease, leading to a reduced entry of cholesterol into mitochondria and lower testosterone production. In addition to preserving its steroidogenic capacity, tumor Leydig cells can also be excellent models for evaluating the mechanisms of action of anticancer agents. In this study, we examined whether polyphenolics having structural similarities to luteolin could promote steroidogenic and cancer-related gene expressions within rat L540 tumor Leydig cells. In this cell model, luteolin activated Star expression and increased progesterone as well as testosterone productions. Interestingly, luteolin decreased gene expression related to cholesterol biosynthesis, possibly inhibiting membrane synthesis and cell proliferation. In addition, increased expression of genes such as Fas, Cdkn1a, Atp7b, and Tp53, as well as increased accumulation of cleaved caspase 3 and PARP, in response to luteolin treatment indicates that apoptosis is being activated. Luteolin also modulated the expression of genes involved in stress response, such as glutathione-S transferases Gsta1 and Gstt2, and the unfolded protein response. Thus, dietary luteolin may be effective in Leydig cell tumor chemoprevention and in maintaining steroidogenesis in aging males.

Stem Leydig Cells in the Adult Testis: Characterization, Regulation and Potential Applications.

Androgen deficiency (hypogonadism) affects males of all ages. Testosterone replacement therapy (TRT) is effective in restoring serum testosterone and relieving symptoms. TRT, however, is reported to have possible adverse effects in part because administered testosterone is not produced in response to the hypothalamic-pituitary-gonadal (HPG) axis. Progress in stem cell biology offers potential alternatives for treating hypogonadism. Adult Leydig cells (ALCs) are generated by stem Leydig cells (SLCs) during puberty. SLCs persist in the adult testis. Considerable progress has been made in the identification, isolation, expansion and differentiation of SLCs in vitro. In addition to forming ALCs, SLCs are multipotent, with the ability to give rise to all 3 major cell lineages of typical mesenchymal stem cells, including osteoblasts, adipocytes, and chondrocytes. Several regulatory factors, including Desert hedgehog and platelet-derived growth factor, have been reported to play key roles in the proliferation and differentiation of SLCs into the Leydig lineage. In addition, stem cells from several nonsteroidogenic sources, including embryonic stem cells, induced pluripotent stem cells, mature fibroblasts, and mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord have been transdifferentiated into Leydig-like cells under a variety of induction protocols. ALCs generated from SLCs in vitro, as well as Leydig-like cells, have been successfully transplanted into ALC-depleted animals, restoring serum testosterone levels under HPG control. However, important questions remain, including: How long will the transplanted cells continue to function? Which induction protocol is safest and most effective? For translational purposes, more work is needed with primate cells, especially human.

Leydig Cell Function in Male Survivors of Childhood Cancer: A Report From the St Jude Lifetime Cohort Study.

PURPOSE: Direct assessment of Leydig cell function in childhood cancer survivors has been limited. The objectives of this study were to describe the prevalence of and risk factors for Leydig cell failure (LCF), Leydig cell dysfunction (LCD), and associated adverse health outcomes. PATIENTS AND METHODS: In this retrospective study with cross-sectional health outcomes analysis, we evaluated 1,516 participants (median age, 30.8 years) at a median of 22.0 years after cancer diagnosis. LCF was defined as serum total testosterone less than 250 ng/dL (or 8.67 nmol/L) and luteinizing hormone greater than 9.85 IU/L, and LCD by testosterone as 250 ng/dL or greater and luteinizing hormone greater than 9.85 IU/L. Polytomous logistic regression evaluated associations with demographic and treatment-related risk factors. Log-binomial regression evaluated associations with adverse physical and psychosocial outcomes. Piecewise exponential models assessed the association with all-cause mortality. RESULTS: The prevalence of LCF and LCD was 6.9% and 14.7%, respectively. Independent risk factors for LCF included an age of 26 years or older at assessment, testicular radiotherapy at any dose, and alkylating agents at cyclophosphamide equivalent doses of 4,000 mg/m2 or greater. The risk increased with older age, higher doses of testicular radiotherapy, and cyclophosphamide equivalent doses. LCF was significantly associated with abdominal obesity, diabetes mellitus, erectile dysfunction, muscle weakness, and all-cause mortality. LCD was associated with unilateral orchiectomy and the same risk factors as LCF; no significant associations were found with adverse physical or psychosocial outcomes. CONCLUSION: Older age, testicular radiotherapy, and exposure to alkylating agents were associated with LCF, which was associated with adverse physical and psychosexual outcomes. LCD, although having similar risk factors, was not associated with adverse health outcomes. Additional studies are needed to investigate the role of sex hormone replacement in mitigating the burden from adverse outcomes in survivors.

CeO2NPs relieve radiofrequency radiation, improve testosterone synthesis, and clock gene expression in Leydig cells by enhancing antioxidation.

Introduction: The ratio of Ce3+/Ce4+ in their structure confers unique functions on cerium oxide nanoparticles (CeO2NPs) containing rare earth elements in scavenging free radicals and protecting against oxidative damage. The potential of CeO2NPs to protect testosterone synthesis in primary mouse Leydig cells during exposure to 1,800 MHz radiofrequency (RF) radiation was examined in vitro. Methods: Leydig cells were treated with different concentrations of CeO2NPs to identify the optimum concentration for cell proliferation. The cells were pretreated with the optimum dose of CeO2NPs for 24 hrs and then exposed to 1,800 MHz RF at a power density of 200.27 µW/cm2 (specific absorption rate (SAR), 0.116 W/kg) for 1 hr, 2 hrs, or 4 hrs. The medium was used to measure the testosterone concentration. The cells were collected to determine the antioxidant indices (catalase [CAT], malondialdehyde [MDA], and total antioxidant capacity [T-AOC]), and the mRNA expression of the testosterone synthase genes (Star, Cyp11a1, and Hsd-3ß) and clock genes (Clock, Bmal1, and Rorα). Results: Our preliminary result showed that 128 µg/mL CeO2NPs was the optimum dose for cell proliferation. Cells exposed to RF alone showed reduced levels of testosterone, T-AOC, and CAT activities, increased MDA content, and the downregulated genes expression of Star, Cyp11a1, Hsd-3ß, Clock, Bmal1, and Rorα. Pretreatment of the cells with 128 µg/mL CeO2NPs for 24 hrs followed by RF exposure significantly increased testosterone synthesis, upregulated the expression of the testosterone synthase and clock genes, and increased the resistance to oxidative damage in Leydig cells compared with those in cells exposed to RF alone. Conclusion: Exposure to 1,800 MHz RF had adverse effects on testosterone synthesis, antioxidant levels, and clock gene expression in primary Leydig cells. Pretreatment with CeO2NPs prevented the adverse effects on testosterone synthesis induced by RF exposure by regulating their antioxidant capacity and clock gene expression in vitro. Further studies of the mechanism underlying the protective function of CeO2NPs against RF in the male reproductive system are required.

Luteinizing hormone signaling is involved in synchronization of Leydig cell's clock and is crucial for rhythm robustness of testosterone production†.

In mammals, circadian clock regulates concentration of many reproductive hormones including testosterone. Previously, we characterized pattern of circadian transcription of core clock genes in testosterone-producing Leydig cells. Here, the potential role of luteinizing hormone receptor (LHR)-cAMP signaling in synchronization of Leydig cell's circadian clock and rhythmic testosterone production were examined. Results showed that activation of LHR-cAMP signaling in primary rat Leydig cell culture increased Star/STAR and changed expression of many clock genes (upregulated Per1/PER1, Dec1/2, and Rorb, and downregulated Bmal1 and Rev-erba/b). Inhibition of protein kinase A prevented LHR-triggered increase in transcription of Per1 and Dec1. Effect of stimulated LHR-cAMP signaling on Leydig cell's clock transcription was also confirmed in vivo, using rats treated with single hCG injection. To analyze in vivo effect of low LH-cAMP activity on rhythmical Leydig cell function, rats with experimental hypogonadotropic hypogonadism were used. Characteristics of hypogonadal rats were decreased LH and testosterone secretion without circadian fluctuation; in Leydig cells decreased arrhythmic cAMP and transcription of steroidogenic genes (Cyp11a1 and Cyp17a1) were observed, while decreased Star/STAR expression retains circadian pattern. However, expression of clock genes, despite changes in transcription levels (increased Bmal1, Per2, Cry1, Cry2, Rora, Rorb, Rev-erba/b/REV-ERBB, Dec1, Csnk1e, and decreased Npas2 and PER1) kept circadian patterns observed in control groups. Altogether, the results strengthened the hypothesis about role of LH-cAMP signaling as synchronizer of Leydig cell's clock. However, clock in Leydig cells is not sufficient to sustain rhythmicity of testosterone production in absence of rhythmic activity of LH-cAMP signaling.

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.

Identification of a Novel Zebrafish Mutant Line that Develops Testicular Germ Cell Tumors.

Testicular tumors are the most common solid malignant tumors in men 20-35 years of age. Although most of testicular tumors are curable, current treatments still fail in 15%-20% of patients. However, insufficient understanding of the molecular basis and lack of animal models limit development of more effective treatments. This study reports the identification of a novel zebrafish mutant line, ns1402, which develops testicular germ cell tumors (TGCTs). While both male and female ns1402 mutants were fertile at young age, male ns1402 mutants became infertile as early as 9 months of age. This infertility was associated with progressive loss of mature sperm. Failure of spermatogenesis was, at least in part, explained by progressive loss of mature Leydig cells, a source of testosterone that is essential for spermatogenesis. Interestingly, TGCTs in ns1402 mutants contained a large number of Sertoli cells and gene expression profiles of Sertoli cells were altered before loss of mature Leydig cells. This suggests that changes in Sertoli cell properties happened first, followed by loss of mature Leydig cells and failure of spermatogenesis. Taken together, this study emphasizes the importance of cell-cell interactions and cell signaling in the testis for spermatogenesis and tissue homeostasis.

The expression of CKLFSF2B is regulated by GATA1 and CREB in the Leydig cells, which modulates testicular steroidogenesis.

CKLFSF is a protein family that serves as a functional bridge between chemokines and members of the transmembrane 4 superfamily (TM4SF). In the course of evolution, CKLFSF2 has evolved as two isoforms, namely CKLFSF2A and CKLFSF2B, in mice. CKLFSF2A, also known as CMTM2A and ARR19, is expressed in the testis and is important for testicular steroidogenesis. CKLFSF2B is also known to be highly expressed in the testis. In the prepubertal stage, CKLFSF2B is expressed only in Leydig cells, but it is highly expressed in haploid germ cells and Leydig cells in adult testis. CKLFSF2B is naturally processed inside the cell at its C-terminus to yield smaller proteins compared to its theoretical size of ≈25 kDa. The Cklfsf2b gene is regulated by GATA-1 and CREB protein, binding to their respective binding elements present in the 2-kb upstream promoter sequence. In addition, the overexpression of CKLFSF2B inhibited the activity of the Nur77 promoter, which consequently represses the promoter activity of Nur77-target steroidogenic genes such as P450c17, 3ß-HSD, and StAR in MA-10 Leydig cells. Adenovirus-mediated overexpression of CKLFSF2B in primary Leydig cells isolated from adult mice shows a repression of steroidogenic gene expression and consequently testosterone production. Moreover, intratesticular injection of CKLFSF2B-expressing adenovirus in adult mice clearly had a repressive effect compared to the control injected with only GFP-expressing adenovirus. Altogether, these findings suggest that CKLFSF2B might be involved in the development and function of Leydig cells and regulate testicular testosterone production by fine-tuning the expression of steroidogenic genes.

Nitric oxide modulates ATP-evoked currents in mouse Leydig cells.

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 µM and 1 mM), L-arginine (10, 100, 300, and 500 µM), ODQ (300 µM), and 8-Br-cGMP (100 µM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 µM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Exposure to cadmium during gestation and lactation affects development and function of Leydig cells in male offspring.

Toxic effects of maternal exposure to Cadmium (Cd) on Leydig cells of male offspring arises much concern recently, but its toxic effects on the development of Leydig cells and androgen synthesis have not been elucidated. In this study, female rats were exposed to Cd during gestation and lactation, and the development of Leydig cells in the first filial-generation (F1) male rats was investigated. The steroidogenic signaling pathway and biomarkers related to the development of Leydig cells were detected to disclose how maternal Cd-exposure caused reproductive damage. F1 male rats with maternal Cd-exposure gained a low relative weight of testis and declined levels of steroid hormones. Maternal Cd-exposure interrupted the development of Leydig cells with high expression of SRD5α and cell morphology of immature Leydig cells in adulthood, inhibited the activation of cyclic adenosine monophosphate/ protein kinase A signaling pathway and down-regulated the steroidogenic enzymes. These results would help to disclose the origin of male sexual dysfunction in the developmental stages of Leydig cells.

Nitric oxide modulates ATP-evoked currents in mouse Leydig cells

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Does signaling of estrogen-related receptors affect structure and function of bank vole Leydig cells?

To get a deeper insight into the function of estrogen-related receptors (ERRs) and dissect underlying mechanism in Leydig cells, ERRs (type α, ß and γ) were blocked or activated in testes of adult bank voles (Myodes glareolus) which show seasonal changes in the intratesticular sex hormones level. Both actively reproducing animals (long day conditions; LD) and those with regression of the reproductive system (short day conditions; SD) received intraperitoneal injections of selective ERRα antagonist 3-[4-(2,4-Bis-trifluoromethylbenzyloxy)-3-methoxyphenyl]-2-cyano-N-(5-trifluoromethyl-1,3,4-thiadiazol-2-yl)acrylamide (XCT 790) or selective ERRß/ERRγ agonist N-(4-(Diethylaminobenzylidenyl)-N'-(4-hydroxybenzoyl)-hydrazine (DY131) (50 µ/kg bw; six doses every other day). Markedly more, XCT 790 (P < 0.05) but also DY131 affected interstitial tissue histology whose volume increased in both LD and SD males while seminiferous epithelium structure was untouched. Ultrastructure analysis revealed alterations in mitochondria number as well as endoplasmic reticulum and Golgi complexes volume and structure especially after ERRα blockage. Diverse and complex ERRs regulation at mRNA level and protein expression (P < 0.05; P < 0.01 and P < 0.001) of steroidogenic (lutropin receptor (LHR), translocator protein (TSPO), steroidogenic acute regulatory protein (StAR)) and secretory (insulin-like protein 3 (INSL3) and relaxin (RLN)) molecules were revealed in relations to endogenous estrogen level in treated males. Notably, immunolocalization of ERRs and above proteins, exclusively in Leydig cells, indicated their involvement in Leydig cell function control based on interactions with endogenous estrogen level and/or estrogen signaling via ERRs. Treatment with XCT 790 or DY131 significantly decreased (P < 0.05; P < 0.01 and P < 0.001) intratesticular estrogens concentration, with exception in SD DY131 males. In addition, androgens level was decreased, but not in LD DY131 voles. Similarly, ERRßγ activation significantly reduced (P < 0.05; P < 0.01 and P < 0.001) cAMP and calcium ions (Ca2+) concentrations particularly in DY131 voles. Overall, for the first time, we have shown that ERRs are involved in maintenance of Leydig cell architecture and supervision of its steroidogenic and secretory activity that is closely related to endogenous estrogen status in the testis. Further understanding of mechanism(s) by which individual types of ERRs can control Leydig cell function is relevant for predicting and preventing steroidogenic and spermatogenic disorders.