Nestin-dependent mitochondria-ER contacts define stem Leydig cell differentiation to attenuate male reproductive ageing.

Male reproductive system ageing is closely associated with deficiency in testosterone production due to loss of functional Leydig cells, which are differentiated from stem Leydig cells (SLCs). However, the relationship between SLC differentiation and ageing remains unknown. In addition, active lipid metabolism during SLC differentiation in the reproductive system requires transportation and processing of substrates among multiple organelles, e.g., mitochondria and endoplasmic reticulum (ER), highlighting the importance of interorganelle contact. Here, we show that SLC differentiation potential declines with disordered intracellular homeostasis during SLC senescence. Mechanistically, loss of the intermediate filament Nestin results in lower differentiation capacity by separating mitochondria-ER contacts (MERCs) during SLC senescence. Furthermore, pharmacological intervention by melatonin restores Nestin-dependent MERCs, reverses SLC differentiation capacity and alleviates male reproductive system ageing. These findings not only explain SLC senescence from a cytoskeleton-dependent MERCs regulation mechanism, but also suggest a promising therapy targeting SLC differentiation for age-related reproductive system diseases.

Bisphenol S exposure induces cytotoxicity in mouse Leydig cells.

Bisphenol S (BPS), an increasingly used alternative to bisphenol A, has been linked to testosterone deficiency and male reproductive dysfunction in laboratory animals. This study aimed to examine the cytotoxicity of BPS exposure to Leydig cells and to investigate its possible mechanisms. After treatment with BPS (100, 200 and 400 µM) for 48 h in vitro, TM3 mouse Leydig cells exhibited a dose-dependent decrease in the viability. Furthermore, BPS challenge triggered oxidative stress manifested by compromised activities of superoxide dismutase and catalase with exaggerated formation of reactive oxygen species. Especially, BPS exposure resulted in augmented mitochondrial permeability transition pore opening, dissipated mitochondrial membrane potential and reduced ATP generation, along with an altered energy metabolism. Moreover, BPS stimulation enhanced BAX expression and caspase-3 activity and inhibited BCL-2 expression. In addition, BPS-treated TM3 cells showed an accumulation of autophagic vacuoles, together with increased Beclin1 and P62 expression and elevated LC3B-II/LC3B-I ratio. These results demonstrated that in vitro exposure to BPS exerted cytotoxicity to TM3 Leydig cells through inducing oxidative stress, mitochondrial impairment, autophagic disturbance and apoptosis.

Boosting effect of testosterone on GDNF expression in Sertoli cell line (TM4); comparison between TM3 cells-produced and exogenous testosterone.

The Glial cell-derived neurotrophic factor (Gdnf) and testosterone induce the spermatogonial stem cells (SSCs) self-renewal and spermatogenesis, respectively. In present study the stimulating role of testosterone on Sertoli cells to produce Gdnf, and the possible effect of Gdnf on Gfrα1 and c-RET expressions were investigated. The TM4 cells (line Sertoli cells) were co-cultured with [0.1, 0.2 and 0.4 (ng/ml)] of exogenous and TM3 (line Leydig cells)-produced testosterones, and consequently the TM4-produced Gdnf concentration was evaluated. Next, the SSCs were co-cultured with the TM-4 derived media (endogenous Gdnf) and exogenous Gdnf [0.1, 0.2, and 0.4 ng/ml)]. The 0.1 and 0.2 ng/ml endogenous and 3 concentrations of exogenous testosterone up-regulated the Gdnf expression versus non-treated Sertoli cells. The TM4-produced and exogenous Gdnfs, in all concentrations, up-regulated the receptors expression. In conclusion, the testosterone, solely, stimulates the Gdnf synthesis and the Gdnf, individually, amplifies its receptor's expression at mRNA and protein levels.

A Protein from Dioscorea polystachya (Chinese Yam) Improves Hydrocortisone-Induced Testicular Dysfunction by Alleviating Leydig Cell Injury via Upregulation of the Nrf2 Pathway.

Leydig cell injury has been described as a primary driver of testicular dysfunction and is affected by oxidative stress. Dioscorea polystachya (Chinese yam) is used to improve testicular dysfunction in clinical and pharmacological research via its antioxidative activity, but the mechanisms underlying the beneficial effect of Chinese yam on testicular dysfunction and its suppression of Leydig cell oxidative damage remain unclear. In this study, we obtained a Chinese yam protein (DP1) and explored its effectiveness and possible mechanism in improving testicular dysfunction in vivo and in vitro. We established a testicular dysfunction model in rats using hydrocortisone (HCT). DP1 increased body weight and organ index, improved the deterioration in testicular morphology (including increasing the diameter of seminiferous tubules and thickness of germinal cell layers, inhibiting testicular cell apoptosis by increasing the Bcl-2/Bax ratio, and impeding collagen leakage by downregulating TGF-ß1 and p-SMAD2/3 expression), and restored the testosterone content. In addition, DP1 enhanced the number of Leydig cells in rats and H2O2-induced TM3 Leydig cells, and the effect of DP1 on the apoptosis, fibrosis, and testosterone content of TM3 cells was similar to that observed in vivo. These changes were dependent on the regulation of oxidative stress, including significantly reduced intracellular 8-hydroxy-2-deoxyguanosine levels, enhanced superoxide dismutase activities, and decreased superoxide anion levels, which were confirmed via a superoxide overexpression system. Furthermore, we observed that DP1 promoted Nrf2 nuclear import and upregulated antioxidant factor expression in vivo and in vitro. However, Nrf2 silencing eliminated the ability of DP1 to increase the Bcl-2/Bax ratio, reduce the expression levels of TGF-ß1 and p-SMAD2/3, and increase testosterone contents in H2O2-induced TM3 cells. In conclusion, DP1 reversed the HCT-induced testicular apoptosis and fibrosis and decreased testosterone contents by alleviating Leydig cell oxidative damage via upregulation of the Nrf2 pathway.

Stress-induced glucocorticoids alter the Leydig cells' timing and steroidogenesis-related systems.

The study aimed to analyze the time-dependent consequences of stress on gene expression responsible for diurnal endocrine Leydig cell function connecting them to the glucocorticoid-signaling. In the first 24h after the stress event, a daily variation of blood corticosterone increased, and testosterone decreased; the testosterone/corticosterone were lowest at the end of the stress session overlapping with inhibition of Leydig cells' steroidogenesis-related genes (Nr3c1/GR, Hsd3b1/2, Star, Cyp17a1) and changed circadian activity of the clock genes (the increased Bmal1/BMAL1 and Per1/2/PER1 and decreased Cry1 and Rev-erba). The glucocorticoid-treated rats showed a similar response. The principal-component-analysis (PCA) displayed an absence of significant differences between treatments especially on Per1 and Rev-erba, the findings confirmed by the in vivo blockade of the testicular glucocorticoid receptor (GR) during stress and ex vivo treatment of the Leydig cells with hydrocortisone and GR-blocker. In summary, stressful stimuli can entrain the clock in the Leydig cells through glucocorticoid-mediated communication.

The role of StAR2 gene in testicular differentiation and spermatogenesis in Nile tilapia (Oreochromis niloticus).

Sex steroids play critical roles in sex differentiation and gonadal development in teleosts. Steroidogenic acute regulatory protein (StAR), transporting cholesterol (the substrate for steroidogenesis) from the outer mitochondrial membrane to the inner membrane, is the first rate-limiting factor of steroidogenesis. Interestingly, two StAR genes (named as StAR1 and StAR2) have been isolated from non-mammalian vertebrates. To characterize the functions of the novel StAR2 gene in the gonadal differentiation and fertility, we generated a StAR2 homozygous mutant line in Nile tilapia (Oreochromis niloticus). StAR2 gene knockout in male tilapia impeded meiotic initiation, associate with the down-regulation of meiosis related gene expressions of vasa, sycp3 and dazl at 90 days after hatching (dah). Meanwhile, cyp11b2 expression and serum 11-KT production significantly declined in StAR2-/- XY fish at 90 dah. From 120-300 dah, spermatogenesis gradually recovered, and so did the expressions of vasa, sycp3 and dazl in StAR2-/- XY fish testes. However, seminiferous lobules arranged disorderly in StAR2-/- XY fish testes at 300 dah. The number of Leydig cells and expressions of downstream steroidogenesis enzymes including cyp11a1, 3ß-HSD-I, 3ß-HSD-II, cyp17a1 and cyp17a2 decreased in StAR2-/- XY fish testes at 300 dah. Serum testosterone and 11-KT levels were significantly lower in StAR2-/- XY fish than that of their control counterparts. Furthermore, significantly elevated ar, fsh and lh expressions in StAR2-deficient XY fish testes and pituitaries were found when compared with the control XY fish. Testes degeneration and spermatogenic cell apoptosis were observed, while no sperm were squeezed out in StAR2-/- XY fish testes at 540 dah. Taken together, our results suggest that StAR2 has a role in testicular development, spermatogenesis and spermiation by regulating androgen production in tilapia, but may not be essential and could be compensated.

Generation of a Highly Biomimetic Organoid, Including Vasculature, Resembling the Native Immature Testis Tissue.

The creation of a testis organoid (artificial testis tissue) with sufficient resemblance to the complex form and function of the innate testis remains challenging, especially using non-rodent donor cells. Here, we report the generation of an organoid culture system with striking biomimicry of the native immature testis tissue, including vasculature. Using piglet testis cells as starting material, we optimized conditions for the formation of cell spheroids, followed by long-term culture in an air-liquid interface system. Both fresh and frozen-thawed cells were fully capable of self-reassembly into stable testis organoids consisting of tubular and interstitial compartments, with all major cell types and structural details expected in normal testis tissue. Surprisingly, our organoids also developed vascular structures; a phenomenon that has not been reported in any other culture system. In addition, germ cells do not decline over time, and Leydig cells release testosterone, hence providing a robust, tunable system for diverse basic and applied applications.

PGAM1 regulates the glycolytic metabolism of SCs in tibetan sheep and its influence on the development of SCs.

This study was to explore the regulation effect of PGAM1 on the proliferation, apoptosis and glycolysis pathway of Tibetan sheep Sertoli cells. In this paper, the reproductive organs of male Tibetan sheep before pre-puberty (3 months old), sexual maturity (1 year old) and adult (3 years old) were used as experimental materials. The complete CDS region sequence of PGAM1 gene was cloned for bioinformatics analysis, and had the closest relationship with Tibetan antelope. QRT-PCR, Western blot and immunohistochemical staining were used to detect the expression and localization of PGAM1 in the testis and epididymis tissues of Tibetan sheep at different growth and development stages at the transcription and translation levels. Then the Tibetan sheep primary Sertoli cells (SCs) were isolated to construct PGAM1 gene overexpression and interference vectors, and to transfect primary SCs so as to promote and inhibit PGAM1 gene expression; CCK-8 and flow cytometry were used to detect the proliferation effect of SCs;qRT-PCR technology was employed to detect the changes in the expression of genes related to cell proliferation and apoptosis. Different kits were used to detect pyruvate, lactic acid, ATP production and LDH activity during glycolysis, and to detect the changes in the expression of downstream genes in the glycolysis pathway. The results showed that the CDS region of Tibetan sheep PGAM1 gene was 765 bp in length, which can encode 254 amino acids; and the expression of PGAM1 protein in the testis and epididymis increased at 1Y group and 3Ygroup compared with 3 M group, and that the PGAM1 protein mainly existed in SCs and Leydig cells at different developmental stages. CCK-8 and flow cytometry test results found that compared with the empty vector group (pcDNA3.1(+)), the proliferation rate of the PGAM1 gene overexpression group (pcDNA3.1(+)-PGAM1) decreased. The mRNA expression of the cell proliferation related genes PCNA and Bcl2 was significantly decreased (P < 0.05), and the expression of apoptosis-related genes Bax and caspase3 was significantly increased (P < 0.05). The expression of downstream genes in the glycolysis pathway was significant increased (P < 0.05), pyruvate content, ATP content, lactic acid production and LDH activity increased significantly (P < 0.05). Compared with the interference control group (NC), the proliferation rate of the PGAM1 gene interference group (si-PGAM1) was weakened. The mRNA expression of the cell proliferation-related genes PCNA and Bcl2 was significantly increased (P < 0.05), and the expression of cell apoptosis related genes Bax and caspase3 was significantly decreased (P < 0.05). The expression of downstream genes in the glycolysis pathway was significantly reduced (P < 0.05), and the pyruvate content, ATP content, lactic acid production and LDH activity were significantly decreased (P < 0.05). The PGAM1 gene might regulate the glycolytic metabolism pathway and regulate the sperm formation and maturation process by affecting the proliferation and apoptosis of SCs. This result provides basic data for the study of the function of PGAM1 in sheep testicular development.

LncRNA FENDRR promotes apoptosis of Leydig cells in late-onset hypogonadism by facilitating the degradation of Nrf2.

This study aimed to investigate the role of lncRNA FENDRR in apoptosis of Leydig cells and the further mechanism. The apoptosis of Leydig cells (TM3 cell line) was induced by H2O2-treatment and detected by flow cytometry. The function of FENDRR was determined by in vitro and in vivo silencing experiments. The mechanism of FENDRR in regulating the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) was assessed by RNA immunoprecipitation, RNA pull-down, and ubiquitination assays. FENDRR expression was up-regulated in H2O2-treated TM3 cells. Knockdown of FENDRR augmented Nrf2 and HO-1 protein levels and testosterone production in H2O2-treated TM3 cells, whereas the apoptosis rate and caspase 3 activity were decreased. Mechanically, FENDRR bound to Nrf2 and promoted its ubiquitination and degradation. Nrf2 overexpression reversed the effects FENDRR overexpression on apoptosis, caspase 3 activity, and testosterone concentration in H2O2-treated TM3 cells. The in vivo experiments showed that FENDRR silence increased serum testosterone level and improved testosterone-related anti-depression behaviors of late-onset hypogonadism (LOH) mice. Our findings suggested that FENDRR could promote apoptosis of Leydig cells in LOH partly through facilitating Nrf2 degradation.

An autofluorescence-based isolation of Leydig cells for testosterone deficiency treatment.

Effective procedures for the purification of Leydig cells (LCs) can facilitate functional studies and transplantation therapies. However, current methods to purify LCs from testes are still far from satisfactory. Here, we found that testicular autofluorescence existed in the interstitium along with the gradual maturation of LCs from birth to adulthood. These autofluorescent cells were further isolated by fluorescence-activated cell sorting (FACS) and determined to be composed of LCs and macrophages. To further purify LCs, we combined two fluorescence channels of FACS and successfully separated LCs and macrophages. Of note, we confirmed that the obtained LCs not only possessed high purity, viability and quantity but also had intact steroidogenic activity and excellent responsiveness to luteinizing hormone. Moreover, subcutaneous transplantation of isolated LCs could alleviate the symptoms of testosterone deficiency in castrated mice. In summary, we established an effective autofluorescence-based method for isolating LCs. This method will aid in the future success of using LCs for basic and translational applications.

Advances in stem cell research for the treatment of primary hypogonadism.

In Leydig cell dysfunction, cells respond weakly to stimulation by pituitary luteinizing hormone, and, therefore, produce less testosterone, leading to primary hypogonadism. The most widely used treatment for primary hypogonadism is testosterone replacement therapy (TRT). However, TRT causes infertility and has been associated with other adverse effects, such as causing erythrocytosis and gynaecomastia, worsening obstructive sleep apnoea and increasing cardiovascular morbidity and mortality risks. Stem-cell-based therapy that re-establishes testosterone-producing cell lineages in the body has, therefore, become a promising prospect for treating primary hypogonadism. Over the past two decades, substantial advances have been made in the identification of Leydig cell sources for use in transplantation surgery, including the artificial induction of Leydig-like cells from different types of stem cells, for example, stem Leydig cells, mesenchymal stem cells, and pluripotent stem cells (PSCs). PSC-derived Leydig-like cells have already provided a powerful in vitro model to study the molecular mechanisms underlying Leydig cell differentiation and could be used to treat men with primary hypogonadism in a more specific and personalized approach.

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice.

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Androgen and Luteinizing Hormone Stimulate the Function of Rat Immature Leydig Cells Through Different Transcription Signals.

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.

Perfluorododecanoic acid delays Leydig cell regeneration from stem cells in adult rats.

Perfluorododecanoic acid (PFDoA) is an endocrine-damaging compound in contaminated food and water. However, the potential role and underlying mechanism of PFDoA in Leydig cell regeneration from stem Leydig cells remain unclear. The current study aims to investigate the effect of PFDoA on the regeneration of Leydig cells in the testis of rats treated with ethylene dimethane sulfonate (EDS). PFDoA (0, 5 or 10 mg/kg/day) was gavaged to adult Sprague-Dawley male rats for 8 days, and 75 mg/kg EDS was intraperitoneally injected to eliminate Leydig cells to initiate its regeneration from day 21-56 after EDS. The serum testosterone levels in the 5 and 10 mg/kg/day PFDoA groups were significantly reduced at day 21 after EDS and the levels of serum luteinizing hormone and follicle-stimulating hormone were significantly decreased in the 10 mg/kg/day PFDoA groups at day 56 after EDS. PFDoA significantly reduced Leydig cell number and proliferation at a dose of 10 mg/kg at days 21 and 56 after EDS. PFDoA significantly down-regulated the expression of Leydig cell-specific genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1 and Cyp17a1) and their proteins at both doses at days 21 and 56 after EDS. PFDoA significantly down-regulated the gene expression of Sertoli cells (Fshr, Dhh, and Sox9) at 5 mg/kg or higher at days 21 and 56 after EDS. In addition, we found that PFDoA significantly inhibited EdU incorporation into putative stem Leydig cells and their differentiation into the Leydig cell lineage in vitro. In conclusion, short-term PFDoA exposure in adulthood delayed the regeneration of Leydig cells by preventing Leydig cells from stem cells via multiple mechanisms.

LncRNA LOC102176306 plays important roles in goat testicular development.

Long ncRNAs regulate a complex array of fundamental biological processes, while its molecular regulatory mechanism in Leydig cells (LCs) remains unclear. In the present study, we established the lncRNA LOC102176306/miR-1197-3p/peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) regulatory network by bioinformatic prediction, and investigated its roles in goat LCs. We found that lncRNA LOC102176306 could efficiently bind to miR-1197-3p and regulate PPARGC1A expression in goat LCs. Downregulation of lncRNA LOC102176306 significantly supressed testosterone (T) synthesis and ATP production, decreased the activities of antioxidant enzymes and mitochondrial complex I and complex III, caused the loss of mitochondrial membrane potential, and inhibited the proliferation of goat LCs by decreasing PPARGC1A expression, while these effects could be restored by miR-1197-3p inhibitor treatment. In addition, miR-1197-3p mimics treatment significantly alleviated the positive effects of lncRNA LOC102176306 overexpression on T and ATP production, antioxidant capacity and proliferation of goat LCs. Taken together, lncRNA LOC102176306 functioned as a sponge for miR-1197-3p to maintain PPARGC1A expression, thereby affecting the steroidogenesis, cell proliferation and oxidative stress of goat LCs. These findings extend our understanding of the molecular mechanisms of T synthesis, cell proliferation and oxidative stress of LCs.

Diabetes Type 1 Negatively Influences Leydig Cell Function in Rats, Which is Partially Reversible By Insulin Treatment.

Type 1 diabetes mellitus (T1DM) is associated with impaired spermatogenesis and lower testosterone levels and epididymal weight. However, the underlying processes in the testis are unknown and remain to be elucidated. Therefore, the present study focused on the effects of T1DM on testicular function in a spontaneously diabetic rat model. BB/OKL rats after diabetes manifestation were divided into 3 groups: those without insulin treatment and insulin treatment for a duration of 2 and of 6 weeks. Anthropometrical data, circulating levels of gonadotrophins, testosterone, and inhibin B were measured. Intratesticular testosterone, oxidative stress, inflammation, and apoptosis were analyzed. Key enzymes of steroidogenesis were evaluated in the testis. Untreated diabetic rats had significantly lower serum follicle-stimulating hormone and luteinizing hormone levels. Serum and intratesticular testosterone levels significantly decreased in untreated diabetic rats compared to healthy controls. Key markers of Leydig cell function were significantly downregulated at the RNA level: insulin-like factor 3 (Insl3) by 53% (P = .006), Star by 51% (P = .004), Cyp11A1 by 80% (P = .003), 3Beta-Hsd2 by 61% (P = .005), and Pbr by 52% (P = .002). In the insulin-treated group, only Cyp11A1 and 3Beta-Hsd2 transcripts were significantly lower. Interestingly, the long-term insulin-treated group showed significant upregulation of most steroidogenic enzymes without affecting testosterone levels. Tumor necrosis factor α and apoptosis were significantly increased in the long-term insulin-treated rats. In conclusion T1DM, with a severe lack of insulin, has an adverse action on Leydig cell function. This is partially reversible with well-compensated blood glucose control. Long-term T1DM adversely affects Leydig cell function because of the process of inflammation and apoptosis.

Differentiation of seminiferous tubule-associated stem cells into leydig cell and myoid cell lineages.

Peritubular stem Leydig cells (SLCs) have been identified from rat testicular seminiferous tubules. However, no stem cells for peritubular myoid cells have been reported in the adult testis so far. In the present study, we tested the hypothesis that the peritubular SLCs are multipotent and able to form either Leydig or myoid cells. Using cultured tubules, we show that in the presence of PDGFAA and luteinizing hormone, SLCs became testosterone-producing Leydig cells, while in the presence of PDGFBB and TGFB, the cells formed α-smooth muscle actin-expressing myoid cells. This multipotency was also confirmed by culture of isolated CD90+ SLCs. These results suggest that these stem cells outside the myoid layer are multipotent and give rise to either Leydig or myoid cells, depending on the inducing factors. These cells may serve as a common precursor population for maintaining homeostasis of both Leydig and myoid cell populations in the adult testis.

Bisphenol A alters differentiation of Leydig cells in the rabbit fetal testis.

The endocrine disruptor Bisphenol A (BPA) crosses the placental barrier and reaches the fetal organs, including the gonads. In the testis, fetal Leydig cells (FLC) produce testosterone required for the male phenotype and homeostatic cell-cell signaling in the developing testis. Although it is known that BPA affects cell proliferation and differentiation in FLC, results concerning the mechanism involved are contradictory, mainly due to differences among species. Fast developing fetal gonads of rodents lack cortex and medulla, whereas species with more extended gestation periods form these two tissue compartments. The rabbit provides a good subject for studying the disruptive effect of BPA in fetal Leydig and possible postnatal endocrine consequences in adult Leydig cells. Here, we investigated the impact of BPA administered to pregnant rabbits on the FLC population of the developing testes. Using qRT-PCR, we assessed the levels of SF1, CYP11A1, 3ß-HSD, and androgen receptor genes, and levels of fetal serum testosterone were measured by ELISA. These levels correlated with both the mitotic activity and the ultrastructural differentiation of the FLC by confocal and electron microscopy, respectively. Results indicate that BPA alters the expression levels of essential genes involved in androgen paracrine signaling, modifies the proliferation and differentiation of the FLCs, and alters the levels of serum testosterone after birth. Thus, BPA may change the postnatal levels of serum testosterone due to the impaired FLC population formed by the proliferating stem and non-proliferating cytodifferentiated FLC.

Jagged1 intracellular domain modulates steroidogenesis in testicular Leydig cells.

Leydig cells represent the steroidogenic lineage of mammalian testis, which produces testosterone. Genetic evidence indicates the requirement of Notch signaling in maintaining a balance between differentiated Leydig cells and their progenitors during fetal development. In primary Leydig cells, Notch1 expression decreases with testicular development, while the expression of its ligand, Jagged1, remains relatively unchanged, suggesting that the roles of Jagged1 extend beyond Notch signaling. In addition, Jagged1 is known to be processed into its intracellular domain, which then translocate to the nucleus. In this study, we investigated the effect of Jagged1 intracellular domain (JICD) on steroidogenesis in Leydig cells. The independent overexpression of JICD in MA-10 Leydig cells was found to inhibit the activity of cAMP-induced Nur77 promoter. In addition, JICD suppressed Nur77 transactivation of the promoter of steroidogenic genes such as P450scc, P450c17, StAR, and 3ß-HSD. Further, adenovirus-mediated overexpression of JICD in primary Leydig cells repressed the expression of steroidogenic genes, consequently lowering testosterone production. These results collectively suggest that steroidogenesis in testicular Leydig cells, which is regulated by LH/cAMP signaling, is fine-tuned by Jagged1 during testis development.