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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.

Perfluorotridecanoic acid (PFTrDA) is a long-chain perfluoroalkyl substance, and its effect on the differentiation of fetal Leydig cells remains unclear. The objective of this study is to explore the effect of in utero PFTrDA exposure on the differentiation of fetal Leydig cells and investigate its underlying mechanisms. Pregnant Sprague-Dawley female rats were daily administered by gavage of PFTrDA at doses of 0, 1, 5, and 10 mg/kg from gestational day 14 to 21. PFTrDA had no effect on the body weight of dams, but significantly reduced the body weight and anogenital distance of male pups at birth at a dose of 10 mg/kg. PFTrDA significantly decreased serum testosterone levels as low as 1 mg/kg. PFTrDA did not affect fetal Leydig cell number, but promoted abnormal aggregation of fetal Leydig cells at doses of 5 and 10 mg/kg. PFTrDA down-regulated the expression of Insl3, Lhcgr, Scarb1, Star, Hsd3b1, Cyp17a1, Nr5a1, and Dhh as well as their proteins. PFTrDA lowered the levels of antioxidants (SOD1, CAT, and GPX1), induced autophagy as shown by increased levels of LC3II and beclin1, and reduced the phosphorylation of mTOR. In conclusion, PFTrDA inhibits the differentiation of fetal Leydig cells in male pups after in utero exposure mainly through increasing oxidative stress and inducing autophagy.

Neurotrophin‐3 (NT‐3) acts as an important growth factor to stimulate and control tissue development. The NT‐3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT‐3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT‐3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT‐3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle‐stimulating hormone levels. NT‐3 increased CYP11A1‐positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT‐3 significantly down‐regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT‐3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT‐3 dose‐dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT‐3 antagonist Celitinib can antagonize NT‐3 action in vitro. In conclusion, the present study demonstrates that NT‐3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.

Background Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro. Here we extend the model to human first-trimester embryonic gonadal tissue. Results Testicular cell suspensions reorganised into testis-like organoids with distinct seminiferous-like cords situated within an interstitial environment after 7 days. In contrast, tissue reorganisation failed to occur when mesonephros, which promotes testicular development in vivo, was included in the tissue digest. Organoids generated from dissociated female gonad cell suspensions formed loosely organised cords after 7 days. In addition to displaying testis-specific architecture, testis-like organoids demonstrated evidence of somatic cell differentiation. Within the 3-LGS, we observed the onset of AMH expression in the cytoplasm of SOX9-positive Sertoli cells within reorganised testicular cords. Leydig cell differentiation and onset of steroidogenic capacity was also revealed in the 3-LGS through the expression of key steroidogenic enzymes StAR and CYP17A1 within the interstitial compartment. While the 3-LGS generates a somatic cell environment capable of supporting germ cell survival in ovarian organoids germ cell loss was observed in testicular organoids. Conclusion The 3-LGS can be used to generate organised whole gonadal organoids within 7 days. The 3-LGS brings a new opportunity to explore gonadal organogenesis and contributes to the development of more complex in vitro models in the field of developmental and regenerative medicine.

The nuclear receptor chicken ovalbumin upstream promoter–transcription factor type II (COUP-TFII)/NR2F2 is expressed in adult Leydig cells, and conditional deletion of the Coup-tfii/Nr2f2 gene impedes their differentiation. Steroid production is also reduced in COUP-TFII–depleted Leydig cells, supporting an additional role in steroidogenesis for this transcription factor. COUP-TFII action in Leydig cells remains to be fully characterized. In the present work, we report that COUP-TFII is an essential regulator of the gene encoding the anti-Müllerian hormone receptor type 2 (Amhr2), which participates in Leydig cell differentiation and steroidogenesis. We found that Amhr2 mRNA levels are reduced in COUP-TFII–depleted MA-10 Leydig cells. Consistent with this, COUP-TFII directly activates a −1486 bp fragment of the mouse Amhr2 promoter in transient transfection assays. The COUP-TFII responsive region was localized between −67 and −34 bp. Chromatin immunoprecipitation assay confirmed COUP-TFII recruitment to the proximal Amhr2 promoter whereas DNA precipitation assay revealed that COUP-TFII associates with the −67/−34 bp region in vitro. Even though the −67/−34 bp region contains an imperfect nuclear receptor element, COUP-TFII–mediated activation of the Amhr2 promoter requires a GC-rich sequence at −39 bp known to bind the specificity protein (SP)1 transcription factor. COUP-TFII transcriptionally cooperates with SP1 on the Amhr2 promoter. Mutations that altered the GCGGGGCGG sequence at −39 bp abolished COUP-TFII–mediated activation, COUP-TFII/SP1 cooperation, and reduced COUP-TFII binding to the proximal Amhr2 promoter. Our data provide a better understanding of the mechanism of COUP-TFII action in Leydig cells through the identification and regulation of the Amhr2 promoter as a novel target.

Spermatogenesis is a complex process requiring intricate cellular interactions between multiple cell types to produce viable sperm. Peritubular myoid cells (PTMs) are smooth muscle cells that line the seminiferous tubules and play a critical role in sperm production by providing mechanical support and molecular signaling factors. In vitro investigation of their contribution to spermatogenesis and their dysfunction in infertility is currently limited by the rare accessibility of human testicular tissue for research. Therefore, this study set forth to generate an alternative source of PTMs using human induced pluripotent stem cells (hiPSCs) - adult cells that have been reprogrammed into a pluripotent state, making them capable of indefinite expansion and the regeneration of any cell type in the body. PTMs and Leydig cells arise from a common progenitor, so we hypothesized that PTMs could be derived by modifying an existing differentiation protocol for Leydig cell differentiation from hiPSCs. These hiPSC-derived cells, or hPTMs, were characterized and compared to hiPSC-derived Leydig cells (hLCs) and human primary Sertoli cells as a negative control. Our findings show that the substitution of the molecular patterning factor Platelet-Derived Growth Factor Subunit B (PDGF-BB) for Platelet-Derived Growth Factor Subunit A (PDGF-AA) in a molecule-based differentiation protocol for deriving Leydig-like cells, is sufficient to derive peritubular myoid-like cells. This study describes a method for generating PTM-like cells from hiPSCs. These cells will allow for ongoing understanding of the cellular interactions required for normal spermatogenesis in an in vitro setting.

Background The mammalian testis is an important male exocrine gland and spermatozoa-producing organ that usually lies in extra-abdominal scrotums to provide a cooler environment for spermatogenesis and sperm storage. Testicles sometimes fail to descend, leading to cryptorchidism. However, certain groups of mammals possess inherently ascrotal testes (i.e. testes that do not descend completely or at all) that have the same physiological functions as completely descended scrotal testes. Although several anatomical and hormonal factors involved in testicular descent have been studied, there is still a paucity of comprehensive research on the genetic mechanisms underlying the evolution of testicular descent in mammals and how mammals with ascrotal testes maintain their reproductive health. Results We performed integrative phenotypic and comparative genomic analyses of 380 cryptorchidism-related genes and found that the mammalian ascrotal testes trait is derived from an ancestral scrotal state. Rapidly evolving genes in ascrotal mammals were enriched in the Hedgehog pathway—which regulates Leydig cell differentiation and testosterone secretion—and muscle development. Moreover, some cryptorchidism-related genes in ascrotal mammals had undergone positive selection and contained specific mutations and indels. Genes harboring convergent/parallel amino acid substitutions between ascrotal mammals were enriched in GTPase functions. Conclusions Our results suggest that the scrotal testis is an ancestral state in mammals, and the ascrotal phenotype was derived multiple times in independent lineages. In addition, the adaptive evolution of genes involved in testicular descent and the development of the gubernaculum contributed to the evolution of ascrotal testes. Accurate DNA replication, the proper segregation of genetic material, and appropriate autophagy are the potential mechanisms for maintaining physiological normality during spermatogenesis in ascrotal mammals. Furthermore, the molecular convergence of GTPases is probably a mechanism in the ascrotal testes of different mammals. This study provides novel insights into the evolution of the testis and scrotum in mammals and contributes to a better understanding of the pathogenesis of cryptorchidism in humans.

Humans are exposed to a multitude of endocrine disruptor chemicals (EDCs) that can interfere with the action of endogenous hormones and the normal development of reproductive organs. Bisphenol A (BPA) is one of the most common EDCs found in the environment. Here, we evaluated BPA toxicity on fetal testes using an in vitro organ culture system. Mouse fetal testes sampled at 15.5 days post coitus were cultured in a medium containing BPA for 5 days. The number of germ cells was reduced by BPA treatment, whereas the number of Sertoli cells was slightly increased by BPA at the highest dose (100 μM). Consistently, BPA treatment reduced the protein and gene expression levels of germ cell markers, but it increased the expression levels of Sertoli cell markers. The expression levels of fetal Leydig cell markers such as Cyp11a1, Thbs2, Cyp17a1, and Pdgf-α were significantly increased, whereas those of adult Leydig cell markers such as Hsd17b3, Ptgds, Sult1e1, Vcam1, and Hsd11b1 were decreased in the testes exposed to BPA. Generally, Notch signaling restricts Leydig cell differentiation from progenitor cells during fetal testis development. The expression levels of Notch1, Notch2, Notch3, Hes1, Ptch1, Jag1, Jag2, c-Myc, Hey1, and Hey2, which are involved in Notch signaling, were markedly higher in BPA-treated fetal testes than in the controls, indicating that BPA interrupts fetal Leydig cell development. BPA also disrupted steroidogenesis in the fetal testis organ culture system. In conclusion, our study showed that BPA inhibits fetal germ cell growth, Leydig cell development, and steroidogenesis.

Purpose Recombinant human growth hormone (rhGH) has been used to treat short stature and rhGH-related syndromes. However, there are concerns that rhGH-treatment may cause precocious puberty. We investigated the effects of rhGH-treatment on the puberty onset, sexual maturation, androgen production, and hypothalamic gene expression in prepubertal male rats. Materials and methods Sprague-Dawley male rats were injected subcutaneously daily with 1 or 2 IU/kg/d rhGH or 0.1 mL saline from postnatal day (PND) 21 to 30. At PND 31 bodyweight, reproductive organs weight, preputial separation, testis histology, circulating testosterone, and expression of testicular steroidogenic pathway genes and hypothalamic Kiss1 were examined. Results By day 4 of injection bodyweights of rhGH groups were significantly higher than those of controls. rhGH 2 IU group showed earlier preputial separation compared to the control group. At PND 31, the weights of testes, epididymides, seminal vesicles, prostates, and preputial glands of the 2 IU-rhGH group were significantly higher than control group. Serum testosterone levels of the 2 IU-rhGH group were significantly higher than control group. Testicular steroidogenic pathway gene Hsd17b3 and Nr5a1 mRNA and cell counts and areas of Leydig cells in rhGH groups were significantly higher than control group, suggesting functional differentiation of Leydig cells. Hypothalamic Kiss1 mRNA levels of the 1 IU-rhGH group were significantly lower than control group, suggesting negative feedback of Kiss1 by elevated testosterone. Conclusions Prepubertal rhGH-treatment in male rats may induce early onset of puberty, sexual maturation, elevation of testosterone, and spermatogenesis, and accompanies downregulation of hypothalamic KISS1.

Androgens responsible for male sexual differentiation in utero are produced by Leydig cells in the fetal testicular interstitium. Leydig cells rarely proliferate and, hence, rely on constant differentiation of interstitial progenitors to increase their number during fetal development. The cellular origins of fetal Leydig progenitors and how they are maintained remain largely unknown. Here we show that Notch-active, Nestin-positive perivascular cells in the fetal testis are a multipotent progenitor population, giving rise to Leydig cells, pericytes, and smooth muscle cells. When vasculature is disrupted, perivascular progenitor cells fail to be maintained and excessive Leydig cell differentiation occurs, demonstrating that blood vessels are a critical component of the niche that maintains interstitial progenitor cells. Additionally, our data strongly supports a model in which fetal Leydig cell differentiation occurs by at least two different means, with each having unique progenitor origins and distinct requirements for Notch signaling to maintain the progenitor population., Leydig cells are steroidogenic cells in the testes and produce the androgens required for male development and spermatogenesis. Here the authors show that a multipotent progenitor population producing Leydig cells, pericytes and smooth muscle cells is maintained in a perivascular niche within the mouse fetal testis.

Leydig cells in fetal and adult testes play pivotal roles in eliciting male characteristics by producing androgen. Although numerous studies of Leydig cells have been performed, the mechanisms for differentiation of the two cell types (fetal Leydig and adult Leydig cells), their developmental and functional relationship, and their differential characteristics remain largely unclear. Based on recent technical progress in genome-wide analysis and in vitro investigation, novel and fascinating observations concerning the issues above have been obtained. Focusing on fetal and adult Leydig cells, this review summarizes the recent progress that has advanced our understanding of the cells.

Background It is an established fact that excess of glucocorticoids could cause the harmful effects, such as suppression on the male reproduction. Although glucocorticoids pharmacologically inhibit the Leydig cell function, their roles in Leydig cell development are unclear. Therefore, the present study was designed to investigate effects of synthetic glucocorticoid dexamethasone (DEX) on rat stem Leydig cell proliferation and differentiation. Methods Male Sprague-Dawley rats received a single intraperitoneal injection of 75 mg/kg EDS to eliminate Leydig cells and an in vitro culture system of the seminiferous tubules was established from Leydig cell-depleted testis. Using basal medium and Leydig cell differentiation-inducing medium (LIM) in the culture system, we examined the effects of DEX (0–100 nM) on the proliferation and differentiation of the stem Leydig cells in vitro, respectively. Results Results showed that LIM is a good agent to induce stem Leydig cell differentiation into Leydig cells that produce testosterone in vitro. DEX inhibited the differentiation of stem Leydig cells by reducing the expression levels of Cyp17a1 and Scarb1 and that NR3C1 antagonist RU38486 reversed the DEX-mediated effects. However, DEX are not involved with the proliferation of stem Leydig cells. Conclusions DEX suppressed the differentiation of rat Leydig cells in vitro and glucocorticoid-induced effects acted through NR3C1. This suppression partially targets on Cyp17a1 and Scarb1 gene expression.

Paraquat is a fast and non-selective herbicide that is widely used in crop cultivation and conservation tillage systems. Animal experiments have shown that paraquat decreases sperm quality and testicular organ coefficient, but its effects on the development of Leydig cells remain unclear. The objective of the current study was to investigate the effects of paraquat exposure on the Leydig cell development in rats during puberty. Twenty-eight male 35-day-old Sprague-Dawley rats were divided into 4 groups: 0, 0.5, 2.0, and 8 mg kg−1 d−1 paraquat. Paraquat was gavaged for 10 d. Adult Leydig cells were isolated and treated with paraquat for 24 h. Paraquat in vivo significantly decreased body and testis weights at 8 mg kg−1 and lowered serum testosterone levels at 2 and 8 mg kg−1 without affecting the levels of serum luteinizing hormone and follicle-stimulating hormone. Paraquat did not alter Leydig cell number and PCNA labeling index. Real-time PCR showed that paraquat down-regulated the expression of Lhcgr, Scarb1, Cyp11a1, Cyp17a1, and Hsd17b3 genes and their proteins at 2 or 8 mg kg−1, while it up-regulated the expression of Srd5a1 at 8 mg kg−1. Paraquat increased ROS and decreased testosterone production by Leydig cells at 1 and 10 μM after in vitro 24-h exposure. Vitamin E (40 μg/ml) reversed paraquat-induced ROS and suppression of testosterone synthesis in vitro. In conclusion, paraquat directly delays Leydig cell differentiation to block testosterone synthesis via down-regulating the expression of critical testosterone synthesis-related genes and up-regulating the expression of testosterone metabolic enzyme (Srd5a1) gene and possibly via increasing ROS production.

Desert hedgehog (dhh) is a gene that is crucial for spermatogenesis and Leydig cell differentiation, but little is known regarding its influence on gonadal differentiation and development in fish. To understand its function, we cloned and characterized the dhh gene from Cynoglossus semilaevis (csdhh). The full length csdhh cDNA was 2473 bp, including a 1386 bp open reading frame (ORF), a 475 bp 5'-UTR, and a 612 bp 3'-UTR, encoding a predicted protein of 461 amino acid residues. Phylogenetic analysis showed that the putative protein belongs to the hedgehog (HH) family, and contains typical HH-N and HH-C domains. Amino acid sequence analysis revealed that CsDhh shares many features with Dhh analogues in other teleost species. Real-time quantitative PCR showed that csdhh was detected in eight different tissues in male and female tongue sole. During early embryonic development, the relative expression of the csdhh was significantly higher in the neural stage than in other embryonic developmental stages (P 0.05). csdhh was detected at 20 days after hatching (dah) and at the critical period of male gonadal differentiation (80-95 dah), the relative expression of the csdhh was significantly higher in the male gonads than the female gonads. In 5, 8, and 12 month old gonads, the relative expression of the csdhh was significantly higher in male and pseudo-male than in female fish. The in situ hybridization (ISH) results showed that the hybridization signal was strongly expressed in primary and secondary spermatocytes, spermatids, and sertoli cells of the 1-year-old fish testis, with only weak signal expression in the corresponding ovarian tissue. These results suggest that csdhh is highly conserved in evolution and plays an important role in spermatogenesis in males and pseudo-males.

Leydig cells, located within the interstitial compartment of the testis, are major contributors of androgen synthesis and secretion, which play an important role in testis development, normal masculinization, maintenance of spermatogenesis, and general male fertility. Accordingly, dysfunction of Leydig cells may lead to various male reproductive maladies, including primary hypogonadism, cryptorchidism, and hypospadias. A better understanding of how cell interactions and gene regulation contribute to testicular Leydig cell development and differentiation may therefore help limit the incidence of such male reproductive pathologies. Several hormones and signaling molecules have been identified as important regulators of Leydig cell differentiation and function. Recent work on the regulation of testis development, especially of Leydig cells, has focused on the Desert hedgehog and platelet-derived growth factor signaling pathways. This review outlines recent findings regarding cell interactions and gene regulation involved in the development and regulation of fetal and adult Leydig cell populations. Mol. Reprod. Dev. 83: 470-487, 2016. © 2016 Wiley Periodicals, Inc.

A multicellular organism's development is coupled with cellular self-organization, which is regulated by cell-cell interactions and cell-extracellular matrix (ECM) crosstalk. Testicular cells from different species such as mouse, rat, and porcine can self-organize into seminiferous tubules both in vitro and in vivo, but the understanding of the functional role of the ECM during this process is limited. Here, it is shown that mouse testicular cells encapsulated with the biomaterial Matrigel can self-organize into seminiferous tubules with blood-testis barrier (BTB) formation and Leydig cell differentiation. By varying the encapsulation method, a combination of sodium alginate and collagen is used to promote reorganization of seminiferous tubules, which resemble those in vivo. In addition, the self-organization ability of testicular cells declines with advanced cell age, and those germ cells play a pivotal role in this process. These findings will be helpful to understand the self-organization process of testicular cells and provide insights for the reconstruction of testes.

The desert hedgehog (Dhh) is crucial for spermatogenesis and Leydig cell differentiation, but little is known regarding its physiological function in cartilage. In this study, Dhh mRNA was abundant in antler chondrocytes, where it advanced cell proliferation concomitant with accelerated transition from the G1 to the S phase and induced elevation of the hypertrophic chondrocyte markers, Col X and Runx2. Silencing of Ptch1 resulted in appreciable Smo accumulation and enhanced rDhh stimulation of Smo, whose impediment by cyclopamine obscured the proliferative function of Dhh and alleviated its guidance of chondrocyte differentiation. Further analysis evidenced the noteworthy positive action of Smo in the bridging between Dhh and Gli transcription factors. Obstruction of Gli1 by GANT58 caused the failed stimulation of Col X and Runx2 by rDhh. Analogously, siRNA against Gli1-3 hindered chondrocyte differentiation in the context of rDhh. Simultaneously, Gli transcription factors mediated the regulation of Dhh on Foxa1, Foxa2, and Foxa3, whose knockdown impaired chondrocyte differentiation. Attenuation of Foxa antagonized the augmentation of Col X and Runx2 generated by rDhh. Collectively, Dhh signaling through its target Foxa appears to induce antler chondrocyte proliferation and differentiation.

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.

Proliferation and differentiation of adult Leydig cells are mainly completed in puberty. In many studies, apart from normal postnatal development process, it is widely indicated that, through administrating EDS, Leydig cell population is eliminated and regenerated. It is believed that osteocalcin released from osteoblasts, which is responsible for modulating bone metabolism, induces testosterone production in Leydig cells, independent of the HPG axis. In addition, INSL3 produced by Leydig cells, such as testosterone, plays a critical role in bone metabolism and is known to reflect the development process and functional capacities of Leydig cells. This study is aimed at investigating OC-mediated testosterone regulation and INSL3 synthesis during differentiation of adult Leydig cells that are independent of LH. For this purpose, male rats were divided into 2 groups: prepubertal normal rats and adult EDS-injected rats. Each group was divided into 4 subgroups in which GnRH antagonist or OC was applied. After adult Leydig cells completed their development, testicular tissue samples obtained from the sacrificed rats were examined by light-electron microscopic, immunohistochemical, and biochemical methods. Slight upregulation in 3βHSD, INSL3, and GPRC6A expressions along with the increase in serum testosterone levels was observed in groups treated with osteocalcin against GnRH antagonist. In addition, biochemical and microscopic findings in osteocalcin treated groups were similar to those in control groups. While there was no significant difference in the number of Leydig cells reported, the presence of a significant upregulation in INSL3 and GPRC6A expressions and the increase in serum testosterone and ucOC levels were observed. After evaluation of findings altogether, it is put forward that, for the first time in this study, although osteocalcin treatment made no significant difference in the number of Leydig cells, it increased the level of testosterone through improving the function of existing adult Leydig cells during normal postnatal development process and post-EDS regeneration. This positive correlation between osteocalcin-testosterone and osteocalcin-INSL3 is concluded to be independent of LH at in vivo conditions.

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

Nicotine, the main toxic substance in cigarette smoke, significantly reduced the differentiation and maturation ratio of Leydig cell in murine testes. To investigate the underlying mechanism, C57BL/6J mice were divided into control (CT) and nicotine treated (NT) groups. Next generation RNA sequencing and bio-informatics analysis were carried out to analysis the effects of nicotine on the RNA profile of Leydig cells. Expression level of 7 pathways remarkably changed after nicotine treatment. As the positive regulating pathway of Leydig cell differentiation, Hedgehog signaling pathway was found among these pathways. PTCH1 and β-TrCP were down-regulated in nicotine treated mice Leydig cells, while GSK3β, Gli2 and Gli2 fragments increased significantly. Nicotine stimulated the destabilization of Gli2 via β-TrCP induced ubiquitination and degradation. Gli2 was phosphorylated by up-expressed GSK3β during this process. Destabilization of Gli2 reduced the activation rate of target genes of Hedgehog signaling pathway such as Ptch1. The differentiation of Leydig cell positively regulated by Hh pathway was thus inhibited by nicotine exposure. Consequently, the male reproduction process powered by Leydic cell-mediated androgen secretion was thus influenced. In conclusion, we find that nicotine inhibits murine Leydig cell differentiation and maturation via regulating Hedgehog signal pathway.

Vitamin A (retinol) is important for multiple functions in mammals. In testis, the role of vitamin A in the regulation of testicular functions is clearly involved in rodents. It is essential for sperm production. Vitamin A deficiency adversely affects testosterone secretion. Adult Leydig cells are responsible for testosterone production in male. The role of vitamin A in regulating the differentiation of Leydig cells is still unknown. In this study, we explored the roles and underlying mechanisms of vitamin A in Leydig cell differentiation. We found that vitamin A could regulate the Leydig cells differentiation. Leydig cell differentiation is adversely affected in mice maintained on a vitamin A-free diet. This effect is mediated by alcohol dehydrogenase 1 (ADH1). ADH1 could increase retinoic acid (RA) synthesis, then RA facilitates Leydig cell differentiation by activating the steroidogenic factor 1 gene (Nr5a1) promoter activity, which consequently promotes Leydig cell specific gene expression, resulting in progenitor Leydig cells differentiation into functional Leydig cells. This is the first study connecting a metabolic enzyme of retinol (ADH1) to the the regulation of Leydig cell differentiation, which will provide experimental evidence for the development of therapeutics to promote Leydig regeneration through the administration of a RA signaling regulator or a vitamin A supplement.

Decabromodiphenyl ether (BDE-209), a congener of polybrominated diphenyl ethers (PBDEs), is used as flame retardant and affects thyroid homeostasis. Thyroid hormones (THs) play crucial role in Leydig cell differentiation and steroidogenesis during early life. Present study examined the effect of maternal BDE-209 exposure during lactation on testicular steroidogenesis and spermatogenesis in relation to thyroid hormone receptor alpha 1 (THRα1) and possible mechanism(s) of its action in prepubertal Parkes mice offspring. Lactating female Parkes mice were orally gavaged with 500, and 700 mg/kg body weight of BDE-209 in corn oil from postnatal day (PND) 1 to PND 28. Lactating mothers and male pups were sacrificed on PND 28. Maternal BDE-209 exposure markedly affected testicular histopathology, steroidogenesis and germ cell dynamics with downregulated expressions of various steroidogenic markers in mice offspring. Serum THs levels were markedly reduced in both pups and lactating mothers compared to controls. Expression of proliferating cell nuclear antigen and THRα1 also deceased in testes of BDE-209-exposed mice offspring. In silico analysis by molecular docking was performed successfully for steroidogenic facor-1 (SF-1) and THRα1 with BDE-209 and T3. Maternal BDE-209 exposure during lactation affects testicular steroidogenesis, spermatogenesis and expression of THRα1 in prepubertal mice offspring through downregulation of SF-1.

The tumor suppressor p53, and the cyclin-dependent kinase inhibitor Ink4c, have been both implicated in spermatogenesis control. Both p53-/- and Ink4c-/- single knockout male mice are fertile, despite testicular hypertrophy, Leydig cell differentiation defect, and increased sperm count in Ink4c-/- males. To investigate their collaborative roles, we studied p53-/- Ink4c-/- dual knockout animals, and found that male p53-/- Ink4c-/- mice have profoundly reduced fertility. Dual knockout male mice show a marked decrease in sperm count, abnormal sperm morphology and motility, prolongation of spermatozoa proliferation and delay of meiosis entry, and accumulation of DNA damage. Genetic studies showed that the effects of p53 loss on fertility are independent of its downstream effector Cdkn1a. Absence of p53 also partially reverses the hyperplasia seen upon Ink4c loss, and normalizes the Leydig cell differentiation defect. These results implicate p53 in mitigating both the delayed entry into meiosis and the secondary apoptotic response that occur in the absence of Ink4c. We conclude that the cell cycle genes p53 and Ink4c collaborate in sperm cell development and differentiation, and may be important candidates to investigate in human male infertility conditions.

In the early stages of organogenesis in mammalian embryos, the genital ridges initially form as a thickening of the coelomic epithelia along the ventral side of the mesonephros within the posterior trunk. After integration of the primordial germ cells, they develop into indifferent gonads, consisting of germ cells, and two major gonadal somatic cell types, epithelial supporting cells and interstitial steroidogenic cells. In male XY embryos, sex-determining region Y ( SRY ) is transiently expressed in the bipotential supporting cells, where it activates the testis-specific genes SRY-box 9 ( SOX9 ) and fibroblast growth factor 9 ( FGF9 ) in a positive feedback manner, leading to Sertoli cell differentiation in the testis cords and Leydig cell differentiation in the interstitium. In female XX embryos lacking the SRY gene, the bipotential gonads develop into ovaries. The first sign of ovarian differentiation is the loss of sexual bipotentiality in the XX supporting cells, which can be monitored by SRY-dependent Sox9 inducibility in the experimental SRY-inducible mouse system. R-spondin homolog 1 (RSPO1)/WNT4/β-catenin signals act in the female-specific expansion of the ovarian cortex region, in addition to antagonizing the SRY-derived SOX9/FGF9 pathway. Forkhead box protein L2 (FOXL2) is also involved in the differentiation and maintenance of the female fate of granulosa cells in the developing ovary. The oogonia proliferate and enter meiotic prophase due to retinoic acid signaling. During the progression of meiotic prophase, each oocyte is surrounded by FOXL2-positive pregranulosa cells within the ovigerous cords, leading to the formation of primordial follicles. In the inner cortex and medullary regions, newly formed primordial follicles appear to directly undergo an initial round of follicular growth, together with the differentiation of the surrounding theca cells, via a growth differentiation factor 9 (GDF9)/Hedgehog signaling pathway. In the outer cortical region, oocytes recruit granulosa cells from the surface epithelia, and form a resting primordial follicle pool that undergoes follicular growth in a sexual cycle-dependent manner.

The regulatory factors for stem Leydig cell development are largely unknown. Herein, we reported that parathyroid hormone-related protein (PTHrP) may be a factor to regulate this process. The effects of PTHrP on rat stem Leydig cell proliferation and differentiation were investigated using a stem Leydig cell culture system and an ethane dimethane sulfonate (EDS)-treated in vivo Leydig cell regeneration model. PTHrP (1,000 pg/ml) significantly increased medium testosterone level and up-regulated STAR, CYP17A1, and 17-HSD3 expressions. Co-treatment with PKA inhibitor H-89 or PKC inhibitor U73122 reversed PTHrP-mediated increase of testosterone production in vitro. Intratesticular injection of PTHrP (100 ng/testis) into the Leydig cell-depleted testis from post-EDS day 7 to day 21 significantly increased serum testosterone level, up-regulated LHCGR, SCARB1, CYP11A1, 11-HSD1, and CYP17A1 expressions. It also enlarged Leydig cell size without affecting PCNA-labeled Leydig cell number. This indicates that PTHrP promotes stem Leydig cell differentiation. PTHrP in vivo increased CREB and p-CREB levels, suggesting that PTHrP acts via a PKA-CREB signaling pathway. In conclusion, PTHrP stimulates stem Leydig cell differentiation without affecting its proliferation, showing its novel action and mechanism on rat stem Leydig cell development.

Bisphenol A (BPA) is widely used in consumer products and a potential endocrine disruptor linked with sexual precocity. However, its action and underlying mechanisms on male sexual maturation is unclear. In the present study, we used a unique in vivo ethane dimethane sulfonate (EDS)-induced Leydig cell regeneration model that mimics the pubertal development of Leydig cells and an in vitro stem Leydig cell differentiation model to examine the roles of BPA in Leydig cell development in rats. Intratesticular exposure to doses (100 and 1000 pmol/testis) of BPA from post-EDS day 14–28 stimulated Leydig cell developmental regeneration process by increasing serum testosterone level and Leydig cell-specific gene (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Hsd11b1) and their protein expression levels. BPA did not alter serum luteinizing hormone and follicle-stimulating hormone levels as well as the proliferative capacity of Leydig cells in vivo. In vitro study demonstrated that BPA (100 nmol/L) stimulated the differentiation of stem Leydig cells by increasing medium testosterone levels and up-regulating Leydig cell-specific gene (Lhcgr, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and their proteins but did not affect their proliferation measured by EdU incorporation. In conclusion, BPA stimulates the differentiation of stem Leydig cells in rat testes, thus possibly causing sexual precocity in the male.

Insulin-like factor 3 (INSL3) is a promising marker of Leydig cell function with potentially high clinical relevance. Limited data of INSL3 levels in relation to other reproductive hormones in healthy pubertal boys exist. In this study, we aimed to evaluate longitudinal serum changes in INSL3 compared with LH, FSH, testosterone, inhibin B, and anti-Müllerian hormone (AMH) during puberty in healthy boys. Ten boys were included from the longitudinal part of the COPENHAGEN Puberty Study. Pubertal evaluation, including testicular volume, was performed and blood samples were drawn every 6 months for 5 years. Serum concentrations of testosterone were determined by a newly developed LC–MS/MS method, and serum concentrations of INSL3, AMH, inhibin B, FSH, and LH respectively were determined by validated immunoassays. The results showed that serum INSL3 levels increased progressively with increasing age, pubertal onset, and testicular volume. In six of the ten boys, LH increased before the first observed increase in INSL3. In the remaining four boys, the increase in LH and INSL3 was observed at the same examination. The increases in serum concentrations of LH, testosterone, and INSL3 were not parallel or in ordered succession and varied interindividually. We demonstrated that INSL3 concentrations were tightly associated with pubertal onset and increasing testicular volume. However, the pubertal increases in LH, INSL3, and testosterone concentrations were not entirely parallel, suggesting that INSL3 and testosterone may be regulated differently. Thus, we speculate that INSL3 provides additional information on Leydig cell differentiation and function during puberty compared with traditional markers of testicular function.

Data provided in this article is RNA profile represented in RPKM and RPKM based TPM value for the research article titled Nicotine inhibits Murine Leydig cell differentiation and maturation via regulating Hedgehog signal pathway Jiajie et al., 2019. Nicotine treatment changes the RNA profile of Murine Leydig cells. RNA of 12 control group Leydig cells and 12 nicotine treated Leydig cells are sequenced and the data of 29943 genes are achieved. The information of the gene symbol, gene description, gene type, position and transcript length are provided.

We recently created a mouse model displaying precocious Sertoli cell (SC) and spermatogenic development induced by SC-specific transgenic androgen receptor expression (TgSCAR). Here we reveal that TgSCAR regulates the development, function, and absolute number of Leydig cells (LCs). Total fetal and adult type LC numbers were reduced in postnatal and adult TgSCAR vs control testes, despite normal circulating LH levels. Normal LC to SC ratios found in TgSCAR testes indicate that SC androgen receptor (SCAR)-mediated activity confers a quorum-dependent relationship between total SC and LC numbers. TgSCAR enhanced LC differentiation, shown by elevated ratios of advanced to immature LC types, and reduced LC proliferation in postnatal TgSCAR vs control testes. Postnatal TgSCAR testes displayed up-regulated expression of coupled ligand-receptor transcripts (Amh-Amhr2, Dhh-Ptch1, Pdgfa-Pdgfra) for potential SCAR-stimulated paracrine pathways, which may coordinate LC differentiation. Neonatal TgSCAR testes displayed normal T and dihydrotestosterone levels despite differential changes to steroidogenic gene expression, with down-regulated Star, Cyp11a1, and Cyp17a1 expression contrasting with up-regulated Hsd3b1, Hsd17b3, and Srd5a1 expression. TgSCAR males also displayed elevated postnatal and normal adult serum testosterone levels, despite reduced LC numbers. Enhanced adult-type LC steroidogenic output was revealed by increased pubertal testicular T, dihydrotestosterone, 3α-diol and 3β-diol levels per LC and up-regulated steroidogenic gene (Nr5a1, Lhr, Cyp11a1, Cyp17a1, Hsd3b6, Srd5a1) expression in pubertal or adult TgSCAR vs control males, suggesting regulatory mechanisms maintain androgen levels independently of absolute LC numbers. Our unique gain-of-function TgSCAR model has revealed that SCAR activity controls temporal LC differentiation, steroidogenic function, and population size.

Sertoli cells play a significant role in regulating fetal testis compartmentalization to generate testis cords and interstitium during development. The Sertoli cell Wilms’ tumor 1 (Wt1) gene, which encodes ~24 zinc finger-containing transcription factors, is known to play a crucial role in fetal testis cord assembly and maintenance. However, whether Wt1 regulates fetal testis compartmentalization by modulating the development of peritubular myoid cells (PMCs) and/or fetal Leydig cells (FLCs) remains unknown. Using a Wt1-/flox; Amh-Cre mouse model by deleting Wt1 in Sertoli cells (Wt1SC-cKO) at embryonic day 14.5 (E14.5), Wt1 was found to regulate PMC and FLC development. Wt1 deletion in fetal testis Sertoli cells caused aberrant differentiation and proliferation of PMCs, FLCs and interstitial progenitor cells from embryo to newborn, leading to abnormal fetal testis interstitial development. Specifically, the expression of PMC marker genes α-Sma, Myh11 and Des, and interstitial progenitor cell marker gene Vcam1 were down-regulated, whereas FLC marker genes StAR, Cyp11a1, Cyp17a1 and Hsd3b1 were up-regulated, in neonatal Wt1SC-cKO testes. The ratio of PMC:FLC were also reduced in Wt1SC-cKO testes, concomitant with a down-regulation of Notch signaling molecules Jag 1, Notch 2, Notch 3, and Hes1 in neonatal Wt1SC-cKO testes, illustrating changes in the differentiation status of FLC from their interstitial progenitor cells during fetal testis development. In summary, Wt1 regulates the development of FLC and interstitial progenitor cell lineages through Notch signaling, and it also plays a role in PMC development. Collectively, these effects confer fetal testis compartmentalization.

Cadmium is an endocrine disruptor, impairing male reproduction. The objective of this study is to investigate whether cadmium affects rat Leydig cell regeneration and to dissect the underlying mechanism. Adult male Sprague-Dawley rats received a single intraperitoneal injection (i.p.) of 0, 0.5 or 1.0 mg/kg of cadmium chloride, followed by ethane dimethane sulfonate (EDS) treatment to eliminate adult Leydig cells 20 days later. Compared to control (0 dose), cadmium treatment reduced serum testosterone levels by days 21, 35, and 56 after EDS treatment. Serum luteinizing hormone (LH) levels were also affected by day 56, the only time point examined. There were fewer regenerated Leydig cells in the cadmium-treated testis on days 35 and 56 after EDS treatment. Further studies demonstrated that the mRNA or protein levels of Leydig (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Hsd11b1), non-Leydig (Fshr and Dhh), and gonadotroph (Lhb) cells were also significantly lower in cadmium-treated animals. Since LH and desert hedgehog (DHH) are critical factors for Leydig cell differentiation, our result demonstrated that the lower doses of cadmium exposure, even briefly, may permanently damage Leydig cell regeneration.

Testicular Leydig cells play pivotal roles in masculinization of organisms by producing androgens. At least two distinct Leydig cell populations sequentially emerge in the mammalian testis. Leydig cells in the fetal testis (fetal Leydig cells) appear just after initial sex differentiation and induce masculinization of male fetuses. Although there has been a debate on the fate of fetal Leydig cells in the postnatal testis, it has been generally believed that fetal Leydig cells regress and are completely replaced by another Leydig cell population, adult Leydig cells. Recent studies revealed that gene expression patterns are different between fetal and adult Leydig cells and that the androgens produced in fetal Leydig cells are different from those in adult Leydig cells in mice. Although these results suggested that fetal and adult Leydig cells have distinct origins, several recent studies of mouse models support the hypothesis that fetal and adult Leydig cells arise from a common progenitor pool. In this review, we first provide an overview of previous knowledge, mainly from mouse studies, focusing on the cellular origins of fetal Leydig cells and the regulatory mechanisms underlying fetal Leydig cell differentiation. In addition, we will briefly discuss the functional differences of fetal Leydig cells between human and rodents. We will also discuss recent studies with mouse models that give clues for understanding how the progenitor cells in the fetal testis are subsequently destined to become fetal or adult Leydig cells.

Endocannabinoids control male reproduction acting at central and local level via cannabinoid receptors. The cannabinoid receptor CB1 has been characterized in the testis, in somatic and germ cells of mammalian and non-mammalian animal models, and its activity related to Leydig cell differentiation, steroidogenesis, spermiogenesis, sperm quality, and maturation. In this short review, we provide a summary of the insights concerning neuroendocrine CB1 activity in male reproduction focusing on adult Leydig cell ontogenesis and steroid biosynthesis.

In mammalian embryogenesis, the gonadal primordium arises from the thickening of the coelomic epithelium, which results in a pair of extremely long and narrow gonadal structures along the anteroposterior axis. These gonadal structures are conserved in various mammalian species, suggesting a great advantage in properly receiving migrating primordial germ cells (PGCs) that are widely scattered throughout the hindgut tube. Soon after the PGCs settle, the bipotential gonads undergo sex determination into testes or ovaries by the sex-determining gene, Sry, which is expressed in supporting cell precursors in a center-to-pole manner. Such a long, narrow gonadal structure bestows a considerable time lag on Sry expression between the center and pole regions, but testiculogenesis with cord formation and Leydig cell differentiation occurs synchronously throughout the whole organ. This synchronous testiculogenesis could be explained by a positive-feedback mechanism between SOX9 (another SRY-related transcription factor) and FGF9 downstream of Sry. FGF signals are likely secreted from the center region, rapidly diffuse into the poles, and then induce the establishment of SOX9 expression in Sertoli cells in the pole domains. This work focuses on recent knowledge of the molecular and cellular events of PGC migration, gonadogenesis, and testiculogenesis, and their biological significance in mammalian embryogenesis.

The manner by which endocrine-disrupting xenobiotics, such as phthalates, can induce changes in the development of the male reproductive system still remains largely unknown. Herein, we have explored the application of ethane dimethane sulphonate (EDS) to eliminate adult-type Leydig cells in the mature rat testis, leading to their regeneration from resident stem cells, as a novel system to investigate the effects of dibutyl phthalate (DBP) and diethylstilbestrol (DES) on adult-type Leydig cell differentiation. The advantage of this model is that one can study adult-type Leydig cell differentiation in vivo divorced from the concomitant endocrine development of puberty. In these preliminary studies, we show that both DBP and/or DES, given for 2 or 4 days following EDS application, indeed affect Leydig cell differentiation in the adult testis, largely by increasing early Leydig cell proliferation and possibly thereby delaying early differentiation. In particular, on day 27 post-EDS, a time-point when the differentiation trajectory appears to be most discriminating, we observe that both DBP and/or DES cause a fourfold increase in Leydig cell density, and a significant increase in the expression of the Leydig cell-specific marker transcripts INSL3, LH receptor, Cyp17a1 and Cyp 11a1. In conclusion, both DBP and DES are able to affect adult-type Leydig cells during their differentiation to cause a significant perturbation in their ultimate functional capacity.

Appearance of mouse fetal Leydig cells requires activation of the Hedgehog pathway. Upon binding to the membrane-bound receptor patched, Hedgehog ligands induce intracellular responses via a combined effect of Gli transcription factors. Szczepny et al. (Biol Reprod 2009; 80:258–263) found that Gli1, one of the three Gli transcription factors, is present in the fetal testis and that its expression is suppressed by the Hedgehog inhibitor cyclopamine. In this study, we investigated the involvement of the Gli1 and Gli2 factors in mouse fetal Leydig cell differentiation. The Gli1 and Gli2 transcription factors showed an overlapping expression pattern in the testis interstitium at the time when fetal Leydig cells appear. Despite their similar expression, Gli1 and Gli2 patterns were differentially regulated. Initial Gli1 and Gli2 expression depends upon an active Hedgehog pathway; however, maintenance of only Gli1, but not Gli2, expression requires activation of the pathway. Inactivation of either the Gli1 or Gli2 gene did not affect fetal Leydig cell development and testis morphology, suggesting a functional redundancy. When the transcriptional activity of both GLI1 and GLI2 was suppressed by a selective inhibitor, GANT61, in cultured fetal testes before the appearance of fetal Leydig cells, Gli1 and Gli2 expression and steroidogenic marker activity were completely abolished. However at later stages when Leydig cells were already present, GANT61 treatment inhibited Gli1 expression but had no effects on Gli2 expression and fetal Leydig cell appearance. Our results reveal overlapping and redundant Gli1 and Gli2 roles in fetal Leydig cell differentiation and a novel regulation of Gli2 expression in the fetal testis.

Several signals, such as hormones and signaling molecules, have been identified as important regulators of Leydig cell differentiation and function. Conveying these signals and translating them into a genomic response to ensure an accurate physiological output requires the action of a network of transcription factors, including those belonging to the nuclear receptor superfamily. Nuclear receptors regulate expression of genes important for growth, differentiation, development, and homeostasis. Several nuclear receptors, such as steroid hormone receptors (NR3A and NR3C families), are activated upon ligand binding, whereas others, including members of the NR2C, NR2F, and NR4A families, either do not require a ligand or ligands have yet to be identified. Several nuclear receptors (e.g., NR2F2 and NR5A1) have been shown to play essential roles in Leydig cells, whereas for others (e.g., NR2B1 and NR4A1), the assessment of their function has been precluded by the early embryonic lethality associated with null mice or by redundancy mechanisms by other family members. This is now being overcome with the generation of novel approaches, including Leydig cell-specific knockout models. This review provides an overview of the nuclear receptor family of transcription factors as they relate to Leydig cell gene expression and function.

Knockout of the gene encoding insulin-like factor 3 (INSL3) results in cryptorchidism in mice due to disruption of the transabdominal phase of testicular descent. This finding was essential for understanding the complete course of testis descensus, and wound up years of speculations regarding the endocrine regulation of this process. INSL3 is, along with testosterone, a major secretory product of testicular Leydig cells. In addition to its crucial function in testicular descent, INSL3 is suggested to play a paracrine role in germ cell survival and an endocrine role in bone metabolism. INSL3 is produced in human prenatal and neonatal, and in adult Leydig cells to various extents, and is in a developmental context regulated like testosterone, with production during second trimester, an early postnatal peak and increasing secretion during puberty, resulting in high adult serum levels. INSL3 production is entirely dependent on the state of Leydig cell differentiation, and is stimulated by the long-term trophic effects mediated by luteinizing hormone (LH). Once differentiated, Leydig cells apparently express INSL3 in a constitutive manner, and the hormone is thereby insensitive to the acute, steroidogenic effects of LH, which for example is an important factor in the regulation of testosterone. Clinically, serum INSL3 levels can turn out to be a usable tool to monitor basal Leydig cell function in patients with various disorders affecting Leydig cell function. According to animal studies, foetal INSL3 production is, directly or indirectly, sensitive to oestrogenic or anti-androgenic compounds. This provides important insight into the mechanism by which maternal exposure to endocrine disrupters can result in cryptorchidism in the next generation. Conclusively, INSL3 is an interesting testicular hormone with potential clinical value as a marker for Leydig cell function. It should be considered on a par with testosterone in the evaluation of testicular function and the consequences of Leydig cell dysfunction.

Luteinizing hormone (LH) is a key regulator of male fertility through its effects on testosterone secretion by Leydig cells. Transcriptional control of this is, however, currently poorly understood. Mice in which the LH receptor is knocked out (LuRKO) show reduced testicular size, reduced testosterone, elevated serum LH, and a spermatogenic arrest that can be rescued by the administration of testosterone. Using genome-wide transcription profiling of LuRKO and control testes during postnatal development and following testosterone treatment, we show that the transcriptional effects of LH insensitivity are biphasic, with an early testosterone-independent phase and a subsequent testosterone-dependent phase. Testosterone rescue reenables the second, testosterone-dependent phase of the normal prepubertal transcription program and permits the continuation of spermatogenesis. Examination of the earliest responses to testosterone highlights six genes that respond rapidly in a dose-dependent fashion to the androgen and that are therefore candidate regulatory genes associated with the testosterone-driven progression of spermatogenesis. In addition, our transcriptional data suggest a model for the replacement of fetal-type Leydig cells by adult-type cells during testicular development in which a testosterone feedback switch is necessary for adult Leydig cell production. LH signaling affects the timing of the switch but is not a strict requirement for Leydig cell differentiation.

Mammalian sex determination is the genetic process that commits the undifferentiated bipotential gonads to develop as either testes or ovaries. The differentiation of SOX9-expressing Sertoli cells is assumed to be necessary to initiate testis development. Insectivorous moles of the genus Talpa represent a unique case of generalized true hermaphroditism, as XX female moles constitutively develop two ovotestes instead of normal ovaries. In this work, we have investigated the expression patterns of a number of genes known to play key roles in gonad organogenesis, throughout the entire process of ovotestis development in female moles. Molecular and morphological evidence are provided that these ovotestes contain primary medullary testis-like cords, Leydig cells, peritubular myoid cells, and a testis-specific vasculature, but no Sertoli cells. Our results show for the first time that SOX9 is not required for the formation of the primary testis cords, but it is necessary for the maintenance and subsequent development of these cords. In addition, the expression pattern of WNT4 in male and female moles indicates that this gene inhibits Leydig cell differentiation and, contrary to the proposed scenario in the mouse, it is not required for the colonization and survival of primordial germ cells. According to our data, mole ovotestes result from a process of PDGFRalpha-mediated mesonephric cell migration, which occurs simultaneously in both sexes. The fact that FST remains inactive during the critical stages of female gonad development, explains the lack of migration inhibition, and may be a consequence of improper WNT4 signalling in the mole.

Some cellular events are crucial in testis organogenesis, including Sertoli and Leydig cell differentiation, mesonephric cell migration and testis cord formation. These processes are controlled by transcription factors, paracrine signalling and hormones. Using the mole species Talpa occidentalis as an alternative animal model, we report the expression patterns of nine genes during testis differentiation and analyse their implications in the above-mentioned cellular processes. We show that: 1) Sertoli cell differentiation occurs very early and precedes mesonephric cell migration, indicating that the latter is not needed for the endocrine cytodifferentiation of Sertoli cells; 2) the time of Leydig cell differentiation is consistent with the participation of PDGFR-alpha in promoting the migration and/or proliferation of Leydig cell precursors, and with that of WNT4 signalling in inhibiting Leydig cell differentiation and 3) the formation of the tunica albuginea involves intragonadal cell migration/movement. These results demonstrate that testicular organogenesis in the mole differs from that in the mouse in some particular aspects, thus providing evidence that the spatio-temporal pattern of testis development is not highly conserved during mammalian evolution.

Endocannabinoids are lipidic modulators able to bind cannabinoid receptors (CNRs). Two types of CNRs have been cloned, CNR1 (central) and CNR2 (peripheral). The objectives of the present study were to investigate the expression pattern of CNR1 in the rat testis during prepubertal development and to define the CNR1 spatiotemporal pattern. From 31 to 60 days of age, CNR1 was immunolocalized in round elongating spermatids and spermatozoa, suggesting an important role for this receptor in spermatogenesis. From 14 to 60 days of age, adult Leydig cells (ALCs) at different developmental stages were positive for CNR1. In particular, CNR1 expression in differentiating ALCs was negatively correlated to cell division. Bromodeoxyuridine uptake experiments on serial sections showed that immature Leydig cells in mitosis were negative for CNR1; in contrast, immature nonmitotic Leydig cells were positive for CNR1. A further observation of few ALCs in CNR1KO mice validates the role of CNR1 during proliferative activity involved in ALC differentiation. In addition, starting from 41 days of age, a faint CNR1 signal was also observed in Sertoli cells. Taken together, these results demonstrate the first clear evidence (to our knowledge) of CNR1 in mammalian germinal epithelium, ALCs, and Sertoli cells and indicate that differentiation of ALCs may depend on the endocannabinoid system.

Testicular development is an androgen-dependent process, and fetal exposure to antiandrogens disrupts male sexual differentiation. A variety of testicular disorders may result from impaired development of fetal Leydig and Sertoli cells. We hypothesized that antiandrogenic exposure during fetal development interferes with desert hedgehog (Dhh) signaling in the testis and results in impaired Leydig cell differentiation. Fetal rats were exposed in utero to the antiandrogen flutamide from 10.5 d post conception (dpc) until they were killed or delivery. Fetal testes were isolated at different time points during gestation and gene expression levels of Dhh, patched-1 (Ptc1), steroidogenic factor 1 (Sf1), cytochrome P450 side-chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase type 1 (Hsd3b1), and insulin-like factor 3 (Insl3) were analyzed. To study direct effects of hedgehog signaling on testicular development, testes from 14.5 dpc fetuses were cultured for 3 d in the presence of cyclopamine, sonic hedgehog, or vehicle, and gene expression levels and testosterone secretion were analyzed. Organ cultures were also analyzed histologically, and cleaved-caspase 3 immunohistochemistry was performed to assess apoptosis. In utero exposure to flutamide decreased expression levels of Dhh, Ptc1, Sf1, P450scc, Hsd3b1, and Insl3, particularly from 17.5 dpc onward. Inhibition of hedgehog signaling in testis cultures resulted in similar effects on gene expression levels. Apoptosis in Wolffian ducts was increased by cyclopamine compared with sonic hedgehog- or vehicle-treated cultures. We conclude that exposure to the antiandrogen flutamide interferes with Dhh signaling resulting in an impaired differentiation of the fetal Leydig cells and subsequently leading to abnormal testicular development and sexual differentiation.Antiandrogenic exposure of rat fetuses in utero affects genes that are involved in sexual differentiation of the testis and suppresses sexual maturation.

Anti-Müllerian hormone (AMH) plays a major role in sexual differentiation, Leydig cell differentiation, and folliculogenesis. In addition, AMH has clinical value in equine practice. In stallions, AMH can serve as an endocrine marker for equine cryptorchidism and as an immunohistochemical marker for Sertoli cell tumors. Considering that AMH is also an ovarian specific product, intact mares can be differentiated from ovariectomized mares. Peripheral AMH concentrations reflect the follicular population in mares, and therefore, are useful in the assessment of ovarian reserve and reproductive life-span of aged mares. Last, AMH is particularly suitable as a diagnostic marker for equine granulosa cell tumors.

LH and FSH act on gonadal cells to regulate steroidogenesis and gametogenesis. To model human reproductive disorders involving loss of LH function and to define LH-responsive genes, we produced knockout mice lacking the hormone-specific LHβ subunit. LHβ null mice are viable but demonstrate postnatal defects in gonadal growth and function resulting in infertility. Mutant males have decreased testes size, a block in Leydig cell differentiation, and a reduction in serum and intra-testicular testosterone levels. Furthermore, spermatogenesis is blocked at the round spermatid stage resulting in a total absence of the elongated spermatids. Mutant female mice are hypogonadal and demonstrate decreased levels of serum estradiol and progesterone. Ovarian histology reveals normal thecal layer, defects in folliculogenesis including many degenerating antral follicles and absence of corpora lutea. The defects in both sexes are not secondary to aberrant FSH regulation, since FSH levels were unaffected in null mice. Finally, the null mice can be pharmacologically rescued by exogenous hCG indicating that LH-responsiveness of the target cells is not irreversibly lost. Thus, LHβ null mice provide a useful model to study the consequences of an isolated deficiency of LH ligand in reproduction, while retaining normal LH-responsiveness in target cells.

Primary cilia are sensory organelles that coordinate numerous cellular signalling pathways during development and adulthood. Defects in ciliary assembly or function lead to a series of developmental disorders and diseases commonly referred to as ciliopathies. Still, little is known about the formation and function of primary cilia in the mammalian testis. Here, we characterized primary cilia in adult human testis and report a constitutive expression of cilia in peritubular myoid cells and a dynamic expression of cilia in differentiating Leydig cells. Primary cilia are generally absent from cells of mature seminiferous epithelium, but present in Sertoli cell-only tubules in Klinefelter syndrome testis. Peritubular cells in atrophic testis produce overly long cilia. Furthermore cultures of growth-arrested immature mouse Leydig cells express primary cilia that are enriched in components of Hedgehog signalling, including Smoothened, Patched-1 and GLI2, which are involved in regulating Leydig cell differentiation. Stimulation of Hedgehog signalling increases the localization of Smoothened to the cilium, which is followed by transactivation of the Hedgehog target genes, Gli1 and Ptch1. Our findings provide new information on the spatiotemporal formation of primary cilia in the testis and show that primary cilia in immature Leydig cells mediate Hedgehog signalling.

The transcription factor GATA-4 is expressed in Sertoli cells, steroidogenic Leydig cells, and other testicular somatic cells. Previous studies have established that interaction between GATA-4 and its cofactor FOG-2 is necessary for proper Sry expression and all subsequent steps in testicular organogenesis, including testis cord formation and differentiation of both Sertoli and fetal Leydig cells. Since fetal Leydig cell differentiation depends on Sertoli cell-derived factors, it has remained unclear whether GATA-4 has a cell autonomous role in Leydig cell development. We used two experimental systems to explore the role of GATA-4 in the ontogeny of testicular steroidogenic cells. First, chimeric mice were generated by injection of Gata4-/- ES cells into Rosa26 blastocysts. Analysis of the resultant chimeras showed that in developing testis Gata4-/- cells can contribute to fetal germ cells and interstitial fibroblasts but not fetal Leydig cells. Second, wild-type or Gata4-/- ES cells were injected into the flanks of intact or gonadectomized nude mice and the resultant teratomas examined for expression of steroidogenic markers. Wild-type but not Gata4-/- ES cells were capable of differentiating into gonadal-type steroidogenic lineages in teratomas grown in gonadectomized mice. In chimeric teratomas derived from mixtures of GFP-tagged Gata4+/+ ES cells and unlabeled Gata4-/- ES cells, sex steroidogenic cell differentiation was restricted to GFP-expressing cells. Collectively these data suggest that GATA-4 plays an integral role in the development of testicular steroidogenic cells.