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Spermatogenesis is a complex biological process crucial for male reproduction and is characterized by intricate interactions between testicular somatic cells and germ cells. Due to the cellular heterogeneity of the testes, investigating different cell types across developmental stages has been challenging. Single-cell RNA sequencing (scRNA-seq) has emerged as a valuable approach for addressing this limitation. Here, we conducted an unbiased transcriptomic study of spermatogenesis in sexually mature 4-month-old Hezuo pigs using 10× Genomics-based scRNA-seq. A total of 16,082 cells were collected from Hezuo pig testes, including germ cells (spermatogonia (SPG), spermatocytes (SPCs), spermatids (SPTs), and sperm (SP)) and somatic cells (Sertoli cells (SCs), Leydig cells (LCs), myoid cells (MCs), endothelial cells (ECs), and natural killer (NK) cells/macrophages). Pseudo-time analysis revealed that LCs and MCs originated from common progenitors in the Hezuo pig. Functional enrichment analysis indicated that the differentially expressed genes (DEGs) in the different types of testicular germ cells were enriched in the PI3K-AKT, Wnt, HIF-1, and adherens junction signaling pathways, while the DEGs in testicular somatic cells were enriched in ECM-receptor interaction and antigen processing and presentation. Moreover, genes related to spermatogenesis, male gamete generation, sperm part, sperm flagellum, and peptide biosynthesis were expressed throughout spermatogenesis. Using immunohistochemistry, we verified several stage-specific marker genes (such as UCHL1 , WT1 , SOX9 , and ACTA2 ) for SPG, SCs, and MCs. By exploring the changes in the transcription patterns of various cell types during spermatogenesis, our study provided novel insights into spermatogenesis and testicular cells in the Hezuo pig, thereby laying the foundation for the breeding and preservation of this breed.

Over the years, the origin of ovarian Leydig cells has been, and still is, a topic subject to deep debate. Seven years ago, we proposed that this origin resided in intraneural elements that came from a possible reservoir of neural crest cells, a reservoir that may be located in the ganglia of the celiac plexus. We believe we have found the evidence necessary to prove this hypothesis., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Although the order Rodentia does not present a high risk of extinction compared to mammals as a whole, several families demonstrate high levels of threat and/or data deficiency, therefore highlighting the need for targeted research and the application of ecological and reproductive data to the development of conservation actions. The order Rodentia, the largest among mammals, includes 9 families, and the family Cricetidae is the most diverse of the Brazilian rodents. In Brazil, 12 of the 16 genera of Oecomys are found. Oecomys bicolor is known in Brazil as the 'arboreal rat' and is, found in dry, deciduous and tropical forests. The mean body weight of Oecomys bicolor was 35.8 g and the gonadal, tubular and epithelial somatic indexes were, 0.53%, 0.47% and 0.37%, respectively. Seminiferous tubules volume density was 89.72% and the mitotic and meiotic indexes corresponded to 8.59 and 2.45 cells, respectively, and the yield of spermatogenesis was 23.83 cells. The intertubular compartment represented 10.28% of the testis parenchyma and around 5% of the interstitial space was occupied by Leydig cells, whose number per gram of testis was 11.10 × 10 7 cells. By evaluating the biometric and histomorphometric characteristics of the testis, there is evidence that this species has a high investment in reproduction. Due to the high contribution of the seminiferous epithelium and the intertubular compartment in this species, compared to the others of the same family, it is possible to infer that the species Oecomys bicolor has a promiscuous reproductive behaviour., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Sex-specific gonadal differentiation is directed by complex signalling promoting development in either male or female direction, while simultaneously inhibiting the opposite pathway. In mice, the WNT/β-catenin pathway promotes ovarian development and the importance of actively inhibiting this pathway to ensure normal testis development has been recognised. However, the implications of alterations in the tightly regulated WNT/β-catenin signalling during human fetal gonad development has not yet been examined in detail. Thus, the aim of this study was to examine the consequences of dysregulating the WNT/β-catenin signalling pathway in the supporting cell lineage during sex-specific human fetal gonad development using an established and extensively validated ex vivo culture model. Inhibition of WNT/β-catenin signalling in human fetal ovary cultures resulted in only minor effects, including reduced secretion of RSPO1 and reduced cell proliferation although this was not consistently found in all treatment groups. In contrast, promotion of WNT/β-catenin signalling in testes severely affected development and function. This included disrupted seminiferous cord structures, reduced cell proliferation, reduced expression of SOX9/AMH, reduced secretion of Inhibin B and AMH as well as loss of the germ cell population. Additionally, Leydig cell function was markedly impaired with reduced secretion of testosterone, androstenedione and INSL3. Together, this study suggests that dysregulated WNT/β-catenin signalling during human fetal gonad development severely impairs testicular development and function. Importantly, our study highlights the notion that sufficient inhibition of the opposite pathway during sex-specific gonadal differentiation is essential to ensure normal development and function also applies to human fetal gonads., (© 2024. The Author(s).)

Differentiation of Leydig cells plays a key role in male reproductive function. Bone marrow mesenchymal stem cells (BMSCs) have emerged as a potential cell source for generating Leydig-like cells due to their multipotent differentiation capacity and accessibility. This study aimed to investigate the morphological and genetic expression changes of BMSCs during differentiation into Leydig-like cells. Testicular extract liquid, which simulates the microenvironment in vivo, induced the third passage BMSCs differentiated into Leydig-like cells. Changes in cell morphology were observed by microscopy, the formation of lipid droplets of androgen precursor was identified by Oil Red Staining, and the expression of testicular specific genes 3β-HSD and SF-1 in testicular stromal cells was detected by RT-qPCR. BMSCs isolated from the bone marrow of Sprague-Dawley (SD) rats were cultured for 3 generations and identified as qualified BMSCs in terms of morphology and cell surface markers. After 14 days of induction with testicular tissue lysate, lipid droplets appeared in the cytoplasm of P3 BMSCs by Oil Red O staining. RT-qPCR detection was performed on BMSCs on the 3 rd , 7 th , 14 th , and 21 st day after induction. Relative expression levels of 3β-HSD mRNA significantly increased after 14 days of induction, while the relative expression of SF-1 mRNA increased after 14 days of induction but was not significant. BMSCs can differentiate into testicular interstitial cells with reserve androgen precursor lipid droplets after induction by testicular tissue lysate. The differentiation ability of BMSCs provides the potential to reconstruct the testicular microenvironment and is expected to fundamentally improve testicular function and provide new treatment options for abnormal spermatogenesis diseases., (© 2024. The Society for In Vitro Biology.)

Deoxynivalenol (DON) is a common food contaminant that can impair male reproductive function. This study investigated the effects and mechanisms of DON exposure on progenitor Leydig cell (PLC) development in prepubertal male rats. Rats were orally administrated DON (0-4 mg/kg) from postnatal days 21-28. DON increased PLC proliferation but inhibited PLC maturation and function, including reducing testosterone levels and downregulating biomarkers like HSD11B1 and INSL3 at ≥2 mg/kg. DON also stimulated mitochondrial fission via upregulating DRP1 and FIS1 protein levels and increased oxidative stress by reducing antioxidant capacity (including NRF2, SOD1, SOD2, and CAT) in PLCs in vivo. In vitro, DON (2-4 μM) inhibited PLC androgen biosynthesis, increased reactive oxygen species production and protein levels of DRP1, FIS1, MFF, and pAMPK, decreased mitochondrial membrane potential and MFN1 protein levels, and caused mitochondrial fragmentation. The mitochondrial fission inhibitor mdivi-1 attenuated DON-induced impairments in PLCs. DON inhibited PLC steroidogenesis, increased oxidative stress, perturbed mitochondrial homeostasis, and impaired maturation. In conclusion, DON disrupts PLC development in prepubertal rats by stimulating mitochondrial fission.

Testicular macrophages are the principal immune cells in the testis. In addition to their classical immune roles, they regulate male hormone synthesis by Leydig cells, regeneration of Leydig cells, spermatogonia proliferation and differentiation, maintenance of testis-specific environment for sperm formation, and testis development. The juvenile and adult testes contain two distinct macrophage populations with unique tissue localization, genetic markers, morphology, and function. The interstitial macrophages are physically and functionally connected to Leydig cells, while the peritubular macrophages localize around the seminiferous tubules and are crucial for spermatogonia differentiation. Macrophages in the fetal testes regulate testis vasculature formation and clearance of mislocated cells. The origin of testicular macrophages is unclear. Some studies suggest their origin from the yolk sac and others from the bone marrow-derived monocytes. We discuss this issue at the end of this review article., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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., (© 2022. The Author(s).)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021. Published by Elsevier B.V.)

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- β 1 and p-SMAD2/3 expression), and restored the testosterone content. In addition, DP1 enhanced the number of Leydig cells in rats and H 2 O 2 -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- β 1 and p-SMAD2/3, and increase testosterone contents in H 2 O 2 -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., Competing Interests: All authors report no conflicts of interest., (Copyright © 2021 Shiting Yu et al.)

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 H 2 O 2 -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 H 2 O 2 -treated TM3 cells. Knockdown of FENDRR augmented Nrf2 and HO-1 protein levels and testosterone production in H 2 O 2 -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 H 2 O 2 -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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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, 3β-HSD-I, 3β-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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2021. Published by Elsevier B.V.)

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., (© 2021. Springer Nature Limited.)

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.

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 TCF21 lin 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, TCF21 lin 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.

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., (Copyright © 2021. Published by Elsevier Ltd.)

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.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Zhu, Wen, Yuan, Su, Wang, Zhong and Ge.)

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 S F1 , CYP11A1 , 3β-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.

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 3β-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., Competing Interests: The authors have declared that no competing interests exist.

Kisspeptin and its receptor KISS1R have been proven as pivotal regulators on controlling the hypothalamus-pituitary-gonad axis. Inactivating mutations in one of them cause idiopathic hypogonadotropic hypogonadism in human as well as rodent models. Notably, gonadotropin insensitivity, failure in hCG response, was presented in the male patients with loss-function-mutations in KISS1R gene; this reveals the essential role of KISS1R signaling in regulating testosterone production beyond the hypothalamic functions of kisspeptin. In this study, we hypothesized that the autocrine action of kisspeptin on Leydig cells may modulate steroidogenesis. Based on the mouse cell model, we first demonstrated that the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) signaling pathway mediated gonadotropin-induced kisspeptin expression. By using siRNA interfering technique, knockdown of Kiss1r in MA-10 cells, a mouse Leydig tumor cell line, significantly reduced progesterone productions in both basal and hCG-treated conditions. Integrating the results from both quantitative real-time PCR and steroidogenic enzyme-activity assay, we found that this steroidogenic defect was associated with decreased luteinizing hormone/choriogonadotropin receptor (Lhcgr) and StAR protein (Star) expressions. Furthermore, exogenous expression of human LHCGR completely rescued hCG-stimulated progesterone production in the KISS1R-deficient cells. In conclusion, we proposed that the reproductive functions of KISS1R signaling in Leydig cell include modulating Lhcgr and steroidogenic gene expressions, which may shed the light on the pathophysiology of gonadotropin insensitivity.

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

Background: Gold nanoparticles (AuNPs) have shown great promise in various biomedical applications, but their effects on male reproductive function remain to be ascertained. The aim of this study was to investigate the uptake, cytotoxicity and testosterone production inhibition of AuNPs in mouse Leydig cells, as well as their accumulation in the testes of male mice and their effects on male reproductive function., Results: AuNPs (5 nm) were able to be internalized into the endosomes/lysosomes of TM3 Leydig cells, induce the formation of autophagosomes, increase the production of reactive oxygen species (ROS), and disrupt the cell cycle in S phase, resulting in concentration-dependent cytotoxicity and DNA damage. Interestingly, AuNPs significantly reduced testosterone production in TM3 cells by inhibiting the expression of 17α-hydroxylase, an important enzyme in androgen synthesis. After repeated intravenous injection, AuNPs gradually accumulated and retained in the testes of male BALB/c mice in a dose-dependent manner. One week after withdrawal, the level of plasma testosterone in the 0.5 mg/kg AuNPs group was significantly reduced compared to that in the PBS control group, accompanied by the decreased expression of 17α-hydroxylase in the testes. In addition, AuNPs treatment significantly increased the rate of epididymal sperm malformation, but without affecting fertility., Conclusion: Our results suggest that AuNPs can accumulate in the testes and reduce testosterone production in Leydig cells by down-regulating the expression of 17α-hydroxylase, thus affecting the quality of epididymal sperm., Competing Interests: The authors declare that they have no conflicts of interest., (© 2020 Liu et al.)

A systematic phytochemical study on the components in the seeds of Allium tuberosum was performed, leading to the isolation of 27 steroidal glycosides (SGs 1 - 27 ). The structures of SGs were identified mainly by nuclear magnetic resonance and mass spectrometries as well as the necessary chemical evidence. In the SGs, 1 - 10 and 22 - 26 are new steroidal saponin analogues. An in vitro bioassay indicates that 1 , 2 , 7 , 8 , 10 , 13 - 15 , 20 , 23 , and 26 display promotional roles in testosterone production of rat Leydig cells with the EC 50 values of 1.0 to 4.5 μM, respectively.

Bisphenol A (BPA) [2,2-bis(4-hydroxyphenyl) propane] has attracted increasing attention over the past few decades as an endocrine-disrupting chemicals that causes low testosterone levels. Linoleic acid (LA) is an essential fatty acid and GPR120 agonist. Herein, we are the first to report that LA induces the expression of GPR120 in mouse Leydig cells to directly promote testosterone production. In addition, we demonstrated that the activated GPR120 / ERK signaling pathway was involved in upregulating the expression of 3β-HSD and StAR for testosterone production by stimulation of LA. Interestingly, although BPA failed to affect GPR120 expression, LA restored the testosterone levels decreased by BPA in Leydig cells in vitro. Furthermore, the in vivo restoration of testosterone levels and testicular structure was also observed in BPA-impaired mice fed LA. As a result, the sperm functions of BPA-impaired mice returned to normal levels. At the same time, the damaged blood-testis barrier and infertility were also resolved by LA. Our study indicates a novel and safe strategy that utilizes LA to repair reproductive damage caused by low testosterone levels through activating the GPR120/ERK pathway in Leydig cells., (Copyright © 2020. Published by Elsevier Inc.)

Background: It has recently been suggested that the hypergonadotropic hypogonadism characterizing Klinefelter syndrome (KS) might not be due to a steroidogenic dysfunction per se, but mainly to an altered testosterone (T) secretion into the bloodstream. However, the Leydig cell functionality remains incompletely studied in KS, and new markers should be considered. Previous data indicated that chronic hCG stimulation influences the production of both insulin-like peptide 3 (INSL3) and 25-hydroxy-vitamin D (25-VD) in eugonadal men., Aim of the Study: To evaluate INSL3 and 25-VD serum levels, as markers of Leydig cell functionality, in association with sex steroids, after an acute hCG test in a group of KS patients and healthy volunteers., Methods: A retrospective analysis of a prospective case-control clinical trial was carried out. KS patients (n = 11) and age-matched healthy controls (n = 11) provided a basal blood sample (V0) immediately followed by a single intramuscular injection of hCG 5000 IU. Blood samples were taken in the following five days (V1-V5)., Results: At baseline, INSL3 was lower in KS patients compared with controls (P = .007). When adjusted for INSL3 levels, the production of steroids was similar between KS patients and controls. 25-VD was in the insufficient range both in KS patients and in controls and was not different (P = .064). Acute hCG stimulation increased neither INSL3 nor 25-VD in both KS patients and controls. In controls, an inverse correlation was detected between INSL3 levels and body mass index (P = .020) and waist circumference (P = .020)., Conclusions: INSL3 secretion is independent from steroidogenesis, and its production is mostly not influenced by acute hCG stimulation both in KS men and in controls. INSL3 serum levels should be considered as a marker of Leydig cell differentiation and numbers rather than steroidogenesis. 25-VD serum levels are also not increased by a single acute hCG administration, which was not able to restore the normal concentrations of 25-VD., (© 2020 American Society of Andrology and European Academy of Andrology.)

Background: Monocyte chemoattractant protein-1(MCP-1) is a chemokine secreted by Leydig cells and peritubular myoid cells in the rat testis. Its role in regulating the development of Leydig cells via autocrine and paracrine is still unclear. The objective of the current study was to investigate the effects of MCP-1 on Leydig cell regeneration from stem cells in vivo and on Leydig cell development in vitro., Results: Intratesticular injection of MCP-1(10 ng/testis) into Leydig cell-depleted rat testis from post-EDS day 14 to 28 significantly increased serum testosterone and luteinizing hormone levels, up-regulated the expression of Leydig cell proteins, LHCGR, SCARB1, CYP11A1, HSD3B1, CYP17A1, and HSD17B3 without affecting progenitor Leydig cell proliferation, as well as increased ERK1/2 phosphorylation. MCP-1 (100 ng/ml) significantly increased medium testosterone levels and up-regulated LHCGR, CYP11A1, and HSD3B1 expression without affecting EdU incorporation into stem cells after in vitro culture for 7 days. RS102895, a CCR2 inhibitor, reversed MCP-1-mediated increase of testosterone level after culture in combination with MCP-1., Conclusion: MCP-1 stimulates the differentiation of stem and progenitor Leydig cells without affecting their proliferation.

Estrogen signaling plays important roles in testicular functions and tumorigenesis. Fifteen years ago, it was discovered that a member of the G protein-coupled receptor family, GPR30, which binds also with high affinity to estradiol and is responsible, in part, for the rapid non-genomic actions of estrogens. GPR30, renamed as GPER, was detected in several tissues including germ cells (spermatogonia, spermatocytes, spermatids) and somatic cells (Sertoli and Leydig cells). In our previous review published in 2014, we summarized studies that evidenced a role of GPER signaling in mediating estrogen action during spermatogenesis and testis development. In addition, we evidenced that GPER seems to be involved in modulating estrogen-dependent testicular cancer cell growth; however, the effects on cell survival and proliferation depend on specific cell type. In this review, we update the knowledge obtained in the last years on GPER roles in regulating physiological functions of testicular cells and its involvement in neoplastic transformation of both germ and somatic cells. In particular, we will focus our attention on crosstalk among GPER signaling, classical estrogen receptors and other nuclear receptors involved in testis physiology regulation.

Background: Origin of human adult Leydig cells (ALCs) is not well understood. This might be partly due to limited data available on the identification and location of human precursor and stem Leydig cells (SLCs) which hampers the study on the development of ALCs., Objectives: The aim of the present study was to investigate whether described human (PDGFRα, NGFR) and rodent (NES, PDGFRα, THY1, NR2F2) SLC markers are expressed by a common cell population within human adult testicular interstitial cells in vivo and before and after in vitro propagation., Materials and Methods: Immunohistochemical analyses were used to identify localization of human adult testicular interstitial cells expressing described SLC markers. Next, interstitial cells were isolated and cultured. The percentage of cells expressing one or more SLC markers was determined before and after culture using flow cytometry., Results: NR2F2 and PDGFRα were present in peritubular, perivascular, and Leydig cells, while THY1 was expressed in peritubular and perivascular cells. Although NES and NGFR were expressed in endothelial cells, co-localization with PDGFRα was found for both in vitro, although for NGFR only after culture. All marker positive cells were able to undergo propagation in vitro., Discussion: The partly overlap in localization and overlap in expression in human testicular cells indicate that PDGFRα, NR2F2, and THY1 are expressed within the same ALC developmental lineage from SLCs. Based on the in vitro results, this is also true for NES and after in vitro propagation for NGFR., Conclusion: Our results that earlier described SLC markers are expressed in overlapping human interstitial cell population opens up further research strategies aiming for a better insight in the Leydig cell lineage and will be helpful for development of strategies to cure ALC dysfunction., (© 2020 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Background: Recently we described two outbred mouse lines which have been selected for high fertility. These mouse models doubled the number of offspring per litter., Objectives: Although selected for a primarily female-trait of high fertility (increased litter size), we were interested whether also males of the fertility lines show differences within their reproductive organs., Materials and Methods: We investigated males from two outbred mouse lines which have been selected for the phenotype "high fertility" for more than 170 generations. In the present study, we analysed the testicular cell type composition by flow cytometry. We further investigated the weights of reproductive organs, histomorphometry of testis as well as studied sperm motility parameters using a thermal stress assay as well as a sperm hyperactivation assay., Results: Here, we describe that males of the fertility line (FL) 1 show an increased percentage of diploid cells within the testis. Flow cytometric analysis identified this enlarged cell population as Leydig cells. Testis weights were unaffected whereas the weights of seminal vesicles of FL1 and FL2 were increased compared to Ctrl bucks. FL2 males show decreased diameter of tubulus seminiferi and an enhanced spermatid/Sertoli cell index. Sperm motility parameters of FL1 and Ctrl males are initially indistinguishable but FL1 spermatozoa show a better performance in a thermal stress experiment over a 5 hours observation period., Discussion: These data indicate that although selected for a primarily female-trait of high fertility also males from the fertility lines are effected by defined alterations in their reproductive organs., Conclusion: Some of these alterations are FL1-specific others are FL2-associated, indicating that different molecular strategies warrant the high-fertility phenotype on the female as well as on the male side., (© 2020 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

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., (© 2020 Wiley Periodicals, Inc.)

Recent studies have demonstrated that fibroblasts can be directly converted into functional Leydig cells by transcription factors. However, the transgenic approach used in these studies raises safety concerns for its future application. Here, we report that fibroblasts can be directly reprogrammed into Leydig-like cells by exposure to a combination of forskolin, 20α-hydroxycholesterol, luteinizing hormone, and SB431542. These chemical compound-induced Leydig-like cells (CiLCs) express steroidogenic genes and have a global gene expression profile similar to that of progenitor Leydig cells, although not identical. In addition, these cells can survive in testis and produce testosterone in a circadian rhythm. This induction strategy is applicable to reprogramming human periodontal ligament fibroblasts toward Leydig-like cells. These findings demonstrated fibroblasts can be directly converted into Leydig-like cells by pure chemical compounds. This strategy overcomes the limitations of conventional transgenic-based reprogramming and provides a simple, effective approach for Leydig cell-based therapy while simultaneously preserving the hypothalamic-pituitary-gonadal axis., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: Stem Leydig cells (SLCs) transplantation can restore testosterone production in rodent models and is thus a potential solution for treating testosterone deficiency (TD). However, it remains unknown whether these favorable effects will be reproduced in more clinically relevant large-animal models. Therefore, we assessed the feasibility, safety and efficacy of autologous SLCs transplantation in a testosterone-deficient non-human primate (NHP) model. Methods: Cynomolgus monkey SLCs (CM-SLCs) were isolated from testis biopsies of elderly (> 19 years) cynomolgus monkeys by flow cytometry. Autologous CM-SLCs were injected into the testicular interstitium of 7 monkeys. Another 4 monkeys were injected the same way with cynomolgus monkey dermal fibroblasts (CM-DFs) as controls. The animals were then examined for sex hormones, semen, body composition, grip strength, and exercise activity. Results: We first isolated CD271 + CM-SLCs which were confirmed to expand continuously and show potential to differentiate into testosterone-producing Leydig cells (LCs) in vitro . Compared with CM-DFs transplantation, engraftment of autologous CM-SLCs into elderly monkeys could significantly increase the serum testosterone level in a physiological pattern for 8 weeks, without any need for immunosuppression. Importantly, CM-SLCs transplantation recovered spermatogenesis and ameliorated TD-related symptoms, such as those related to body fat mass, lean mass, bone mineral density, strength and exercise capacity. Conclusion: For the first time, our short-term observations demonstrated that autologous SLCs can increase testosterone levels and ameliorate relevant TD symptoms in primate models. A larger cohort with long-term follow-up will be required to assess the translational potential of autologous SLCs for TD therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Zearalenone (ZEA) is a mycotoxin that contaminates crops worldwide and is toxic to the reproductive systems of mammals, however, the toxicological mechanism by which ZEA affects germ cells is not fully understood. In this study, proteomic analysis using iTRAQ technology was adopted to determine the cellular response of Leydig cells of rats to ZEA exposure. The results were used to elucidate the mechanisms responsible for the toxicity of the ZEA towards germ cells. After 24 h of exposure to ZEA at a concentration of 30 μmol/L, a total of 128 differentially expressed proteins (DEPs) were identified. Of these, 70 DEPs were up-regulated and 58 DEPs were down-regulated. The DEPs associated with ZEA toxicology were then screened by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The results show that these DEPs are involved in a number of important ZEA toxicological pathways including apoptosis, immunotoxicity, DNA damage, and signaling pathways. The complex regulatory relationships between the DEPs and ZEA toxicological signaling pathways are also explicitly demonstrated in the form of a protein-protein interaction network. This study thus provides a theoretical molecular basis for understanding the toxicological mechanisms by which ZEA affects germ cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Epidermal growth factor (EGF) has many physiological roles. However, its effects on stem and progenitor Leydig cell development remain unclear. Rat stem and progenitor Leydig cells were cultured with different concentrations of EGF alone or in combination with EGF antagonist, erlotinib or cetuximab. EGF (1 and 10 ng/mL) stimulated the proliferation of stem Leydig cells on the surface of seminiferous tubules and isolated CD90 + stem Leydig cells and progenitor Leydig cells but it blocked their differentiation. EGF also exerted anti-apoptotic effects of progenitor Leydig cells. Erlotinib and cetuximab are able to reverse EGF-mediated action. Gene microarray and qPCR of EGF-treated progenitor Leydig cells revealed that the down-regulation of steroidogenesis-related proteins (Star and Hsd3b1) and antioxidative genes. It was found that EGF acted as a proliferative agent via increasing phosphorylation of AKT1. In conclusion, EGF stimulates the proliferation of rat stem and progenitor Leydig cells but blocks their differentiation., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Propofol is an anesthetic agent moderating GABA receptors in the nervous system. A number of studies have demonstrated that propofol exerts a negative effect on neural stem cell development in the neonatal mouse hippocampus. However, to the best of our knowledge, there is no study available to date illustrating whether neonatal exposure to propofol affects Leydig stem/progenitor cell development for normal male reproductive development and functions, and the regulatory mechanism remains elusive. In the present study, TM3 cells, a mouse Leydig stem/progenitor cell line, was treated with propofol. The data illustrated that propofol significantly reduced TM3 cell viability. TM3 subG1 phase cell numbers were significantly increased by propofol assayed by flow cytometric analysis. Annexin V/PI double staining assay of the TM3 Leydig cells also demonstrated that propofol increased TM3 cell apoptosis. In addition, cleaved caspase‑8, ‑9 and ‑3 and/or poly(ADP‑ribose) polymerase (PARP) were significantly activated by propofol in the TM3 cells. Furthermore, the expression levels of phospho‑JNK, phospho‑ERK1/2 and phospho‑p38 were activated by propofol in the TM3 cells, indicating that propofol induced apoptosis through the mitogen‑activated protein kinase (MAPK) pathway. Additionally, propofol diminished the phosphorylation of Akt to increase the apoptosis of TM3 cells. On the whole, the findings of the present study demonstrate that propofol induces TM3 cell apoptosis by activating caspases and MAPK pathways, as well as by inhibiting the Akt pathway in TM3 cells. These findings illustrate that propofol affects the viability of Leydig stem/progenitor cells possibly related to the development of the male reproductive system.

Background: Testicular architecture and sperm production are supported by a complex network of communication between various cell types. These signals ensure fertility by: regulating spermatogonial stem/progenitor cells; promoting steroidogenesis; and driving male-specific differentiation of the gonad. Sertoli cells have long been assumed to be the major cellular player in testis organogenesis and spermatogenesis. However, cells in the interstitial compartment, such as Leydig, vascular, immune, and peritubular cells, also play prominent roles in the testis but are less well understood., Objectives: Here, we aim to outline our current knowledge of the cellular and molecular mechanisms by which interstitial cell types contribute to spermatogenesis and testicular development, and how these diverse constituents of the testis play essential roles in ensuring male sexual differentiation and fertility., Methods: We surveyed scientific literature and summarized findings in the field that address how interstitial cells interact with other interstitial cell populations and seminiferous tubules (i.e., Sertoli and germ cells) to support spermatogenesis, male-specific differentiation, and testicular function. These studies focused on 4 major cell types: Leydig cells, vascular cells, immune cells, and peritubular cells., Results and Discussion: A growing number of studies have demonstrated that interstitial cells play a wide range of functions in the fetal and adult testis. Leydig cells, through secretion of hormones and growth factors, are responsible for steroidogenesis and progression of spermatogenesis. Vascular, immune, and peritubular cells, apart from their traditionally acknowledged physiological roles, have a broader importance than previously appreciated and are emerging as essential players in stem/progenitor cell biology., Conclusion: Interstitial cells take part in complex signaling interactions with both interstitial and tubular cell populations, which are required for several biological processes, such as steroidogenesis, Sertoli cell function, spermatogenesis, and immune regulation. These various processes are essential for testicular function and demonstrate how interstitial cells are indispensable for male fertility., (© 2019 American Society of Andrology and European Academy of Andrology.)

This study reports encapsulation-vitrification of Leydig cells. The Leydig cells were encapsulated in sodium alginate beads of different sizes and cryopreserved by vitrification or slow freezing. Physico-chemical characterization of beads was done by Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), Fluorescence Recovery after Photobleaching (FRAP) and in vitro biodegradation study. Surface morphology of cryopreserved cell-encapsulated beads was evaluated by Environmental Scanning Electron Microscopy (E-SEM), encapsulation efficiency and viability of cells were assessed by Trypan blue assay, mitochondrial activity (MTT assay) and cytoplasmic esterase enzyme activity (FDA assay), respectively. Results showed that vitrification gives better results than slow freezing with respect to surface morphology as well as cell viability of the cell-encapsulated beads (86.94 ± 2.20% vs. 67.94 ± 2.30%; p < 0.05). Encapsulation of cells in small diameter beads (1.8 mm) gave a better cell proliferation rate than large (2.1 mm and 2.7 mm). There was a significant difference in the population doubling time (47.9 ± 1.7 h vs. 67.1 ± 2.5 h) and cell proliferation rate (0.50 ± 0.24 vs. 0.36 ± 0.24 per day) of vitrified-warmed cell encapsulated beads with different diameter (p < 0.05). Encapsualtion in sodium alginate beads is a promising method for cryopreservation of Leydig cells by slow freezing as well as vitrification., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mutations in Foxn1 and Prkdc genes lead to nude and severe combined immunodeficiency (scid) phenotypes, respectively. Besides being immunodeficient, previous reports have shown that nude mice have lower gonadotropins and testosterone levels, while scid mice present increased pachytene spermatocyte (PS) apoptosis. Therefore, these specific features make them important experimental models for understanding Foxn1 and Prkdc roles in reproduction. Hence, we conducted an investigation of the testicular function in nude and scid BALB/c adult male mice and significant differences were observed, especially in Leydig cell (LC) parameters. Although the differences were more pronounced in nude mice, both immunodeficient strains presented a larger number of LC, whereas its cellular volume was smaller in comparison to the wild type. Besides these alterations in LC, we also observed differences in androgen receptor and steroidogenic enzyme expression in nude and scid mice, suggesting the importance of Foxn1 and Prkdc genes in androgen synthesis. Specifically in scid mice, we found a smaller meiotic index, which represents the number of round spermatids per PS, indicating a greater cell loss during meiosis, as previously described in the literature. In addition and for the first time, Foxn1 was identified in the testis, being expressed in LC, whereas DNA-PKc (the protein produced by Prkdc) was observed in LC and Sertoli cells. Taken together, our results show that the changes in LC composition added to the higher expression of steroidogenesis-related genes in nude mice and imply that Foxn1 transcription factor may be associated to androgen production regulation, while Prkdc expression is also important for the meiotic process.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In the past few years, Leydig cell (LC) transplantation has been regarded as an effective strategy for providing physiological patterns of testosterone in vivo. Recently, we have successfully converted human foreskin fibroblasts (HFFs) into functional Leydig-like cells (iLCs) in vitro by using the CRISPR/dCas9 system, which shows promising potential for seed cells. However, it is not known whether the reprogrammed iLCs can survive or restore serum testosterone levels in vivo. Therefore, in this study, we evaluate whether reprogrammed iLCs can restore the serum testosterone levels of castrated rats when they are transplanted into the fibrous capsule. We first developed the castrated Sprague Dawley rat model through bilateral orchiectomy and subsequently injected extracellular matrix gel containing transplanted cells into the fibrous capsule of castrated rats. Finally, we evaluated dynamic serum levels of testosterone and luteinizing hormone (LH) in castrated rats, the survival of implanted iLCs, and the expression levels of Leydig steroidogenic enzymes by immunofluorescence staining and Western blotting. Our results demonstrated that implanted iLCs could partially restore the serum testosterone level of castrated rats, weakly mimic the role of adult Leydig cells in the hypothalamic-pituitary-gonadal axis for a short period, and survive and secrete testosterone, through 6 weeks after transplantation. Therefore, this study may be valuable for treating male hypogonadism in the future., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The current study evaluates potential applications of Sertoli cell (SC)-conditioned medium (CM) and explores the effects of the conditioned medium on the spermatogenesis process in azoospermic mice. For this study, 40 adult mice (28-30 g) were divided into 4 experimental groups: (1) control, (2) DMSO 2% (10 μl), (3) busulfan (40 mg/kg single dose) and (4) busulfan/CM (10 μl). SCs were isolated from 4-week-old mouse testes. After using anesthetics, 10 μl of CM was injected over 3-5 min into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. The data showed that CM notably increased the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocytes, round spermatids, SCs and Leydig cells compared with the control, DMSO and busulfan groups. Furthermore, the results showed that expression of c-kit and STRA8 was significantly decreased in the busulfan and busulfan/SC groups at 8 weeks after the last injection (p < 0.001) but no significant difference was found for PCNA compared with the control and DMSO groups (p < 0.05). These findings suggest that the Sertoli cell-conditioned medium may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine.

As a plasticizer, di(2-ethylhexyl) phthalate (DEHP) is the most usually used phthalate. Leydig cell is a male-specific cell, which plays a principal role in spermatogenesis and masculinization by the androgens of synthesis and secretion. Numerous researchers have indicated that DEHP can result in testicular toxicity by inducing oxidative stress. Lycopene (LYC) is a possible treatment option for male infertility due to its natural antioxidant properties. Our study was aimed to investigate whether LYC could rescue DEHP-induced Leydig cell damage. The mice were treated with DEHP (500 mg/kg BW/day or 1000 mg/kg BW/day) and/or LYC (5 mg/kg BW/day) for 28 days. We found that LYC attenuated DEHP-induced Leydig cell damage. Moreover, the protective role of LYC was verified by the histopathological and ultrastructural analysis of the Leydig cell. LYC suppressed oxidative stress that was induced by DEHP. In the Leydig cell, the expressions of the nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target genes were improved through LYC-mediated protection in DEHP-induced Leydig cell damage. Our findings indicated that LYC could increase the antioxidant capacity via mediating Nrf2 signaling pathway, thereby attenuating DEHP-induced Leydig cell damage.