Per- and polyfluoroalkyl substances (PFAS) are persistent environmental pollutants associated with neurodevelopmental toxicity. However, the combined effects of PFAS subclasses and the modifying role of endogenous hormones remain poorly understood. Using data from the Maoming birth cohort (n = 543), we longitudinally assessed associations between prenatal exposure to perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkyl sulfonic acids (PFSAs) and developmental delay in children aged 3-60 months, employing generalized linear mixed models for individual PFAS and grouped weighted quantile sum regression for effects of PFAS mixtures. We further examined the effect modification by cord blood estradiol and progesterone, classified via latent profile analysis, and conducted molecular docking to explore potential receptor interactions. Both individual compounds [e.g., perfluorooctanoic acid and perfluorooctane sulfonic acid (PFOS)] and PFCA mixtures were associated with increased odds of developmental delay, particularly in total-ASQ, communication, and motor domains. These associations were significantly modified by latent profiles of cord blood hormones. For example, in the low-hormone profile, perfluorodecanoic acid was associated with fine motor delay (Odds ratio [OR] = 2.04; 95% CI: 1.18, 3.49), whereas linear-PFOS was associated with gross motor delay (OR = 4.76; 95% CI: 1.96, 11.54) in the high-hormone profile. Molecular docking indicated that most PFCAs have a preferential binding affinity for the ligand-binding domains of estradiol and progesterone receptors, supporting the biological plausibility of the observed hormonal effect modification. Our findings demonstrate that the association between prenatal PFAS exposure and neurodevelopment is subclass-specific and critically shaped by the in utero hormonal environment.

Human aging is associated with testosterone decline and spermatogenic defects, impacting fertility and health, yet the underlying cellular mechanisms remain poorly understood. This study aimed to establish a regulatory cell atlas of Leydig cells in aging rat testes and characterize intercellular interactions. Using single-cell RNA sequencing and immunohistochemistry, interstitial cells from young (3-month) and aged (21-month) BN rat testes were analyzed. Among 8 identified interstitial cell types, aging was not synchronous, but with remarkable heterogeneity and stochasticity. Mesenchymal cells exhibited reduced expression of genes involved in detoxification and extracellular matrix maintenance, alongside increased immune regulation genes. Endothelial cells, the most communicatively active population, showed decreased angiogenesis and cell adhesion/migration but enhanced immune responses and leukocyte chemotaxis. In immune populations, dendritic cells decreased while lymphocytes increased, with no significant change in macrophages. Transcriptomic shifts indicated a transition from innate to adaptive immunity. The loss of dendritic cells and gain of lymphocytes may be linked to CCL7 and CXCL9-12, as receptors Ccr1/Ccr5 and Cxcr3 were upregulated in lymphocytes but downregulated in dendritic cells. Overall, inflammatory activation and functional losses occurred in both immune and non-immune cells during testicular aging. This innate-to-adaptive immune shift may further compromise non-immune cell functions, accelerating testicular aging.

Male infertility is a growing global health concern increasingly linked to environmental exposure to endocrine-disrupting chemicals (EDCs). However, the specific molecular mechanisms by which EDCs contribute to impaired reproductive function remain unclear. In this study, we systematically identified EDC-related genes using curated chemical-gene interaction databases and assessed their causal roles in male infertility through Mendelian randomization (MR) and colocalization analyses, utilizing large-scale cis-eQTL and GWAS datasets. A total of six genes, RHEB, PARP1, SLTM, PLIN1, PEX11A, and SDCBP, showed strong evidence of causal relationships and shared genetic variants associated with both gene expression and infertility traits. Single-cell RNA sequencing of human testicular tissue revealed that these genes are predominantly expressed in germ cells and are significantly dysregulated in non-obstructive azoospermia (NOA) samples, supporting their functional relevance. Additionally, environmental mapping indicated that several widely encountered EDCs, including bisphenol A (BPA) and its analogs, triphenyl phosphate (TPP), and sodium arsenite, interact with multiple candidate genes. These findings provide mechanistic insight into how chemical exposures can dysregulate gene expression in testicular cells and contribute to male infertility, highlighting the need for targeted environmental risk assessments and regulatory strategies.

BACKGROUND:Serum testosterone (T) concentration declines with aging in men, potentially affecting reproduction, mental and physical well-beings. A role of immune factors in Leydig cell (LC) function is well-known, but the specific factors involved, especially these playing roles in LC aging, are still unclear. This study investigated effects of interleukin 33 (IL-33) on LC function and its expression during testicular aging. METHODS:Immunohistochemistry and Western blotting were used to determine IL-33 and its receptor IL1RL1 expressions in testes of young (3-month-old) and old (19-24-month-old) Wistar rats. In vitro, the effects of IL-33 on sex steroid hormone productions were evaluated in primary and MLTC-1 LCs over 2-24 h. Different steroidogenic stimulators or signaling molecules (luteinizing hormone [LH], 8-Br-cAMP, Forskolin, pertussis toxin, and MAPK activators) were compared with elucidate mechanisms. Steroidogenic pathway proteins and potential signaling molecules were explored by Western blotting. RESULTS:IL-33 is expressed by mesenchymal cells, with the number increasing significantly with aging. IL1RL1, its receptor, is expressed by LCs and remains unchanged. In vitro, IL-33 acutely inhibited LC steroidogenesis in a dose-dependent manner (1-100 ng/mL) within 2-24 h. The effect was LH-dependent; replacing LH with either 8-Br-cAMP or Forskolin abolished the inhibition. IL-33 mainly affected STAR in the steroidogenic pathway. Signaling molecules involving STAR regulation (AKT and MAPK) were down-regulated while PKA phosphorylation was increased. P38 MAPK involvement was confirmed as increased Tyr182 phosphorylation of P38 by SB203580 partly reversed the IL-33-induced steroidogenesis inhibition. CONCLUSION:Testicular mesenchymal cells can synthesize IL-33, and LCs express the receptor IL1RL1. IL-33 inhibits LC steroidogenesis in vitro, partially via inhibiting P38 MAPK phosphorylation. As IL-33-expressing cell numbers rise significantly with aging, its role in age-related LC T production decline warrants further study.

Aging is a complex biological process driven by genetic and immune-mediated mechanisms, yet the causal roles of immune-cell-specific gene regulation remain unclear. In this study, we integrate single-cell expression quantitative trait loci (sc-eQTL) data with Mendelian randomization (MR) and colocalization analyses to identify immune-mediated regulatory mechanisms and therapeutic targets for aging. Using data from 14 immune cell types, we systematically evaluated 8733 eGenes for causal effects on telomere length (TL), facial aging (FA), and frailty index (FI). We identified 27 immune-cell-specific eGenes with significant causal associations and strong colocalization evidence (posterior probability for a shared causal variant, PP.H4 > 50 %). Key regulators include FUBP1, TUFM, ATIC, and SLC22A5, with distinct effects across cell types and aging traits. Phenome-wide association studies (PheWAS) demonstrated minimal off-target associations for most genes, supporting their safety as therapeutic targets. Drug repurposing analysis revealed several approved or investigational compounds, such as Irofulven, zinc-based agents, and acetylcarnitine, with potential for aging-related interventions. Our findings provide new insights into the immune-genetic architecture of aging and establish a scalable framework for identifying cell-type-specific causal genes and repurposable drug targets. This approach enhances precision medicine strategies aimed at promoting healthy aging and delaying age-related decline.

Environmental endocrine-disrupting chemicals (EDCs) are increasingly implicated in male infertility, yet the gene-level mechanisms by which EDCs contribute to non-obstructive azoospermia (NOA) remain unclear. This study aimed to identify EDC-related genes that are causally linked to NOA and uncover their potential roles in reproductive dysfunction. We integrated transcriptomic analysis of GEO datasets, EDC-associated gene data from the Comparative Toxicogenomics Database (CTD), and Mendelian randomization (MR) to prioritize candidate genes with causal relevance to male infertility. Phenome-wide association study (PheWAS) was conducted to assess systemic effects. EDC-gene interactions were visualized, and single-cell RNA sequencing (scRNA-seq) was used to validate cell-type-specific gene expression in the testis. A total of 136 genes were identified at the intersection of differentially expressed genes in NOA and EDC-related genes. MR analysis highlighted PPP6R1 and AP1M2 as causally linked to male infertility. PheWAS revealed a significant association between PPP6R1 and cardiovascular traits (p < 1 × 10−6), suggesting pleiotropic effects. Interaction analysis identified 26 EDCs targeting these genes, including thiram, bisphenol A, and sodium arsenite. The scRNA-seq confirmed downregulation of both genes in germ cells of NOA patients. Our multi-omics approach suggests a potential EDC-gene-NOA axis, identifying PPP6R1 and AP1M2 as candidate genes whose expression may be influenced by environmental exposure in the context of male infertility. These findings deepen mechanistic understanding of NOA pathogenesis and support future research into environmental and molecular targets for diagnosis and prevention.

PURPOSE:To investigate the relationship between blood lead levels (BLLs) and IVF clinical outcomes in infertile females and to further explore the possible involvement of granulosa cell (GC) endoplasmic reticulum (ER) stress in the process.METHODS:One hundred twenty-three infertile women undergoing IVF cycles were included in the current study. All participants were divided into three (low, medium, and high) groups determined by BLL tertiles. Gonadotropin releasing hormone (GnRH) agonist regimen for ovarian stimulation was used for all patients, with follicular fluids being collected on the day of oocyte retrieval. Lactate dehydrogenase (LDH) levels in follicular fluid and the endoplasmic reticulum stress-signaling pathway of granulosa cells (GCs) were examined.RESULTS:The oocyte maturation rate and high-quality embryo rate on cleaved stage decreased significantly as BLL increased. For lead levels from low to high, live birth rate (68.29%, 56.10%, 39.02%; P=0.028) showed negative correlations with BLLs. Also, follicular fluid Pb level and LDH level was significantly higher in the high lead group versus the low group. Binomial regression analysis revealed significant negative correlation between BLLs and live birth rate (adjusted OR, 0.38; 95% CI, 0.15-0.95, P=0.038). Further analysis of the endoplasmic reticulum stress (ER stress) signaling pathway of GCs found that expressions of GRP78, total JNK, phosphorylated JNK, and CHOP increased and BCL-2 decreased with increasing BLLs.CONCLUSIONS:BLLs are negatively associated with final clinical outcomes in IVF patients that may be related to increased ER stress response and GC apoptosis. Thus, reducing Pb exposure before IVF procedures may improve final success rates.

BackgroundThe mammalian testicular interstitial cells are not well-defined. The present study characterized the interstitial cell types and their turnover dynamics in adult rats. Additionally, the heterogeneity of the mesenchymal population and the effects of Leydig cell elimination on interstitial homeostasis were further analyzed by scRNA-seq datasets and immunocytochemical techniques.MethodsInterstitial cells were defined at the transcriptomic level by scRNA-seq and then confirmed and quantified with protein markers. The dividing activity of the major cell types was determined by continuous EdU labeling of the animals for one week. Some of the rats were also treated with a dose of ethylenedimethylsulfonate (EDS) to examine how the loss of Leydig cells (LCs) could affect interstitial homeostasis for three weeks.ResultsSeven interstitial cell types were identified, including cell types (percentage of the whole interstitial population) as follows: Leydig (44.6%), macrophage and dendritic (19.1%), lymphoid (6.2%), vascular endothelial (7.9%), smooth muscle (10.7%), and mesenchymal (11.5%) cells. The EdU experiment indicated that most cell types were dividing at relatively low levels (<9%) except for the mesenchymal cells (MCs, 17.1%). Further analysis of the transcriptome of MCs revealed 4 subgroups with distinct functions, including 1) glutathione metabolism and xenobiotic detoxification, 2) ROS response and AP-1 signaling, 3) extracellular matrix synthesis and binding, and 4) immune response and regulation. Stem LCs (SLCs) are primarily associated with subgroup 3, expressing ARG1 and GAP43. EDS treatment not only eliminated LCs but also increased subgroup 3 and decreased subgroups 1 and 2 of the mesenchymal population. Moreover, EDS treatment increased the division of immune cells by more than tenfold in one week.ConclusionSeven interstitial cell types were identified and quantified for rat testis. Many may play more diversified roles than previously realized. The elimination of LCs led to significant changes in MCs and immune cells, indicating the importance of LCs in maintaining testicular interstitial homeostasis.

In brief:Progenitor cells with ovulation-related tissue repair activity were identified with defined markers (LGR5, EPCR, LY6A, and PDGFRA), but their potentials to form steroidogenic cells were not known. This study shows that the cells can generate progenies with different steroidogenic activities. Abstract:Adult mammalian ovaries contain stem/progenitor cells necessary for folliculogenesis and ovulation-related tissue rupture repair. Theca cells are recruited and developed from progenitors during the folliculogenesis. Theca cell progenitors were not well defined. The aim of current study is to compare the potentials of four ovarian progenitors with defined markers (LY6A, EPCR, LGR5, and PDGFRA) to form steroidogenic theca cells in vitro. The location of the progenitors with defined makers was determined by immunohistochemistry and immunofluorescence staining of ovarian sections of adult mice. Different progenitor populations were purified by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) techniques from ovarian cell preparation and were tested for their abilities to generate steroidogenic theca cells in vitro. The cells were differentiated with a medium containing LH, ITS, and DHH agonist for 12 days. The results showed that EPCR+ and LGR5+ cells primarily distributed along the ovarian surface epithelium (OSE), while LY6A+ cells distributed in both the OSE and parenchyma. However, PDGFRA+ cells were exclusively located in interstitial compartment. When the progenitors were purified by these markers and differentiated in vitro, LY6A+ and PDGFRA+ cells formed steroidogenic cells expressing both CYP11A1 and CYP17A1 and primarily producing androgens, showing characteristics of theca-like cells, while LGR5+ cells generated steroidogenic cells devoid of CYP17A1 expression and androgen production, showing a characteristic of progesterone-producing cells (granulosa- or lutea-like cells). In conclusion, progenitors from both OSE and parenchyma of adult mice are capable of generating steroidogenic cells with different steroidogenic capacities, showing a possible lineage preference.

The effect of heavy metal cadmium (Cd) on testicular function is recognized. However, the mechanism involved is not well-established. In the present study, we analyzed the testicular transcriptomic changes induced by acute Cd exposure of adult rats with and without supplementation of antioxidants selenium (Se) and/or coenzyme Q10 (CoQ). Cd significantly decreased serum testosterone and two steroidogenic proteins SCARB1 and STAR. RNA-Seq analyses of testicular RNAs revealed specific activation of oxidative stress-, inflammation-, MAPK- and NF-κB-related signaling molecules. In addition, Cd treatment down-regulated gene for I, III and IV complexes of mitochondrial electron transport chain and up-regulated genes for NADPH-oxidase, major cascade in ROS production. The decrease in steroidogenesis and increase in inflammation may result from oxidative stress since supplementation of Se and CoQ, but not with either alone, almost completely prevented these changes, including overall alterations in transcriptome. Cd exposure induced total of 1192 differentially expressed genes (DEGs), which was reduced to 29 without considering confounding factors associated with Se/CoQ, a 97.6% protection rate. In conclusion, Cd exposure inhibited Leydig cell steroidogenesis by down-regulating SCARB1 and STAR through increasing oxidative stress and inflammation, but Se plus CoQ synergistically prevented all the changes induced by the Cd exposure.

Correction to: Asian Journal of Andrology 2022; 24 (4): 335-44. Doi: 10.4103/aja202193. In the published article by Li et al.,1 an error occurred in the Acknowledgments. The funding agency should be “the Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515010947)”, not “the Natural Science Foundation of Guangdong Province (No. 2021A1515010947)”. The authors sincerely apologize for the error.

BackgroundTestosterone plays a critical role in maintaining reproductive functions and well-beings of the males. Adult testicular Leydig cells (LCs) produce testosterone and are generated from stem Leydig cells (SLCs) during puberty through adulthood. In addition, macrophages are critical in the SLC regulatory niche for normal testicular function. Age-related reduction in serum testosterone contributes to a number of metabolic and quality-of-life changes in males, as well as age-related changes in immunological functions. How aging and testicular macrophages may affect SLC function is still unclear.MethodsSLCs and macrophages were purified from adult and aged mice via FACS using CD51 as a marker protein. The sorted cells were first characterized and then co-cultured in vitro to examine how aging and macrophages may affect SLC proliferation and differentiation. To elucidate specific aging effects on both cell types, co-culture of sorted SLCs and macrophages were also carried out across two ages.ResultsCD51+ (weakly positive) and CD51++ (strongly positive) cells expressed typical SLC and macrophage markers, respectively. However, with aging, both cell types increased expression of multiple cytokine genes, such as IL-1b, IL-6 and IL-8. Moreover, old CD51+ SLCs reduced their proliferation and differentiation, with a more significant reduction in differentiation (2X) than proliferation (30%). Age matched CD51++ macrophages inhibited CD51+ SLC development, with a more significant reduction in old cells (60%) than young (40%). Crossed-age co-culture experiments indicated that the age of CD51+ SLCs plays a more significant role in determining age-related inhibitory effects. In LC lineage formation, CD51+ SLC had both reduced LC lineage markers and increased myoid cell lineage markers, suggesting an age-related lineage shift for SLCs.ConclusionThe results suggest that aging affected both SLC function and their regulatory niche cell, macrophages.

EDITORIAL article Front. Endocrinol., 19 May 2023Sec. Reproduction Volume 14 - 2023 | https://doi.org/10.3389/fendo.2023.1207907

Epidemic studies showed that lead exposures are associated with various female reproductive dysfunctions, including infertility, miscarriage, preterm delivery, and early menopause. However, the mechanism involved is still unclear. In the current study, SD rats were exposed to lead at doses of 0, 5, 25, 50 or 250 mg/L through drinking water from postnatal day 21-56. Lead exposures did not affect the body weight or ovary weight. However, the puberty initiation (ages by which vagina opens and estrous cycle occurs) was significantly delayed by as many as 5.8 and 6.8 days respectively (P < 0.05). Also, lead exposures disrupted the estrous cycles, reduced the numbers of primordial and primary follicles and increased the number of atretic follicles by adult. Furthermore, for the highest does group, serum levels of progesterone and testosterone decreased by 80.2% (P < 0.01) and 49.9% (P < 0.05) respectively, while estradiol level increased by 69.8% (P < 0.01). Western blot analyses indicated that lead exposures specifically down-regulated the expressions of steroidogenic protein STAR, CYP17A1, and HSD3B1, while up-regulated FSHR and CYP19A1. Also, the exposure stimulated the endoplasmic reticulum stress (ERS)-related IRE1a-JNK signaling pathway members. Such activation may also result in apoptosis since the death-signaling molecules CHOP and cleaved-CASP3 were up-regulated while BCL2 was down-regulated. In conclusion, lead exposure during juvenile and puberty significantly affected ovary development and functions. The effects may relate to ERS response since the 6 members related to the pathway were all consistently activated.

Advanced paternal age has increasingly been recognized as a risk factor for male fertility and progeny health. While underlying causes are not well understood, aging is associated with a continuous decline of blood and tissue NAD + levels, as well as a decline of testicular functions. The important basic question to what extent ageing-related NAD + decline is functionally linked to decreased male fertility has been difficult to address due to the pleiotropic effects of aging, and the lack of a suitable animal model in which NAD + levels can be lowered experimentally in chronologically young adult males. We therefore developed a transgenic mouse model of acquired niacin dependency (ANDY), in which NAD + levels can be experimentally lowered using a niacin-deficient, chemically defined diet. Using ANDY mice, this report demonstrates for the first time that decreasing body-wide NAD + levels in young adult mice, including in the testes, to levels that match or exceed the natural NAD + decline observed in old mice, results in the disruption of spermatogenesis with small testis sizes and reduced sperm counts. ANDY mice are dependent on dietary vitamin B3 (niacin) for NAD + synthesis, similar to humans. NAD + -deficiency the animals develop on a niacin-free diet is reversed by niacin supplementation. Providing niacin to NAD + -depleted ANDY mice fully rescued spermatogenesis and restored normal testis weight in the animals. The results suggest that NAD + is important for proper spermatogenesis and that its declining levels during aging are functionally linked to declining spermatogenesis and male fertility. Functions of NAD + in retinoic acid synthesis, which is an essential testicular signaling pathway regulating spermatogonial proliferation and differentiation, may offer a plausible mechanism for the hypospermatogenesis observed in NAD + -deficient mice.

Bisphenol A (BPA) is widely used by manufacturers and in consumer products. Its release in the environment may affect male reproductive function. In this study, we examined the effect of low dose (0.1 mg/kg BW), short term exposure during puberty (PD21-35) on adult rat male reproduction. The results indicated that such exposure reset growth hormone (GH) and follicular stimulating hormone (FSH) homeostasis and resulted in a significantly higher level of serum testosterone without affecting serum luteinizing hormone level. QPCR and Western blot results showed that BPA significantly up-regulated selective genes/proteins in the Leydig cell steroidogenic pathway, including steroidogenic acute regulatory protein, cytochrome P450 11A1, cytochrome P450 17A, and low-density lipoprotein receptor. RNA-Seq analysis of testicular RNAs showed that BPA significantly affected the gene profiles of multiple testicular interstitial populations without affecting germ cells. Also, GO- and KEGGanalysis suggested that IGF1-related PI3K/AKT signaling was activated, which was confirmed by the increased phosphorylation of IRS1, AKT1 and CREB. The results indicated that a low-dose, short-term BPA exposure during puberty affected the adult male rat pituitary (GH and FSH) and testis (testosterone) homeostasis.

Spermatogenesis is an efficient, complex, and highly organized proliferation and differentiation process that relies on multiple factors including testosterone produced by the Leydig cells. Although the critical role played by testosterone in spermatogenesis is well recognized, the mechanism by which it works is still not completely understood, partially due to the inability to specifically and precisely monitor testosterone-dependent changes within developing germ cells. Here we present single-cell RNA sequencing data from10,983 adult rat testicular cells after the rats were treated with ethanedimethanesulfonate, which temporarily eliminates Leydig cells. The elimination and recovery of Leydig cells represented a complete testosterone depletion and restoration cycle. The dataset, which includes all developing germ cells from spermatogonia to spermatozoa, should prove useful for characterizing developing germ cells, their regulatory networks, and novel cell-specific markers. The dataset should be particularly useful for exploring the effects of the androgen environment on the regulation of spermatogenesis. As this is the first single-cell RNA-Seq dataset for rat testes, it can also serve as a reference for future studies.

Stem Leydig cells (SLCs) play a critical role in the development and maintenance of the adult Leydig cell (ALC) population. SLCs also are present in the adult testis. Their identification, characteristics, and regulation in the adult testis remain uncertain. Using single-cell RNA-seq, we found that the mesenchymal stromal population may be involved in ALC regeneration. Upon ALC elimination, a fraction of stromal cells begins to proliferate while a different fraction begins to differentiate to ALCs. Transcriptomic analysis identified five stromal clusters that can be classified into two major groups representing proliferation and differentiation populations. The proliferating group represents stem cells expressing high levels of CD90, Nes, Lum, Fn and Gap43. The differentiating group represents a progenitor stage that is ready to form ALCs, and specifically expresses Vtn, Rasl11a, Id1 and Egr2. The observation that the actively dividing cells after ALC loss were not those that formed ALCs suggests that stem cell proliferation and differentiation are regulated separately, and that the maintenance of the stromal stem cell pool occurs at the population level. The study also identified specific markers for the major interstitial cell groups and potential paracrine factors involved in the regulation of SLCs. Our data suggest a new theory about SLC identity, proliferation, differentiation, and regulation.

Testosterone production by Leydig cells (LCs) plays a crucial role in male reproduction. The functional degeneration of LCs can cause testosterone deficiency, ultimately resulting in primary male hypogonadism. Transplantation of exogenous LCs with the ability to produce testosterone in response to the regulation of the hypothalamus–pituitary–gonad axis could be a promising alternative option to treat male primary hypogonadism. Recent studies have shown that it is possible to generate Leydig-like cells from stem cells by various approaches. In addition, somatic cells, such as embryonic or adult fibroblasts, have also been successfully reprogrammed into Leydig-like cells. In this review, we summarized the recent advances in the generation of Leydig-like cells, with an emphasis on comparing the effectiveness and safety of different protocols used and the cells generated. By further analyzing the characteristics of Leydig-like cells generated from fibroblasts based on small signaling molecules and regulatory factors, we found that although the cells may produce testosterone, they are significantly different from real LCs. For future in vivo applications, it is important that the steroidogenic cells generated be evaluated not only for their steroidogenic functions but also for their overall cell metabolic state by proteomics or transcriptomic tools.

Androgen deficiency is a common medical conditions that affects males of all ages. Transplantation of testosterone-producing cells is a promising treatment for male hypogonadism. However, getting a cell source with the characteristics of Leydig cells (LCs) is still a challenge. Here, a high-efficiency reprogramming of skin-derived fibroblasts into functional Leydig-like cells (LLCs) based on epigenetic mechanism was described. By performing an integrated analysis of genome-wide DNA methylation and transcriptome profiling in LCs and fibroblasts, the potentially epigenetic-regulating steroidogenic genes and signaling pathways were identified. Then by using CRISPR/dCas9 activation system and signaling pathway regulators, the male- or female-derived fibroblasts were reprogrammed into LLCs with main LC-specific traits. Transcriptomic analysis further indicated that the correlation coefficients of global genes and transcription factors between LLCs and LCs were higher than 0.81 and 0.96, respectively. After transplantation in the testes of hypogonadal rodent models, LLCs increased serum testosterone concentration significantly. In type 2 diabetic rats model, LLCs which were transplanted in armpit, have the capability to restore the serum testosterone level and improve the hyperglycemia status. In conclusion, our approach enables skin-derived fibroblasts reprogramming into LLCs with high fidelity, providing a potential cell source for the therapeutics of male hypogonadism and metabolic-related comorbidities.