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Stem cells are specialized cells that can renew themselves through cell division and can differentiate into multi-lineage cells. Mesenchymal stem cells are adult stem cells that exist in animal and human tissues. Mesenchymal stem cells have the ability to differentiate into mesodermal lineages, such as Leydig cells, adipocytes, osteocytes, and chondrocytes. Mesenchymal stem cells express cell surface markers, such as cluster of differentiation (CD) 29, CD44, CD73, CD90, CD105, and lack the expression of CD14, CD34, CD45 and HLA (human leukocyte antigen)-DR. Stem Leydig cells are one kind of mesenchymal stem cells, which are present in the interstitial compartment of testis. Stem Leydig cells are multipotent and can differentiate into Leydig cells, adipocytes, osteocytes, and chondrocytes. Stem Leydig cells have been isolated from rodent and human testes. Stem Leydig cells may have potential therapeutic values in several clinical applications, such as the treatment of male hypogonadism and infertility. In this review, we focus on the latest research on stem Leydig cells of both rodents and human, the expression of cell surface markers, culture, differentiation potential, and their applications.

BACKGROUND Oncostatin M (OSM) is a member of the interleukin-6 group of cytokines, which can regulate cell proliferation, growth, and function. Immature Leydig cells have the ability to proliferate and differentiate, and adult Leydig cells have the function of testosterone synthesis. However, the role and underlying mechanisms of OSM on the proliferation and function of Leydig cells remain unclear. METHODS The effects of OSM on the proliferation, apoptosis, and function of immature Leydig cells isolated from 35-day-old rats and the function of adult Leydig cells isolated from 63-day-old rats in vitro. RESULTS OSM stimulated immature Leydig cell proliferation after up-regulating the expression of Ccnd1 and Cdk4 to drive the transition of G1 phase to M2 phase in the cell cycle at 10 and 100 ng/ml. OSM did not affect the apoptosis of immature Leydig cells up to 100 ng/ml. OSM inhibited testosterone production in immature and adult Leydig cells by down-regulating the expression of Lhcgr, Star, Cyp11a1, Hsd3b1, and Cyp17a1 at 1-100 ng/ml. OSM induced reactive oxygen species and down-regulated the expression of antioxidant genes and lowered mitochondrial membrane potential at 10 and 100 ng/ml in both Leydig cells. Janus kinase 1 (JAK1) antagonist filgotinib and signal transducer and activator of transcription 3 (STAT3) antagonist S3I-201 reversed the effect of OSM, indicating that it acts on JAK1/STAT3 signaling. CONCLUSION Oncostatin M stimulates immature Leydig cell proliferation while inhibiting the function of immature and adult Leydig cells.

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

Collagen α3 (IV) chains are one of the major constituent components of the basement membrane in the mammalian testis. Studies have shown that biologically active fragments, such as noncollagenase domain (NC1)-peptide, can be released from the C-terminal region of collagen α3 (IV) chains, possibly through the proteolytic action of metalloproteinase 9 (MMP9). NC1-peptide was shown to promote blood-testis barrier (BTB) remodeling and fully developed spermatid (e.g., sperm) release from the seminiferous epithelium because this bioactive peptide was capable of perturbing the organization of both actin- and microtubule (MT)-based cytoskeletons at the Sertoli cell-cell and also Sertoli-spermatid interface, the ultrastructure known as the basal ectoplasmic specialization (ES) and apical ES, respectively. More importantly, recent studies have shown that this NC1-peptide-induced effects on cytoskeletal organization in the testis are mediated through an activation of mammalian target of rapamycin complex 1/ribosomal protein S6/transforming retrovirus Akt1/2 protein (mTORC1/rpS6/Akt1/2) signaling cascade, involving an activation of cell division control protein 42 homolog (Cdc42) GTPase, but not Ras homolog family member A GTPase (RhoA), and the participation of end-binding protein 1 (EB1), a microtubule plus (+) end tracking protein (+TIP), downstream. Herein, we critically evaluate these findings, providing a critical discussion by which the basement membrane modulates spermatogenesis through one of its locally generated regulatory peptides in the testis.

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

Actin cytoskeleton is crucial to support spermatogenesis in the mammalian testis. However, the molecular mechanism(s) underlying changes of actin cytoskeletal organization in response to cellular events that take place across the seminiferous epithelium (e.g., self-renewal of spermatogonial stem cells, germ cell differentiation, meosis, spermiogenesis, spermiation) at specific stages of the epithelial cycle of spermatogenesis remain largely unexplored. This, at least in part, is due to the lack of suitable study models to identify the crucial regulatory proteins and to investigate how these proteins work in concert to support actin dynamics. Much of the information on the role of actin binding proteins in the literature, namely the actin bundling proteins, actin nucleation proteins and motor proteins, are either findings based on genetic models or morphological analyses. While this information is helpful to delineate the function of these proteins to support spermatogenesis, they are not helpful to identify the regulatory signaling proteins, the signaling pathways and the cascade of events to modulate actin cytoskeleton dynamics. Recent studies based on the use of toxicant models, both in vitro and in vivo, however, have bridged this gap by identifying putative regulatory and signaling proteins of actin cytoskeleton. Herein, we summarize and critically evaluate these findings. We also provide a hypothetical model by which actin cytoskeletal dynamics in Sertoli cells are regulated, which in turn supports spermatid transport across the seminiferous epithelium, and at the blood-testis barrier (BTB) during the epithelial cycle of spermatogenesis.

Studies have shown that mammalian testes, in particular the Sertoli cells, are highly susceptible to exposure of environmental toxicants, such as cadmium, perfluorooctanesulfonate, phthalates, 2,5-hexanedione and bisphenol A. However, important studies conducted by reproductive toxicologists and/or biologists in the past have been treated as toxicology reports per se. Yet, many of these studies provided important mechanistic insights on the toxicant-induced testis injury and reproductive dysfunction, relevant to the biology of the testis and spermatogenesis. Furthermore, recent studies have shown that findings obtained from toxicant models are exceedingly helpful tools to unravel the biology of testis function in particular spermatogenesis, including specific cellular events associated with spermatid transport to support spermiogenesis and spermiation. In this review, we critically evaluate some recent data, focusing primarily on the molecular structure and role of microtubules in cellular function, illustrating the importance of toxicant models to unravel the biology of microtubule cytoskeleton in supporting spermatogenesis, well beyond information on toxicology. These findings have opened up some potential areas of research which should be carefully evaluated in the years to come.

95% of the body's testosterone is produced by the Leydig Cells (LCs) in adult testis, and LC functional degradation can cause testosterone deficiency ultimately leading towards hypogonadism. The transplantation of LCs derived from stem cells is a very promising therapy to overcome the testosterone deficiency. The isolated umbilical cord mesenchymal stem cells (UMSCs) were identified by flow cytometry and adipogenic and osteogenic differentiation. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were used for the differentiated Leydig-like cell identification. The comparisons of the testosterone levels, gene expression levels, and cyclic adenosine monophosphate (cAMP) productions were performed through radioimmunoassay, quantitative polymerase chain reaction (qPCR), and cAMP assay kit, respectively. Here, it is stated that our isolated human UMSCs, which could positively express CD29, CD44, CD59, CD90, CD105, and CD166 but negatively express CD34 as well as could be differentiated into adipocytes and osteocytes, could be differentiated into Leydig-like cells (UMSC-LCs) using a novel differentiation method based on molecular compounds. The enrichment UMSC-LCs could secrete testosterone into the medium supernatant and produce considerable cAMP at the stimulation of luteinizing hormone (LH), and positively expressed LC lineage-typical markers LHCGR, SCARB1, SATR, CYP11A1, CYP17A1, HSD3B1, HSD17B3, and SF-1 as well as negatively expressed mesenchymal stem cell typical markers CD29, CD44, and CD105. The expression levels of NR3C4, PDGFRA, and NR3A1 in UMSC-LCs were higher than those of UMSCs and were comparable with LCs. These results illuminated that UMSCs could be differentiated into Leydig-like cells using the defined molecular compounds, which might further support MSC-derived Leydig cell transplantation therapy for testosterone insufficiency.

Spermatogenesis is supported by intricate crosstalk between Sertoli cells and germ cells including spermatogonia, spermatocytes, haploid spermatids, and spermatozoa, which takes place in the epithelium of seminiferous tubules. Sertoli cells, also known as 'mother' or 'nurse' cells, provide nutrients, paracrine factors, cytokines, and other biomolecules to support germ cell development. Sertoli cells facilitate the generation of several biologically active peptides, which include F5-, noncollagenous 1 (NC1)-, and laminin globular (LG)3/4/5-peptide, to modulate cellular events across the epithelium. Here, we critically evaluate the involvement of these peptides in facilitating crosstalk between Sertoli and germ cells to support spermatogenesis and thus fertility. Modulating or mimicking the activity of F5-, NC1-, and LG3/4/5-peptide could be used to enhance the transport across the blood-testis barrier (BTB) of contraceptive drugs or to treat male infertility.

During spermatogenesis, preleptotene spermatocytes and haploid spermatids, lacking lamellipodia and filopodia to initiate cell movement per se, but rely on Sertoli cells for transport across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. Tracks provided by microtubules (MTs) that lay across the epithelium are essential to support germ cell and other cargo transports, but the mechanism(s) remain elusive. Studies have provided insightful information through the use of toxicant models. Herein, we summarize findings based on studies of the microtubule plus (+)-end tracking proteins (+TIPs) and the microtubule minus (-)-end targeting proteins (-TIPs), at the corresponding plus (+)-end and minus (-)-end of the polarized MTs in rat testes. We also provide a model by which + TIPs and -TIPs that work in concert with microtubule-associated proteins (MAPs; e.g., MAP-1a), MARKs (microtubule affinity-regulating kinases), and microtubule-specific motor proteins (e.g., dynein 1) to support germ cell and cargo transports. This thus provides a framework to design experiments for future studies.

Epidemiological studies have demonstrated that the genetic factors partly influence the development of same-sex sexual behavior, but most genetic studies have focused on people of primarily European ancestry, potentially missing important biological insights. Here, we performed a two-stage genome-wide association study (GWAS) with a total sample of 1478 homosexual males and 3313 heterosexual males in Han Chinese populations and identified two genetic loci (rs17320865, Xq27.3, FMR1NB, P

Epidemiological studies have demonstrated that the genetic factors partly influence the development of same-sex sexual behavior, but most genetic studies have focused on people of primarily European ancestry, potentially missing important biological insights. Here, we performed a two-stage genome-wide association study (GWAS) with a total sample of 1478 homosexual males and 3313 heterosexual males in Han Chinese populations and identified two genetic loci (rs17320865, Xq27.3, FMR1NB, Pmeta = 8.36 × 10−8, OR = 1.29; rs7259428, 19q12, ZNF536, Pmeta = 7.58 × 10−8, OR = 0.75) showing consistent association with male sexual orientation. A fixed-effect meta-analysis including individuals of Han Chinese (n = 4791) and European ancestries (n = 408,995) revealed 3 genome-wide significant loci of same-sex sexual behavior (rs9677294, 2p22.1, SLC8A1, Pmeta = 1.95 × 10−8; rs2414487, 15q21.3, LOC145783, Pmeta = 4.53 × 10−9; rs2106525, 7q31.1, MDFIC, Pmeta = 6.24 × 10−9). These findings may provide new insights into the genetic basis of male sexual orientation from a wider population scope. Furthermore, we defined the average ZNF536-immunoreactivity (ZNF536-ir) concentration in the suprachiasmatic nucleus (SCN) as lower in homosexual individuals than in heterosexual individuals (0.011 ± 0.001 vs 0.021 ± 0.004, P = 0.013) in a postmortem study. In addition, compared with heterosexuals, the percentage of ZNF536 stained area in the SCN was also smaller in the homosexuals (0.075 ± 0.040 vs 0.137 ± 0.103, P = 0.043). More homosexual preference was observed in FMR1NB-knockout mice and we also found significant differences in the expression of serotonin, dopamine, and inflammation pathways that were reported to be related to sexual orientation when comparing CRISPR-mediated FMR1NB knockout mice to matched wild-type target C57 male mice.

Triadimefon is a broad-spectrum fungicide widely applied in the agriculture. It is believed to be an endocrine disruptor. Whether triadimefon can inhibit the development of fetal Leydig cells and the underlying mechanisms are unknown. Thirty-two female pregnant Sprague-Dawley rats were randomly assigned into four groups and were dosed via gavage of triadimefon (0, 25, 50, and 100 mg/kg/day) for 9 days from gestational day (GD) 12–20. Triadimefon significantly reduced serum testosterone level in male fetuses at 100 mg/kg. The double immunofluorescence staining of proliferating cell nuclear antigen (PCNA) and cytochrome P450 cholesterol side-chain cleavage (a biomarker for fetal Leydig cells) was used to measure PCNA-labeling in fetal Leydig cells. It markedly increased fetal Leydig cell number primarily via increasing single cell population and elevated the PCNA-labeling of fetal Leydig cells in male fetuses at 100 mg/kg while it induced abnormal aggregation of fetal Leydig cells. The expression levels of fetal Leydig cell genes, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3 and Nr5a1, were determined to explore its effects on fetal Leydig cell development. We found that triadimefon markedly down-regulated the expression of Leydig cell genes, Hsd17b3, Insl3, and Nr5a1 as low as 25 mg/kg and Scarb1 and Cyp11a1 at 100 mg/kg. It did not affect Sertoli cell number but markedly down-regulated the expression of Sertoli cell gene Amh at 50 and 100 mg/kg. Triadimefon significantly down-regulated the expression of antioxidant genes Sod1, Gpx1, and Cat at 25–100 mg/kg, suggesting that it can induce oxidative stress in fetal testis, and it reduced the phosphorylation of ERK1/2 and AKT2 at 100 mg/kg, indicating that it can inhibit the development of fetal Leydig cells. In conclusion, gestational exposure to triadimefon inhibits the development of fetal Leydig cells in male fetuses by inhibiting its differentiation.

Phthalates are plasticizers widely found in the environment. They are potential endocrine disruptors. Bis(2-butoxyethyl) phthalate (BBOP) is a unique phthalate that contains oxygen atoms in the carbon backbone. Little is known about its reproductive and developmental toxicity. The objective of this study was to determine the effect of BBOP on fetal Leydig cell development after in utero exposure to rats. Sprague Dawley pregnant dams were randomly allocated into 6 groups, and were gavaged with BBOP (0, 10, 100, 250, 500, and 1000 mg/kg body weight/day) from gestational day (GD) 14–21. Seven of the 8 dams in the 1000 mg/kg BBOP group died before giving birth. Twelve of the 20 dams in the 500 mg/kg BBOP group had whole litter loss. BBOP significantly reduced the body weight of dams and male offspring and serum testosterone level and anogenital distance of male fetus on GD 21 at 500 mg/kg. BBOP markedly increased fetal Leydig cell proliferation and number at 500 mg/kg while inducing their abnormal aggregation at 250 and 500 mg/kg. BBOP down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, and Nr5a1 at various doses while up-regulating the expression of Sertoli cell gene Fshr and Sox9. The phosphorylation of AKT1, AKT2, and ERK1/2 was also markedly reduced by BBOP. In conclusion, BBOP in utero exposure can disrupt fetal Leydig cell development, possibly via the mechanism that may include inhibiting the phosphorylation of AKT1, AKT2, and ERK1/2.

Polybrominated diphenyl ethers (PBDEs) are a group of flame retardants with two or more bromines attached. They are endocrine disruptors. PBDEs photodegrade into 4-bromodiphenyl ether (BDE3). Whether BDE3 impairs adrenal cortical cell function during postnatal development still remains unknown. The aim of the current study was to investigate the influence of BDE3 on adrenal cortical cell function. Sprague-Dawley rats (35 days of age, male) were orally administered with BDE3 (0, 50, 100, and 200 mg/kg/day body weight) for 21 days. BDE3 significantly increased serum aldosterone and corticosterone levels at 200 mg/kg without affecting adrenocorticotropic hormone level. Further study showed that BDE3 up-regulated Cyp11b1 at 100 and 200 mg/kg and Scarb1, Star, Cyp11b2, Cyp21, and Nr5a1 mRNA levels in the 200 mg/kg group. BDE3 also decreased the phosphorylation of AMP-activated protein kinase (AMPK) at 200 mg/kg and increased PGC-1α and phosphorylated cyclic AMP-responsive element-binding protein (CREB)/CREB at 200 mg/kg. Taken together, these findings demonstrate that BDE3 stimulates adrenal cell function likely through decreasing phosphorylation of AMPK and increasing phosphorylation of CREB.

During spermatogenesis, cell organelles, and germ cells, most notably haploid spermatids, are transported across the seminiferous epithelium so that fully developed spermatids line-up at the edge of the tubule lumen to undergo spermiation at stage VIII of the cycle. Studies have suggested that the microtubule (MT)-based cytoskeleton is necessary to support these cellular events. However, the regulatory molecule(s) and underlying mechanism(s) remain poorly understood. Herein, we sought to better understand this event by using an adjudin-based animal model. Adult rats were treated with adjudin at low-dose (10 mg/kg b.w.) which by itself had no notable effects on spermatogenesis. Rats were also treated with low-dose adjudin combined with overexpression of 2 endogenously produced blood-testis barrier (BTB) modifiers, namely rpS6 (ribosomal protein S6, the downstream signaling protein of mammalian target of rapamycin complex 1 [mTORC1]) and F5-peptide (a biological active peptide released from laminin-γ3 chain at the Sertoli-spermatid interface) versus the 2 BTB modifiers alone. Overexpression of these 2 BTB modifiers in the testis was shown to enhance delivery of adjudin to the testis, effectively inducing disruptive changes in MT cytoskeletons, causing truncation of MT conferred tracks that led to their collapse across the epithelium. The net result was massive germ cell exfoliation in the tubules, disrupting germ cell transport and cell adhesion across the seminiferous epithelium that led to aspermatogenesis. These changes were the result of disruptive spatial expression of several MT-based regulatory proteins. In summary, MT cytoskeleton supported by the network of MT regulatory proteins is crucial to maintain spermatogenesis.

Leydig cells (LCs) are the primary source of testosterone in the testis, and testosterone deficiency caused by LC functional degeneration can lead to male reproductive dysfunction. LC replacement transplantation is a very promising approach for this disease therapy. Here, we report that human adipose derived stem cells (ADSCs) can be differentiated into Leydig‐like cells using a novel differentiation method based on molecular compounds. The isolated human ADSCs expressed positive CD29, CD44, CD59 and CD105, negative CD34, CD45 and HLA‐DR using flow cytometry, and had the capacity of adipogenic and osteogenic differentiation. ADSCs derived Leydig‐like cells (ADSC‐LCs) acquired testosterone synthesis capabilities, and positively expressed LC lineage‐specific markers LHCGR, STAR, SCARB1, SF‐1, CYP11A1, CYP17A1, HSD3B1 and HSD17B3 as well as negatively expressed ADSC specific markers CD29, CD44, CD59 and CD105. When ADSC‐LCs labelled with lipophilic red dye (PKH26) were injected into rat testes which were selectively eliminated endogenous LCs using ethylene dimethanesulfonate (EDS, 75 mg/kg), the transplanted ADSC‐LCs could survive and function in the interstitium of testes, and accelerate the recovery of blood testosterone levels and testis weights. These results demonstrated that ADSCs could be differentiated into Leydig‐like cells by few defined molecular compounds, which might lay the foundation for further clinical application of ADSC‐LC transplantation therapy.

Human placental CYP19A1 catalyzes the estrogen synthesis from androgens. The enzyme is encoded by CYP19A1 gene located in chromosome 15q21. This enzyme is a monooxygenase in the smooth endoplasmic reticulum. The various promoters of the CYP19A1 gene determine its expression in different tissues and the distal promoter I.1 controls its expression in the placenta and retinoids can regulate the expression. Many food components and environmental chemicals inhibit CYP19A1 activity via different modes of action. These chemicals include gossypol, flavones, flavanones, chalconoids, resveratrol, and tobacco alkaloids derived from foods as well as phthalates, insecticides, fungicides, and biocides in the contaminated foods. The inhibition of placental CYP19A1 could impair pregnancy.

Bisphenol A (BPA) is widely used in consumer products and is a potential endocrine disruptor linked with abnormal development of male reproductive tract. However, its action and its effects on the pathways in the development of male gonad are still unclear. Here we report that effects of BPA exposure during gestation on male gonad development. Sprague-Dawley rats were gavaged daily with BPA (0, 4, 40, and 400 mg/kg body weight) from gestational day 12 to day 21. BPA dose-dependently decreased serum testosterone levels (0.45 ± 0.08 ng/ml and 0.32 ± 0.08 ng/ml for 40 and 400 mg/kg BPA, respectively) versus the control level (1.11 ± 0.22 ng/ml, Mean ± SE). BPA lowered Leydig cell Insl3 and Hsd17b3 mRNA and their protein levels at doses of 40 and 400 mg/kg. BPA also lowered Leydig cell (Lhcgr, Cyp11a1, and Cyp17a1) and Sertoli cell (Amh) mRNA and their protein levels at 400 mg/kg. BPA decreased fetal Leydig cell number via inhibiting their proliferation, but it did not affect fetal Sertoli cell number. In conclusion, the current study shows that in utero exposure to BPA inhibits fetal Leydig and Sertoli cell differentiation, possibly disrupting the development of male reproductive tract.

Leydig cells (LCs) play crucial roles in producing testosterone, which is critical in the regulation of male reproduction and development. Low levels of testosterone will lead to male hypogonadism. LC transplantation is a promising alternative therapy for male hypogonadism. However, the source of LCs limits this strategy for clinical applications. Thus far, others have reported that LCs can be derived from stem cells by gene transfection, but the safe and effective induction method has not yet been reported. Here, we report that Leydig-like cells can be derived from human induced pluripotent stem cells (iPSCs) using a novel differentiation protocol based on molecular compounds. The iPSCs-derived Leydig-like cells (iPSC-LCs) acquired testosterone synthesis capabilities, had the similar gene expression profiles with LCs, and positively expressed Leydig cell lineage-specific protein markers LHCGR, STAR, SCARB1, SF-1, CYP11A1, HSD3B1, and HSD17B3 as well as negatively expressed iPSC-specific markers NANOG, OCT4, and SOX2. When iPSC-LCs labeled with lipophilic red dye (PKH26) were transplanted into rat testes that were selectively eliminated endogenous LCs using EDS (75 mg/kg), the transplanted iPSC-LCs could survive and function in the interstitium of testes, and accelerate the recovery of serum testosterone levels and testis weights. Collectively, these findings demonstrated that the iPSCs were able to be differentiated into Leydig-like cells by few defined molecular compounds, which may lay the safer groundwork for further clinical application of iPSC-LCs for hypogonadism.

Lambda-cyhalothrin (LCT) is a widely used broad-spectrum pyrethroid insecticide and is expected to cause deleterious effects on the male reproductive system. However, the effects of LCT on Leydig cell development during puberty are unclear. The current study addressed these effects. Twenty-eight-day-old male Sprague Dawley rats orally received LCT (0, 0.25, 0.5 or 1 mg/kg body weight/day) for 30 days. The levels of serum testosterone, luteinizing hormone, and follicle-stimulating hormone, Leydig cell number, and its specific gene and protein expression were determined. LCT exposure lowered serum testosterone levels at doses of 0.5 and 1 mg/kg and luteinizing hormone levels at a dose of 1 mg/kg, but increased follicle-stimulating hormone levels at doses of 0.5 and 1 mg/kg. LCT lowered Star and Hsd3b1 mRNA or their protein levels at a dose of 1 mg/kg. Immature Leydig cells were purified from pubertal rats and treated with different concentrations of LCT for 24 h and medium androgen levels, Leydig cell mRNA and protein levels, the mitochondrial membrane potential (△Ψm), and the apoptotic rate of immature Leydig cells were investigated. LCT inhibited androgen production at 5 μM and downregulated Scarb1 at 0.05 μM, Hsd3b1 and Hsd11b1 at 0.5 μM, and Cyp11a1 at 5 μM. LCT also decreased △Ψm at 0.5 and 50 μM. In conclusion, LCT can influence the function of Leydig cells.

Background: Diisoheptyl phthalate (DIHP) is a phthalate plasticizer, which is a branched phthalate. Here, we reported the effects of gestational exposure to DIHP on testis development in male rats.Methods: Pregnant Sprague-Dawley rats were orally fed with vehicle (corn oil, control) or DIHP (10, 100, 500, and 1,000 mg/kg) from gestational day (GD) 12–21. At GD21, serum testosterone levels, the number and distribution of fetal Leydig cells, and testicular mRNA and protein levels, the incidence of multinucleated gonocytes, and focal testicular hypoplasia in the neonatal testis were measured.Results: DIHP increased the fetal Leydig cell cluster size and decreased the fetal Leydig cell size with LOAEL of 10 mg/kg. DIHP did not affect the fetal Leydig cell number. DIHP significantly lowered serum testosterone levels, down-regulated the expression of steroidogenesis-related genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and testis descent-related gene (Insl3) as well as protein levels of cholesterol side-chain cleavage enzyme (CYP11A1) and insulin-like 3 (INSL3). DIHP dose-dependently increased the percentage of multinucleated gonocytes with the low observed adverse-effect level (LOAEL) of 100 mg/kg. DIHP induced focal testicular hypoplasia.Conclusion: Gestational exposure to DIHP causes testis dysgenesis in rats.

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.

Bisphenol B (BPB) has been used as a substitute for bisphenol A (BPA) in plastic materials. Whether BPB disrupts the male reproductive system remains unknown. Here, we report the effect of BPB on Leydig cell maturation in late puberty. Male Sprague-Dawley (35 days old) rats were gavaged with BPB at 0, 10, 100, and 200 mg/kg/day for 21 days. BPB significantly reduced body and epididymis weight at 200 mg/kg. BPB markedly decreased serum testosterone levels at 100 and 200 mg/kg and serum luteinizing hormone and follicle-stimulating hormone levels at 200 mg/kg. BPB significantly increased Leydig cell number at 100 and 200 mg/kg, while down-regulating the expression of Leydig cell genes (Cyp11a1 and Hsd3b1) at ≥100 mg/kg and up-regulating the expression of Sertoli cell genes (Pdgfra, Fshr, Sox9) and cell cycle regulators (Pcna, Ccnb1, Cdk2, and Cdk4) at 10–200 mg/kg. BPB markedly increased the phosphorylation of AKT1, AKT2, and ERK1/2 at 200 mg/kg. BPB increased the proliferation of rat immature Leydig cells via promoting the S/M2 phase shift at 100 and 1000 nM after 24-h culture in vitro. In conclusion, BPB disrupts Leydig cell maturation in late puberty by increasing Leydig cell number while inhibiting its maturation.

Leydig cells of the testis have the capacity to synthesize androgen (mainly testosterone) from cholesterol. Adult Leydig cells are the cell type for the synthesis of testosterone, which is critical for spermatogenesis. At least four steroidogenic enzymes take part in testosterone synthesis: cytochrome P450 cholesterol side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase/17,20-lyase and 17β-hydroxysteroid dehydrogenase isoform 3. Testosterone metabolic enzyme steroid 5α-reductase 1 and 3α-hydroxysteroid dehydrogenase are expressed in some precursor Leydig cells. Androgen is transported by androgen-binding protein to Sertoli cells, where it binds to androgen receptor to regulate spermatogenesis.

Adjudin, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (formerly called AF-2364), is a nonhormonal male contraceptive, since it effectively induces reversible male infertility without perturbing the serum concentrations of follicle stimulating hormone (FSH), testosterone, and inhibin B based on studies in rats and rabbits. Adjudin was shown to exert its effects preferentially by perturbing the testis-specific actin-rich adherens junction (AJ) at the Sertoli–spermatid interface known as apical ectoplasmic specialization (apical ES), thereby effectively inducing spermatid exfoliation. Adjudin did not perturb germ cell development nor germ cell function. Also, it had no effects on Sertoli cell–cell AJ called basal ectoplasmic specialization (basal ES), which, together with tight junction constitute the blood-testis barrier (BTB), unless an acute dose of adjudin was used. Adjudin also did not perturb the population of spermatogonial stem cells nor Sertoli cells in the testis. However, the downstream signaling protein(s) utilized by adjudin to induce transient male infertility remains unexplored. Herein, using adult rats treated with adjudin and monitored changes in the phenotypes across the seminiferous epithelium between 6 and 96 h in parallel with the steady-state protein levels of an array of signaling and cytoskeletal regulatory proteins, recently shown to be involved in apical ES, basal ES and BTB function. It was shown that adjudin exerts its contraceptive effects through changes in microtubule associated proteins (MAPs) and signaling proteins mTORC1/rpS6 and p-FAK-Y407. These findings are important to not only study adjudin-mediated male infertility but also the biology of spermatogenesis.

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.

4-Bromodiphenyl ether (BDE3) is a photodegradation product of higher polybrominated diphenyl ether flame retardants and is known as an endocrine disruptor. However, it is unclear whether and how BDE3 affects the development of fetal testes. This study aimed to investigate the effect of in utero exposure to BDE3 on fetal testicular development in rats. From gestational day (GD) 12 to 21, BDE3 (0, 50, 100, and 200 mg/kg) was daily gavaged to female pregnant Sprague Dawley rats. BDE3 significantly reduced serum testosterone levels of male pups starting at 50 mg/kg. BDE3 reduced fetal Leydig cell number at a dose of 200 mg/kg without affecting fetal Leydig cell cluster frequency and Sertoli cell number. In addition, BDE3 down-regulated the expression of fetal Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and their proteins at 100 and/or 200 mg/kg. RNA-seq analysis showed that genes responsive to cAMP (Ass1, Gpd1, Rpl13a) were down-regulated and hypoxia-related genes (Egln3 and P4ha1) were up-regulated at 200 mg/kg. In utero exposure to BDE3 can promote autophagy and apoptosis of fetal Leydig cells via increasing the levels of Beclin1, LC3-II, BAX, and by decreasing the levels of p62 and BCL2. In conclusion, in utero exposure to BDE3 blocks the development of fetal rat testes.

Bisphenol A (BPA) is a ubiquitous environmental pollutant, mainly from the production and use of plastics and the degradation of wastes related to industrial plastics. Evidence from laboratory animal and human studies supports the view that BPA has an endocrine disrupting effect on Leydig cell development and function. To better understand the adverse effects of BPA, we reviewed its role and mechanism by analyzing rodent data in vivo and in vitro and human epidemiological evidence. BPA has estrogen and anti-androgen effects, thereby destroying the development and function of Leydig cells and causing related reproductive diseases such as testicular dysgenesis syndrome, delayed puberty, and subfertility/infertility. Due to the limitation of BPA production, the increased use of BPA analogs has also attracted attention to these new chemicals. They may share actions and mechanisms similar to or different from BPA.

Tebuconazole (TEB) is one of the widely used broad-spectrum triazole fungicides. Its accumulation in mammals leads to various endocrine disruptions. However, it is unclear whether the exposure of TEB during pregnancy affects the growth and development of fetus and placenta. Here, TEB was exposed to pregnant Sprague-Dawley female rats from gestational days 12–21 of 0, 25, 50 or 100 mg/kg for 10 days. TEB reduced placental estradiol levels. TEB disrupted the structure and function of the placenta, leading to hypertrophy, fibrin exudation, edema, calcification, arterial fibroblast proliferation, and trophoblastic infarction. RNA-seq analysis showed that TEB mainly down-regulated the expression of iron transport genes and up-regulated the expression of genes for immune/inflammatory responses. Further qPCR showed that TEB down-regulated Tfrc, Hamp, Eif2ak2 and up-regulated the expression of Cd34, Cd36, Jag1, Pln, Cyp1a1, Esrra, and Aqp1 at 50 and 100 mg/kg. Western blot and semi-quantitative immunohistochemical staining also demonstrated that TEB lowered the levels of TFRC and EIF2AK2 and increased the levels of CD34, CD36, JAG1, CYP1A1, and ESRRA at 50 and 100 mg/kg. In conclusion, TEB severely damages the structure and function of the placenta, leading to hypertrophy of the placenta, low birth weight and feminization of the male fetus possibly via several pathways including iron transport and TNF signaling.

Tebuconazole is a triazole fungicide, used in agriculture to treat phytopathogenic fungi, and as a biocide, has been reported to be related to reproductive and developmental toxicity. The purpose of this study was to investigate the effect of tebuconazole exposure on rat fetal Leydig cells and fetal testis during pregnancy. Pregnant Sprague-Dawley rats were randomly divided into 4 groups, daily gavaged with corn oil (as a control), 25, 50, and 100 mg/kg body weight tebuconazole for 10 days (from the 12th day of pregnancy). Tebuconazole increased fetal serum testosterone and progesterone levels at a dose of 100 mg/kg. Exposure to 100 mg/kg tebuconazole significantly caused an increase in the number of fetal Leydig cells per testis without inducing cell aggregation. Tebuconazole up-regulated the expression of Star, Cyp11a1, Hsd17b3, and Fshr and their proteins. Further investigation found that tebuconazole caused increased phosphorylation of AKT1, ERK1/2, and mTOR, the level of BCL2, as well as the decrease of Beclin1, LC3B, and BAX, which may contribute to the fetal Leydig cell autophagy and proliferation. In conclusion, in utero exposure of tebuconazole causes the proliferation of fetal Leydig cells.

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.

Studies on cell polarity proteins and planar cell polarity (PCP) proteins date back to almost 40 years ago in Drosophila and C. elegans when these proteins were shown to be crucial to suppo...

Myocardial ischemic postconditioning- (IPo-) mediated cardioprotection against myocardial ischemia-reperfusion (IR) injury needs the activation of signal transducer and activator of transcription 3 (STAT3), which involves adiponectin (APN). APN confers its biological effects through AMP-activated protein kinase- (AMPK-) dependent and AMPK-independent pathways. However, the role of AMPK in APN-mediated STAT3 activation in IPo cardioprotection is unknown. We hypothesized that APN-mediated STAT3 activation in IPo is AMPK-independent and that APN through AMPK-dependent STAT3 activation facilitates IPo cardioprotection. Here, Sprague-Dawley rats were subjected to myocardial IR without or with IPo and/or APN. APN or IPo significantly improved postischemic cardiac function and reduced myocardial injury and oxidative stress, and their combination further attenuated postischemic myocardial injuries. APN or its combination with IPo but not IPo alone significantly increased AMPK activation and both nuclear and mitochondrial STAT3 activation, while IPo significantly enhanced mitochondrial but not nuclear STAT3 activation. In primarily isolated cardiomyocytes, recombined globular APN (gAd), hypoxic postconditioning (HPo), or their combination significantly attenuated hypoxia/reoxygenation-induced cell injury and increased nuclear and/or mitochondrial STAT3 activation. STAT3 inhibition had no impact on gAd or gAd in combination with HPo-induced AMPK activation but abolished their cellular protective effects. AMPK inhibition did not affect HPo cardioprotection but abolished gAd cardioprotection and disabled gAd to facilitate/enhance HPo cardioprotection and STAT3 activation. These results suggest that APN confers cardioprotection through AMPK-dependent and AMPK-independent STAT3 activation, while IPo confers cardioprotection through AMPK-independent mitochondrial STAT3 activation. Joint use of APN and IPo synergistically attenuated myocardial IR injury by activating STAT3 via distinct signaling pathways.

Perfluoroalkyl substances (PFASs) are a group of man-made organic substances. Some of PFASs have been classified as persistent organic pollutants and endocrine disruptors. They might interfere with the male sex endocrine system, causing the abnormal development of the male reproductive tract and failure of pubertal onset and infertility. The present review discusses the development and function of two generations of Leydig cells in rodents and the effects of PFASs on Leydig cell development after their exposure in gestational and postnatal periods. We also discuss human epidemiological data for the effects of PFASs on male sex hormone levels. The structure-activity relationship of PFASs on Leydig cell steroidogenesis and enzyme activities are also discussed.

Oncostatin M (OSM) is a pleiotropic cytokine within the interleukin six family of cytokines, which regulate cell growth and differentiation in a wide variety of biological systems. However, its action and underlying mechanisms on stem Leydig cell development are unclear. The objective of the present study was to investigate whether OSM affects the proliferation and differentiation of rat stem Leydig cells. We used a Leydig cell regeneration model in rat testis and a unique seminiferous tubule culture system after ethane dimethane sulfonate (EDS) treatment to assess the ability of OSM in the regulation of proliferation and differentiation of rat stem Leydig cells. Intratesticular injection of OSM (10 and 100 ng/testis) from post‐EDS day 14 to 28 blocked the regeneration of Leydig cells by reducing serum testosterone levels without affecting serum luteinizing hormone and follicle‐stimulating hormone levels. It also decreased the levels of Leydig cell‐specific mRNAs (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins by the RNA‐Seq and Western blotting analysis. OSM had no effect on the proliferative capacity of Leydig cells in vivo. In the seminiferous tubule culture system, OSM (0.1, 1, 10 and 100 ng/mL) inhibited the differentiation of stem Leydig cells by reducing medium testosterone levels and downregulating the expression of Leydig cell‐specific genes (Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1 and Hsd11b1) and their proteins. OSM‐mediated action was reversed by S3I‐201 (a STAT3 antagonist) or filgotinib (a JAK1 inhibitor). These data suggest that OSM is an inhibitory factor of rat stem Leydig cell development.

During spermatogenesis, head-tail cell polarity, apico-basal cell polarity and planar cell polarity (PCP) are remarkably noted in the seminiferous epithelium in which the heads of developing haploid spermatids are pointed to the basement membrane, and with their tails to the tubule lumen. Furthermore, these polarized spermatids are laid unidirectionally across the plane of the seminiferous epithelium, mimicking PCP noted in hair cells of the inner ear. Treatment of rodents with environmental toxicants that lead to germ cell exfoliation, however, are associated with notable changes in spermatid polarity, and defects in spermatid polarity always precede spermatid loss from the epithelium. Studies have also shown that environmental toxicant-induced Sertoli cell or testis injury is mediated through changes in actin and/or microtubule (MT) cytoskeletons. Emerging evidence has illustrated that cell polarity and PCP also exert their regulatory effects through changes in cytoskeletal organization. Herein, we discuss and critically evaluate these recent findings, hoping that better efforts can be coordinated by investigators to address this rapidly developing field regarding the role of cell polarity and PCP proteins in toxicant-induced male reproductive dysfunction.

As the theory of cancer stem cells (CSCs) is maturing, CSC-targeted therapy is emerging as an important therapeutic strategy and seeking the ideal method for rapid enrichment and purification of CSCs has become crucial. So far, based on the known CSC phenotypes and biological characteristics, the methods for enrichment CSCs mainly include low adhesion culture, low oxygen culture, chemotherapy drug stimulation and side population (SP) sorting but these methods cannot realize quick enrichment of the desired CSCs. Herein, we adopt a novel method that efficiently enriches a certain amount of CSCs through agarose multi-well dishes using rubber micro-molds to make cancer cells into cell spheroids (3D). These 3D cancer cell spheroids in the proportions of expression of CSC biomarkers (single stain of CD44, CD44v6 and CD133 or double stain of both CD44 and CD133) were significantly higher than those of the conventional adherent culture (2D) using flow cytometry analysis. In addition, the expression levels of stemness transcription factors such as OCT4, NANOG and SOX2 in 3D were also significantly higher than that in 2D through Western blot (WB) and quantitative polymerase chain reaction (qPCR) assays. In addition, the CSCs in 3D could form colonies with different sizes in soft agar. In conclusion, we developed a new method to enrich some kinds of CSCs, which might be a benefit for future CSC-targeted therapy studies and anti-CSC drug screening applications.

Inflammation causes male hypogonadism. Several inflammatory cytokines, including interleukin 6 (IL-6), are released into the blood and may suppress Leydig cell development. The objective of the present study was to investigate whether IL-6 affected the proliferation and differentiation of rat stem Leydig cells. Leydig cell-depleted rat testis (in vivo) and seminiferous tubules (in vitro) with ethane dimethane sulfonate (EDS) were used to explore the effects of IL-6 on stem Leydig cell development. Intratesticular injection of IL-6 (10 and 100 ng/testis) from post-EDS day 14 to 28 blocked the regeneration of Leydig cells, as shown by the lower serum testosterone levels (21.6% of the control at 100 ng/testis dose), the down-regulated Leydig cell gene (Lhcgr, Star, Cyp11a1, Cyp17a1, and Hsd17b3) expressions, and the reduced Leydig cell number. Stem Leydig cells on the surface of the seminiferous tubules were induced to enter the Leydig cell lineage in vitro in the medium containing luteinizing hormone and lithium. IL-6 (1, 10, and 100 ng/ml) concentration-dependently decreased testosterone production and Lhcgr, Cyp11a1, Cyp17a1, Hsd17b3 and Insl3 mRNA levels. The IL-6 mediated effects were antagonized by Janus kinase 1 (JAK) inhibitor (filgotinib) and Signal Transducers and Activators of Transcription 3 (STAT3) inhibitor (S3I-201), indicating that a JAK-STAT3 signaling pathway is involved. In conclusion, our results demonstrated that IL-6 was an inhibitory factor of stem Leydig cell development.

It is conceivable that spermatid apico-basal polarity and spermatid planar cell polarity (PCP) are utmost important to support spermatogenesis. The orderly arrangement of developing germ cells in particular spermatids during spermiogenesis are essential to obtain structural and nutrient supports from the fixed number of Sertoli cells across the limited space of seminiferous epithelium in the tubules following Sertoli cell differentiation by ∼17 day postpartum (dpp) in rodents and ∼12 years of age at puberty in humans. Yet few studies are found in the literature to investigate the role of these proteins to support spermatogenesis. Herein, we briefly summarize recent findings in the field, in particular emerging evidence that supports the concept that apico-basal polarity and PCP are conferred by the corresponding polarity proteins through their effects on the actin- and microtubule (MT)-based cytoskeletons. While much research is needed to bridge our gaps of understanding cell polarity, cytoskeletal function, and signaling proteins, a critical evaluation of some latest findings as summarized herein provides some important and also thought-provoking concepts to design better functional experiments to address this important, yet largely expored, research topic.

Di-n-butyl phthalate (DBP) is widely used in consumer products as a plasticizer. Here, we report a natural product taxifolin that can attenuate developmental and reproductive toxicity of DBP. Pregnant rats were daily gavaged with 500 mg/kg DBP alone or together with taxifolin (10 and 20 mg/kg) from gestational day (GD) 12–21. At GD21, sera and testes of male fetus were collected. DBP significantly lowered serum testosterone level at 500 mg/kg and taxifolin can completely reverse its action. DBP caused abnormal aggregation of fetal Leydig cells and taxifolin can reverse it. DBP down-regulated the expression of the genes of cholesterol side-chain cleavage enzyme (Cyp11a1), 17β-hydroxysteroid dehydrogenase 3 (Hsd17b3), and insulin-like 3 (Insl3) and taxifolin can reverse its action. DBP increased malondialdehyde levels and decreased superoxide dismutase and glutathione peroxidase expression and taxifolin can reverse it. DBP increased incidence of multinucleated gonocytes and taxifolin can prevent it. Moreover, DBP lowered sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and phosphorylated AMP-activated protein kinase (pAMPK) signalling and taxifolin antagonized DBP. In conclusion, in utero exposure to DBP caused developmental/reproductive toxicity of male offspring via increasing reactive oxygen species and taxifolin is an effective food component that completely reverses DBP-mediated action.

Zearalenone is a phenolic Fusarium mycotoxin, which is ubiquitous in human and animal feedstuff and often co-occurs with other mycotoxins. ZEA has been reported to disturb Leydig cell function and even cause the apoptosis to the Leydig cells. However, the effects of gestational exposure to zearalenone on fetal Leydig cells and the underlying mechanism remain unknown. Sprague Dawley dams were daily gavaged with 0, 2.5, 5, 10, and 20 mg/kg body weight ZEA from gestational day 14–21. On gestational day 21, rats were euthanized and serum testosterone levels were measured, and testes were collected for further evaluation of Leydig cell number, cell size, gene, and protein expression. Zearalenone significantly decreased anogenital distance and its index of male fetus, serum testosterone levels, Leydig cell proteins (SCARB1, STAR, CYP11A1, CYP17A1, and INSL3), and fetal Leydig cell number at 10 and/or 20 mg/kg by delaying the commitment of stem Leydig cells into the Leydig cell lineage and proliferation. Further study found that Notch signaling (RFNG, PSEN1, NOTCH1, and NOTCH3) was up-regulated by zearalenone. In conclusion, gestational exposure to high doses of zearalenone (10 and 20 mg/kg) blocks fetal Leydig cell development, thus possibly causing the anomalies of the male reproductive tract.

Male fetal Leydig cells in the testis secrete androgen and insulin-like 3, determining the sexual differentiation. The abnormal development of fetal Leydig cells could lead to the reduction of androgen and insulin-like 3, thus causing the male reproductive tract anomalies in male neonates, including cryptorchidism and hypospadias. Environmental pollutants, such as phthalic acid esters (phthalates), can perturb the development and differentiated function of Leydig cells, thereby contributing to the reproductive toxicity in the male. Here, we review the epidemiological studies in humans and experimental investigations in rodents of various phthalates. Most of phthalates disturb the expression of various genes encoded for steroidogenesis-related proteins and insulin-like 3 in fetal Leydig cells and the dose-additive effects are exerted after exposure in a mixture.

Ziram is an endocrine disruptor and may cause birth abnormality of the male reproductive system. However, the effects of ziram on fetal Leydig cell (FLC) development are still unknown. The objective of the present study was to determine the endocrine-disrupting effect of ziram on rat FLC development after gestational exposure. Pregnant Sprague Dawley dams were randomly divided into 5 groups and were gavaged with 0 (corn oil, the control), 1, 2, 4, or 8 mg/kg ziram from gestational day 12 (GD12) to GD21. FLC development was evaluated by measuring serum testosterone, FLC number and distribution, and the expression levels of Leydig and Sertoli cell genes. Ziram significantly increased serum testosterone level at 1 mg/kg (1.350 ± 0.099 ng/ml vs. 0.989 ± 0.106 ng/ml in the control), while it remarkably lowered it at 8 mg/kg (0.598 ± 0.086 ng/ml). Quantitative immunohistochemical staining showed that ziram increased FLC number via stimulating cell proliferation at 1 mg/kg and lowered it via inhibiting its proliferation at 8 mg/kg without affecting Sertoli cell number. Further study demonstrated that the expression of Nr5a1, Lhcgr, Scarb1, Star, Cyp11a1, and Cyp17a1 genes and proteins in the testis was upregulated at 1 mg/kg and the expression of Leydig (Nr5a1, Lhcgr, Scarb1, Star, Cyp11a1, Cyp17a1, and Insl3) and Sertoli cell (Fshr, Hsd17b3, Dhh, Amh, and Sox9) genes and proteins was downregulated by ziram at 8 mg/kg. In conclusion, ziram had biphasic effects on FLC development with low dose to increase FLC number and function and high dose to decrease them.

Leydig cells are the main endogenous testosterone synthesis cells in the body. Testosterone is an essential hormone in males that affects metabolism, emotion, and pubertal development. However, little is known about the development of Leydig cells and relationship between fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). The aims of this study were to investigate the effect of (FLCs) on ALC development. Our study showed that FLCs in neonatal rat testis can be eliminated by 100 mg/kg ethane dimethane sulfonate (EDS) treatment without affecting the health of newborn rats. Immunohistological results showed that eliminating FLCs led to early re-generation of the ALC population (progenitor Leydig cells [PLCs] and ALCs) accompanied at first by increased and then by decreased serum testosterone, indicating that ALCs which appeared after neonatal EDS treatment were degenerated or had attenuated functions. Our results showed that FLCs were eliminated 4 days after EDS treatment, the ALC population regenerated by 21 days, and serum testosterone levels dramatically decreased at 56 days. Collectively, our results indicate that the ablation of FLCs in neonatal rat results in abnormal development of ALCs. Our study further indicates that abnormal development of Leydig cells in the fetal stage leads to steroid hormone disorders, such as testosterone deficiency, in the adult stage. Therefore, studies of Leydig cell development are important for understanding the pathogenesis of testosterone deficiency or pubertas praecox.

Perfluorotetradecanoic acid (PFTeDA) is one of perfluoroalkyl substances widely found in the environment. PFTeDA may cause the dysfunction of male reproductive system. However, whether PFTeDA affects the regeneration of Leydig cells remains unclear. The objective of this study was to examine the effects of short-term exposure of PFTeDA on the late-stage maturation of Leydig cells. Fifty-four adult Sprague-Dawley male rats were daily gavaged with PFTeDA (0, 10, or 20 mg/kg body weight) for 10 days, and then were injected intraperitoneally with ethylene dimethane sulfonate (EDS, 75 mg/kg body weight/once) to ablate Leydig cells to induce their regeneration. On day 21 (early stage) and 56 (late stage) after EDS, hormone levels, gene expression, and protein levels were measured. PFTeDA did not affect the early stage of Leydig cell regeneration, because it had no effect on serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels, Leydig cell number, and its gene and protein expression. PFTeDA significantly reduced serum testosterone level and down-regulated the expression of Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, and Insl3) and their proteins (CYP11A1, HSD3B1, CYP17A1, HSD17B3, and INSL3), decreased the phosphorylation of AKT1 and ERK1/2, as well as lowered sperm count in the epididymis at 20 mg/kg. In conclusion, short-term exposure to PFTeDA blocks the late-stage maturation of Leydig cells.

Placenta produces progesterone and estradiol for maintaining pregnancy. Two critical enzymes are responsible for their production: 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) that catalyzes the formation of progesterone from pregnenolone and aromatase that catalyzes the production of estradiol from testosterone. Fungicide ziram may disrupt the placental steroid production. In the present study, we investigated the effects of ziram on steroid formation in human placental cell line JEG-3 cells and on HSD3B1 and aromatase in the human placenta. Ziram did not inhibit progesterone production in JEG-3 cells and HSD3B1 activity at 100 μM. Ziram was a potent aromatase inhibitor with the half maximal inhibitory concentration (IC50) value of 333.8 nM. When testosterone was used to determine the mode of action, ziram was found to be a competitive inhibitor. Docking study showed that ziram binds to the testosterone binding pocket of the aromatase. In conclusion, ziram is a potent inhibitor of human aromatase.

Apigenin, a common flavonoid, has extensive pharmacological activities. Apigenin inhibits some steroid biosynthetic enzymes, suggesting that it may block neurosteroid synthesis. Neurosteroids play many important roles in neurological functions. The objective of the present study is to investigate effects of apigenin on neurosteroidogenic enzymes, 5α-reductase 1 (SRD5A1), 3α-hydroxysteroid dehydrogenase (AKR1C9), and retinol dehydrogenase 2 (RoDH2), in rats. SRD5A1, AKR1C9, and RoDH2 were expressed in COS-1 cells and the effects of apigenin on these enzymes and modes of action were explored using radiolabeled substrates and thin-layer chromatographic separation coupled with radiometry. Apigenin inhibited SRD5A1, AKR1C9, and RoDH2 activities with IC50 values of 100, 0.891 ± 0.065, and >100 μM, respectively. Apigenin competitively inhibited rat AKR1C9 when its substrate 5α-dihydrotestosterone was used and uncompetitively inhibited the enzyme when cofactor NADPH was used. In conclusion, apigenin is a potent inhibitor of rat AKR1C9, thereby controlling the rate of neurosteroid biosynthesis.

Phthalates as plasticizers are widely used in many consumer products. Dipentyl phthalate (DPeP) is one of phthalates. However, there are currently few data on whether DPeP exposure affects rat Leydig cell development. In this study, we investigated the effects of in utero DPeP exposure on Leydig cell development in the testes of male newborn and adult rats. From gestational days 14 to 21, Sprague-Dawley pregnant rats were gavaged vehicle (corn oil, control) or DPeP (10, 50, 100, and 500 mg/kg body weight/day). Testosterone and the expression of Leydig cell genes and proteins in the testis at birth and at postnatal day 56 were examined. DPeP dose-dependently reduced serum testosterone levels of male offspring at birth and at postnatal day 56 at 100 and 500 mg/kg and lowered serum luteinizing hormone levels at adult males at ≥10 mg/kg when compared with the control. In addition, DPeP increased number of fetal Leydig cells by inducing their proliferation but down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Insl3 in fetal Leydig cells per se. DPeP reduced number of adult Leydig cells by inducing cell apoptosis and down-regulated the expression of Lhcgr and Star in adult Leydig cells at postnatal day 56. DPeP lowered SIRT1 and BCL2 levels in the testis of adult rats. In conclusion, DPeP adversely affects both fetal and adult Leydig cell development after in utero exposure.

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.