Isolation, culture, characterization and the functional study of testicular Leydig cells from Hezuo pig.

Testosterone, an important sex hormone, regulates sexual maturation, testicular development, spermatogenesis and the maintenance of secondary sexual characteristics in males. Testicular Leydig cells are the primary source of testosterone production in the body. Hezuo pigs, native to the southern part of Gansu, China, are characterized by early sexual maturity, strong disease resistance and roughage tolerance. This study employed type IV collagenase digestion combined with cell sieve filtration to isolate and purify Leydig cells from the testicular tissue of 1-month-old Hezuo pigs. We also preliminarily investigated the functions of these cells. The results indicated that the purity of the isolated and purified Leydig cells was as high as 95%. Immunofluorescence analysis demonstrated that the isolated cells specifically expressed the 3ß-hydroxysteroid dehydrogenase antibody. Enzyme-linked immunosorbent assay results showed that the testosterone secretion of the Leydig cells cultured in vitro (generations 5-9) ranged between 1.29-1.67 ng/mL. Additionally, the content of the cellular autophagy signature protein microtubule-associated protein 1 light chain 3 was measured at 230-280 pg/mL. Through this study, we established an in vitro system for the isolation, purification and characterization of testicular Leydig cells from 1-month-old Hezuo pigs, providing a reference for exploring the molecular mechanism behind precocious puberty in Hezuo pigs.

Loss of PBX1 function in Leydig cells causes testicular dysgenesis and male sterility.

Leydig cells are essential components of testicular interstitial tissue and serve as a primary source of androgen in males. A functional deficiency in Leydig cells often causes severe reproductive disorders; however, the transcriptional programs underlying the fate decisions and steroidogenesis of these cells have not been fully defined. In this study, we report that the homeodomain transcription factor PBX1 is a master regulator of Leydig cell differentiation and testosterone production in mice. PBX1 was highly expressed in Leydig cells and peritubular myoid cells in the adult testis. Conditional deletion of Pbx1 in Leydig cells caused spermatogenic defects and complete sterility. Histological examinations revealed that Pbx1 deletion impaired testicular structure and led to disorganization of the seminiferous tubules. Single-cell RNA-seq analysis revealed that loss of Pbx1 function affected the fate decisions of progenitor Leydig cells and altered the transcription of genes associated with testosterone synthesis in the adult testis. Pbx1 directly regulates the transcription of genes that play important roles in steroidogenesis (Prlr, Nr2f2 and Nedd4). Further analysis demonstrated that deletion of Pbx1 leads to a significant decrease in testosterone levels, accompanied by increases in pregnenolone, androstenedione and luteinizing hormone. Collectively, our data revealed that PBX1 is indispensable for maintaining Leydig cell function. These findings provide insights into testicular dysgenesis and the regulation of hormone secretion in Leydig cells.

Effect of IL-1ß on the Development of Spermatogenesis In Vitro in Normal and Busulfan-Treated Immature Mice.

Gonadotoxic agents could impair spermatogenesis and may lead to male infertility. The present study aimed to evaluate the effect of IL-1ß on the development of spermatogenesis from cells isolated from seminiferous tubules (STs) of normal and busulfan-treated immature mice in vitro. Cells were cultured in a 3D in vitro culture system for 5 weeks. We examined the development of cells from the different stages of spermatogenesis by immunofluorescence staining or qPCR analyses. Factors of Sertoli and Leydig cells were examined by qPCR analysis. We showed that busulfan (BU) treatment significantly reduced the expression of testicular IL-1ß in the treated mice compared to the control group (CT). Cultures of cells from normal and busulfan-treated immature mice induced the development of pre-meiotic (Vasa), meiotic (Boule), and post-meiotic (acrosin) cells. However, the percentage of developed Boule and acrosin cells was significantly lower in cultures of busulfan-treated mice compared to normal mice. Adding IL-1ß to both cultures significantly increased the percentages of Vasa, Boule, and acrosin cells compared to their controls. However, the percentage of Boule and acrosin cells was significantly lower from cultures of busulfan-treated mice that were treated with IL-1ß compared to cultures treated with IL-1ß from normal mice. Furthermore, addition of IL-1ß to cultures from normal mice significantly increased only the expression of androgen receptor and transferrin but no other factors of Sertoli cells compared to their CT. However, the addition of IL-1ß to cultures from busulfan-treated mice significantly increased only the expression of androgen-binding protein and the FSH receptor compared to their CT. Adding IL-1ß to cultures of normal mice did not affect the expression of 3ßHSD compared to the CT, but it significantly reduced its expression in cultures from busulfan-treated mice compared to the CT. Our findings demonstrate the development of different stages of spermatogenesis in vitro from busulfan-treated mice and that IL-1ß could potentiate this development in vitro.

Radio frequency electromagnetic radiations interfere with the Leydig cell functions in-vitro.

A growing threat to male infertility has become a major concern for the human population due to the advent of modern technologies as a source of radiofrequency radiation (RFR). Since these technologies have become an integral part of our daily lives, thus, it becomes necessary to know the impression of such radiations on human health. In view of this, the current study aims to focus on the biological effects of radiofrequency electromagnetic radiations on mouse Leydig cell line (TM3) in a time-dependent manner. TM3 cells were exposed to RFR emitted from 4G cell phone and also exposed to a particular frequency of 1800 MHz and 2450 MHz from RFR exposure system. The cells were then evaluated for different parameters such as cell viability, cell proliferation, testosterone production, and ROS generation. A considerable reduction in the testosterone levels and proliferation rate of TM3 cells were observed at 120 min of exposure as compared to the control group in all exposure settings. Conversely, the intracellular ROS levels showed a significant rise at 60, 90 and 120 min of exposure in both mobile phone and 2450 MHz exposure groups. However, RFR treatment for different time durations (15, 30, 45, 60, 90, and 120 min) did not have significant effect on cell viability at any of the exposure condition (2450 MHz, 1800 MHz, and mobile phone radiation). Therefore, our findings concluded with the negative impact of radiofrequency electromagnetic radiations on Leydig cell's physiological functions, which could be a serious concern for male infertility. However, additional studies are required to determine the specific mechanism of RFR action as well as its long-term consequences.

Androgen synthesis cell-specific CREBZF deficiency alters adrenal cortex steroid secretion and develops behavioral abnormalities in adult male mice.

The global challenge of male infertility is escalating, notably due to the decreased testosterone (T) synthesis in testicular Leydig cells under stress, underscoring the critical need for a more profound understanding of its regulatory mechanisms. CREBZF, a novel basic region-leucine zipper transcription factor, regulates testosterone synthesis in mouse Leydig cells in vitro; however, further validation through in vivo experiments is essential. Our study utilized Cyp17a1-Cre to knock out CREBZF in androgen-synthesis cells and explored the physiological roles of CREBZF in fertility, steroid hormone synthesis, and behaviors in adult male mice. Conditional knockout (cKO) CREBZF did not affect fertility and serum testosterone level in male mice. Primary Leydig cells isolated from CREBZF-cKO mice showed impaired testosterone secretion and decreased mRNA levels of Star, Cyp17a1, and Hsd3b1. Loss of CREBZF resulted in thickening of the adrenal cortex, especially X-zone, with elevated serum corticosterone and dehydroepiandrosterone levels and decreased serum dehydroepiandrosterone sulfate levels. Immunohistochemical staining revealed increased expression of StAR, Cyp11a1, and 17ß-Hsd3 in the adrenal cortex of CREBZF-cKO mice, while the expression of AR was significantly reduced. Along with the histological changes and abnormal steroid levels in the adrenal gland, CREBZF-cKO mice showed higher anxiety-like behavior and impaired memory in the elevated plus maze and Barnes maze, respectively. In summary, CREBZF is dispensable for fertility, and CREBZF deficiency in Leydig cells promotes adrenal function in adult male mice. These results shed light on the requirement of CREBZF for fertility, adrenal steroid synthesis, and stress response in adult male mice, and contribute to understanding the crosstalk between testes and adrenal glands.

Hexafluoropropylene oxide trimer acid (HFPO-TA) exerts cytotoxic effects on leydig cells via the ER stress/JNK/ß-trcp/mcl-1 axis.

Hexafluoropropylene oxide trimer acid (HFPO-TA) is an alternative to perfluorooctanoic acid (PFOA) and is widely used in various industries. The effects of HFPO-TA on the male reproductive system and the underlying mechanisms are still not fully understood. In this study, TM3 mouse Leydig cells were used as the main model to evaluate the cytotoxicity of HFPO-TA in vitro. HFPO-TA inhibited the viability and expression of multiple biomarkers of Leydig cells. HFPO-TA also induced Leydig cell apoptosis in a caspase-dependent manner. Moreover, HFPO-TA induced the ubiquitination and degradation of Mcl-1 in a ß-TrCP-dependent manner. Further investigations showed that HFPO-TA treatment led to the upregulation of ROS, which activated the ER stress/JNK/ß-TrCP axis in Leydig cells. Overall, our study provides novel insights into the cytotoxic effects of HFPO-TA on the male reproductive system.

Mitigating effect of pomegranate peel extract against the furan induced testicular injury by apoptosis, steroidogenic enzymes and oxidative stress.

Furan is generated in a wide array of heat-treated foods through thermal degradation, leading to severe impairments in the male reproductive system. The main objective of this study was to investigate the potential of pomegranate peel extract (PGPE) in mitigating testicular dysfunctions induced by furan. Male rats were categorized into four groups: control/untreated, PGPE, furan, and PGPE + furan group. The study results revealed that furan-treated rats exhibited significantly elevated aminotransferase and phosphatase activity, and also generated increased oxidative stress, and reduced antioxidative stress protein activity. Additionally, protein content levels (ALT, AST, ALP, and ACP) and activities of steroidogenic Leydig cell hydroxysteroid dehydrogenase (3ß-HSD and 17ß-HSD) enzymes were significantly decreased. Significant variations in testicular parameters, apoptotic genes (Bcl-2, P53, and Caspase3), inflammatory and anti-inflammatory cytokines (IL1ß, IL10), male sex hormones follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and sperm quality were also observed. Furthermore, testicular histological abnormalities were confirmed by biochemical and molecular modifications. Notably, PGPE pre-treated furan-intoxicated animals exhibited significant improvements in most of the assessed parameters compared to furan-treated groups. In conclusion, PGPE presents essential preventive measures and a novel pharmacological potential therapy against furan-induced testicular injury.

Comparative steroidogenic effects of hexafluoropropylene oxide trimer acid (HFPO-TA) and perfluorooctanoic acid (PFOA): Regulation of histone modifications.

As a widely used alternative to perfluorooctanoic acid (PFOA), hexafluoropropylene oxide trimer acid (HFPO-TA) has been detected in the environment and humans; however, little is known regarding its male reproductive toxicity. To compare the effects of HFPO-TA on steroid hormone synthesis with PFOA, we exposed Leydig cells (MLTC-1) to non-lethal doses (0.1, 1, and 10 µM) of PFOA and HFPO-TA for 48 h. It was found that the levels of steroid hormones, 17α-hydroxyprogesterone (OHP), androstenedione (ASD), and testosterone (T) were significantly increased in 1 and 10 µM of PFOA and HFPO-TA groups, with greater elevation being observed in the HFPO-TA groups than in the PFOA groups at 10 µM. We further showed that the two rate-limiting steroidogenic genes (Star and Cyp11a1) were up-regulated, while Hsd3b, Cyp17a1, and Hsd17b were down-regulated or unchanged after PFOA/HFPO-TA exposure. Moreover, PFOA exposure significantly up-regulated histone H3K4me1/3 and H3K9me1, while down-regulated H3K4me2 and H3K9me2/3 levels. By contrast, H3K4me2/3 and H3K9me2/3 were enhanced, while H3K4me1 and H3K9me1 were repressed after HFPO-TA treatment. It was further confirmed that H3K4me1/3 were increased and H3K9me2 was decreased in Star and Cyp11a1 promoters by PFOA, while HFPO-TA increased H3K4me2/3 and decreased H3K9me1 in the two gene promoters. Therefore, we propose that low levels of PFOA/HFPO-TA enhance the expression of Star and Cyp11a1 by regulating H3K4 and H3K9 methylation, thus stimulating the production of steroid hormones in MLTC-1 cells. Collectively, HFPO-TA exhibits stronger effects on steroidogenesis compared to PFOA, which may be ascribed to the distinct regulation of histone modifications. These data suggest that HFPO-TA does not appear to be a safer alternative to PFOA on the aspect of male reproductive toxicity.

Ameliorative effects of elderberry (Sambucus nigra L.) extract and extract-derived monosaccharide-amino acid on H2O2-induced decrease in testosterone-deficiency syndrome in a TM3 Leydig cell.

With aging, men develop testosterone-deficiency syndrome (TDS). The development is closely associated with age-related mitochondrial dysfunction of Leydig cell and oxidative stress-induced reactive oxygen species (ROS). Testosterone-replacement therapy (TRT) is used to improve the symptoms of TDS. However, due to its various side effects, research on functional ingredients derived from natural products that do not have side effects is urgently needed. In this study, using the mitochondrial dysfunction TM3 (mouse Leydig) cells, in which testosterone biosynthesis is reduced by H2O2, we evaluated the effects of elderberry extract and monosaccharide-amino acid (fructose-leucine; FL) on mRNA and protein levels related to steroidogenesis-related enzymes steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1(CYP11A1, cytochrome P450 17A1(CYP17A1), cytochrome P450 19A1(CYP19A1, aromatase), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and 17ß-hydroxysteroid dehydrogenase(17ß-HSD). We analyzed elderberry extract and extract-derived FL for changes in ROS scavenging activity and testosterone secretion. Elderberry extract and FL significantly reduced H2O2-induced intracellular ROS levels, improved testosterone secretion, and increased the mRNA and protein expression levels of steroidogenesis-related enzymes (StAR, 3b-HSD, 17b-HSD, CYP11A1, CYp17A1). However, the conversion of testosterone to estradiol was inhibited by elderberry extract and extract-derived FL, which reduced the mRNA and protein expression of CYP19A1. In conclusion, elderberry extract and FL are predicted to have value as novel functional ingredients that may contribute to the prevention of TDS by ameliorating reduced steroidogenesis.

Deoxynivalenol Inhibits Progenitor Leydig Cell Development by Stimulating Mitochondrial Fission in Rats.

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

Environmental cadmium inhibits testicular testosterone synthesis via Parkin-dependent MFN1 degradation.

Low testosterone (T) levels are associated with many common diseases, such as obesity, male infertility, depression, and cardiovascular disease. It is well known that environmental cadmium (Cd) exposure can induce T decline, but the exact mechanism remains unclear. We established a murine model in which Cd exposure induced testicular T decline. Based on the model, we found Cd caused mitochondrial fusion disorder and Parkin mitochondrial translocation in mouse testes. MFN1 overexpression confirmed that MFN1-dependent mitochondrial fusion disorder mediated the Cd-induced T synthesis suppression in Leydig cells. Further data confirmed Cd induced the decrease of MFN1 protein by increasing ubiquitin degradation. Testicular specific Parkin knockdown confirmed Cd induced the ubiquitin-dependent degradation of MFN1 protein through promoting Parkin mitochondrial translocation in mouse testes. Expectedly, testicular specific Parkin knockdown also mitigated testicular T decline. Mito-TEMPO, a targeted inhibitor for mitochondrial reactive oxygen species (mtROS), alleviated Cd-caused Parkin mitochondrial translocation and mitochondrial fusion disorder. As above, Parkin mitochondrial translocation induced mitochondrial fusion disorder and the following T synthesis repression in Cd-exposed Leydig cells. Collectively, our study elucidates a novel mechanism through which Cd induces T decline and provides a new treatment strategy for patients with androgen disorders.

Chlorpyrifos impairs sperm parameters and number of Sertoli and Leydig cells in rats after exposure during the peripubertal period.

Chlorpyrifos is an organophosphate insecticide used to control pests in crops. Thus, humans are constantly exposed through ingestion of contaminated food or water, inhalation of contaminated air, and through the skin. The juvenile and peripubertal periods comprise a window of development of the reproductive system, sensitive to toxic agents. Considering the scarcity of data on exposure to the insecticide during these periods, the aim of this study was to evaluate the effects of chlorpyrifos on the testis during the juvenile and peripubertal periods. Thirty Wistar rats with an initial age of 25 days were distributed into 3 groups: control, which received corn oil (vehicle); CPS5, which received 5 mg/Kg b.w. of chlorpyrifos; and CPS15, which received 15 mg/Kg b.w. of chlorpyrifos. The groups were treated via gavage daily for 40 days and on the 41st experimental day, the animals were anesthetized and submitted to euthanasia to collect the organs. Blood was collected to obtain plasma and testosterone measurement. The testicles were removed, weighed and used for sperm count analyses, histopathological and morphometric analyzes and for oxidative stress analyses. Spermatozoa from the vas deferens were collected for analyzes of sperm morphology and acrosome integrity. The results showed that the two concentrations of chlorpyrifos caused a decrease in the number of Leydig and Sertoli cells and germ cells and increased the number of morphologically abnormal sperm and sperm with acrosomal damage. Furthermore, a decrease in lipid peroxidation was observed in the CPS5 and CPS15 groups, and a decrease in glutathione-S-transferase activity in the CPS5 group. We conclude that exposure to chlorpyrifos harms the daily production of sperm, as well as their quality, in addition to causing an imbalance in the oxidoreductive balance of the testicle.

Fetal Leydig cells: What we know and what we don't.

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehog signal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gαs -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal.

Testosterone is closely related to Leydig cell activity, environmental factors, and androgen receptor distribution in adult male lizards of Liolaemus cuyanus (Reptilia: Liolaemidae) during the reproductive cycle.

Testosterone, the primary sex hormone in male lizards, is closely linked to Leydig cell activity (the cells where steroidogenesis occurs) throughout the reproductive cycle, but testosterone action is related to androgen receptors (ARs) distribution in the seminiferous epithelium. In temperate zones, environmental factors detected through the hypothalamic-pituitary-gonadal axis, downregulate plasma testosterone, resulting in a seasonal reproductive cycle. The aim of this work is to study plasma testosterone in adult male lizards of Liolaemus cuyanus, an oviparous species, throughout its reproductive cycle and its relationship with Leydig cell histology, TotalLeydigCell/ActiveLeydigCell (TLC/ALC) ratio, environmental factors (temperature, relative humidity and solar irradiation) and ARs distribution in seminiferous epithelium. Specimens (N = 27) were captured (October to March) in a semi-arid zone (Valle de Matagusanos, San Juan, Argentina) and grouped into three relevant reproductive periods: pre-reproductive (PrR), reproductive (R), and post-reproductive (PsR). Significant differences in plasma testosterone were found among these periods, highest during R than in PsR. A significant positive correlation between plasma testosterone and TLC/ALC ratio was also observed. Plasma testosterone has a significant positive correlation only with solar irradiation, but not with the other variables. In PrR and R, ARs distribution was cytoplasmic and nuclear, shifting to only cytoplasmic in PsR. These results highlight the close correspondence between plasma testosterone, Leydig cell histology and activity, environmental factors, and ARs distribution, resulting in a synchronization that allows males of L. cuyanus to coordinate their reproductive cycle with the most favorable environmental conditions, probably for mating and birth of offspring.

Cyclophosphamide activates ferroptosis-induced dysfunction of Leydig cells via SMAD2 pathway†.

Cyclophosphamide (CP) is a widely used chemotherapeutic drug and immunosuppressant in the clinic, and the hypoandrogenism caused by CP is receiving more attention. Some studies found that ferroptosis is a new mechanism of cell death closely related to chemotherapeutic drugs and plays a key role in regulating reproductive injuries. The purpose of this study is to explore ferroptosis' role in testicular Leydig cell dysfunction and molecular mechanisms relating to it. In this study, the level of ferroptosis in the mouse model of testicular Leydig cell dysfunction induced by CP was significantly increased and further affected testosterone synthesis. The ferroptosis inhibitors ferrostatin-1 (Fer-1) and iron chelator deferoxamine (DFO) can improve injury induced by CP. The results of immunohistochemistry showed that Fer-1 and DFO could improve the structural disorder of seminiferous tubules and the decrease of the number of Leydig cells in testicular tissue induced by CP. Immunofluorescence and western blot confirmed that Fer-1 and DFO could improve the expression of key enzymes in testosterone synthesis. The activation of SMAD family member 2 (Smad2)/cyclin-dependent kinase inhibitor 1A (Cdkn1a) pathway can improve the ferroptosis of Leydig cells induced by CP and protect the function of Leydig cells. By inhibiting the Smad2/Cdkn1a signal pathway, CP can regulate ferroptosis, resulting in testicular Leydig cell dysfunction. In this study, CP-induced hypoandrogenism is explained theoretically and a potential therapeutic strategy is provided.

WNT5A regulates the proliferation, apoptosis and stemness of human stem Leydig cells via the ß-catenin signaling pathway.

Stem Leydig cells (SLCs) are essential for maintaining normal spermatogenesis as the significant component of testis microenvironment and gonadal aging. Although progress has been achieved in the regulation of male germ cells in mammals and humans, it remains unknown about the genes and signaling pathways of human SLCs. Here we have demonstrated, for the first time, that WNT5A (Wnt family member 5a) mediates the proliferation, apoptosis, and stemness of human SLCs, namely NGFR+ Leydig cells. We revealed that NGFR+ Leydig cells expressed NGFR, PDGFRA, NES, NR2F2, and THY1, hallmarks for SLCs. RNA-sequencing showed that WNT5A was expressed at a higher level in human SLCs than non-SLCs, while immunohistochemistry and Western blots further illustrated that WNT5A was predominantly expressed in human SLCs. Notably, CCK-8, EdU and Western blots displayed that WNT5A enhanced the proliferation and DNA synthesis and retained stemness of human SLCs, whereas flow cytometry and TUNEL analyses demonstrated that WNT5A inhibited the apoptosis of these cells. WNT5A knockdown caused an increase in LC lineage differentiation of human SLCs and reversed the effect of WNT5A overexpression on fate decisions of human SLCs. In addition, WNT5A silencing  resulted in the decreases in nuclear translocation of ß-catenin and expression levels of c-Myc, CD44, and Cyclin D1. Collectively, these results implicate that WNT5A regulates the proliferation, apoptosis and stemness of human SLCs through the activation of the ß-catenin signaling pathway. This study thus provides a novel molecular mechanism underlying the fate determinations of human SLCs, and it offers a new insight into the niche regulation of human testis.

Sirt3 Regulates Proliferation and Progesterone Production in Leydig Cells via Suppression of Reactive Oxygen Species.

Sirt3 is a mitochondrial protein deacetylase functioning in energy metabolism, regulation of intracellular reactive oxygen species (ROS) levels, and aging. Although Sirt3 loss has negative effects on fertility of oocytes during in vitro fertilization and on progesterone production in granulosa cells, Sirt3's function in Leydig cells remains unclear. Therefore, we investigated Sirt3 activity in Leydig cells, focusing on androgen production. To do so, we performed immunohistochemistry to confirm Sirt3 localization in gonads and observed strong Sirt3 immunostaining in Leydig cells of human testes and of Sirt3+/+ and Sirt3+/- mouse testes, while Sirt3-/- mouse testis tissue was negative. In human ovary, hilus cells were strongly Sirt3-positive, theca cells showed weak positivity, and granulosa cells showed very weak or almost no immunostaining. Next, we used the murine Leydig tumor cell line MA-10 as a model. We overexpressed Sirt3 but observed no changes in proliferation, expression of Star, Cyp11a1 (p450scc gene), and Hsd3b, or progesterone production in MA-10 cells. Sirt3 knockdown significantly reduced proliferation, suppressed expressions of steroidogenic enzymes and of transcription factors Ad4bp (Sf-1 gene) and Gata4, and decreased progesterone production. Sirt3 knockdown in MA-10 cells also increased intracellular ROS levels based on CM-H2DCFDA fluorescence dye analysis and increased the proportion of both early and late apoptotic (necrotic) cells based on Annexin V/7AAD assays. These results indicate that Sirt3 has a potential function in androgen production in Leydig cells by regulating intracellular ROS levels.

Long noncoding RNA XIST inhibition promotes Leydig cell apoptosis by acting as a competing endogenous RNA for microRNA-145a-5p that targets SIRT1 in late-onset hypogonadism.

Leydig cell (LCs) apoptosis is responsible for decreased serum testosterone levels during late-onset hypogonadism (LOH). Our study was designed to illustrate the regulatory effect of lncRNA XIST on LCs and to clarify its molecular mechanism of action in LOH. The Leydig cells (TM3) was treated by 300 µM H2O2 for 8 h to establish Leydig cell oxidative stress model in vitro. The expression levels of lncRNA XIST in the testicular tissues of patients with LOH were measured using fluorescence in situ hybridization (FISH). The interaction between lncRNA XIST/SIRT1 and miR-145a-5p was assessed using starBase and dual-luciferase reporter gene assays. Apoptotic cells and Caspase3 activity were determined by flow cytometry (FCM) assay. Testosterone concentration was determined by ELISA. Moreover, histological assessment of testicles in mice was performed by using HE staining and the TUNEL assay was used to determine apoptosis. We found that the lncRNA XIST was downregulated in the testicular tissues of LOH patients and mice and in H2O2-induced TM3 cells. XIST siRNA significantly promoted apoptosis, enhanced Caspase3 activity and reduced testosterone levels in H2O2-stimulated TM3 cells. Further studies showed that the miR-145a-5p inhibitor reversed the effect of XIST-siRNA on H2O2-induced Leydig cell apoptosis. MiR-145a-5p negatively regulated SIRT1 expression, and SIRT1-siRNA reversed the effects of the miR-145a-5p inhibitor on H2O2 stimulated TM3 cells. The in vivo experiments indicated that silencing of the lncRNA XIST aggravated LOH symptoms in mice. Inhibition of lncRNA XIST induces Leydig cell apoptosis through the miR-145a-5p/SIRT1 axis in the progression of LOH.

ARTS is essential for di-2-ethylhexyl phthalate (DEHP)-induced apoptosis of mouse Leydig cells.

As an extensively employed plasticizer in industrial applications, di-2-ethylhexyl phthalate (DEHP) can induce apoptosis of mouse Leydig cells, yet the precise mechanism remains elusive. In the current study, we identified that DEHP could specially induced apoptosis in the Leydig cells of the testis tissue, accompanied with the upregulation of apoptosis-related protein in the TGF-ß signaling pathway (ARTS) in the cells. Overexpression of ARTS significantly induced apoptosis of TM3 cells, while knockdown of ARTS inhibited apoptosis. Furthermore, DEHP-induced apoptosis of TM3 cells could be alleviated by knockdown of ARTS, which indicated that ARTS was involved in DEHP-induced apoptosis of mouse Leydig cells. Bioinformation assay predicts that there are four potential p53-responsive elements (p53-REs) located at - 6060, - 5726, - 5631 and - 5554 before the transcription start site of ARTS gene, implying that gene transcription of ARTS could be regulated by p53. Interestingly, DEHP was shown to specifically upregulate the expression of p53 in the Leydig cells of the testis tissue and TM3 cells. Consistently, p53 was proved to bind to the RE4 site of the ARTS gene promoter and transcriptionally activated the promoter-driven expression of the luciferase reporter gene. Overexpression of p53 could induce apoptosis of TM3 cells; while knockdown of p53 could not only rescue DEHP-induced apoptosis of the cells, but also inhibit DEHP-caused upregulation of ARTS. Meanwhile, we showed that oxidative stress could induce apoptosis of TM3 cells, accompanied with the increased protein levels of p53 and ARTS; while inhibition of oxidative stress dramatically alleviated DEHP-induced apoptosis and the up-regulation of p53 and ARTS. Taken together, these results indicated that DEHP-induced oxidative stress activates the p53-ARTS cascade to promote apoptosis of mouse Leydig cells.

Testicular expression of heat SHOCK proteins 60, 70, and 90 in cryptorchid horses.

Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in their contralateral descended normally testes, and in testes of normal horses were investigated by Western blot and immunohistochemistry. The histomorphological investigation of retained and scrotal testes was also investigated. The seminiferous epithelium of the retained testes showed a vacuolized appearance and displayed a completely blocked spermatogenesis for lacking meiotic and spermiogenetic cells. On the contrary, the contralateral scrotal testes did not show morphological damage and the seminiferous epithelium displayed all phases of the spermatogenetic cycle as in the normal testes. The morphology of Leydig cells was not affected by the cryptorchid state. Western blot and immunohistochemistry evidenced that equine testis (both scrotal and retained) expresses the three investigated HSPs. More in detail, the Western blot evidenced that HSP70 is the more expressed chaperone and that together with HSP90 it is highly expressed in the retained gonad (P < 0.05). The immunohistochemistry revealed the presence of the three HSPs in the spermatogonia of normal and cryptorchid testes. Spermatogonia of retained testes showed the lowest expression of HSP60 and the highest expression of HSP90. Spermatocytes, spermatids of scrotal testes, and the Sertoli cells of retained and scrotal testes did not display HSP60 whereas expressed HSP70 and HSP90. These two proteins were also localized in the nucleus of the premeiotic cells. The Leydig cells displayed the three HSPs with the higher immunostaining of HSP70 and 90 in the cryptorchid testes. The results indicate that the heat stress condition occurring in the cryptorchid testis influences the expression of HSPs.