A Protein from Dioscorea polystachya (Chinese Yam) Improves Hydrocortisone-Induced Testicular Dysfunction by Alleviating Leydig Cell Injury via Upregulation of the Nrf2 Pathway.

Leydig cell injury has been described as a primary driver of testicular dysfunction and is affected by oxidative stress. Dioscorea polystachya (Chinese yam) is used to improve testicular dysfunction in clinical and pharmacological research via its antioxidative activity, but the mechanisms underlying the beneficial effect of Chinese yam on testicular dysfunction and its suppression of Leydig cell oxidative damage remain unclear. In this study, we obtained a Chinese yam protein (DP1) and explored its effectiveness and possible mechanism in improving testicular dysfunction in vivo and in vitro. We established a testicular dysfunction model in rats using hydrocortisone (HCT). DP1 increased body weight and organ index, improved the deterioration in testicular morphology (including increasing the diameter of seminiferous tubules and thickness of germinal cell layers, inhibiting testicular cell apoptosis by increasing the Bcl-2/Bax ratio, and impeding collagen leakage by downregulating TGF-ß1 and p-SMAD2/3 expression), and restored the testosterone content. In addition, DP1 enhanced the number of Leydig cells in rats and H2O2-induced TM3 Leydig cells, and the effect of DP1 on the apoptosis, fibrosis, and testosterone content of TM3 cells was similar to that observed in vivo. These changes were dependent on the regulation of oxidative stress, including significantly reduced intracellular 8-hydroxy-2-deoxyguanosine levels, enhanced superoxide dismutase activities, and decreased superoxide anion levels, which were confirmed via a superoxide overexpression system. Furthermore, we observed that DP1 promoted Nrf2 nuclear import and upregulated antioxidant factor expression in vivo and in vitro. However, Nrf2 silencing eliminated the ability of DP1 to increase the Bcl-2/Bax ratio, reduce the expression levels of TGF-ß1 and p-SMAD2/3, and increase testosterone contents in H2O2-induced TM3 cells. In conclusion, DP1 reversed the HCT-induced testicular apoptosis and fibrosis and decreased testosterone contents by alleviating Leydig cell oxidative damage via upregulation of the Nrf2 pathway.

An autofluorescence-based isolation of Leydig cells for testosterone deficiency treatment.

Effective procedures for the purification of Leydig cells (LCs) can facilitate functional studies and transplantation therapies. However, current methods to purify LCs from testes are still far from satisfactory. Here, we found that testicular autofluorescence existed in the interstitium along with the gradual maturation of LCs from birth to adulthood. These autofluorescent cells were further isolated by fluorescence-activated cell sorting (FACS) and determined to be composed of LCs and macrophages. To further purify LCs, we combined two fluorescence channels of FACS and successfully separated LCs and macrophages. Of note, we confirmed that the obtained LCs not only possessed high purity, viability and quantity but also had intact steroidogenic activity and excellent responsiveness to luteinizing hormone. Moreover, subcutaneous transplantation of isolated LCs could alleviate the symptoms of testosterone deficiency in castrated mice. In summary, we established an effective autofluorescence-based method for isolating LCs. This method will aid in the future success of using LCs for basic and translational applications.

Advances in stem cell research for the treatment of primary hypogonadism.

In Leydig cell dysfunction, cells respond weakly to stimulation by pituitary luteinizing hormone, and, therefore, produce less testosterone, leading to primary hypogonadism. The most widely used treatment for primary hypogonadism is testosterone replacement therapy (TRT). However, TRT causes infertility and has been associated with other adverse effects, such as causing erythrocytosis and gynaecomastia, worsening obstructive sleep apnoea and increasing cardiovascular morbidity and mortality risks. Stem-cell-based therapy that re-establishes testosterone-producing cell lineages in the body has, therefore, become a promising prospect for treating primary hypogonadism. Over the past two decades, substantial advances have been made in the identification of Leydig cell sources for use in transplantation surgery, including the artificial induction of Leydig-like cells from different types of stem cells, for example, stem Leydig cells, mesenchymal stem cells, and pluripotent stem cells (PSCs). PSC-derived Leydig-like cells have already provided a powerful in vitro model to study the molecular mechanisms underlying Leydig cell differentiation and could be used to treat men with primary hypogonadism in a more specific and personalized approach.

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice.

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Diabetes Type 1 Negatively Influences Leydig Cell Function in Rats, Which is Partially Reversible By Insulin Treatment.

Type 1 diabetes mellitus (T1DM) is associated with impaired spermatogenesis and lower testosterone levels and epididymal weight. However, the underlying processes in the testis are unknown and remain to be elucidated. Therefore, the present study focused on the effects of T1DM on testicular function in a spontaneously diabetic rat model. BB/OKL rats after diabetes manifestation were divided into 3 groups: those without insulin treatment and insulin treatment for a duration of 2 and of 6 weeks. Anthropometrical data, circulating levels of gonadotrophins, testosterone, and inhibin B were measured. Intratesticular testosterone, oxidative stress, inflammation, and apoptosis were analyzed. Key enzymes of steroidogenesis were evaluated in the testis. Untreated diabetic rats had significantly lower serum follicle-stimulating hormone and luteinizing hormone levels. Serum and intratesticular testosterone levels significantly decreased in untreated diabetic rats compared to healthy controls. Key markers of Leydig cell function were significantly downregulated at the RNA level: insulin-like factor 3 (Insl3) by 53% (P = .006), Star by 51% (P = .004), Cyp11A1 by 80% (P = .003), 3Beta-Hsd2 by 61% (P = .005), and Pbr by 52% (P = .002). In the insulin-treated group, only Cyp11A1 and 3Beta-Hsd2 transcripts were significantly lower. Interestingly, the long-term insulin-treated group showed significant upregulation of most steroidogenic enzymes without affecting testosterone levels. Tumor necrosis factor α and apoptosis were significantly increased in the long-term insulin-treated rats. In conclusion T1DM, with a severe lack of insulin, has an adverse action on Leydig cell function. This is partially reversible with well-compensated blood glucose control. Long-term T1DM adversely affects Leydig cell function because of the process of inflammation and apoptosis.

Jagged1 intracellular domain modulates steroidogenesis in testicular Leydig cells.

Leydig cells represent the steroidogenic lineage of mammalian testis, which produces testosterone. Genetic evidence indicates the requirement of Notch signaling in maintaining a balance between differentiated Leydig cells and their progenitors during fetal development. In primary Leydig cells, Notch1 expression decreases with testicular development, while the expression of its ligand, Jagged1, remains relatively unchanged, suggesting that the roles of Jagged1 extend beyond Notch signaling. In addition, Jagged1 is known to be processed into its intracellular domain, which then translocate to the nucleus. In this study, we investigated the effect of Jagged1 intracellular domain (JICD) on steroidogenesis in Leydig cells. The independent overexpression of JICD in MA-10 Leydig cells was found to inhibit the activity of cAMP-induced Nur77 promoter. In addition, JICD suppressed Nur77 transactivation of the promoter of steroidogenic genes such as P450scc, P450c17, StAR, and 3ß-HSD. Further, adenovirus-mediated overexpression of JICD in primary Leydig cells repressed the expression of steroidogenic genes, consequently lowering testosterone production. These results collectively suggest that steroidogenesis in testicular Leydig cells, which is regulated by LH/cAMP signaling, is fine-tuned by Jagged1 during testis development.

Leydig stem cells and future therapies for hypogonadism.

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

KISS1R signaling modulates gonadotropin sensitivity in mouse Leydig cell.

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

Role of GPER-Mediated Signaling in Testicular Functions and Tumorigenesis.

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

Restorative functions of Autologous Stem Leydig Cell transplantation in a Testosterone-deficient non-human primate model.

Rationale: Stem Leydig cells (SLCs) transplantation can restore testosterone production in rodent models and is thus a potential solution for treating testosterone deficiency (TD). However, it remains unknown whether these favorable effects will be reproduced in more clinically relevant large-animal models. Therefore, we assessed the feasibility, safety and efficacy of autologous SLCs transplantation in a testosterone-deficient non-human primate (NHP) model. Methods: Cynomolgus monkey SLCs (CM-SLCs) were isolated from testis biopsies of elderly (> 19 years) cynomolgus monkeys by flow cytometry. Autologous CM-SLCs were injected into the testicular interstitium of 7 monkeys. Another 4 monkeys were injected the same way with cynomolgus monkey dermal fibroblasts (CM-DFs) as controls. The animals were then examined for sex hormones, semen, body composition, grip strength, and exercise activity. Results: We first isolated CD271+ CM-SLCs which were confirmed to expand continuously and show potential to differentiate into testosterone-producing Leydig cells (LCs) in vitro. Compared with CM-DFs transplantation, engraftment of autologous CM-SLCs into elderly monkeys could significantly increase the serum testosterone level in a physiological pattern for 8 weeks, without any need for immunosuppression. Importantly, CM-SLCs transplantation recovered spermatogenesis and ameliorated TD-related symptoms, such as those related to body fat mass, lean mass, bone mineral density, strength and exercise capacity. Conclusion: For the first time, our short-term observations demonstrated that autologous SLCs can increase testosterone levels and ameliorate relevant TD symptoms in primate models. A larger cohort with long-term follow-up will be required to assess the translational potential of autologous SLCs for TD therapy.

Epidermal growth factor regulates the development of stem and progenitor Leydig cells in rats.

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

Propofol induces apoptosis by activating caspases and the MAPK pathways, and inhibiting the Akt pathway in TM3 mouse Leydig stem/progenitor cells.

Propofol is an anesthetic agent moderating GABA receptors in the nervous system. A number of studies have demonstrated that propofol exerts a negative effect on neural stem cell development in the neonatal mouse hippocampus. However, to the best of our knowledge, there is no study available to date illustrating whether neonatal exposure to propofol affects Leydig stem/progenitor cell development for normal male reproductive development and functions, and the regulatory mechanism remains elusive. In the present study, TM3 cells, a mouse Leydig stem/progenitor cell line, was treated with propofol. The data illustrated that propofol significantly reduced TM3 cell viability. TM3 subG1 phase cell numbers were significantly increased by propofol assayed by flow cytometric analysis. Annexin V/PI double staining assay of the TM3 Leydig cells also demonstrated that propofol increased TM3 cell apoptosis. In addition, cleaved caspase­8, ­9 and ­3 and/or poly(ADP­ribose) polymerase (PARP) were significantly activated by propofol in the TM3 cells. Furthermore, the expression levels of phospho­JNK, phospho­ERK1/2 and phospho­p38 were activated by propofol in the TM3 cells, indicating that propofol induced apoptosis through the mitogen­activated protein kinase (MAPK) pathway. Additionally, propofol diminished the phosphorylation of Akt to increase the apoptosis of TM3 cells. On the whole, the findings of the present study demonstrate that propofol induces TM3 cell apoptosis by activating caspases and MAPK pathways, as well as by inhibiting the Akt pathway in TM3 cells. These findings illustrate that propofol affects the viability of Leydig stem/progenitor cells possibly related to the development of the male reproductive system.

Novel insights into Dhh signaling in antler chondrocyte proliferation and differentiation: Involvement of Foxa.

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

Sertoli cell-conditioned medium restores spermatogenesis in azoospermic mouse testis.

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

Stem Leydig Cells in the Adult Testis: Characterization, Regulation and Potential Applications.

Androgen deficiency (hypogonadism) affects males of all ages. Testosterone replacement therapy (TRT) is effective in restoring serum testosterone and relieving symptoms. TRT, however, is reported to have possible adverse effects in part because administered testosterone is not produced in response to the hypothalamic-pituitary-gonadal (HPG) axis. Progress in stem cell biology offers potential alternatives for treating hypogonadism. Adult Leydig cells (ALCs) are generated by stem Leydig cells (SLCs) during puberty. SLCs persist in the adult testis. Considerable progress has been made in the identification, isolation, expansion and differentiation of SLCs in vitro. In addition to forming ALCs, SLCs are multipotent, with the ability to give rise to all 3 major cell lineages of typical mesenchymal stem cells, including osteoblasts, adipocytes, and chondrocytes. Several regulatory factors, including Desert hedgehog and platelet-derived growth factor, have been reported to play key roles in the proliferation and differentiation of SLCs into the Leydig lineage. In addition, stem cells from several nonsteroidogenic sources, including embryonic stem cells, induced pluripotent stem cells, mature fibroblasts, and mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord have been transdifferentiated into Leydig-like cells under a variety of induction protocols. ALCs generated from SLCs in vitro, as well as Leydig-like cells, have been successfully transplanted into ALC-depleted animals, restoring serum testosterone levels under HPG control. However, important questions remain, including: How long will the transplanted cells continue to function? Which induction protocol is safest and most effective? For translational purposes, more work is needed with primate cells, especially human.

Lycopene Prevents DEHP-Induced Leydig Cell Damage with the Nrf2 Antioxidant Signaling Pathway in Mice.

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

Generation of Organized Porcine Testicular Organoids in Solubilized Hydrogels from Decellularized Extracellular Matrix.

Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity.

Characterization of stem cells associated with seminiferous tubule of adult rat testis for their potential to form Leydig cells.

Adult testicular Leydig cells arise from stem cells in the neonatal and adult testis. The nature of these stem Leydig cells (SLCs) have not been well characterized. We have found previously that a group cells expressing CD90, a cell surface glycoprotein that may play roles in cell-cell and cell-matrix interactions and associated with the seminiferous tubule surface, have the ability to form Leydig cells. As yet, the relationship between this CD90+ cell population and SLCs reported previously by other groups is still unknown. In the present study, we systematically characterized these CD90+ cells by their ability to express multiple potential SLC markers and to proliferate and differentiate into Leydig cells in vitro. First, we have found by qPCR and immunohistochemical staining that the CD90+ cells do not express any of the markers of the common seminiferous tubular cells, including myoid, Sertoli, germ and Leydig cells, as well as macrophages. Moreover, when the CD90+ cells were isolated by fluorescent-sorting, the cells expressed high levels of all the potential SLC marker genes, including Nestin, Cd51, Coup-tf2, Arx, Pdgfra and Tcf21. Also, CD90-positive, but not -negative, cells were able to form Leydig cells in vitro with the proper inducing medium. Overall, the results indicated that the tubule-associated CD90+ cells represent a population of SLC in adult testis.

Human Adipose-derived Pericytes Display Steroidogenic Lineage Potential in Vitro and Influence Leydig Cell Regeneration in Vivo in Rats.

Exogenous androgen replacement is used to treat symptoms associated with low testosterone in males. However, adverse cardiovascular risk and negative fertility impacts impel development of alternative approaches to restore/maintain Leydig cell (LC) androgen production. Stem Leydig cell (SLC) transplantation shows promise in this regard however, practicality of SLC isolation/transplantation impede clinical translation. Multipotent human adipose-derived perivascular stem cells (hAd-PSCs) represent an attractive extragonadal stem cell source for regenerative therapies in the testis but their therapeutic potential in this context is unexplored. We asked whether hAd-PSCs could be converted into Leydig-like cells and determined their capacity to promote regeneration in LC-ablated rat testes. Exposure of hAd-PSCs to differentiation-inducing factors in vitro upregulated steroidogenic genes but did not fully induce LC differentiation. In vivo, no difference in LC-regeneration was noted between Sham and hAd-PSC-transplanted rats. Interestingly, Cyp17a1 expression increased in hAd-PSC-transplanted testes compared to intact vehicle controls and the luteinising hormone/testosterone ratio returned to Vehicle control levels which was not the case in EDS + Sham animals. Notably, hAd-PSCs were undetectable one-month after transplantation suggesting this effect is likely mediated via paracrine mechanisms during the initial stages of regeneration; either directly by interacting with regenerating LCs, or through indirect interactions with trophic macrophages.

Curcumin protects against palmitic acid-induced apoptosis via the inhibition of endoplasmic reticulum stress in testicular Leydig cells.

BACKGROUND: Palmitic acid (PA) is a common saturated fatty acid that induces apoptosis in various types of cells, including testicular Leydig cells. There is evidence suggesting that PA is increased in patients with obesity and that PA-induced cell apoptosis may play an important role in obesity-related male infertility. Curcumin, a natural polyphenol, has been reported to exert cytoprotective effects in various cell types. However, the cytoprotective effect of curcumin against PA-induced apoptosis in Leydig cells remains unknown. Therefore, the current study was performed to investigate the protective effects of curcumin in response to PA-induced toxicity and apoptosis in murine Leydig tumor cell line 1 (MLTC-1) cells and explore the mechanism underlying its anti-apoptotic action. METHODS: MLTC-1 cells were cultured in Roswell Park Institute-1640 medium and divided into five groups. First four groups were treated with 50-400 µM PA, 400 µM PA + 5-40 µM curcumin, 400 µM PA + 500 nM 4-phenylbutyric acid (4-PBA, an endoplasmic reticulum (ER) stress inhibitor), and 500 nM thapsigargin (TG, an ER stress inducer) + 20 µM curcumin, respectively, followed by incubation for 24 h. Effects of PA and/or curcumin on viability, apoptosis, and ER stress in MLTC-1 cells were then determined by cell proliferation assay, flow cytometry, and western blot analysis. The fifth group of MLTC-1 cells was exposed to 400 µM of PA and 5 IU/mL of human chorionic gonadotropin (hCG) for 24 h in the absence and presence of curcumin, followed by measurement of testosterone levels in cell-culture supernatants by enzyme-linked immunosorbent assay (ELISA). Rats fed a high-fat diet (HFD) were treated with or without curcumin for 4 weeks, and the testosterone levels were detected by ELISA. RESULTS: Exposure to 100-400 µM PA reduced cell viability, activated caspase 3, and enhanced the expression levels of the apoptosis-related protein BCL-2-associated X protein (BAX) and ER stress markers glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP) in MLTC-1 cells. Treating cells with 500 nM 4-PBA significantly attenuated PA-induced cytotoxicity through inhibition of ER stress. Curcumin (20 µM) significantly suppressed PA- or TG-induced decrease in cell viability, caspase 3 activity, and the expression levels of BAX, CHOP, and GRP78. In addition, treating MLTC-1 cells with 20 µM curcumin effectively restored testosterone levels, which were reduced in response to PA exposure. Similarly, curcumin treatment ameliorated the HFD-induced decrease in serum testosterone level in vivo. CONCLUSIONS: The present study suggests that PA induces apoptosis via ER stress and curcumin ameliorates PA-induced apoptosis by inhibiting ER stress in MLTC-1 cells. This study suggests the application of curcumin as a potential therapeutic agent for the treatment of obesity-related male infertility.