Effects of perfluorododecanoic acid on testicular function in mice.

Perfluorodecanoic acid (PFDoA) is a widely distributed environmental pollutant that can affect the functions of many organs. However, systematic evaluations of the effects of PFDoA on testicular functions are lacking. The aim of this study was to investigate the effects of PFDoA on mouse testicular functions, including spermatogenesis, testosterone synthesis, and stem Leydig cells (SLCs) in the interstitial tissue of the testis. PFDoA (0, 2, 5, 10 mg/kg/d) was administered via gavage to 2-month-old mice for 4 weeks. Serum hormone levels and sperm quality were assayed. Furthermore, to investigate the mechanisms by which PFDoA affects testosterone synthesis and spermatogenesis in vivo, the expression of StAR and P450scc in testicular tissue was measured by immunofluorescence staining and quantitative real-time PCR. In addition, the levels of SLC markers, including nestin and CD51, were studied. PFDoA decreased the luteinizing hormone concentration and sperm quality. Although the difference was not statistically significant, mean testosterone levels showed a downward trend. The expression of StAR, P450scc, CD51, and nestin was also suppressed in the PFDoA-treated groups compared with the control group. Our study suggested that PFDoA exposure can decrease testosterone biosynthesis, and even reduce the number of SLCs. These results indicated that PFDoA suppressed the main functions of testis, and further researches are required to identify strategies for preventing or reducing the effect of PFDoA on testicular function.

Current status and reflections on stem cell transplantation in the treatment of testicular aging / 器官移植

Testicular aging is mainly characterized by a gradual decline in the capability of testosterone synthesis and spermatogenesis, which not only affects male fertility, but also correlates with aging-related chronic diseases intimately. Therefore, delaying testicular aging plays a significant role in improving the health and quality of life of middle-aged and elderly men. Stem cells are a cell group with potent self-renewal capability and multi-directional differentiation potential. In recent years, the research of stem cells in basic and clinical application has been carried out in-depth, which has accelerated the development of cell therapy. Currently, stem cell transplantation has been employed to treat multiple diseases, which has captivated widespread attention in the field of aging and regenerative medicine. Stem cell transplantation has demonstrated promising prospects in the treatment of testicular aging. In this article, research profile and progress of stem cell transplantation in the treatment of testicular aging were reviewed, and bottleneck issues encountered in clinical translation and strategies for optimizing clinical efficacy were discussed, aiming to provide novel ideas for the research and development and clinical translation of stem cell therapy for testicular aging.

Identification of Rat Testicular Leydig Precursor Cells by Single-Cell-RNA-Sequence Analysis.

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

Effects of Midazolam on the Development of Adult Leydig Cells From Stem Cells In Vitro.

Background: Midazolam is a neurological drug with diverse functions, including sedation, hypnosis, decreased anxiety, anterograde amnesia, brain-mediated muscle relaxation, and anticonvulsant activity. Since it is frequently used in children and adolescents for extended periods of time, there is a risk that it may affect their pubertal development. Here, we report a potential effect of the drug on the development of Leydig cells (LCs), the testosterone (T)-producing cells in the testis. Methods: Stem LCs (SLCs), isolated from adult rat testes by a magnetic-activated cell sorting technique, were induced to differentiate into LCs in vitro for 3 weeks. Midazolam (0.1-30 µM) was added to the culture medium, and the effects on LC development were assayed. Results: Midazolam has dose-dependent effects on SLC differentiation. At low concentrations (0.1-5 µM), the drug can mildly increase SLC differentiation (increased T production), while at higher concentrations (15-30 µM), it inhibits LC development (decreased T production). T increases at lower levels may be due to upregulations of scavenger receptor class b Member 1 (SCARB1) and cytochrome P450 17A1 (CYP17A1), while T reductions at higher levels of midazolam could be due to changes in multiple steroidogenic proteins. The uneven changes in steroidogenic pathway proteins, especially reductions in CYP17A1 at high midazolam levels, also result in an accumulation of progesterone. In addition to changes in T, increases in progesterone could have additional impacts on male reproduction. The loss in steroidogenic proteins at high midazolam levels may be mediated in part by the inactivation of protein kinase B/cAMP response element-binding protein (AKT/CREB) signaling pathway. Conclusion: Midazolam has the potential to affect adult Leydig cell (ALC) development at concentrations comparable with the blood serum levels in human patients. Further studies are needed to test the effects on human cells.

Phthalate inhibits Leydig cell differentiation and promotes adipocyte differentiation.

Studies have shown that phthalates are capable of affecting the development and functions of male reproductive system. The effect of phthalates on Leydig cell functions is well documented. However, little is known about their potential effects on the functions of stem Leydig cells (SLC). In the present study, we have examined the effects of mono-(2-ethylhexyl) phthalate (MEHP) on SLC functions in vitro by culturing seminiferous tubules and isolated SLCs. The results indicate that MEHP can significantly inhibit the proliferation and differentiation of SLCs in both the organ and cell culture systems. Interestingly, the minimal effective concentration that is able to affect SLC function was lower in the tubule culture system (1 µM) than in the isolated cells (10 µM), suggesting a possible involvement of the niche cells. Also, MEHP appeared to affect both the efficiency of SLCs to form Leydig cells and a selected group of Leydig cell-specific genes, including Lhcgr, Scarb1, Hsd3b1, Cyp17a1, Star, Srd5a1, Akr1c14, Insl3, Hao2 and Pah. Since SLCs are multipotent, we also tested the effect of MEHP on the differentiation of SLCs to adipocytes. Though MEHP by itself can not specify SLCs into adipocyte lineage, it indeed significantly increased the adipogenic activity of SLCs if used with an adipocyte inducing medium by up-regulation of multiple adipogenic-related genes, including Pparg and Cebpa. Overall, the results indicate that MEHP inhibits SLCs differentiating into Leydig lineage while stimulates the differentiating potential of SLCs to adipocytes.

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

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.

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.

Maternal exposure to zearalenone in masculinization window affects the fetal Leydig cell development in rat male fetus.

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.

Primary human testicular PDGFRα+ cells are multipotent and can be differentiated into cells with Leydig cell characteristics in vitro.

STUDY QUESTION: Is it possible to differentiate primary human testicular platelet-derived growth factor receptor alpha positive (PDGFRα+) cells into functional Leydig cells? SUMMARY ANSWER: Although human testicular PDGFRα+ cells are multipotent and are capable of differentiating into steroidogenic cells with Leydig cell characteristics, they are not able to produce testosterone after differentiation. WHAT IS KNOWN ALREADY: In rodents, stem Leydig cells (SLCs) that have been identified and isolated using the marker PDGFRα can give rise to adult testosterone-producing Leydig cells after appropriate differentiation in vitro. Although PDGFRα+ cells have also been identified in human testicular tissue, so far there is no evidence that these cells are true human SLCs that can differentiate into functional Leydig cells in vitro or in vivo. STUDY DESIGN, SIZE, DURATION: We isolated testicular cells enriched for interstitial cells from frozen-thawed fragments of testicular tissue from four human donors. Depending on the obtained cell number, PDGFRα+-sorted cells of three to four donors were exposed to differentiation conditions in vitro to stimulate development into adipocytes, osteocytes, chondrocytes or into Leydig cells. We compared their cell characteristics with cells directly after sorting and cells in propagation conditions. To investigate their differentiation potential in vivo, PDGFRα+-sorted cells were transplanted in the testis of 12 luteinizing hormone receptor-knockout (LuRKO) mice of which 6 mice received immunosuppression treatment. An additional six mice did not receive cell transplantation and were used as a control. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human testicular interstitial cells were cultured to Passage 3 and FACS sorted for HLA-A,B,C+/CD34-/PDGFRα+. We examined their mesenchymal stromal cell (MSC) membrane protein expression by FACS analyses. Furthermore, we investigated lineage-specific staining and gene expression after MSC trilineage differentiation. For the differentiation into Leydig cells, PDGFRα+-sorted cells were cultured in either proliferation or differentiation medium for 28 days, after which they were stimulated either with or without hCG, forskolin or dbcAMP for 24 h to examine the increase in gene expression of steroidogenic enzymes using qPCR. In addition, testosterone, androstenedione and progesterone levels were measured in the culture medium. We also transplanted human PDGFRα+-sorted testicular interstitial cells into the testis of LuRKO mice. Serum was collected at several time points after transplantation, and testosterone was measured. Twenty weeks after transplantation testes were collected for histological examination. MAIN RESULTS AND THE ROLE OF CHANCE: From primary cultured human testicular interstitial cells at Passage 3, we could obtain a population of HLA-A,B,C+/CD34-/PDGFRα+ cells by FACS. The sorted cells showed characteristics of MSC and were able to differentiate into adipocytes, chondrocytes and osteocytes. Upon directed differentiation into Leydig cells in vitro, we observed a significant increase in the expression of HSD3B2 and INSL3. After 24 h stimulation with forskolin or dbcAMP, a significantly increased expression of STAR and CYP11A1 was observed. The cells already expressed HSD17B3 and CYP17A1 before differentiation but the expression of these genes were not significantly increased after differentiation and stimulation. Testosterone levels could not be detected in the medium in any of the stimulation conditions, but after stimulation with forskolin or dbcAMP, androstenedione and progesterone were detected in culture medium. After transplantation of the human cells into the testes of LuRKO mice, no significant increase in serum testosterone levels was found compared to the controls. Also, no human cells were identified in the interstitium of mice testes 20 weeks after transplantation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed using tissue from only four donors because of limitations in donor material. Because of the need of sufficient cell numbers, we first propagated cells to passage 3 before FACS of the desired cell population was performed. We cannot rule out this propagation of the cells resulted in loss of stem cell properties. WIDER IMPLICATIONS OF THE FINDINGS: A lot of information on Leydig cell development is obtained from rodent studies, while the knowledge on human Leydig cell development is very limited. Our study shows that human testicular interstitial PDGFRα+ cells have different characteristics compared to rodent testicular PDGFRα+ cells in gene expression levels of steroidogenic enzymes and potential to differentiate in adult Leydig cells under comparable culture conditions. This emphasizes the need for confirming results from rodent studies in the human situation to be able to translate this knowledge to the human conditions, to eventually contribute to improvements of testosterone replacement therapies or establishing alternative cell therapies in the future, potentially based on SLCs. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Amsterdam UMC, location AMC, Amsterdam, the Netherlands. All authors declare no competing interests.

Fibroblast Growth Factor 1 Promotes Rat Stem Leydig Cell Development.

Fibroblast growth factor 1 (FGF1) is reported to be expressed in the testis. How FGF1 affects stem Leydig cell development remains unclear. Here, we report the effects of FGF1 on rat stem Leydig cell development in an ethane dimethane sulfonate (EDS)-treated model. FGF1 (100 ng/testis) significantly increased serum testosterone level, increased PCNA-positive Leydig cell percentage and Leydig cell number, but down-regulated the expression of Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd11b1 in Leydig cells per se, after its daily intratesticular injection from post-EDS day 14 for 14 days. Primary culture of the seminiferous tubules showed that FGF1 stimulated EdU incorporation to stem Leydig cells but blocked the differentiation into the Leydig cell lineage, possibly via FGFR1-mediated mechanism. In conclusion, FGF1 promotes stem Leydig cell proliferation but blocks its differentiation.

Stem Leydig cell regeneration in the adult rat testis is inhibited after a short-term triphenyltin exposure.

Triphenyltin (TPT) is an organotin compound and may be an endocrine disruptor, impairing the male reproductive system. However, the effect of short-term TPT exposure on stem Leydig cell regeneration later on remains unknown. Here, we show that TPT affects stem Leydig cell regeneration in the adult rat testis. Adult male Sprague Dawley rats were gavaged with TPT (0, 0.5, 1.0, 2.0 mg/kg body weight/day) for 10 days, followed by a single intraperitoneal injection of ethane dimethane sulfonate (EDS, 75 mg/kg body weight) to eliminate Leydig cells. Testis parameters and hormone levels were investigated on post-EDS days 21, 35, and 56. TPT significantly reduced serum testosterone levels, decreased Leydig cell number and cell size, and down-regulated its specific gene and protein expression at 1.0 and 2.0 mg/kg even 56 days after cession of treatment. TPT lowered PCNA-labeling index of progenitor Leydig cells on post-EDS day 21. TPT also lowered AKT1 and AKT2, and ERK1/2 phosphorylation on post-EDS day 56. This study reveals that a short-term exposure to TPT blocks stem Leydig cell regeneration in the long term thus delaying spermatogenesis.

Parathyroid Hormone-Related Protein Promotes Rat Stem Leydig Cell Differentiation.

The regulatory factors for stem Leydig cell development are largely unknown. Herein, we reported that parathyroid hormone-related protein (PTHrP) may be a factor to regulate this process. The effects of PTHrP on rat stem Leydig cell proliferation and differentiation were investigated using a stem Leydig cell culture system and an ethane dimethane sulfonate (EDS)-treated in vivo Leydig cell regeneration model. PTHrP (1,000 pg/ml) significantly increased medium testosterone level and up-regulated STAR, CYP17A1, and 17ß-HSD3 expressions. Co-treatment with PKA inhibitor H-89 or PKC inhibitor U73122 reversed PTHrP-mediated increase of testosterone production in vitro. Intratesticular injection of PTHrP (100 ng/testis) into the Leydig cell-depleted testis from post-EDS day 7 to 21 significantly increased serum testosterone level, up-regulated LHCGR, SCARB1, CYP11A1, 11ß-HSD1, and CYP17A1 expressions. It also enlarged Leydig cell size without affecting PCNA-labeled Leydig cell number. This indicates that PTHrP promotes stem Leydig cell differentiation. PTHrP in vivo increased CREB and p-CREB levels, suggesting that PTHrP acts via a PKA-CREB signaling pathway. In conclusion, PTHrP stimulates stem Leydig cell differentiation without affecting its proliferation, showing its novel action and mechanism on rat stem Leydig cell development.

Regulation of development of rat stem and progenitor Leydig cells by activin.

Stem Leydig cells have been demonstrated to differentiate into adult Leydig cells via intermediate stages of progenitor and immature Leydig cells. However, the exact regulatory mechanisms are unclear. We hypothesized that the development of stem or progenitor Leydig cells depends upon locally produced growth factors. Microarray analysis revealed that the expression levels of activin type I receptor (Acvr1) and activin A receptor type II-like 1 (Acvrl1) were stem > progenitor = immature = adult Leydig cells. This indicates that their ligand activin might play an important role in stem and progenitor Leydig cell proliferation and differentiation. When seminiferous tubules were incubated with 1 or 10 ng/mL activin A for 3 days, it concentration-dependently increased EdU incorporation into stem Leydig cells by up to 20-fold. When progenitor Leydig cells were incubated with 1 or 10 ng/mL activin A for 2 days, it concentration-dependently increased 3 H-thymidine incorporation into progenitor Leydig cells by up to 200%. Real-time PCR analysis showed that activin A primarily increased Pcna expression but reduced Star, Hsd3b1, and Cyp17a1 expression levels. Activin A also significantly inhibited the basal and luteinizing hormone-stimulated androgen production. In conclusion, activin A primarily stimulates the proliferation of stem and progenitor Leydig cells, but inhibits the differentiation of stem and progenitor Leydig cells into the Leydig cell lineage in rat testis.