Stem Leydig cells: Current research and future prospects of regenerative medicine of male reproductive health.

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

Bisphenol S stimulates Leydig cell proliferation but inhibits differentiation in pubertal male rats through multiple mechanisms.

Bisphenol S (BPS) is a novel bisphenol A (BPA) analogue, a ubiquitous environmental pollutant that disrupts male reproductive system. Whether BPS affects Leydig cell maturation in male puberty remains unclear. Male Sprague-Dawley rats (age of 35 days) were daily gavaged to 0, 1, 10, 100, and 200 mg/kg/day from postnatal days 35-56. BPS at 1-10 mg/kg/day and higher doses markedly reduced serum testosterone and progesterone levels but it at 200 mg/kg/day significantly increased estradiol level. BPS at 100 and 200 mg/kg/day significantly elevated serum luteinizing hormone (LH) levels. BPS at 1-10 mg/kg/day and higher doses significantly reduced inhibin A and inhibin B levels. BPS at 100 and 200 mg/kg/day markedly increased CYP11A1+ Leydig cell number, but did not affect HSD11B1+ (a mature Leydig cell marker) cell number. BPS at 10 mg/kg/day and higher doses significantly downregulated the expression of Cyp11a1 and at 100 and 200 mg/kg/d significantly lowered Cyp17a1, Hsd11b1, and Nr5a1 in the testes. BPS at 100 and/or 200 mg/kg/day significantly elevated Lhb in the pituitary. BPS at 100 and 200 mg/kg/day significantly increased the phosphorylation of AKT1, AKT2, and CREB without affecting total AKT1, AKT2, and CREB levels. BPS at 1-100 µM significantly suppressed testosterone production and induced proliferation of primary immature Leydig cells after 24 h of treatment and these actions were reversed by estrogen receptor α antagonist, ICI 182780, and partially reversed by vitamin E. BPS at 0.1-10 µM significantly increased oxidative stress of Leydig cells in vitro. BPS also directly inhibited 17ß-hydroxysteroid dehydrogenase 3 activity at 10-100 µM. In conclusion, BPS causes hypergonadotropic androgen deficiency in male rats during pubertal exposure via activating ESR1 and inducing ROS in immature Leydig cells and directly inhibiting 17ß-hydroxysteroid dehydrogenase 3 activity.

Inhibition of human sperm motility and capacitation by ziram is mediated by decreasing tyrosine protein kinase.

Many endocrine disruptors may interfere with sperm motility, hyperactivation, and capacitation, thereby leading to male infertility. In the current study, we screened 14 endocrine disruptors, including plant ingredients, cigarette ingredients, minerals, insecticides and fungicides, plastics, and plasticizers, to inhibit human sperm motility and forward motility. Only ziram, a dithiocarbamate fungicide, can effectively inhibit sperm motility, forward motility, hyperactivation, capacitation, and spontaneous acrosome reaction of normal human spermatozoa. Its half maximum inhibitory concentration (IC50) values were less than 4 µM. Ziram also inhibited sperm motility and forward motility of asthenozoospermia spermatozoa and IC50 values were about 6-8 µM. In addition, ziram inhibited normal sperm motility, calcium influx, reactive oxygen species, and mitochondrial membrane potential at 2.5 and/or 5 µM, with IC50 values ​​exceeding 100 µM, although it did not affect sperm DNA fragmentation up to 5 µM. Ziram-mediated inhibition of sperm motility and forward motility was irreversible. Forskolin, 8Br-cAMP, pentoxifylline, progesterone, vitamin E, and A23187 cannot prevent ziram-mediated inhibition of sperm motility and forward motility. Further studies have shown that ziram inhibited the level of tyrosine protein kinase with an IC50 value of about 10 µM, without affecting p21-activated kinase 4, and it caused damage to the mitochondrial structure of normal spermatozoa at 2.5 and 5 µM. In conclusion, ziram irreversibly inhibits human sperm motility, forward motility, and capacitation by reducing the level of tyrosine protein kinase and damaging the ultrastructure of mitochondria.

Oncostatin M inhibits differentiation of rat stem Leydig cells in vivo and in vitro.

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

Association study between polymorphisms of PRMT6, PEX10, SOX5, and nonobstructive azoospermia in the Han Chinese population.

A previous genomewide association study of nonobstructive azoospermia (NOA) in the Han Chinese population identified three risk loci (rs12097821, rs2477686, and rs10842262) and provided strong evidence for a genetic influence in male infertility. However, recently, a follow-up study of these single nucleotide polymorphism (SNP) loci in the Japanese population showed that none of them were significantly associated with NOA. Therefore, we conducted an association study, consisting of 550 NOA cases and 555 normal controls, to further validate whether the risk of those three SNPs still existed in an independent Han Chinese male population. The association studies did not support the association of rs12097821 and rs2477686 with NOA for both genotype and allele distributions, but rs10842262 in the SOX5 gene was significantly associated with NOA (chi square = 9.31; P = 0.0095 and chi square = 9.27; P = 0.0023, respectively). Our study provides genetic evidence for SOX5 polymorphism in NOA, contributing to predicting males at high risk of NOA in Han Chinese population. Considering genetic differences among populations, future validating studies in independent samples are suggested.

A novel mutation in CFAP47 causes male infertility due to multiple morphological abnormalities of the sperm flagella.

Introduction: A previous study suggested that loss of CFAP47 function is involved in multiple morphological abnormalities of the sperm flagella (MMAF) in humans and mice. However, the comprehensive role of CFAP47 in spermatogenesis is largely unknown. Methods: Whole-exome sequencing (WES) was conducted to identify pathogenic variant in two patients with MMAF. The functional effect of the identified mutations was investigated by immunofluorescence staining and western blotting. Intracytoplasmic sperm injection (ICSI) was used to assist fertilization for the patient with MMAF. Results: In this study, we identified a novel missense mutation (c.1414G>A; p.V472M) in CFAP47 in two unrelated patients with oligoasthenoteratozoospermia. Intriguingly, in addition to the MMAF phenotype very analogous to the previous report, the two patients notably presented abnormal morphology of sperm heads, the sperm mitochondrial sheath was obviously disorganized, and the sperm annulus were almost defective. Further functional experiments confirmed that the expression of CFAP47 was markedly reduced in the spermatozoa of the patients. Mechanism analysis suggested that CFAP47 might regulate the expression of CFAP65, CFAP69 and SEPTIN4 through their physical interactions and thus modulating sperm morphogenesis. Conclusion: we revealed a novel mutation in CFAP47 and further expanded the phenotype and mutation spectrum of CFAP47, as well as the potential mechanism of CFAP47 manipulating spermatogenesis, finally providing important guidance for genetic counseling and targeted treatment for CFAP47 mutation-related male infertility.

Loss-of-function CFTR p.G970D missense mutation might cause congenital bilateral absence of the vas deferens and be associated with impaired spermatogenesis.

Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells.

Human Umbilical Cord Mesenchymal Stem Cells Encapsulated with Pluronic F-127 Enhance the Regeneration and Angiogenesis of Thin Endometrium in Rat via Local IL-1ß Stimulation.

Thin endometrium (< 7 mm) could cause low clinical pregnancy, reduced live birth, increased spontaneous abortion, and decreased birth weight. However, the treatments for thin endometrium have not been well developed. In this study, we aim to determine the role of Pluronic F-127 (PF-127) encapsulation of human umbilical cord mesenchymal stem cells (hUC-MSCs) in the regeneration of thin endometrium and its underlying mechanism. Thin endometrium rat model was created by infusion of 95% ethanol. Thin endometrium modeled rat uterus were treated with saline, hUC-MSCs, PF-127, or hUC-MSCs plus PF-127 separately. Regenerated rat uterus was measured for gene expression levels of angiogenesis factors and histological morphology. Angiogenesis capacity of interleukin-1 beta (IL-1ß)-primed hUC-MSCs was monitored via quantitative polymerase chain reaction (q-PCR), Luminex assay, and tube formation assay. Decreased endometrium thickness and gland number and increased inflammatory factor IL-1ß were achieved in the thin endometrium rat model. Embedding of hUC-MSCs with PF-127 could prolong the hUC-MSCs retaining, which could further enhance endometrium thickness and gland number in the thin endometrium rat model via increasing angiogenesis capacity. Conditional medium derived from IL-1ß-primed hUC-MSCs increased the concentration of angiogenesis factors (basic fibroblast growth factor (bFGF), vascular endothelial growth factors (VEGF), and hepatocyte growth factor (HGF)). Improvement in the thickness, number of glands, and newly generated blood vessels could be achieved by uterus endometrium treatment with PF-127 and hUC-MSCs transplantation. Local IL-1ß stimulation-primed hUC-MSCs promoted the release of angiogenesis factors and may play a vital role on thin endometrium regeneration.

[Clinical efficacy of vardenafil on ED patients with kidney-yang or -yin deficiency or liver-qi stasis].

OBJECTIVE: To study the clinical efficacy of vardenafil on erectile dysfunction (ED) patients with kidney-yang deficiency, kidney-yin deficiency or liver-qi stasis. METHODS: Based on the syndromes of Traditional Chinese Medicine, 124 ED patients were divided into Groups A (kidney-yang deficiency, n=44), B (kidney-yin deficiency, n=41) and C (liver-qi stasis, n = 39). All the patients were treated with vardenafil at 5 mg daily for 8 weeks, and the therapeutic effects were evaluated by comparing the scores on IIEF-5 and Erection Quality Scale (EQS) before and after the treatment. RESULTS: After vardenafil treatment, the IIEF-5 and EQS scores of the ED patients were markedly increased, with statistically significant differences among the three groups (P < 0.01). The success rate of sexual intercourse was significantly improved in Groups A, B (P < 0.01) and C (P < 0.05). And the hardness of penile erection was enhanced by 81.82%, 73.17% and 43.59% respectively in the three groups of patients. CONCLUSION: Vardenafil is more effective for ED patients with kidney-yang or kidney-yin deficiency than for those with liver-qi stasis.

[Effects of prepubertal continuous exposure to dibutyl phthalate on testicular development in rats].

OBJECTIVE: To investigate the effects of prepubertal continuous exposure to dibutyl phthalate (DBP) on the testis development in SD rats. METHODS: Twenty-one-day-old weanling prepubertal male SD rats were randomly divided into a control (n = 24) and an experiment group (n = 54), gavaged daily with corn oil vehicle or corn oil + DBP at the repeated dose of 0 mg/(kg x d) (control), 50 mg/(kg x d) (low-dose), 200 mg/(kg x d) (medium-dose) and 600 mg/(kg x d) (high-dose) for 14, 21 and 28 days, and then sacrificed by decapitation on PND35, PND42 and PND49. The body weight gain, the testis weight and volume and the weight of accessory sex organs were measured, the serum testosterone level assayed by chemoluminescence technique, the testis tissues stained by H&E and observed under the light microscope for morphological alteration, the mean diameter of the seminiferous tubules determined and testicular biopsy scores obtained. RESULTS: Disordered arrangement of spermatogenic cells was found in some seminiferous tubules on PND35 in the low-dose group, but testis development and spermatogenesis were normal on PND42 and PND49. In the medium-dose group, disordered arrangement and decreased number of spermatogenic cells were observed on PND35 and PND42, but without testicular atrophy, and various grades of spermatogenic cells and sperm were seen on PND49. High-dose DBP slowed down the body weight gain, decreased serum T levels and induced degeneration of seminiferous tubules, arrest of spermatogenic epithelium development and necrosis of spermatogenic cells. The pubertal rats (PND49) showed testicular atrophy, azoospermia and delayed development of accessory sex organs. CONCLUSION: Prepubertal continuous exposure to DBP induces damages to testicular development and spermatogenesis in a dose-dependent manner, and those induced by high-dose DBP cannot be recuperated in the phase of prepubertal development, while the slight adverse effects on the testis induced by low- and medium-dose DBP could be completely or partly reversible before PND49.

Taxifolin suppresses rat and human testicular androgen biosynthetic enzymes.

Taxifolin is a flavonoid. It has been used as a chemopreventive agent and supplement. It may have some beneficial effects to treat prostate cancer by suppressing androgen production in Leydig cells. The objective of the present study was to study the effects of taxifolin on androgen production of rat Leydig cells isolated from immature testis and some rat and human testosterone biosynthetic enzyme activities. Rat Leydig cells were incubated with 100µM taxifolin without (basal) or with 10ng/ml luteinizing hormone (LH), 10mM 8-bromoadenosine 3',5'-cyclic monophosphate (8BR), and steroid enzyme substrates (20µM): 22R-hydroxychloesterol, pregnenolone, progesterone, and androstenedione. The medium concentrations of 5α-androstane-3α, 17ß-diol (DIOL) and testosterone were measured. Taxifolin significantly suppressed basal, LH-stimulated, 8BR-stimulated, pregnenolone-mediated, and progesterone-mediated androgen production by Leydig cells. Further study demonstrated that taxifolin inhibited rat 3ß-hydroxysteroid dehydrogenase and 17α-hydroxylase/17, 20-lyase with IC50 values of 14.55±0.013 and 16.75±0.011µM, respectively. Taxifolin also inhibited these two enzyme activities in human testis with IC50 value of about 100µM. Taxifolin was a competitive inhibitor for these two enzymes when steroid substrates were used. In conclusion, taxifolin may have benefits for the treatment of prostate cancer.

4-Bromodiphenyl ether delays pubertal Leydig cell development in rats.

Polybrominated diphenyl ethers are a class of brominated flame retardants that are potential endocrine disruptors. 4-Bromodiphenyl ether (BDE-3) is the most abundant photodegradation product of higher polybrominated diphenyl ethers. However, whether BDE-3 affects Leydig cell development during puberty is still unknown. The objective of this study was to explore effects of BDE-3 on the pubertal development of rat Leydig cells. Male Sprague Dawley rats (35 days of age) were gavaged daily with BDE-3 (0, 50, 100, and 200 mg/kg body weight/day) for 21 days. BDE-3 decreased serum testosterone levels (1.099 ±â€¯0.412 ng/ml at a dose of 200 mg/kg BDE-3 when compared to the control level (2.402 ±â€¯0.184 ng/ml, mean ±â€¯S.E.). BDE-3 decreased Leydig cell size and cytoplasmic size at a dose of 200 mg/kg, decreased Lhcgr, Star, Dhh, and Sox9 mRNA levels at ≥ 100 mg/kg and Scarb1, Cyp11a1, Hsd17b3, and Fshr at 200 mg/kg. BED-3 also decreased the phosphorylation of AKT1, AKT2, ERK1/2, and AMPK at 100 or 200 mg/kg. BDE-3 in vitro induced ROS generation, inhibited androgen production, down-regulated Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Srd5a1, and Akr1c14 expression in immature Leydig cells after 24-h treatment. In conclusion, the current study indicates that BDE-3 disrupts Leydig cell development via suppressing AKT, ERK1/2, and AMPK phosphorylation and inducing ROS generation.

[Advances in researches on the relationship of Y chromosome with male infertility].

The Y chromosome contains genes and gene families that play critical roles in the process of testis determination and differentiation. Male infertility can be induced by many factors, and extensive studies have strongly indicated that Y chromosome microdeletions are closely related to male reproductive dysfunction. Because most of the Y chromosome does not participate in sexual recombination, it has degenerated both in size and gene content, in comparison with the X chromosome. Consequently males may be faced with survival problems in the future. This article reviews the role of the Y chromosome in male infertility and the fate of the male in the future.