Leydig Cell Proliferation Research Articles

Tannin-Rich Fraction of Cyperus esculentus Protects against Lead-Induced Testicular Toxicity in Male Wistar Rats through ITS Antioxidant Properpty

Aims: Lead as a common environmental toxic metal, causes many histological, physiological and biomedical abnormalities in human and animals. This study evaluated the antioxidant potential of tannins on serum level of testosterone, LH and FSH and histology of the testes of male Wistar rats. Materials and Methods: Twenty-five adult male Wistar rats were divided into five (5) groups, (n=5). Group NS was administered normal saline only, Group PBO was administered with 30mg/kg body weight (BW) of lead, group LDTPB was administered with 50 mg/kg BW of tannins and 30mg/kg BW of lead, group MDTPB was administered with 100mg/kg BW of tannins and 30mg/kg BW of lead, group HDTPB was administered 150mg/kg BW of tannins and 30mg/kg BW of lead orally for 28 days. The animals were sacrificed and testes were harvested on day 29 of the experiment and histological and histochemistry studies done using the H&E and VVG staining respectively. Sperm parameters (motility, concentration), sex hormones (Testosterone, LH, FSH) and antioxidant activities were also determined. Results: There was Leydig cell proliferation and an increase series of spermatogenesis of the testes in the rats of groups administered with lead and tannins different doses (LDTPB, MDTPB and HDTPB) when compared with rats administered with lead only. Rats in groups LDTPB, MDTPB and HDTPB had a significant increase in levels of serum testosterone (p<0.05) when compared with positive control group (2). There was increase in levels of FSH in MDTPB and HDTPB groups when compared with positive control (2). Increased MDA levels were observed in the rats given lead only, PBO when compared to NS group. The rats given lead and tannins significant growth of seminiferous epithelium, improved sperm quality, and had decreased MDA levels. Conclusion: This study demonstrated the protective role of tannins fraction of Cyperus esculentus on lead-induced testicular toxicity in male Wistar rats.

Diaph1 knockout inhibits mouse primordial germ cell proliferation and affects gonadal development

BackgroundExploring the molecular mechanisms of primordial germ cell (PGC) migration and the involvement of gonadal somatic cells in gonad development is valuable for comprehending the origins and potential treatments of reproductive-related diseases.MethodsDiaphanous related formin 1 (Diaph1, also known as mDia1) was screened by analyzing publicly available datasets (ATAC-seq, DNase-seq, and RNA-seq). Subsequently, the CRISPR-Cas9 technology was used to construct Diaph1 knockout mice to investigate the role of Diaph1 in gonad development.ResultsBased on data from public databases, a differentially expressed gene Diaph1, was identified in the migration of mouse PGC. Additionally, the number of PGCs was significantly reduced in Diaph1 knockout mice compared to wild type mice, and the expression levels of genes related to proliferation (Dicer1, Mcm9), adhesion (E-cadherin, Cdh1), and migration (Cxcr4, Hmgcr, Dazl) were significantly decreased. Diaph1 knockout also inhibited Leydig cell proliferation and induced apoptosis in the testis, as well as granulosa cell apoptosis in the ovary. Moreover, the sperm count in the epididymal region and the count of ovarian follicles were significantly reduced in Diaph1 knockout mice, resulting in decreased fertility, concomitant with lowered levels of serum testosterone and estradiol. Further research found that in Diaph1 knockout mice, the key enzymes involved in testosterone synthesis (CYP11A1, 3β-HSD) were decreased in Leydig cells, and the estradiol-associated factor (FSH receptor, AMH) in granulosa cells were also downregulated.ConclusionsOverall, our findings indicate that the knockout of Diaph1 can disrupt the expression of factors that regulate sex hormone production, leading to impaired secretion of sex hormones, ultimately resulting in damage to reproductive function. These results provide a new perspective on the molecular mechanisms underlying PGC migration and gonadal development, and offer valuable insights for further research on the causes, diagnosis, and treatment of related diseases.

VD3/VDR attenuates bisphenol A-induced impairment in mouse Leydig cells via regulation of autophagy.

Bisphenol A (BPA) is an endocrine disruptor with reproductive toxicity. Further, 1,25-dihydroxyvitamin D3 (VD3) plays an important role in male reproduction by binding vitamin D receptor (VDR) and mediating the pleiotropic biological actions that include spermatogenesis. However, whether VD3/VDR regulates the effect of BPA on Leydig cells (LCs) injury remains unknown. This study aimed to explore the effects of VD on BPA-induced cytotoxicity in mouse LCs. Hereby, LCs treated with BPA, VD3, or both were subjected to the assays of cell apoptosis, proliferation, autophagy, and levels of target proteins. This study unveiled that cell viability was dose-dependently reduced after exposure to BPA. BPA treatment significantly inhibited LC proliferation, induced apoptosis, and also downregulated VDR expression. By jointly analyzing transcriptome data and Comparative Toxicogenomics Database (CTD) data, autophagy signaling pathways related to testicular development and male reproduction were screened out. Therefore, the autophagy phenomenon of cells was further detected. The results showed that BPA treatment could activate cell autophagy, Vdr-/- inhibits cell autophagy, and active VD3 does not have a significant effect on the autophagy of normal LCs. After VD3 and BPA were used in combination, the autophagy of cells was further enhanced, and VD3 could alleviate BPA-induced damage of LCs. In conclusion, this study found that supplementing VD3 could eliminate the inhibition of BPA on VDR expression, further enhance LCs autophagy effect, and alleviate the inhibition of LCs proliferation and induction of apoptosis by BPA, playing a protective role in cells. The research results will provide valuable strategies to alleviate BPA-induced reproductive toxicity.

Multi-omics reveals the testosterone promotion effect mechanism of Cordyceps Sobolifera on Leydig cells

Ethnopharmacological relevanceCordyceps sobolifera (CS) has been traditionally utilized as an ethnic remedy for various health conditions, including chronic kidney diseases, anti-fatigue interventions, and management of chronic inflammation. Notably, CS is recognized for its substantial content of bioactive compounds, among which nucleosides prominently feature as constituents with diverse therapeutic advantages.Aim of the study: This study aims to investigate the effects of CS on testosterone secretion in Leydig cells and explore the underlying mechanism. Materials and methodsLeydig cells were isolated from rat testes to establish a primary rat Leydig cells model. Cell proliferation and testosterone secretion were assessed via the methyl-piperidino-pyrazole (MTT) assay and enzyme-linked immunosorbent assay (ELISA), respectively. Samples earmarked for RNA sequencing (RNA-Seq) analysis facilitated the identification of significantly differentially expressed genes (DEGs), and we conducted Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation and enrichment analyses. The veracity of our findings was validated through quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. ResultsThe results showed that CS and guanosine could promote Leydig cell proliferation and bolster testosterone secretion. Our integrative analysis of metabolomics and transcriptomics has unveiled the potential mechanisms governing testosterone synthesis. Specifically, metabolomics has illuminated striking correlations within cholesterol metabolism, and bile secretion. Concurrently, transcriptomics has underscored the pivotal roles played by the cyclic adenosine monophosphate (cAMP) signaling pathway and steroid hormone biosynthesis. Furthermore, our investigation has demonstrated CS's aptitude in elevating the expression of proteins and genes. Notably, our findings have elucidated that these effects can be mitigated by protein kinase A (PKA) and adenylate cyclase (AC) specific inhibitors. ConclusionThis study delineates the cAMP-PKA pathways as plausible mechanisms underpinning the testosterone-enhancing properties of CS, with guanosine emerging as a fundamental bioactive constituent.

Patulin Stimulates Progenitor Leydig Cell Proliferation but Delays Its Differentiation in Male Rats during Prepuberty.

Patulin is a mycotoxin with potential reproductive toxicity. We explored the impact of patulin on Leydig cell (LC) development in male rats. Male Sprague Dawley rats (21 days postpartum) were gavaged patulin at doses of 0.5, 1, and 2 mg/kg/day for 7 days. Patulin markedly lowered serum testosterone at ≥0.5 mg/kg and progesterone at 1 and 2 mg/kg, while increasing LH levels at 2 mg/kg. Patulin increased the CYP11A1+ (cholesterol side-chain cleavage, a progenitor LC biomarker) cell number and their proliferation at 1 and 2 mg/kg. Additionally, patulin downregulated Lhcgr (luteinizing hormone receptor), Scarb1 (high-density lipoprotein receptor), and Cyp17a1 (17α-hydroxylase/17,20-lyase) at 1 and 2 mg/kg. It increased the activation of pAKT1 (protein kinase B), pERK1/2 (extracellular signal-related kinases 1 and 2), pCREB (cyclic AMP response binding protein), and CCND1 (cyclin D1), associated with cell cycle regulation, in vivo. Patulin increased EdU incorporation into R2C LC and stimulated cell cycle progression in vitro. Furthermore, patulin showed a direct inhibitory effect on 11β-HSD2 (11β-hydroxysteroid dehydrogenase 2) activity, which eliminates the adverse effects of glucocorticoids. This study provides insights into the potential mechanisms via which patulin affects progenitor LC development in young male rats.

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 17β-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 17β-hydroxysteroid dehydrogenase 3 activity.

The role of platelet-derived growth factor BB signaling pathway in the regulation of stem and progenitor Leydig cell proliferation and steroidogenesis in male rats

Platelet-derived growth factor BB (BB) regulates cell proliferation and function. However, the roles of BB on proliferation and function of Leydig stem (LSCs) and progenitor cells (LPCs) and the underlying signaling pathways remain unclear. This study aimed to analyze the roles of PI3K and MAPK pathways in the regulation of proliferation-related and steroidogenesis-related gene expression in rat LSCs/LPCs. In this experiment, BB receptor antagonist, tyrosine kinase inhibitor IV (PKI), the PI3K inhibitor, LY294002, and the MEK inhibitor, U0126, were used to measure the effects of these pathways on the expression of cell cycle-related genes (Ccnd1 and Cdkn1b) and steroidogenesis-related genes (Star, Cyp11a1, Hsd3b1, Cyp17a1, and Srd5a1), as well as Leydig cell maturation gene Pdgfra [1]. These results showed that BB (10 ng/mL)-stimulated EdU-incorporation into LSCs and BB-mediated inhibition on its differentiation was mediated through the activation of its receptor, PDGFRB, as well as MAPK and PI3K pathways. The results of LPC experiment also showed that LY294002 and U0126 decreased BB (10 ng/mL)-upregulated Ccnd1 expression while only U0126 reversed BB (10 ng/mL)-downregulated Cdkn1b expression. U0126 significantly reversed BB (10 ng/mL)-mediated downregulation of Cyp11a1, Hsd3b1, and Cyp17a1 expression. On the other hand, LY294002 reversed the expression of Cyp17a1 and Abca1. In conclusion, BB-mediated induction of proliferation and suppression of steroidogenesis of LSCs/LPCs are dependent on the activation of both MAPK and PI3K pathways, which show distinct regulation of gene expression.

Melatonin alleviates cisplatin-induced mice spermatogenesis defects

Cisplatin (CDDP) is a chemotherapeutic drug that is used to treat many different types of tumors. However, it also has significant adverse effects on male reproduction, which are partially mediated oxidative damage. Melatonin (MLT) is a promising antioxidant that can be used for reproductive protection. In this paper, we investigated the effect of CDDP on spermatogenesis, as well as MLT’s potential role in reproductive protection. CDDP (5 mg/kg BW) significantly reduced male mice testosterone levels and decreased sperm vitality and progressive motility. Additionally, a lower percentage of stage VII and VIII seminiferous tubules were observed in CDDP-treated mice. MLT administration significantly alleviated CDDP-induced testicular damages, CDDP-induced lowered male fertility in vivo, and enhanced in vitro embryonic development of two cells and blastocysts. These changes may be due to CDDP-mediated spermatogenesis defects in germ cell and Leydig cell proliferation, which are reflected in abnormal PCNA, SYCP3, and CYP11A1 expression levels and can be improved by MLT. CDDP treatment significantly decreased the total antioxidant capacity (TAC), as well as SOD and GSH levels, and increased MDA levels in mice testis, leading to increased apoptosis of germ cells and increased BAX/BCL2 ratios in mice testis. MLT treatment may reduce germ cell apoptosis by reducing oxidative damage in mice testis. This study demonstrated that CDDP affects sperm fertility by altering germ cell and Leydig cell proliferation via increased oxidative damage and that MLT can attenuate these damages. Our work provides potential information for further research on the toxic effects of CDDP and the protective effects of MLT on male reproduction.

Status of estrogen receptor expression and epigenetic methylation in Leydig cells after exposure to metalloestrogen - selenium

The trace element selenium (Se) is essential for the maintenance of spermatogenesis and fertility. A growing volume of evidence shows that Se is necessary for testosterone synthesis, and Se can stimulate Leydig cell proliferation. However, Se can also act as a metalloestrogen, which can mimic estrogen and activate the estrogen receptors. This study aimed to investigate Se effect on estrogen signaling and the epigenetic status of Leydig cells. Mouse Leydig cells (MA-10) were cultured in a medium supplemented with different Se concentrations (4, 8 µM) for 24 h. Next, cells were assessed for morphological and molecular (qRT PCR, western blot, immunofluorescence) analyses. Immunofluorescence revealed strong immunosignal for 5-methylcytosine in both control and treated cells, with a stronger signal in the 8 μM treated group. qRT-PCR confirmed an increased expression of methyltransferase 3 beta (Dnmt3b) in 8 μM cells. Analysis of the expression of γH2AX (a marker for double-stranded DNA breaks) revealed an increase in the DNA breaks in cells exposed to 8 μM Se. Selenium exposure did not affect the expression of canonical estrogen receptors (ERα and ERβ), however, an increase in membrane estrogen receptor G-protein coupled (GPER) protein expression was observed.To sum up, in a high concentration (8 μM) Se affects GPER expression (non-genomic estrogen signaling) in Leydig cells possibly via acting on receptor protein and/or its binding. This causes DNA breaks and induces changes in Leydig cell methylation status, especially in de novo methylation which is mediated by Dnmt3b.

Molecular insights underlying the adverse effects of bisphenol A on gonadal somatic cells' steroidogenic activity

Bisphenol A (BPA) exposure may impair gonadal steroidogenesis, although the underlying mechanism is not well known. Hereby, we assessed BPA action on human primary granulosa (hGC) and mouse Leydig cells (BLTK-1) proliferation, cytotoxicity, hormone secretion, and steroidogenic enzyme/receptor gene profile. hGC and BLTK-1 cells were stimulated with increasing concentrations of BPA (10-12 M to 10-4 M for cell proliferation assay, 10-8 M to 10-4 M for LDH-cytotoxicity assay, and 10-9 M to 10-5 M for hormone secretion and genes expression analysis). BPA at low concentrations (pM – nM) did not affect cell proliferation in either cell type, although was toxic at higher (µM) concentrations. BPA stimulation at low nM concentrations decreased the production of estradiol (E2) and testosterone (T) in BLTK-1, E2, and progesterone in hGCs. BPA down-regulated Star, Cyp11a1, and Hsd17b3, but up-regulated Cyp19a1, Esr1, Esr2, and Gpr30 expression in BLTK-1 cells. In hGC, BPA down-regulated STAR, CYP19A1, PGRMC1, and PAQR7 but up-regulated ESR2 expression. Estrogen receptor degrader fulvestrant (FULV) attenuated BPA inhibition of hormone production in both cell lines. FULV also blocked the BPA-induced Gpr30 up-regulation in BLTK-1 cells, whereas in hGC, failed to reverse the down-regulation of PGRMC1, STAR, and CYP19A1. Our findings provide novel mechanistic insights into environmentally-relevant doses of BPA action through both nuclear estrogen receptor-dependent and independent mechanisms affecting cultured granulosa and Leydig cell steroidogenesis.

Ciliary neurotrophic factor stimulates stem/progenitor Leydig cell proliferation but inhibits differentiation into its lineage in rats.

Ciliary neurotrophic factor (CNTF) is a member of the interleukin-6 family of cytokines. CNTF drives many cells for their development. However, its effects on Leydig cells (LCs) development remains unclear. In the current study, we used 3D seminiferous tubule culture system to induce the proliferation and differentiation of tubule-associated stem Leydig cells (SLCs) and primary progenitor Leydig cells (PLCs) culture to address the effects of CNTF. We found that CNTF stimulated the proliferation of SLCs but inhibited its development to the LCs lineage. The CNTF-mediated effects can be reversed by STAT3 inhibitor S3I-201 and PI3K inhibitor wortmannin, indicating that CNTF acts via STAT3-PI3K signaling pathways to increase stem/progenitor Leydig cell proliferation. CNTF at 1 and 10 ng/mL significantly decreased androgen production by PLCs. Microarray analysis for CNTF-treated PLCs showed that CNTF blocked steroidogenic pathways through downregulating Scarb1, Star and Hsd3b1 possibly through the downregulating the transcription factor Nr5a1 expression. CNTF stimulates the proliferation but blocks the differentiation of stem/progenitor Leydig cells. This article is protected by copyright. All rights reserved.

Effects of aging and macrophages on mice stem Leydig cell proliferation and differentiation in vitro.

Testosterone plays a critical role in maintaining reproductive functions and well-beings of the males. Adult testicular Leydig cells (LCs) produce testosterone and are generated from stem Leydig cells (SLCs) during puberty through adulthood. In addition, macrophages are critical in the SLC regulatory niche for normal testicular function. Age-related reduction in serum testosterone contributes to a number of metabolic and quality-of-life changes in males, as well as age-related changes in immunological functions. How aging and testicular macrophages may affect SLC function is still unclear. SLCs and macrophages were purified from adult and aged mice via FACS using CD51 as a marker protein. The sorted cells were first characterized and then co-cultured in vitro to examine how aging and macrophages may affect SLC proliferation and differentiation. To elucidate specific aging effects on both cell types, co-culture of sorted SLCs and macrophages were also carried out across two ages. CD51+ (weakly positive) and CD51++ (strongly positive) cells expressed typical SLC and macrophage markers, respectively. However, with aging, both cell types increased expression of multiple cytokine genes, such as IL-1b, IL-6 and IL-8. Moreover, old CD51+ SLCs reduced their proliferation and differentiation, with a more significant reduction in differentiation (2X) than proliferation (30%). Age matched CD51++ macrophages inhibited CD51+ SLC development, with a more significant reduction in old cells (60%) than young (40%). Crossed-age co-culture experiments indicated that the age of CD51+ SLCs plays a more significant role in determining age-related inhibitory effects. In LC lineage formation, CD51+ SLC had both reduced LC lineage markers and increased myoid cell lineage markers, suggesting an age-related lineage shift for SLCs. The results suggest that aging affected both SLC function and their regulatory niche cell, macrophages.

PPP2R2A promotes testosterone secretion in Hu sheep Leydig cells via activation of the AKT/mTOR signaling pathway.

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that plays a vital role in regulating male reproductive activities. However, as an essential member of the PP2A family, the physiological functions of PP2A regulatory subunit B55α (PPP2R2A) in testis remain inconclusive. Hu sheep are noted for their reproductive precocity and fertility, and are ideal models for the study of male reproductive physiology. Here, we analyzed the expression patterns of PPP2R2A in the male Hu sheep reproductive tract at different developmental stages and further investigated its role in testosterone secretion and its underlying mechanisms. In this study, we found that there were temporal and spatial differences in PPP2R2A protein expression in the testis and epididymis, especially the expression abundance in the testis at 8 months old (8M) was higher than that at 3 months old (3M). Interestingly, we observed that PPP2R2A interference reduced the testosterone levels in the cell culture medium, which is accompanied by a reduction in Leydig cell proliferation and an elevation in Leydig cell apoptosis. The level of reactive oxygen species in cells increased significantly, while the mitochondrial membrane potential (ΔΨm) decreased significantly after PPP2R2A deletion. Meanwhile, the mitochondrial mitotic protein DNM1L was significantly upregulated, while the mitochondrial fusion proteins MFN1/2 and OPA1 were significantly downregulated after PPP2R2A interference. Furthermore, PPP2R2A interference suppressed the AKT/mTOR signaling pathway. Taken together, our data indicated that PPP2R2A enhanced testosterone secretion, promoted cell proliferation, and inhibited cell apoptosis in vitro, all of which were associated with the AKT/mTOR signaling pathway.

Ciprofloxacin and enrofloxacin can cause reproductive toxicity via endocrine signaling pathways.

Ciprofloxacin (CIP) and enrofloxacin (ENR) are veterinary antibiotics commonly utilized to treat and prevent animal diseases. Environmental and dietary antibiotic residues can directly and indirectly affect the reproductive development of animals and humans. This article investigated the reproductive toxicity of CIP in male zebrafish, showing that it could decrease the spermatogonial weight and damage the spermatogonial tissue. The sex hormone assays showed that CIP decreased fshb and lhb gene expression and plasma testosterone (T). In addition, transcriptome analysis indicated that the effect of CIP on zebrafish might be related to the endocrine signaling pathways. ENR, which was selected for further study, inhibited mouse Leydig (TM3) and Sertoli (TM4) cell proliferation and caused cell cycle arrest. The sperm concentration, serum luteotropic hormone (LH) and follicle-stimulating hormone (FSH), and T levels decreased in adolescent mice after ENR treatment for 30d in vivo. Hematoxylin and eosin (H&E) staining showed that ENR exposure potentially induced testicular injury, while the real-time quantitative PCR (qPCR) results indicated that ENR inhibited the mRNA expression of key genes in the Leydig cells (cyp11a1, 3β-HSD, and 17β-HSD), Sertoli cells (Inhbβ and Gdnf) and spermatogenic cells (Plzf, Stra8 and Dmc1). In conclusion, these findings indicated that ENR exposure might influence the development of the testes of pubescent mice.

Improved cell proliferation and testosterone secretion following exposure of TM3 Leydig cells to three-dimensional scaffold and light emitting diode.

Green LED and three-dimensional (3D) scaffolds have recently received extensive attentions due to their impact on cell proliferation and differentiation. Melatonin, a circadian rhythm-regulating hormone, is involved in some physiological phenomena including testosterone biosynthesis. Lower testosterone biosynthesis results in some disorders such as puberty retarding, andropause, and muscle weakness. Therefore, our aim was to investigate the proliferation of Leydig cells and their testosterone-related Gene expression and secretion under the influence of 3D scaffold, green light and melatonin. The experimental groups of TM3 cells embedded in the 3D scaffold, were exposed to green light, melatonin, both and all three factors. Expression of cell cycle genes including PCNA, CYCLIND1, CDC2 and CDKN1B, and testosterone related genes; GATA4 and RORα were also examined. 3D scaffold enhanced Leydig cells proliferation, and testosterone-related genes expression. While melatonin decreased cell proliferation and testosterone-related genes expression. Green light did not significantly change the results but slightly decreased cell proliferation and testosterone synthesis. The combination of green light with melatonin significantly reduced the proliferation rate of TM3 cells and the expression of steroidogenic genes, while the combination of green light with scaffold improved the results. In general, the use of scaffolding enhances proliferation and testosterone-related genes expression of TM3 Leydig cells. Also, application of green light and scaffolding reduces the deleterious effects of melatonin on these cells.

Fluornen-9-bisphenol increases Leydig cell proliferation but inhibits maturation in pubertal male rats via interacting with androgen receptor as an antagonist and estrogen receptor α as an agonist

Fluornen-9-bisphenol (BPFL) is used as one of the alternatives for bisphenol A. However, whether BPFL has deleterious effects to the male reproductive system and the underlying mechanism remain unknown. Here, we report the effects of BPFL on Leydig cell development in male rats in puberty. Male Sprague-Dawley (28 days old) rats were dosed with 0, 10, 100, 200 mg/kg/day BPFL via gavage for 28 days. BPFL significantly decreased serum testosterone levels at 200 mg/kg while increasing serum luteinizing hormone and follicle-stimulating hormone levels at 200 mg/kg. BPFL markedly increased Leydig cell number but down-regulated the expression of Cyp17a1 and its protein level in Leydig cells at 200 mg/kg. Further study showed that BPFL significantly increased Pcna and Cdk2 expression and increased Leydig cell proliferation at 200 mg/kg. BPFL treatment to immature Leydig cells isolated from 28-day-old male rats for 24 h significantly inhibited testosterone biosynthesis at 50 μM, which was completely reversed by the androgen receptor agonist 7α-methyl-nortestosterone and estrogen receptor α antagonist ICI 182,780. In conclusion, BPFL increases Leydig cell proliferation but inhibits its maturation in male rats in puberty by blocking androgen receptor and activating estrogen receptor α.

Disruption of cell proliferation and apoptosis balance in the testes of crossbred cattle-yaks affects spermatogenic cell fate and sterility.

The balance between proliferation, differentiation and apoptosis is well-coordinated in spermatogenesis for the timely production of appropriate numbers of sperm in animals. Disruption or decrease in sperm production is due to many conditions, including changes in testicular cell fate balance. Interspecies hybridization of domestic yaks and cattle results in sterility in males because of spermatogenic arrest; however, the underlying mechanisms involved in sterility are still unclear. In the present study, we investigated the proliferation and apoptosis status during the development of yaks and crossbred cattle-yaks using immunohistochemistry of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays. Testicular tissues from yaks (immature: 1year old, mature: 2-3 years old) and backcrossed hybrids (2year old) were collected and used to investigate the expression of each parameter in testicular cells. During the maturation of yak testes, proliferation and apoptosis became active only in spermatogenic cells, and not in other somatic cells, such as Sertoli cells, myoid cells and Leydig cells. Furthermore, hybrid cattle-yak testes maintained proliferation ability but less apoptotic ability in spermatogenic cells when compared to yaks of the same age, suggesting that normal spermatogenic cell fate control is disrupted by changes in the balance between proliferation and apoptosis. In addition, Leydig cell proliferation rate was higher than apoptosis rate in the cattle-yak testes, indicating an increased number of Leydig cells, which may affect spermatogenesis through changes in steroidogenesis. Although epigenetic changes may be involved in cattle-yak testes, further studies are needed to clarify the modulation of proliferation and apoptosis to elucidate the mechanisms of infertility in hybrid cattle-yak males.

Dietary Fibre Supplementation Improves Semen Production by Increasing Leydig Cells and Testosterone Synthesis in a Growing Boar Model.

Testicular development is imperative to spermatogenesis, and pre-puberty is the key period for testis development. This study, therefore, investigated the effects of fibre supplementation on testis development and its possible mechanism in a growing boar model. Thirty Yorkshire boars were randomly divided into a control group (Control) and a fibre group (Fibre) from day 0 to 90 after weaning, with three pigs per pen and five pens per treatment. Blood and testes were collected for analysis. Dietary fibre supplementation had no significant effect on growth performance, testicular volume, or libido but increased the semen production of boars. Boars fed with fibre had lower serum cholesterol (CHO) and low-density lipoprotein (LDL) levels compared to those on the Control diet; however, testicular CHO, triglyceride (TG), and LDL concentration in the Fibre group were significantly higher than the Control group (P < 0.01). Testicular histological analysis showed that seminiferous tubules and testicular germ cells of 120-day-old boars were densely arranged in the Fibre group, and the number of Leydig cells was significantly higher than that of the Control group (P < 0.001). Furthermore, the diet supplemented with fibre significantly decreased leptin, leptin receptor (Leptor), and luteinising hormone (LH) concentrations in boar serum (P < 0.05), whereas follicle-stimulating hormone (FSH) and testosterone concentrations were significantly increased (P < 0.05). Meanwhile, the expression of AMH, AMHR2, and SYCP3 genes related to proliferation and differentiation, and hormone-related genes STAR and SOCS3, were significantly up-regulated (P < 0.05). OCCLUDIN expression was up-regulated, whereas CDH2 expression was down-regulated. In conclusion, increased fibre intake during the pre-puberty period in growing boar is crucial for Leydig cell proliferation, up-regulating the expression of genes related to hormone synthesis and thereby promoting the secretion of testosterone and semen production.

Novel enhancer for male fertility dysfunction in STZ diabetic rats

This new study evaluated effect of aqueous leaves extracts of Chrozophora tinctoria (L.) Rafin on male reproductive hormones and histological features of the testis diabetic rats. The results obtained showed remarkable increased of sperm count, viability and motility after treatment. Testosterone, FSH and LH hormones also increased with oral administration of the plant extract at dose 70 mg/kg of body weight. The results suggested the anti hyperglycemic property of the extract as well as its positive effect to maintain male hormones concentrations. Histological examination of the testis revealed improvement of spermatogenesis and Leydig cell proliferation with all doses of the extract. However, the histoarchitecture of the testis rats received 70 and 90 mg/kg of the plant extract, was found to be similar to the healthy group. These findings suggest that the extract can maintain the damage fertility in diabetic rats. These promising results may explain the use of this plant in the future for therapeutic treatment of diabetes complications associated subfertility.

Novel enhancer for male fertility dysfunction in STZ diabetic rats

This new study evaluated effect of aqueous leaves extracts of Chrozophora tinctoria (L.) Rafin on male reproductive hormones and histological features of the testis diabetic rats. The results obtained showed remarkable increased of sperm count, viability and motility after treatment. Testosterone, FSH and LH hormones also increased with oral administration of the plant extract at dose 70 mg/kg of body weight. The results suggested the anti hyperglycemic property of the extract as well as its positive effect to maintain male hormones concentrations. Histological examination of the testis revealed improvement of spermatogenesis and Leydig cell proliferation with all doses of the extract. However, the histoarchitecture of the testis rats received 70 and 90 mg/kg of the plant extract, was found to be similar to the healthy group. These findings suggest that the extract can maintain the damage fertility in diabetic rats. These promising results may explain the use of this plant in the future for therapeutic treatment of diabetes complications associated subfertility.