Search Results (13)

Testicular torsion is a common emergency in adolescents, and can lead to severe ischemia reperfusion injury (IRI). LncRNA H19 has been shown to increase during ischemia, but its role in testicular IRI remains unknown. Focusing on this research gap, we utilized H19 biallelic mutant mice and Sertoli cell line (TM4) to construct in vivo and in vitro models of ischemia/reperfusion (I/R) and oxygen–glucose deprivation/reperfusion (OGD/R). Compared to WT I/R mice, H19^−/− I/R mice showed milder tissue disorganization and cell loss, with a more intact blood–testis barrier (BTB). The cell viability decreased, ROS levels and apoptosis-related factors such as Bax/Bcl-2 increased in TM4 cells after OGD/R, whereas these changes were reversed when H19 was knocked down followed by OGD/R (si-H19+OGD/R). In contrast, over-expression of H19 in TM4 cells exacerbates OGD/R-induced cell apoptosis. Through in-depth analysis of KEGG-enriched pathways, the PI3K/AKT pathway was identified as a potential target of H19 modulation. Western blotting confirmed that, in OGD/R cells, elevated H19 levels were accompanied by the excessive AKT phosphorylation and the tight junction marker ZO-1 degradation; and in si-H19+OGD/R cells, the decreased AKT phosphorylation was recovered and the up-regulated ZO-1 expression was weakened simultaneously via using the AKT activator SC79. These results suggest that inhibiting H19 in OGD/R cells might preserve the integrity of the BTB by reversing the excessive phosphorylation of AKT. Moreover, H19 deficiency in si-H19+OGD/R cells alleviated the disturbances in glycolysis, fatty acid biosynthesis, and amino acid metabolism. Our study indicates that H19 might be a potential therapeutic target for clinic testicular I/R treatment. Full article

Blood–testis barrier (BTB) genes are crucial for the cellular mechanisms of spermatogenesis as they protect against detrimental cytotoxic agents, chemicals, and pathogens, thereby maintaining a sterile environment necessary for sperm development. BTB proteins predominantly consist of extensive tight and gap junctions formed between Sertoli cells. These junctions form a crucial immunological barrier restricting the intercellular movement of substances and molecules within the adluminal compartment. Epithelial tight junctions are complex membrane structures composed of various integral membrane proteins, including claudins, zonula occludens-1, and occludin. Inter-testicular cell junction proteins undergo a constant process of degradation and renewal. In addition, the downregulation of genes crucial to the development and preservation of cell junctions could disrupt the functionality of the BTB, potentially leading to male infertility. Oxidative stress and inflammation may contribute to disrupted spermatogenesis, resulting in male infertility. L-cysteine is a precursor to glutathione, a crucial antioxidant that helps mitigate damage and inflammation resulting from oxidative stress. Preclinical research indicates that L-cysteine may offer protective benefits against testicular injury and promote the expression of BTB genes. This review emphasizes various BTB genes essential for preserving its structural integrity and facilitating spermatogenesis and male fertility. Furthermore, it consolidates various research findings suggesting that L-cysteine may promote the expression of BTB-associated genes, thereby aiding in the maintenance of testicular functions. Full article

The yak (Bos grunniens) is a valuable livestock animal endemic to the Qinghai–Tibet Plateau in China with low reproductive rates. Cryptorchidism is one of the primary causes of infertility in male yaks. Compared with normal testes, the tight junctions (TJs) of Sertoli cells (SCs) and the integrity of the blood–testis barrier (BTB) in cryptorchidism are both disrupted. MicroRNAs are hairpin-derived RNAs of about 19–25 nucleotides in length and are involved in a variety of biological processes. Numerous studies have shown the involvement of microRNAs in the reproductive physiology of yak. In this study, we executed RNA sequencing (RNA-seq) to describe the expression profiles of mRNAs and microRNAs in yaks with normal testes and cryptorchidism to identify differentially expressed genes. GO and KEGG analyses were used to identify the biological processes and signaling pathways which the target genes of the differentially expressed microRNAs primarily engaged. It was found that novel-m0230-3p is an important miRNA that significantly differentiates between cryptorchidism and normal testes, and it is down-regulated in cryptorchidism with p < 0.05. Novel-m0230-3p and its target gene CSF1 both significantly contribute to the regulation of cell adhesion and tight junctions. The binding sites of novel-m0230-3p with CSF1 were validated by a dual luciferase reporter system. Then, mimics and inhibitors of novel-m0230-3p were transfected in vitro into SCs, respectively. A further analysis using qRT-PCR, immunofluorescence (IF), and Western blotting confirmed that the expression of cell adhesion and tight-junction-related proteins Occludin and ZO-1 both showed changes. Specifically, both the mRNA and protein expression levels of Occludin and ZO-1 in SCs decreased after transfection with the novel-m0230-3p mimics, while they increased after transfection with the inhibitors, with p < 0.05. These were achieved via the CSF1/CSF1R/Ras signaling pathway. In summary, our findings indicate a negative miRNA-mRNA regulatory network involving the CSF1/CSF1R/Ras signaling pathway in yak SCs. These results provide new insights into the molecular mechanisms of CSF1 and suggest that novel-m0230-3p and its target protein CSF1 could be used as potential therapeutic targets for yak cryptorchidism. Full article

As a pivotal player in spermatogenesis, the blood-testis barrier (BTB) made from junction apparatus coexisting in Sertoli cells (SCs) is impaired with an increase in age and ultimately induces spermatogenic dysfunction or even infertility. It has been corroborated that bone marrow mesenchymal stem cell (BMSC) transplantation can efficiently repair and regenerate the testicular function. As vital mediators of cell-to-cell communication, MSC-derived exosomes (Exos) can directly serve as therapeutic agents for tissue repair and regeneration. However, the therapeutic value of BMSC-Exos in aging-induced BTB damage remains to be confirmed. In this study, we explored that the old porcine testes had defective autophagy, which aggravated BTB disruption in SCs. BMSC-Exos could decrease ROS production and NLRP3 inflammasome activation but enhanced autophagy and tight junction (TJ) function in D-gal-triggered aging porcine SCs and mouse model testes, according to in vitro and in vivo experiments. Furthermore, rapamycin, NAC, MCC950, and IL-1Ra restored the TJ function in D-gal-stimulated aging porcine SCs, while BMSC-Exos’ stimulatory effect on TJ function was inhibited by chloroquine. Moreover, the treatment with BMSC-Exos enhanced autophagy in D-gal-induced aging porcine SCs by means of the AMPK/mTOR signal transduction pathway. These findings uncovered through the present study that BMSC-Exos can enhance the BTB function in aging testes by improving autophagy via the AMPK/mTOR signaling pathway, thereby suppressing ROS production and NLRP3 inflammasome activation. Full article

Yaks, a valuable livestock species endemic to China’s Tibetan plateau, have a low reproductive rate. Cryptorchidism is believed to be one of the leading causes of infertility in male yaks. In this study, we compared the morphology of the normal testis of the yak with that of the cryptorchidism, and found dysplasia of the seminiferous tubules, impaired tightness of the Sertoli cells, and a disruption of the integrity of the blood–testis barrier (BTB) in the cryptorchidism. Previous studies have shown that CAV1 significantly contributes to the regulation of cell tight junctions and spermatogenesis. Therefore, we hypothesize that CAV1 may play a regulatory role in tight junctions and BTB in Yaks Sertoli cells, thereby influencing the development of cryptorchidism. Additional analysis using immunofluorescence, qRT-PCR, and Western blotting confirmed that CAV1 expression is up-regulated in yak cryptorchidism. CAV1 over-expression plasmids and small RNA interference sequences were then transfected in vitro into yak Sertoli cells. It was furthermore found that CAV1 has a positive regulatory effect on tight junctions and BTB integrity, and that this regulatory effect is achieved through the FAK/ERK signaling pathway. Taken together, our findings, the first application of CAV1 to yak cryptorchidism, provide new insights into the molecular mechanisms of cell tight junctions and BTB. This paper suggests that CAV1 could be used as a potential therapeutic target for yak cryptorchidism and may provide insight for future investigations into the occurrence of cryptorchidism, the maintenance of a normal physiological environment for spermatogenesis and male reproductive physiology in the yak. Full article

Cryptorchidism, a condition in which testes fail to descend from the abdomen into the scrotum, is a risk factor for infertility and germ cell cancer. Normally, tight junctions between adjacent Sertoli cells in the testes form a blood–testes barrier that regulates spermatogenesis; however, the effect of cryptorchidism on tight junctions is not well-understood. We established a model of heat-induced testicular damage in dogs using surgical cryptorchidism. We sequenced RNA to investigate whether certain transcripts are expressed at higher rates in heat-damaged versus normally descended testes. Claudins, cell adhesion molecules, were relatively highly expressed in cryptorchid testes: claudins 2, 3, 5, 11, and 18 were significantly increased in cryptorchid testes and reduced by orchiopexy. SOX9-positive Sertoli cells were present in the seminiferous tubules in both cryptorchid and control testes. Using real-time PCR and Western blot analysis to compare Sertoli cells cultured at 34 °C and 37 °C, we found that Sertoli cell claudins 2, 3, 5, 11, and 18 were significantly increased at 37 °C; however, accumulation was higher in the G0/G1 phase in Sertoli cells cultured at 34 °C. These results indicate that testicular hyperthermia caused by cryptorchidism affects claudin expression, regulated germ cell death, and the proliferation of Sertoli cells. Full article

The blood–testis barrier (BTB) is formed from tight junctions (TJs) between Sertoli cells. This dynamic structure, which establishes an immune-privileged environment protecting haploid germ cells formed in puberty from cells of the innate immune system, protects male fertility. Testosterone produced in Leydig cells is one of the main regulators of TJ protein expression and BTB dynamics. Nevertheless, although it has been assumed that testosterone effects on TJs and BTB are mediated through the classical androgen receptor (AR), newer results call the importance of this receptor into question. ZIP9, a recently identified androgen receptor of plasma membranes, mediates testosterone effects that promote the expression of TJ proteins and TJ formation in a rat Sertoli cell line that lacks the classical AR. Although these findings suggest that ZIP9 mediates these testosterone effects, participation of the classical AR in these events cannot be excluded. Here we used immortalized adult rat Sertoli cells that express both ZIP9 and AR and addressed the involvement of these receptors in the stimulation of TJ protein expression and TJ formation in response to testosterone and to the androgenic peptide IAPG that acts via ZIP9. We find that both testosterone and IAPG trigger the so-called non-classical signaling pathway of testosterone and stimulate the expression of TJ-associated proteins and TJ formation. Silencing classical AR expression had no effect on the responses, whereas silencing of ZIP9 expression completely blocked them. Our results demonstrate that ZIP9 is the sole androgen receptor involved in the regulation of TJ protein expression and TJ formation at the BTB. Full article

Spermatogenesis is a complex process occurring in mammalian testes, and constant sperm production depends on the exact regulation of the microenvironment in the testes. Many studies have indicated the crucial role of blood–testis barrier (BTB) junctions and retinoic acid (RA) signaling in the spermatogenesis process. The BTB consists of junctions between adjacent Sertoli cells, comprised mainly of tight junctions and gap junctions. In vitamin A-deficient mice, halted spermatogenesis could be rebooted by RA or vitamin A administration, indicating that RA is absolutely required for spermatogenesis. Accordingly, this manuscript will review and discuss how RA and the BTB regulate spermatogenesis and the interaction between RA signaling and BTB function. Full article

Alkylating agents and irradiation induce testicular damage, which results in prolonged azoospermia. Even very low doses of radiation can significantly impair testis function. However, re-irradiation is an effective strategy for locally targeted treatments and the pain response and has seen important advances in the field of radiation oncology. At present, little is known about the relationship between the harmful effects and accumulated dose of irradiation derived from continuous low-dose radiation exposure. In this study, we examined the levels of mRNA transcripts encoding markers of 13 markers of germ cell differentiation and 28 Sertoli cell-specific products in single- and re-irradiated mice. Our results demonstrated that re-irradiation induced significantly decreased testicular weights with a significant decrease in germ cell differentiation mRNA species (Spo11, Tnp1, Gfra1, Oct4, Sycp3, Ddx4, Boll, Crem, Prm1, and Acrosin). In the 13 Sertoli cell-specific mRNA species decreased upon irradiation, six mRNA species (Claudin-11,Espn, Fshr, GATA1, Inhbb, and Wt1) showed significant differences between single- and re-irradiation. At the same time, different decreases in Sertoli cell-specific mRNA species were found in single-irradiation (Aqp8, Clu, Cst12, and Wnt5a) and re-irradiation (Tjp1, occludin,ZO-1, and ZO-2) mice. These results indicate that long-term aspermatogenesis may differ after single- and re-irradiated treatment. Full article

Androgens stimulate the expression of tight junction (TJ) proteins and the formation of the blood–testis barrier (BTB). Interactions of testosterone with the zinc transporter ZIP9 stimulate the expression of TJ-forming proteins and promote TJ formation in Sertoli cells. In order to investigate androgenic effects mediated by ZIP9 but not by the nuclear androgen receptor (AR), the effects of three tetrapeptides fitting the androgen binding site of ZIP9 were compared with those induced by testosterone in a Sertoli cell line expressing ZIP9 but not the AR. Three tetrapeptides and testosterone displaced testosterone-BSA-FITC from the surface of 93RS2 cells and stimulated the non-classical testosterone signaling pathway that includes the activation of Erk1/2 kinases and transcription factors CREB and ATF-1. The expression of the TJ-associated proteins ZO-1 and claudin-5 was triggered as was the re-distribution of claudin-1 from the cytosol to the membrane and nucleus. Furthermore, TJ formation was stimulated, indicated by increased transepithelial electrical resistance. Silencing ZIP9 expression by siRNA prevented all of these responses. These results are consistent with an alternative pathway for testosterone action at the BTB that does not involve the nuclear AR and highlight the significant role of ZIP9 as a cell-surface androgen receptor that stimulates TJ formation. Full article

Ubiquitination, an essential posttranslational modification, plays fundamental roles during mammalian spermatogenesis. We previously reported the requirement of two Cullin 4 ubiquitin ligase family genes, Cullin 4a (Cul4a) and Cullin 4b (Cul4b), in murine spermatogenesis. Both genes are required for male fertility despite their distinct functions in different cell populations. Cul4a is required in primary spermatocytes to promote meiosis while Cul4b is required in secondary spermatocytes for spermiogenesis. As the two genes encode proteins that are highly homologous and have overlapping expression in embryonic germ cells, they may compensate for each other during germ cell development. In the present study, we directly address the potential functional redundancy of these two proteins by deleting both Cul4 genes, specifically, in the germ cell lineage during embryonic development, using the germ-cell specific Vasa-Cre line. Conditional double-knockout (dKO) males showed delayed homing and impaired proliferation of gonocytes, and a complete loss of germ cells before the end of the first wave of spermatogenesis. The dKO male germ cell phenotype is much more severe than those observed in either single KO mutant, demonstrating the functional redundancy between the two CUL4 proteins. The dKO mutant also exhibited atypical tight junction structures, suggesting the potential involvement of CUL4 proteins in spermatogonial stem cell (SSC) niche formation and blood–testis-barrier (BTB) maintenance. We also show that deleting Cul4b in both germ and Sertoli cells is sufficient to recapitulate part of this phenotype, causing spermatogenesis defects and drastically reduced number of mature sperms, accompanied by defective tight junctions in the mutant testes. These results indicate the involvement of CUL4B in maintaining BTB integrity. Full article

A protocol for the isolation and long-term propagation of adult rat Sertoli cells (SCs) using conditional reprogramming (CR) was developed and the formation of tight junctions as an in vitro model for the blood testis barrier (BTB) was studied. Three pure primary SC lines were isolated successfully and maintained for several months without significant changes in expression levels of SC-typical markers such as SRY-box transcription factor 9 (SOX9), transferrin, clusterin, androgen receptor (AR), and GATA binding protein 1 (GATA1). In addition to AR expression, the tight junction proteins, zonula occludens-1 (ZO-1) and the junctional adhesion molecule-3 (JAM-3), were upregulated and the SC barrier integrity was enhanced by testosterone. Peritubular/myoid cells did not increase the tightness of the SC. The cytokines, interleukin-6 (IL-6), bone morphogenetic protein-2 (BMP2), and transforming growth factor beta-3 (TGF-β3), negatively affected the tightness of the SC barrier. We have established a protocol for the isolation and long-term propagation of highly pure primary adult rat SCs, which are able to respond to androgen treatments, to form tight junctions and to maintain the mRNA expression of SC-specific genes. By applying this new method, adult SCs can now be analyzed in more detail and might serve as an in vitro model for the study of many SC functions. Full article

Androgens are key for pubertal development of the mammalian testis, a phenomenon that is tightly linked to Sertoli cell maturation. In this review, we discuss how androgen signaling affects Sertoli cell function and morphology by concomitantly inhibiting some processes and promoting others that contribute jointly to the completion of spermatogenesis. We focus on the molecular mechanisms that underlie anti-Müllerian hormone (AMH) inhibition by androgens at puberty, as well as on the role androgens have on Sertoli cell tight junction formation and maintenance and, consequently, on its effect on proper germ cell differentiation and meiotic onset during spermatogenesis. Full article