Regulation of testicular cell junction dynamics

Abstract

Spermatogenesis is an important cellular process in which mature haploid spermatozoa are produced from diploid spermatogonia and finally released from the seminiferous epithelium into the tubular lumen. This process involves extensive and timely restructuring of cell junctions at the Sertoli-Sertoli cell interface where the blood-testis barrier (BTB) and basal ectoplasmic specialization (ES) are located and at the Sertoli-germ cell interface where apical ES is present. The restructuring of cell junctions is involved in facilitating the translocation of spermatocytes across the BTB into adluminal compartment of the seminiferous epithelium as well as the detachment of mature elongated spermatids from the epithelium for release.

Turnover of the BTB and Sertoli-mature spermatid contact (apical ES) can be interrupted by environmental toxicant such as cadmium (Cd) and is tightly regulated by cytokines such as transforming growth factor betas (TGF-βs). However, the molecular mechanisms involved in the regulation of testicular cell junction dynamics have not been completely explored. This dissertation focused on examining the mechanisms on how Cd and TGF-β3 regulate junction proteins, nectin-2 and JAM-B respectively.

Nectin-2 is found at the basal and apical ES for the formation of the BTB and Sertoli-spermatid adhesion. Results of this study have demonstrated that CdCl2 reduces nectin-2 mRNA and protein levels and causes the loss of nectin-2 at the cell-cell interface. Inhibitor and shRNA knockdown have shown that CdCl2 speeds up nectin-2 protein degradation through clathrin-mediated endocytosis. Immunofluorescence staining and endocytosis assays further confirmed that nectin-2 internalization is promoted upon Cd treatment. In addition, Cd causes nectin-2 gene repression by reducing the bioavailability of endogenous transcription factors as well as impeding the binding of positive regulators to the promoter region. Collectively, these results have unraveled a novel and direct regulatory mechanism of Cd-mediated male reproductive dysfunction and explain why testis is more susceptible to Cd toxicity than other tissues.

Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells at the BTB as well as between Sertoli and germ cells at the apical ES in the testis. It was found that TGF-β3 downregulates JAM-B mRNA and protein levels, and reduces the bioavailability of JAM-B at the site of cell-cell contact. TGF-β3 promotes JAM-B protein degradation via ubiquitin-proteasome pathway with the activation of Smad signaling cascade. Besides, TGF-β3 destabilizes JAM-B mRNA transcript via ERK1/2 and p54 JNK activation. These results have unraveled the mechanisms by which TGF-β3 exerts negative regulatory effects on JAM-B expression that is crucial for the BTB integrity and Sertoli-germ cell interaction.

Taken together, the findings reported herein demonstrate that testicular cell junction proteins are regulated by environmental toxicant and cytokine at the transcriptional, post-transcriptional and post-translational levels. Data presented herein strengthen the understanding on how testicular cell junction dynamic is regulated and shed new insights for prevention of Cd-induced male infertility.