Elucidating the roles and regulation of coxsackievirus and adenovirus receptor in the testis

Abstract

Spermatogenesis is a highly complex, coordinated cellular process during which highly specialized haploid male gametes are developed from spermatogonial stem cells. Timely restructuring of the cell junctions between Sertoli cells and Sertoli cells, as well as junctions between Sertoli cells and germ cells are required throughout spermatogenesis. Although these cell junctions share common features with cell junctions in other tissues, they have their own unique characteristics. However, the exact components, architecture, and the regulatory mechanism for the unique characteristics of testicular cell junctions remain to be elucidated.

Coxsackievirus and adenovirus receptor (CXADR) is a membrane protein that is expressed by germ cells and Sertoli cells. The canonical membrane-bound CXADR can mediate Sertoli-Sertoli and Sertoli-germ cell interactions in either a homophilic or heterophilic manner. Our previous studies and reports from other investigators have revealed that Sertoli cell (SC)-specific CXADR knockout exhibits impaired spermatogenesis, suggesting that SC-CXADR is indispensable for spermatogenesis. Cytokines such as transforming growth factor-β3 (TGF-β3) have been demonstrated to play essential roles in modulating the disassembly and reassembly of those cell junctions at stage VIII of the cycle of the seminiferous epithelium.

This dissertation focuses on the molecular regulation of CXADR by TGF-β3 and the structure role of CXADR in testicular cells.

The expression patterns of TGF-β3 and CXADR are highly correlated. Our studies showed that TGF-β3 downregulates the expression level of Cxadr mRNA, as well as CXADR protein. The disappearance of CXADR at the site of the Sertoli-Sertoli cell interface was observed following TGF-β3 treatment. Inhibitor treatment and siRNA knockdown assays demonstrated that clathrin-mediated endocytosis is involved in TGF-β3-induced CXADR downregulation and mis-localization. TGF-β3 promotes CXADR protein degradation via ubiquitin-proteasome pathway. Besides, TGF-β3 induces the degradation of Cxadr mRNA partially via p38 mitogen-activated protein kinase signaling pathway.

By combining the proximity-dependent biotin identification assay and glutathione S-transferase pull down assay, an interactome of CXADR was plotted. Several important structural components of cell junctions are identified as potential interacting partners of CXADR, including desmoplakin, cofilin, and filamin-A. Gene ontology enrichment analysis revealed that CXADR relates these proteins to biological processes related to the intermediate filament and actin filament organization.

This thesis study revealed the regulatory mechanism of TGF-β3 on CXADR expression and an interactome of CXADR. Data obtained in this thesis study sheds new insights on the molecular structure of cell junctions as well as the regulation of cell junction dynamics in spermatogenesis.