Abstract
Nectin-like molecule-2 (Necl-2), a junction molecule, is exclusively expressed by spermatogenic cells. It mediates homophilic interaction between germ cells and heterophilic interaction between Sertoli and germ cells. Knockout studies have shown that loss of Necl-2 causes male infertility, suggesting Necl-2-based cell adhesion is crucial for spermatogenesis. Transforming growth factor-βs (TGF-βs) are crucial for regulating cell junction restructuring that are required for spermatogenesis. In the present study, we aim to investigate the mechanism on how TGF-β1 regulates Necl-2 expression to achieve timely junction restructuring in the seminiferous epithelium during spermatogenesis. We have demonstrated that TGF-β1 reduces Necl-2 mRNA and protein levels at both transcriptional and post-translational levels. Using inhibitor and clathrin shRNA, we have revealed that TGF-β1 induces Necl-2 protein degradation via clathrin-dependent endocytosis. Endocytosis assays further confirmed that TGF-β1 accelerates the internalization of Necl-2 protein to cytosol. Immunofluorescence staining also revealed that TGF-β1 effectively removes Necl-2 from cell-cell interface. In addition, TGF-β1 reduces Necl-2 mRNA via down-regulating Necl-2 promoter activity. Mutational studies coupled with knockdown experiments have shown that TGF-β1-induced Necl-2 repression requires activation of Smad proteins. EMSA and ChIP assays further confirmed that TGF-β1 promotes the binding of Smad proteins onto MyoD and CCAATa motifs in vitro and in vivo. Taken together, TGF-β1 is a potent cytokine that provides an effective mechanism in controlling Necl-2 expression in the testis via Smad-dependent gene repression and clathrin-mediated endocytosis.
Highlights
During spermatogenesis, developing germ cells must migrate from the basal to the adluminal compartment, which requires the disassembly and reassembly of cell junctions between Sertoli cells and developing germ cells [1]
These results clearly suggested that Transforming Growth Factor-b1 (TGF-b1) is a potent negative regulator of Nectin-like molecule-2 (Necl-2) expression
Knockout studies indicated that Necl-2 mediates Sertoli-germ and germ-germ cell interactions that are essential for spermatogenesis
Summary
During spermatogenesis, developing germ cells must migrate from the basal to the adluminal compartment, which requires the disassembly and reassembly of cell junctions between Sertoli cells and developing germ cells [1]. Recent animal studies have shown that knockout of individual junction proteins, including nectin-2, nectin-3, or JAM-C cause infertility in male mice, indicating that each of the protein components plays an indispensable role in cell adhesion during spermatogenesis [7,8,9,10]. Knockout studies have shown that Necl-2 null male mice are sterile, and display defects in spermatogenesis with reduced sperm motility and abnormal morphology of spermatids [20,21,22,23]. A significant reduction in the number of mature spermatids has been
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