The Members of the IgSF (immunoglobulin super family) family are multifunctional, transmembranous proteins involved in the regulation of membrane fusion, and cell-to-cell adhesion, and they may act as receptors in processes such as cancer, neurogenesis, myogenesis, and spermatogenesis. VSIG1, which was originally known as A34, consists of two extracellular immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic domain; the Scanlan research group has characterized VSIG1 as a new member of the junctional adhesion molecule (JAM) family. The expression of A34 is highly tissue-restricted, it is expressed predominantly in the stomach and testis. However, this molecule has not been functionally characterized. We were interested in studying the possible role of VSIG1 in spermatogenic cells and sertoli cells. In order to investigate the role(s) of VSIG1 in mammalian spermatogenesis, we first generated a specific antibody against mouse VSIG1 and examined the presence and localization of the protein in tissues. Reverse transcriptase-polymerase chain reaction (RT-RCR) and Western blot analysis of the mouse tissues indicated that VSIG1 was expressed specifically in the testis. Furthermore, our trypsinization and biotinylation assays strongly support the assumption that VSIG1 is localized on the testicular germ cell surface. In order to tetermine whether VSIG1 is capable of participatin in homotypic interactions, we produced glutathione S-transferase (GST) fusion proteins against each domain. Western blot analysis under mild conditions demonstrated that each Ig-like domain could form homophilic complexes, while the cytoplasmic domain could not. To obtain further information on this observation, we performed a GST-pull down assay by using recombinant GST-fusion and His-tagging proteins. The pull-down assay also demonstrated that each GST-fusion Ig-like domain shows homotypic binding. Although the effects of VSIG1 function during spermatogenesis are still largely unknown, these results conclusively show that VSIG1 is a new component of a testicular specific the members of which potentially interact with the sertoli cell surface protein. Further investigations to clarify the role of VSIG1 in the interactions between spermatogenic cells and sertoli cells will be beneficial in elucidating the mechanism of spermatogenesis. (poster)