Immunocytochemistry of extracellular matrix components in the rat seminiferous tubule: electron microscopic localization with improved methodology.

Cell-cell interactions between Sertoli, myoid, Leydig, and germ cells are thought to be essential for spermatogenesis. These cells interact with each other through the extracellular matrices (ECMs) of the testicular lamina propria, which thus may have an important function in spermatogenesis. For an understanding of the role of ECMs in spermatogenesis, it is important to investigate the molecular constitution of the testicular ECM. We examined the distribution of type IV, V, and VI collagens (Cols), laminin (LM), fibronectin (FN), and heparan sulfate proteoglycan (HSPG) in the rat testicular lamina propria. Adult rat testes were fixed with 4% paraformaldehyde and 0.2% glutaraldehyde. Ultrathin frozen sections were made and immunolabeling was performed according to the method of Tokuyasu (1986 J. Microsc., 143:139-149). Alternatively, for preembedding immunocytochemistry, we used labeling with nanogold, followed by silver enhancement and gold toning. The tissues were then fixed with osmium and embedded in Epon (Sawada and Esaki 1994 J. Electron Microsc., 43:361-366). These specimens were examined in a JEOL JEM-100C transmission electron microscope operated at 80 kV. Col IV, LM, and HSPG were localized in the basement membranes of the seminiferous tubule (ST-BM) and in the BM of myoid cells. Cols V and VI, and FN were localized both in the connective tissue between the seminiferous tubule and the myoid cells (tubule-myoid connective tissue) and in the connective tissue between the myoid cells and the lymphatic endothelial cells (myoid-endothelium connective tissue). Furthermore, statistical analysis of the micrographs of immunogold labeling for Col IV, LM, and FN around the ST-BM suggested that the Col IV molecule is located uniformly in the ST-BM, whereas the LM molecule is distributed mainly in the lamina rara of ST-BM, and the FN molecule appears to be present predominantly in the tubule-myoid connective tissue. Distinct distribution patterns were observed for each antigen within the testicular lamina propria at the ultrastructural level. However, localization of some components was not consistent with localizations reported by others.