Size-selective junctional barrier and Ca(2+)-independent cell adhesion in the testis of Cynops pyrrhogaster: expression and function of occludin.

In urodeles which has testicular structure different from that in mammals, blood-testis barrier was reported to exist like in mammals. However, molecular and functional analyses of the components of the blood-testis barrier in urodeles have not been reported yet. Toward elucidation of the barrier functions and their molecular components in newt testis, we aimed to isolate occludin cDNAs and obtained two kinds of occludin partial cDNAs (occludin 1 and 2) encoding the putative second extracellular loop. Immunoblot and immunofluorescence studies using antibodies against peptides each corresponding to a part of the second extracellular loop of occludin 1 and 2, and those against beta-catenin and zonula occludens-1 (ZO-1) showed that occludin, as well as beta-catenin and ZO-1, was expressed not only in Sertoli cells but also in germ cells throughout all the stages from spermatogonia to elongate spermatids. Tracer experiments revealed a size-selective barrier which allows small molecules ( approximately 500 Da) to get into cysts through Sertoli cells' barrier, but not larger ones (>1.9 kDa) in the stages from spermatogonia to almost mature sperm. No occludin peptides corresponding to a part of the second extracellular loop destroyed the junctional barrier, while both the peptides and antibodies significantly inhibited reaggregation of the dissociated testicular cells which was to a large extent Ca(2+)-independent. These results indicate that the second extracellular loop of occludin is involved in cell adhesion rather than in size-selective barrier in newt testis, though the possibility cannot be excluded that the peptides were not long enough to inhibit the barrier function.