Size‐selective junctional barrier and Ca²⁺‐independent cell adhesion in the testis of Cynops pyrrhogaster: Expression and Function of Occludin

Abstract

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.

Promotion of spermatogonial proliferation by neuregulin 1 in newt (Cynops pyrrhogaster) testis

“…Addition of nNRG1-EGF increased the number of BrdU-positive germ cells in a concentration-dependent manner up to 350 nM, whereas 700 nM did not show any significant difference compared to 350 nM. This result indicates that nNRG1 promotes spermatogonial proliferation indirectly through somatic cells, because nNRG1-EGF could not have worked on germ cells directly due to size-selective barrier formed by Sertoli cells that enclose germ cells in a cyst (Jin et al, 2008).…”

“…Hence, to expose germ cells to nNRG1-EGF directly, we performed reaggregate cultures, in which size-selective barrier was destroyed by dissociation of testis with collagenase treatment (Ito and Abé , 1999;Jin et al, 2008) (Fig. 5A).…”

“…In addition, we reported that SCF and IGF-I, both of which are upregulated in response to FSH (unpublished data; Yamamoto et al, 2001), induce spermatogonial proliferation (Abé et al, 2002) and differentiation (Nakayama et al, 1999), respectively, in the organ culture. Recently, we have demonstrated that the blood-testis barrier formed by Sertoli cells show size selectivity, which allows small molecules ($500 Da) to penetrate into the cyst, but not larger ones (>1.9 kDa) in the organ culture (Jin et al, 2008). Therefore, SCF and IGF-I induce spermatogonial proliferation and differentiation indirectly in the organ culture.…”

Testicular structure and germ cells morphology in salamanders

“…Some spermatogonia are also observed between the connective tissue that surrounds lobules in other regions of the testis, not only in the cephalic region but in the direction of the middle part of the testis. The spermatogonia are the biggest germinal cells (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55) (Figs. 3A,B).…”

“…Sertoli cells maintain a permeability barrier to spermatogenic cells during spermatogenesis, determine the endocrine activity that controls spermatogenesis, phagocytose degenerating spermatogenic cells and residual bodies during the spermiogenesis, and form specific antigens. 25,31,37,39,40 During spermiation, when the cysts open and spermatozoa leave the testis, Sertoli cells (Fig. 2D) remain inside the lobule (Fig.…”