Espin Fails to Localize at Sertoli-Spermatid Junctions in Nectin-2LacZ/LacZ Testis
The data presented above and our earlier results suggest that Sertoli-spermatid junctions form improperly in nec-tin-2LacZ/LacZ testis. Furthermore, we have shown that actin assembly in Sertoli cells of nectin-2LacZ/LacZmice is disturbed at their junctions with elongated spermatids. To test whether other components of the junctional complex are affected, we analyzed the expression of the actin-bundling protein espin (ectoplasmic s/ecialization + “in”), a known component of Sertoli cell ectoplasmic specializations (Fig. 7). In wt testis, espin colocalized with nectin-3 at Sertoli-spermatid junctions (Fig. 7C, yellow color). In contrast, the overall expression of espin was lower in nectin-2LacZ/LacZ testis, and remarkably, virtually no espin was found to be associated with Sertoli-spermatid junctions (Fig. 7F). This result suggests that in the absence of nectin-2, ecto-plasmic specializations cannot be assembled.
Expression of Nectin-2 in Epididymis
Interestingly, as soon as spermatozoa were released from the seminiferous epithelium to be transported to the epididymis, no nectin-2 or nectin-3 immunoreactivity was associated with spermatozoan heads (Fig. 8, A and a1 and B and b1, respectively). This was expected for nectin-2, because the protein seems to be exclusively expressed by Sertoli cells (see above). In contrast, spermatid expression of nectin-3 appears to be downregulated, possibly to facilitate spermia-tion by detachment from nectin-2 containing ectoplasmic specialization on Sertoli cells. in detail
Furthermore, we detected nectin-2 expression at apical junctions of epithelial cells lining the epididymal duct (Fig. 8, A, arrowhead, and a2, arrowheads). The localization of nectin-2 staining at the apical region of the epithelium is consistent with a role of nectin-2 as a homophilic adhesion molecule at cadherin-based adherens junction, as has been previously described in other epithelia.
Unlike Bouchard et al., we did not observe nectin-2 staining on the midpiece of spermatozoa at any stage of spermatogenesis (see, e.g., Figs. 6A and 8A), including mature spermatozoa of the cauda epididymis (data not shown).
FIG. 8. Expression of nectin-2 and nec-tin-3 in the caput epididymis. A-D) Sections through the caput epididymis of nec-tin-2wtww mice with spermatozoa present. C and D) Phase-contrast images of the respective immunofluorescence images above. No nectin-2 staining is associated with heads of early spermatozoa (A, a1, and c1). However, abundant nectin-2 protein is found at apical junctions of principal epithelial cells lining the epididymal duct (A, arrowheads in a2). A tangential cut through the apical region of the epithelium lining the epididymal duct reveals intense nectin-2 staining at epithelial cellcell junctions in a typical honeycomb pattern (arrowhead in A). No nectin-3 expression is observed on spermatozoa or epididymal duct epithelium (B, b1, and d1). Bar = 100 ^m (B) and 8 ^m (bl).