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Loss of Nectin-2 at Sertoli-Spermatid: RESULTS(10)

RESULTS(10)

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).

Fig8Loss of Nectin1-9
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).