Expression of Nectin-2 in the Male Reproductive System
To confirm the absence of nectin-2 protein in nectin-2LacZ/ LacZ testis and to relate nectin function to the observed phenotype, we analyzed the expression of nectin-2 and that of its heterophilic binding partner, nectin-3, by immunofluorescence in wt (Fig. 6, A and B) and in nectin-2LacZ/LacZ testis (Fig. 6, C and D). Both nectin-2 and nectin-3 were strongly expressed along the convex curvature of the heads of elongated spermatids (Fig. 6, A and B). In addition, nectin-2 protein, but not nectin-3, was found in an almost ring-like staining pattern encircling the seminiferous tubule throughout the basal compartment (Fig. 6A, arrowheads). The latter staining appears to be associated with inter-Sertoli junctions, and its location near the base of the tubule suggests the presence of nectin-2 at the BTB.
Because in nectin-2LacZ/LacZ mice the nectin-2 gene is interrupted by the in-frame insertion of a LacZ reporter gene, we were interested whether the LacZ protein is expressed and whether analysis of LacZ expression can provide further insight regarding the function of nectin-2 in testis. As expected, no intact nectin-2 protein could be detected in testes of nectin-2LacZ/LacZ males, confirming the successful inactivation of the nectin-2 gene (Fig. 6C). Instead, the targeted nectin-2 locus indeed expressed the LacZ protein as a fusion to the N-terminal 61 amino acids of nectin-2 (Fig. 6, C and D). Because these N-terminal 61 amino acids of nectin-2 only contain a signal sequence and a small portion of the first immunoglobulin-like domain, it is not expected that any residual function of nectin-2 was preserved. Ventolin aerosol inhaler
FIG. 6. Expression nectin-2 and nectin-3 in sections of testis of nectin-2wt/wt and nectin-2LacZ/LacZ mice by coimmunofluorescence. Two adjacent testis sections of nectin-2wt/wt mice (A and B) and nectin-2LacZLacZ mice (C and D) were stained with rat mAbs 6B3/17B10 (red) against nectin-2 (A and C), with rat mAb 103-A1 (red) against nectin-3 (B and D), and with rabbit anti-LacZ (green; A-D). Nuclei were visualized with Hoechst 33258 DNA stain (AD). Strong nectin-2 staining is associated with the convex side of elongated spermatid stages (A inset) and within the basal compartment (A, arrowheads). No nectin-2 expression is detected in testis of nectin-2-deficient mice (C). Whereas no LacZ expression is seen in testis of wt males (A and B), strong reactivity is found in nectin-2LacZ/LacZ testis, in which LacZ is expressed under the control of the endogenous nec-tin-2 gene promoter (C and D). The observed LacZ staining is indicative for the activity of the nectin-2 gene locus in Sertoli cells but not spermatids (C, arrowheads, and D inset). Nectin-3 expression on elongated nectin-2wt/wt spermatids is very similar to that of nectin-2 (B insert); however, no nectin-3 staining is detected in the basal compartment (B). In testis of nectin-2-deficient mice, nectin-3 is still associated with spermatid heads (D insert, asterisk), but protein localization is disturbed. Bar = 100 ^m (A-D) and 12 ^m (all insets).
FIG. 7. Disruption of Sertoli-spermatid ectoplasmic specializations in nectin-2LacZ’ LacZ testis. Testis sections of nectin-2wt/vt mice (A-C and G-I) and nectin-2LacZ/LlicZ mice (D-F) were stained with rat mAb 103-A1 against nectin-3 (A and D, green color) and with mouse mAb 31 against es-pin (B and E, red color). Espin, known to be specifically expressed at Sertoli-sperma-tid ectoplasmic specializations, colocalizes with nectin-3 on elongated spermatid heads in nectin-2vM”t testis (C, yellow color). In nectin-2LacZ/LacZ testes, overall expression of espin is lower, and virtually no espin colocalizes to spermatid heads (F inset), suggesting that ectoplasmic specializations have not formed. Note that the only staining within the lumen of the tubule is specific for espin. The level of nonspecific staining because of endogenous mouse immunoglobulin (Ig) in the basal compartment is depicted (H). Bar = 50 l^m (A-I) and 8 ^m (all insets).