Tag Archives: Spermatid

Loss of Nectin-2 at Sertoli-Spermatid: DISCUSSION(5)

Acrosome-intact mouse sperm bind to the zona pellucida through specific receptor-ligand interactions and subsequently undergo an acrosome reaction. This step appears to have been impaired with sperm of nectin-2-defi-cient mice. Sperm binding to the oolemma is also mediated by a family of gamete receptors and their ligands. In mouse, both acrosome-intact and acrosome-reacted sperm are capable of binding to the oolemma of zona-free hamster eggs. Interestingly, this binding to the oolemma was not impaired in the nectin-2-deficient mice, although subsequent sperm penetration of the oocytes was abnormal. This observation suggests that the ability of knockout sperm to undergo an acrosome reaction may be altered. asthma rescue inhalers

Loss of Nectin-2 at Sertoli-Spermatid: DISCUSSION(4)

DISCUSSION(4)

While this work was ongoing, Bouchard et al. generated a similar nectin-2 knockout mouse line. In theirs as well as in our experiments, only one founder line was obtained. Therefore, our mouse nectin-2LacZ/LacZ line serves as an important validation of the infertility phenotype described earlier. A significant difference in our line is the presence of an in-frame fusion of a LacZ reporter into the nectin-2 gene that provided us with an additional tool for assessing the activity of the nectin-2 promoter by LacZ detection in nectin-2LacZ/LacZ mouse tissue.

Loss of Nectin-2 at Sertoli-Spermatid: DISCUSSION(3)

The differentiating germ cells evade autoimmune recognition by translocating to the immune-privileged adlu-minal compartment, which is separated from the basal compartment by the Sertoli cell-maintained junctional complexes of the BTB. This translocation requires the concerted action of breaking the tight and adherens junctions apically and rejoining them basally of the migrating germ cell, without compromising the BTB. We report here that nectin-2 protein localizes between Sertoli cells near the base of the tubule (Fig. 5). yasmin pills

Loss of Nectin-2 at Sertoli-Spermatid: DISCUSSION(2)

DISCUSSION(2)

Although Sertoli cell-expressed nectin-2 is linked to actin filaments of the apical ectoplasmic specializations, this cortical actin is non-contractile, and no myosin is associated with it. It therefore appears to have a merely structural function and is unlikely to act as a force-generating entity. Although a possible link between microtubule-based transport in Sertoli cells and translocation of the Sertoli-spermatid junctional plaque together with the attached spermatid has been suggested previously, the precise mechanisms of shaping and translocating the differentiating spermatid within the seminiferous epithelium are still elusive.

Loss of Nectin-2 at Sertoli-Spermatid: DISCUSSION(1)

Spermatogenesis, the progression from a diploid sper-matogenic stem cell to a highly specialized, motile, flagellated, haploid cell (i.e., the spermatozoon), presents one of the most dramatic differentiation processes in biology. This metamorphosis depends on a complex relationship between the differentiating germ cell and Sertoli cells that form the seminiferous epithelium. At least two types of cell-cell junctions are critically involved in maintaining the integrity of the seminiferous epithelium. Specialized Ser-toli-Sertoli junctional complexes (basal ectoplasmic specializations) separate the basal and adluminal compartments of the seminiferous epithelium by generating the BTB. Apical ectoplasmic specializations between Sertoli cells and elongated spermatids appear to be critical for morphogenesis and translocation of maturing spermatids, and their disassembly is required for release of spermatozoa into the lumen of the tubule. review

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.

Loss of Nectin-2 at Sertoli-Spermatid: RESULTS(9)

The nectin-2 N-terminal signal peptide will likely target the resulting nectin-2-LacZ fusion protein to the cell surface of cells in which the nectin-2 gene promoter is active. Staining for LacZ protein expression thus provided us with an alternative means of assessing the activity of the nectin-2 gene in the absence of secondary posttranslational events of the nectin-2 protein (e.g., protein-protein interactions). We found LacZ staining to outline the Sertoli cells, whereas no LacZ protein expression was associated with spermatid heads (Fig. 6, C, arrowheads, and D inset, asterisk). This indicates to us that LacZ was expressed by Sertoli cells. It can thus be inferred that the nectin-2 gene is active only in Sertoli cells and not in spermatids. This result supports those of our previous ultra-structural studies and germ cell transplantation experiments. so

Loss of Nectin-2 at Sertoli-Spermatid: RESULTS(8)

RESULTS(8)

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.

Loss of Nectin-2 at Sertoli-Spermatid: RESULTS(7)

Unexpectedly, nectin-2LacZ/LacZ spermatozoa were able to bind to the oolemma at levels comparable those of nectin-2wt/LacZ controls (Table 2). After 90 min of incubation, an average of 42.1 nectin-2LacZ/LacZ versus 47.9 nect^in-2wt^acZ spermatozoa had bound per oocyte (counted as the number of protruding sperm tails at a median focal plane) (Fig. 5). In contrast, penetration of oocytes by nectin-2LacZ/LacZ spermatozoa, as assessed by the presence of decondensing sperm nuclei in the ooplasm, was 75-fold and 32-fold lower after 45 and 90 min of incubation, respectively (Table 2 and Fig. 5). Albuterol Inhaler

Loss of Nectin-2 at Sertoli-Spermatid: RESULTS(6)

RESULTS(6)

Nectin-2-Null Spermatozoa Display Impaired Binding to Zona-Intact Mouse Oocytes

Zona-intact mouse oocytes were recovered from ovulated CD1 females 15 h post-hCG administration and incubated with 5 X 105 nectin-2wt/LacZ or nectin-2LacZ/LacZ sperm per milliliter (Fig. 4). We observed a 6-fold reduction in the binding capacity of nectin-2LacZ/LacZ spermatozoa to oocytes. An average of 40.8 nectin-2LacZ/LacZ spermatozoa bound per oocyte, but only 6.7 r^e^1^^^-2wt/LacZ spermatozoa were found to bind per oocyte (Fig. 4).

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