Localisation of RA175 (Cadm1), a cell adhesion molecule of the immunoglobulin superfamily, in the mouse testis, and analysis of male infertility in the RA175-deficient mouse.

RA175, a member of the immunoglobulin superfamily, plays an important role in cell adhesion, and RA175 gene-deficient mice (RA175(-/-) ) show oligoastheno-teratozoospermia. To understand the function of RA175, location in the testis and the morphological features of its spermatogenic cells in RA175(-/-) mice were investigated. Immunohistochemical studies revealed that RA175 immunoreactivity was observed on the cell surface of the spermatogenic cells at specific stages. A strong reaction was detected from type A spermatogonia to pachytene spermatocytes at stage IV and from step 6 to step 16 spermatids during spermatogenesis. From pachytene spermatocytes at stage VI to step 4 spermatids, the reaction was not detected by the enzyme-labelled antibody method and was faintly detected by the indirect immunofluorescence method. Abnormal vacuoles in the seminiferous epithelium, showing exfoliation of germ cells, and ultrastructural abnormality of the elongate spermatids were revealed in the RA175(-/-) testes. Other members of the immunoglobulin superfamily such as basigin, nectin-2 and nectin-3, which have an important role in spermatogenesis, were immunohistochemically detected in the RA175(-/-) testis. These observations indicate a unique expression pattern of RA175 in the testis and provide clues regarding the mechanism of male infertility in the testis.

Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: a statistical study.

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density +/- SD of large IMP was 379 +/- 64 and 266 +/- 58/microm2, and that of small IMP was 1450 +/- 155 and 672 +/- 252/microm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.

Immunohistochemical localization of a water channel, aquaporin 7 (AQP7), in the rat testis.

Cell volume reduction is one of the most distinct morphological changes during spermiogenesis and may be largely attributable to water efflux from the cell. A strong candidate for a water efflux route, aquaporin 7 (AQP7), which is a water channel, was studied immunohistochemically in the rat testis. Immunoreactivity was restricted within the elongated spermatids, testicular spermatozoa, and residual bodies remaining in the seminiferous epithelium. Weak but distinct immunoreactivity was first observed in the cytoplasmic mass of the spermatid at step 8 of spermiogenesis. The Golgi-like apparatus became steadily immunoreactive at step 10. The plasma membrane covering the cytoplasmic mass showed strong immunoreactivity after step 16. At this step, the middle piece of the tail also showed immunoreactivity at the portion protruding into the lumen. The whole head and distal tail, where the elongated spermatid had only a limited amount of cytoplasm, showed no immunoreactivity throughout spermiogenesis. After spermiation, the immunoreactivity of AQP7 remained at the middle piece and in the cytoplasmic droplet in the testicular spermatozoon. The present observations suggest that AQP7 contributes to the volume reduction of spermatids, since this water channel protein is localized on the plasma membrane covering the condensing cytoplasmic mass of the elongated spermatid, and since the seminiferous tubule fluid is hypertonic.

Stage-specific localization of basigin, a member of the immunoglobulin superfamily, during mouse spermatogenesis.

Ablation of the transmembrane glycoprotein basigin leads to azoospermic mice, indicating that this gene is essential for spermatogenesis. To examine the functions of basigin in the testis, the precise localization of basigin during spermatogenesis was examined immunohistochemically. In the adult mouse testis, basigin immunoreactivity appeared on the cell surface of leptotene spermatocytes and gradually increased in intensity during the meiotic prophase. Cytoplasmic staining, as well as cell surface staining, was detected in spermatids. The most conspicuous reactivity was found in the spermatids at steps 9-11 and in the flagella of spermatids. Immuno-electron microscopic analysis demonstrated that basigin was localized not only on the plasma membranes of spermatocytes and spermatids, but also on the plasma membrane of the Sertoli cell processes which contact the spermatocytes and spermatids. Basigin immunoreactivity was also detected during postnatal development in spermatocytes and spermatids but not in spermatogonia. Experimental cryptorchid testes which contain only spermatogonia and Sertoli cells in the seminiferous epithelium showed no basigin immunoreactivity. Seven days after surgical reversal of the cryptorchid testis, spermatocytes reappeared in the tubules, along with basigin immunoreactivity. Furthermore, in sterile mutant mice, in which neither spermatocytes nor spermatids were generated, no basigin immunoreactivity was detected in the seminiferous tubules. These findings indicate that expression of basigin is concomitant with appearance of spermatocytes in the seminiferous tubule, and suggest that basigin is involved in the interaction between Sertoli cells and germ cells at specific stages of spermatogenesis.

Immuno-colloidal gold labeled surface replica, and its application to detect sp56, the egg recognition and binding protein, on the mouse spermatozoon.

Using immuno-colloidal gold surface replicas (IGSR), we developed a method to demonstrate surface antigens in a whole view of a cell at the EM level. In this study, a monoclonal antibody (MAB) 7C5 directed against sp56, which is a mouse sperm protein (M(r) = 56,000 daltons) and has specific affinity for the zona pellucida, was used as primary antibody. Fixed mouse spermatozoa were incubated with primary antibody and colloidal gold-labeled secondary antibody on a slide glass. Used gold particles were 10, 20 or 30 nm in diameter. The spermatozoa were then critical point dried, and replicated with platinum and carbon. Replicas were examined with a transmission EM. The result suggests that sp56 is located on the outer surface of the plasma membrane covering approximately 2/3 of dorsal edge of the sperm head, but it is virtually absent in the tapered apical tip. When 10 and 20 nm colloidal particles were used, about 500 particles were observed on one side of the flat sperm head. Although the large 30 nm particles were easily recognized in low magnification figures, the attached number on the sperm head was generally lower than the number of 10 or 20 nm particles observed. By this method, we could effectively demonstrate the localization of a surface antigen in a wide cell view with clear landmarks.