Basigin (bsg) is a transmembrane glycoprotein belonging to an immunoglobulin superfamily and is localized on the surface of the sperm tail. The behaviour of bsg during epididymal maturation and its role in fertilization were examined using an anti-bsg antibody. Spermatozoa from caput, corpus and cauda epididymides were immunostained by indirect immunofluorescence (IIF). Immunostaining revealed that bsg is localized on the principal piece of caput spermatozoa and the molecule was found on the middle piece during transit in the corpus and cauda epididymides. Concomitantly, the molecular mass of bsg was reduced from 37 kDa (testis) to 26 kDa (cauda epididymidis). IVF experiments were designed to assess the effect of anti-bsg antibody on the fertilization events. Anti-bsg antibody significantly inhibited primary binding to the cumulus-invested oocytes with intact zonae pellucidae in a dose-dependent manner. Consequently, the fertilization rate of cumulus-invested oocytes with intact zonae pellucidae was also inhibited. The bsg molecule was also detected on the head of live capacitated spermatozoa by IIF under IVF conditions. These findings indicate that testicular bsg is a glycosylated protein that undergoes molecular processing and deglycosylation during its transit in the epididymis. The bsg molecule that was detected on the sperm head after capacitation may facilitate the primary binding or might be involved in distinct events required for primary binding of spermatozoa to the zona pellucida during capacitation and sperm-cumulus interaction.

In this study, we examined the localization and characteristics of an intra-acrosomal protein, acrin2 (MC41), during guinea pig spermiogenesis and post-testicular sperm maturation in the epididymis, using the monoclonal antibody MC41. Immunoelectron microscopy demonstrated not only a specific domain localization of acrin2 in the apical segment of the guinea pig sperm acrosome, but also its dynamic behavior according to the spermatid differentiation and passage through the epididymis, as follows: acrin2 was exclusively localized in the membrane of the endoplasmic reticulum of early-stage spermatids but was not detectable in the developing acrosome until spermatids reached the maturation phase. In the final stage of spermiogenesis, acrin2 became localized in the outer acrosomal membrane (OAM)/matrix-associated materials both in the small region posterior to the dorsal matrix and along the ventral margin of the acrosomal apical segment. The acrosomal location of acrin2 in caput epididymidal sperm was almost identical to that observed in the final step spermatids, but during maturation it became progressively more restricted in area until on distal cauda epididymidal sperm it remained only in the dorsal region. In Western blot analysis, the MC41 antibody recognized a 165-kDa protein in the mature sperm extract. Furthermore, it was demonstrated that molecular weight reduction of the protein occurred during sperm passage through the epididymis. These findings indicate that acrin2 changes progressively in both distribution and size during development and maturation of the acrosome.

The monoclonal antibody mMN9 recognizes an antigenic molecule, equatorin, which is localized at the equatorial segment of the mammalian sperm acrosome. Our previous results using an IVF system indicated that mMN9 blocked sperm-oocyte fusion. Antibody-containing and control solutions were injected directly into the right and left oviductal ampullae, respectively, of anaesthetized female mice to assess the effect of mMN9 on fertilization in vivo. After hCG treatment, the females were mated, and their oviductal eggs and implanted embryos were examined. mMN9 was retained in the oviductal lumen at 20 h after injection. The rates of fertilization and concomitant pregnancy were significantly lower than in the control side (P < 0.05). In addition, histological studies showed no evidence of pathological changes in the female reproductive tract after the injections. These results indicate that mMN9 inhibits mouse fertilization significantly under in vivo conditions and that this injection method should be useful for studying the effects of antibodies and agents on fertilization in vivo.

We isolated the MC31 cDNA clone coding the antigen specifically recognized by the monoclonal antibody mMC31, and found that MC31 was identical to rat CE9. Therefore, this molecule is called MC31/CE9. MC31/CE9, a member of the immunoglobulin superfamily molecules, was localized on the rat sperm flagellar plasma membrane. We analyzed the expression and cellular localization of MC31/CE9 mRNA and protein in the adult rat testis by use of Northern hybridization, in situ hybridization, and immunohistochemical analyses. In the course of spermatogenesis, MC31/CE9 mRNA first appeared in type B spermatogonia. The mRNA signal intensity increased progressively to pachytene spermatocytes and remained constantly at a considerable level throughout the subsequent phases of spermatocytes and round spermatids, and then decreased gradually from step-11 spermatids to disappear in step-15 spermatids. On the other hand, MC31/CE9 protein expression showed a bimodal pattern. Immunohistochemical analysis for the MC31/CE9 protein revealed its most intense immunoreactivity on the flagella of step-8 to step-19 elongated spermatids. The cytoplasmic immunoreactivity of the MC31/CE9 protein also appeared in preleptotene to early pachytene spermatocytes and elongated spermatids, with particularly intense immunoreactivity in the Golgi complexes of zygotene and early pachytene spermatocytes (stage XIII to III) as well as step-8 to step-13 spermatids. Between these two phases, the MC31/CE9 protein proved undetectable in the cytoplasm of any spermatogenic cells. Sertoli cells and Leydig cells were devoid of MC31/CE9 mRNA and its protein. Therefore, the production of MC31/CE9 is thought to be posttranscriptionally regulated during spermiogenesis.

Sperm with a large acrosome such as that of guinea pigs and hamsters have a subdomain structure in the anterior acrosome, but the mouse acrosome looks homogeneous and its matrix has not been precisely analyzed. The intra-acrosomal protein MC41 is localized in the cortical region of the mouse anterior acrosome, suggesting a subdomain structure in the mouse acrosome. Thus, the present study was undertaken to analyze the mouse acrosomal matrix using an anti-MC41 antibody. When mouse sperm were treated with 2% Triton X-100, Triton-insoluble matrix components remained in the acrosomal cortical region. Immunogold for MC41 labeled the Triton X-100 and high-salt-insoluble matrix components, demonstrating that MC41 is a subdomain-specific acrosomal matrix protein. We further examined interactions of MC41 with acrosomal proteases and zona proteins. A serine protease of 75 kDa was associated with MC41 under low-salt conditions, presumably forming a complex. Far Western blotting technique indicated that MC41 bound to both ZP2 and ZP2(f) in the presence of high-salt-soluble sperm proteins. In acrosome-reacting sperm, MC41 was present on the hybrid vesicles formed by the fusion of the plasma and outer acrosomal membranes. Presumably, MC41 has a significant role in secondary sperm-zona binding during the acrosomal reaction.