(l) Role of intra-acrosomal antigen in fertilization: To know the role of intra-acrosomal molecules, MN7, MC41, MC101 and MN9, on fertilization, specific antibodies, mMN7, mMC41, mMC101, mMN9, respectively, were added to the IVF medium and determined the effect of antibodies on the rate of fertilization. mMN7 inhibited completion of acrosome reaction and mMC41 inhibited retention of tight-binding of sperm with zona (Saxena et al, J Reprod Fertil, 1999). mMC101 inhibited sperm penetration to zona, in addition, possibly affected sperm plasma membrane modification during acrosome reaction, causing inhibition of plasma membrane fusion of sperm and oocyte (submitted). mMN9 inhibited membrane fusion process of sperm and oocyte (Toshimori et al, Biol Reprod, 1998).
(2) Interaction of intra-acrosomal antigen with zona proteins and acrosomal proteinases: Far-Western blotting technique was used to determine whether intra-acrosomal molecules interact with zona proteins, MN7 showed binding activity
with ZP3 suggesting that the molecule has a role in fertilization by way of the interaction with zona. We further examined the interaction of intra-acrosomal molecules with acrosomal proteinases by zymography. Proteins coprecipitated with MC41 contained a serine proteinase. MC41 by itself showed no proteolytic activity and was undergone processing to smaller size when incubated in the neutral condition, This could be due to the proteolysis by the serine proteinase bound to MC41 (paper in preparation).
(3) Morphological study of acrosome formation: Addition of brefeldin A, a potent inhibitor of membrane traffic, to the organ culture of seminiferous tubules caused degeneration of the Golgi apparatus in spermatids and disappearance of mMN7-immunoreactivity from the acrosome of early round spermatids. In addition, acrosomal membrane was fragmented. From the results, vesicular transport via coatmer-coated vesicles is presumably main pathway to the acrosome (Tanii et al, J Electron Microsc, 1998), During final steps of spermiogenesis, the acrosome reduces its volume, We demonstrated the involvement of tubulobulbar complexes in the elimination of excess acrosomal contents (Tanii et al, Anat Rec, 1999). Since guinea pig sperm has a markedly large acrosome, we studied acrosomal microdomain formation in the guinea pig. MN7 was incorporated in the early acrosomic granules and changed in localization as acrosome formation proceeded, and finally confined to the dorsal matrix region of the apical segment (Yoshinaga et al, Anat Rec, 2000). After epididymal transit, MN7 restricted in two regions: at the electron-lucent dorsal matrix and on the outer acrosome membrane/matrix associated materials at the dorsal region. The latter is a newly defined acrosomal microdomain (Yoshinaga et al, Cell Tissue Res, 1998). Less