Biochemical and morphological study of molecules localized in the distinct acrosomal microdomains

(l)顶体内抗原在受精中的作用：为了解顶体内分子MN7、MC41、MC101和MN9在受精中的作用，分别在体外受精培养基中加入特异性抗体mMN7、mMC41、mMC101、mMN9，测定抗体对受精率的影响。mMN7抑制顶体反应的完成，mMC41抑制精子与带紧密结合的保留（Saxena et al, J reproductive Fertil, 1999）。此外，mMC101抑制精子穿透带，可能影响顶体反应过程中精子的质膜修饰，导致精子与卵母细胞的质膜融合受到抑制（提交）。mMN9抑制精子和卵母细胞的膜融合过程（Toshimori et al ., Biol repd, 1998）。(2)顶体内抗原与带蛋白和顶体蛋白酶的相互作用：采用Far-Western blotting技术测定顶体内分子是否与带蛋白相互作用，MN7与ZP3的结合活性更强，提示该分子通过与带蛋白相互作用参与受精。我们通过酶谱法进一步研究了顶体内分子与顶体蛋白酶的相互作用。与MC41共沉淀的蛋白含有丝氨酸蛋白酶。MC41本身没有蛋白水解活性，在中性条件下孵育时，被加工成较小的尺寸，这可能是由于与MC41结合的丝氨酸蛋白酶对蛋白质进行了水解（准备论文）。(3)顶体形成的形态学研究：在精小管器官培养中加入有效的膜运输抑制剂brefeldin A，导致早期圆形精子顶体高尔基体变性和mmn7免疫反应性消失。顶体膜碎裂。从研究结果来看，囊泡运输可能是到达顶体的主要途径（Tanii等人，J Electron Microsc, 1998）。在精子发生的最后阶段，顶体体积减小，我们证明了小管球泡复合物参与了顶体多余内容物的消除（Tanii等人，Anat Rec, 1999）。由于豚鼠精子具有明显较大的顶体，我们研究了豚鼠顶体微结构域的形成。MN7被纳入早期顶体颗粒中，并随着顶体形成的进行而改变定位，最终局限于顶节的背基质区域（Yoshinaga et al, Anat Rec, 2000）。附睾过境后，MN7限制在两个区域：电子透明背区基质和背区顶体外膜/基质相关材料。后者是一个新定义的顶体微域（Yoshinaga et al ., Cell Tissue Res, 1998）。少

(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 … More 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