Secretion and Fibril Formation of Vitelline Membrane from Avian Granulosa cells

1.排卵前围绕卵母细胞的细胞外基质在禽类中称为卵黄周膜。卵黄周膜由两种主要糖蛋白ZPC和ZP1构成，它们分别在卵巢颗粒细胞和肝脏中合成。虽然已经研究了卵黄周膜的主要成分的性质，但对细胞中原纤维的形成和分泌模式的本质缺乏了解。在本研究中，我们重点研究了鹌鹑ZPC与ZP1的结合，并比较了不同鸟类的基因结构。此外，验证了ZPC结合蛋白在未成熟卵泡中纤维形成中起重要作用的假设。2.用抗ZPC抗体免疫印迹颗粒细胞部分，并检测到一种新的在200 kDa处迁移的蛋白质。该带不与ZPC抗血清或ZP1抗血清反应，但与ZPC强结合。为了比较不同鸟类ZPC与ZP1结合的亲和力，发现ZPC能与其它ZP1结合。克隆了火鸡和鸭ZP1，测定了它们的核酸序列。结果表明，该蛋白在C端氨基酸序列上存在变异。此外，突变体ZP1在CHO-K1细胞中得到了表达，并与ZPC强烈结合。这些结果表明，ZP1的N末端蛋白降解过程可能发生在ZP1到达卵巢后，其产物以物种特有的方式被结合到卵黄周膜上。

1.The extracellular matrix surrounding the oocyte before ovulation is called perivitelline membrane in avian species. The perivitelline membrane is constructed with two major glycoproteins, ZPC and ZP1 that are synthesized in the ovarian granulosa cells and the liver, respectively. Although the properties of the major components of the perivitelline membrane had been examined, the knowledge of the nature of fibril formation and secretion pattern from the cell is lacking. In this study, we focused on the binding of quail ZPC and quail ZP1, and comparison of gene structure of various avian species. Moreover, the hypothesis that ZPC binding protein shows an important role for the fibril formation in immature follicle was examined.2.The microsomal fraction of the granulosa cell was immunoblotted with anti-ZPC after SDS-polyacrylamido gel electrophoresis, and a novel protein migrated at 200 kDa was detected. This band was not reacted with anti-ZPC antiserum or anti-ZP1 antiserum, but strongly bind to ZPC. These results indicated the existence of ZPC binding protein for the fibril formation of perivitelline membrane.3.In order to compare the binding affinity of quail ZPC to ZP1 of the various avian species, the gel electrophoresis analysis was performed and found that quail ZPC can bind to other partidge ZP1. Turkey and duck ZP1 was cloned their nucleic acid sequences were determined. The results showed that the protein has variance at C terminal amino acids. Moreover, mutant ZP1 was expressed in CHO-K1 cells, and it is bind to ZPC strongly. These results indicated that the N-terminal proteolytic processing of ZP1 might take place after the arrival of ZP1 at the ovary, and the resulting product is incorporated into the perivitelline membrane in species-specific manner.