Study on regulatory factors involved in fertilization and development

受精是由精子-卵子相互作用启动的，卵子与单个精子的融合激活了卵子，随后发生了胚胎发育。受精和发育的进程受许多调节蛋白的调控。我们研究了蛋白酶体和小GTP结合蛋白在这一过程中的生理作用，包括(1)精子穿透卵黄被膜，(2)由于卵子激活而使卵黄被膜升高，以及(3)受精卵的分裂。1.从海囊精子中分离到两种类糜蛋白酶。一种是典型的20 S蛋白酶体，另一种是与抗蛋白酶体抗体发生交叉反应但抑制敏感性与20 S蛋白酶体不同的较大的复合蛋白酶。这些蛋白酶体是在精子反应时从精子中释放出来的，释放的蛋白酶体具有消化卵黄被的活性。2.微量注射ADP-核糖化小分子GTP结合蛋白的肉毒杆菌胞外酶C3可引起海囊卵卵黄膜的升高，而抑制C3催化的ADP-核糖化的单抗可抑制受精后卵黄膜的升高。这些结果表明Rho家族的小GTP结合蛋白参与了腹水虫卵的激活。3.用抗海鞘虫卵蛋白酶体的单抗进行免疫细胞化学研究表明，在海鞘受精卵的卵裂过程中，蛋白酶体经历了依赖于细胞周期的变化。蛋白酶体活性在卵裂过程中也被激活。这些结果表明，蛋白酶体参与了海鞘胚胎发育过程中细胞分裂周期的进程。

Fertilization is initiated by sperm-egg interaction, fusion of egg with a single sperm activates the egg, and embryonic development ensues. The progression of fertilization and development is regulated by many regulatory proteins. We have investigated the physiological roles of proteasome (high molecular weight protease complex) and small GTP-binding protein in the process including (1) sperm penetration through egg vitelline coat, (2) elevation of egg vitelline coat as a result of egg activation, and (3) cleavage of fertilized eggs. 1. We isolated two kinds of chymotrypsin-like enzymes from ascidian sperm. One is characterized as a typical 20 S proteasome, while the other is a larger protease comples that is cross-reacted with anti-proteasome antibody but shows different inhibitor susceptibility from that of 20 S proteasome. These proteasomes were found to be released from sperm upon the sperm reaction and the released proteasomes had the activity to digest the egg vitelline coat. 2. Microinjection of botulinum exoenzyme C3 that ADP-ribosylates small GTP-binding proteins induced elevation of the vitelline coat of ascidian eggs, whereas that of monoclonal antibody that inhibits C3-catalyzed ADP-ribosylation inhibited the elevation induced by insemination. These results suggest the involvement of Rho family of small GTP-binding proteins in the activation of ascidian eggs. 3. Immunocytochemistry using monoclonal antibody against ascidian egg proteasome shows that proteasome undergoes a cell cycle- dependent change during cleavage of ascidian fertilized eggs. Proteasome activity also was activated during cleavage. Theses results suggest that proteasome participates in the progression of cell cleavage cycle during ascidian embryonic development.