Study on regulatory factors involved in fertilization and development

受精是由精卵相互作用开始的，卵子与单个精子融合激活卵子，胚胎发育开始。受精和发育的进程受到许多调节蛋白的调控。我们研究了蛋白酶体（高分子量蛋白酶复合物）和小GTP结合蛋白在精子穿透卵卵黄膜、卵激活引起的卵卵黄膜升高和受精卵分裂过程中的生理作用。1.我们从海鞘精子中分离到两种胰凝乳蛋白酶样酶。一种是典型的20 S蛋白酶体，另一种是较大的蛋白酶复合物，与抗蛋白酶体抗体发生交叉反应，但表现出与20 S蛋白酶体不同的抑制剂敏感性。发现这些蛋白酶体在精子反应时从精子中释放，并且释放的蛋白酶体具有消化卵卵黄膜的活性。2.显微注射肉毒杆菌外切酶C3，ADP核糖基化小GTP结合蛋白诱导的海鞘卵的卵黄外壳的海拔，而单克隆抗体，抑制C3催化的ADP核糖基化抑制海拔诱导授精。这些结果表明Rho家族的小GTP结合蛋白参与了海鞘卵的激活。3.用抗海鞘卵蛋白酶体单克隆抗体进行的免疫细胞化学研究表明，海鞘受精卵卵裂过程中蛋白酶体发生了细胞周期依赖性的变化。蛋白酶体活性也在裂解过程中被激活。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.