Study on regulatory factors involved in fertilization and development

受精发育调控因素研究

• 批准号: 03454490
• 负责人: YOKOSAWA Hideyoshi
• 金额: $ 4.16万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454490/
• 关键词: Fertilization; Development; Proteasome; GTP-binding protein; アネキシン; グルコシダーゼ; プロテア-ゼ; プロテアソ-ム; GTPー結合蛋白質; リポコルチン; グルコシダ-ゼ

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.

受精是由精子与卵子相互作用开始的，卵子与单个精子融合激活卵子，胚胎发育随之而来。受精和发育的过程受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结合蛋白在这一过程中的生理作用，包括：(1)精子穿透卵黄膜，(2)卵黄膜因卵子活化而升高，以及(3)受精卵的裂解。1. 从海鞘精子中分离出两种凝乳胰蛋白酶样酶。一种是典型的20s蛋白酶体，另一种是较大的蛋白酶复合物，与抗蛋白酶体抗体交叉反应，但与20s蛋白酶体表现出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出来，并且释放的蛋白酶体具有消化卵黄膜的活性。2. 微量注射adp -核糖基化小gtp结合蛋白的肉毒杆菌外酶C3可诱导海鞘卵卵黄膜的升高，而抑制C3催化adp -核糖基化的单克隆抗体可抑制受精引起的卵黄膜的升高。这些结果表明Rho家族的小gtp结合蛋白参与了海鞘卵的激活。3. 免疫细胞化学表明，海鞘卵蛋白酶体在受精卵分裂过程中发生细胞周期依赖性变化。蛋白酶体的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。

项目成果

Yoshiko Saitoh: "High molecular weight protease complexes (proteasomes) of sperm of the ascidian,Halocynthia roretzi:" Dev.Biol.in press. (1993)

Yoshiko Saitoh：“海鞘精子的高分子量蛋白酶复合物（蛋白酶体），Halocynthia roretzi：”Dev.Biol.in press。

Y. Saitoh, H. Sawada, and H. Yokosawa: "High molecular weight protease complexes (proteasomes) of sperm of the ascidian, Halocynthia roretzi: Isolation, characterization, and physiological roles in fertilization." Dev. Biol.(1993)

Y. Saitoh、H. Sawada 和 H. Yokosawa：“海鞘、Halocynthia roretzi 精子的高分子量蛋白酶复合物（蛋白酶体）：分离、表征和受精中的生理作用。”

H. Sawada and H. Yokosawa: "Physiological roles of proteases in fertilization and development." Protein, Nucleic acid and Enzyme. 36. 814-819 (1991)

H. Sawada 和 H. Yokosawa：“蛋白酶在受精和发育中的生理作用。”

Hiroyuki Kawahara: "Cell cycle-dependent change of proteasome distribution during embryonic development of the acidian Halocynthia roretzi." Dev.Biol.151. 27-33 (1992)

Hiroyuki Kawahara：“酸根盐藻胚胎发育过程中蛋白酶体分布的细胞周期依赖性变化。”

Yoshiko Saitoh: "Comparative studies on proteasomes(multicatalytic proteinases)isolated from spermatozoa and eggs of sea urchins." Comp.Biochem.Physiol.99. 71-76 (1991)

Yoshiko Saitoh：“从海胆精子和卵中分离的蛋白酶体（多催化蛋白酶）的比较研究。”