Study on regulatory factors involved in fertilization and development

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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.