Role of amphibian egg transglutaminase in the development of secondary cytostatic factor in vitro

Abstract

Fresh cytosols extracted from unfertilized amphibian eggs contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+, and is designated primary CSF (1°CSF). During storage of Ca2+-containing cytosols at 2°C, stable CSF activity appears, designated secondary CSF (2°CSF). In Rana pipiens egg cytosols, the development of 2°CSF coincides with the formation of a protein complex with a molecular weight above 2,000 kDa, and this large molecule exhibits a high 2°CSF activity when purified (Shibuya and Masui, 1989: Development 106:799–808). The present study shows that both the formation of 2°CSF protein complex and the development of its activity are inhibited by ethylamine and glycine-ethyl-ester (GEE), both known as potent transglutaminase (TGase) inhibitors. An affinity-purified polyclonal antibody raised against mammalian transglutaminase reacts with an approximately 68-kDa protein in fresh egg cytosols, as well as with the 2°CSF protein complex. In cytosols deprived of transglutaminase by immunoprecipitation, neither the development of 2°CSF activity nor the formation of its protein complex can occur. These results indicate that transglutaminase of Rana pipiens eggs is responsible for the formation of 2°CSF, and that transglutaminase itself is incorporated into 2°CSF molecules. Mol. Reprod. Dev. 47:302–311, 1997. © 1997 Wiley-Liss, Inc.