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  • Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster.

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster.

Developmental biology (2007-05-15)
Yuichirou Harada, Tamami Matsumoto, Shino Hirahara, Akira Nakashima, Shuichi Ueno, Shoji Oda, Shunichi Miyazaki, Yasuhiro Iwao
ABSTRACT

Eggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) zeta-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-α-Tubulin−FITC antibody, Mouse monoclonal, clone DM1A, purified from hybridoma cell culture
Sigma-Aldrich
Citrate Synthase from porcine heart, ammonium sulfate suspension, ≥100 units/mg protein