Characterization of phospholipases C beta and gamma and their possible roles in Chaetopterus egg activation

Intracellular calcium release from the endoplasmic reticulum is a hallmark at egg activation of both vertebrates and invertebrates. This fertilization-associated calcium release results from generation of the second messenger inositol 1,4,5-trisphosphate (IP(3)) by one or more phospholipases C (PLC). We characterized Chaetopterus PLCbeta and gamma by reverse transcription/degenerate oligonucleotide primed PCR and rapid amplification of cDNA end PCR. Phylogenetic analyses suggested that the deduced PLCbeta protein shared the greatest homology with mammalian PLCbeta4; the deduced PLCgamma protein shared the greatest homology with starfish PLCgamma and diverged from mammalian PLCgamma before mammalian the PLCgamma1 and gamma2 isoforms diverged. Western blot analyses with specific anti-PLCbeta and gamma antibodies, respectively, revealed that 135 and 150 kDa proteins were expressed in eggs. The general PLC antagonist U-73122 blocked fertilization-induced egg activation; however, the inactive analog, U-73343, had no effect on egg activation. We further tested whether egg activation was G protein-PLCbeta and/or protein tyrosine kinase-PLCgamma dependent. Cholera and pertussis toxins, well-known effectors of G proteins, had no effect on egg activation; while two antagonists of PTK, genistein and tyrphostin B42, inhibited both fertilization-induced and artificial egg activation. Taken together, our studies suggested that PLC activity from eggs contributes to Chaetopterus egg activation and PLCgamma might play an important role during this biological process.