Identification of phospholipases C (PLCs) and their possible roles in egg activation in <i>Chaetopterus</i>
Intracellular calcium release is an early and ubiquitous event in egg activation of vertebrates and invertebrates. One key mechanism of intracellular calcium release is that phospholipase C (PLC) hydrolyzes phosphatidylinositol-4, 5-bis-phosphate (PIP2) to generate the second messenger inositol 1, 4, 5-trisphosphate (IP3) which in turn triggers calcium release from the internal stores. Identification of PLC from the egg is a key step to investigate the mechanisms of egg activation. So far, six distinct classes (β, γ, δ, ϵ, ζ and η) and fourteen different PLC isoforms have been identified in mammals, but few PLC isoforms have been identified in invertebrates. In present study, we cloned cDNA sequences encoding full-length PLCβ and PLCγ from Chaetopterus eggs, which we assigned as cp-PLCβ and cp-PLCγ, by reverse transcription/degenerate oligonucleotide primed polymerase chain reaction (RT/DOP-PCR) and rapid amplification of cDNA end polymerase chain reaction (RACE PCR). Phylogenetic analysis indicated that cp-PLCβ shared the greatest homology with mammalian PLCβ4; however, the cp-PLCγ shared the greatest homology with starfish PLCγ and they both might be diverged from the same ancestral PLC that was different from mammalian PLCγ1. Western blot analyses demonstrated that cp-PLCβ and cp-PLCγ were expressed in Chaetopterus egg as 150 kDa and 135 kDa proteins, respectively. Treatment eggs with the general PLC antagonist, U-73122, blocked egg activation; however, the inactive analog of U-73122, U-73443, had no effect on egg activation. We further determined that egg activation was G protein-PLCβ and/or protein tyrosine kinase (PTK)-PLCγ dependent. An agonist and antagonist of G protein, cholera toxin (CTX) and pertussis toxin (PTX), had no significant effects on egg activation; while the antagonists of PTK, genestein and tyrophstin B42 inhibited both normal and artificial egg activation. Taken together, our studies suggested that PLC activity from the eggs was required for Chaetopterus egg activation and PLCγ might play an important role during this biological process.