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Characterization of phospholipases C β and γ and their possible roles in Chaetopterus egg activation
Author(s) -
Yin Xunqin,
Eckberg William R.
Publication year - 2009
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.20961
Subject(s) - biology , oocyte activation , phospholipase c , complementary dna , microbiology and biotechnology , diacylglycerol kinase , g protein , protein kinase c , signal transduction , biochemistry , oocyte , gene , embryo
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 PLCβ and γ by reverse transcription/degenerate oligonucleotide primed PCR and rapid amplification of cDNA end PCR. Phylogenetic analyses suggested that the deduced PLCβ protein shared the greatest homology with mammalian PLCβ4; the deduced PLCγ protein shared the greatest homology with starfish PLCγ and diverged from mammalian PLCγ before mammalian the PLCγ1 and γ2 isoforms diverged. Western blot analyses with specific anti‐PLCβ and γ 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‐PLCβ and/or protein tyrosine kinase‐PLCγ 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 PLCγ might play an important role during this biological process. Mol. Reprod. Dev. 76: 460–470, 2009. © 2008 Wiley‐Liss, Inc.