CpZPC, a newt ZPC molecule, localizes to the inner surface of the egg envelope.
Zona pellucida-3 is an essential molecule for the binding of sperm to the egg envelope and for induction of the acrosome reaction in mice. Its homologous molecules, ZPCs, have been widely identified in the eggs of many vertebrates, except for urodeles. In this study, to investigate the participation of ZPC in newt fertilization, we cloned the cDNA of newt ZPC from the ovary of Cynops pyrrhogaster by reverse transcription polymerase chain reaction (RT-PCR). The cDNA was constructed from 1,397 nucleotides and included one open reading frame corresponding to a sequence of 439 amino acids. The deduced amino acid sequence had identities at 52, 47 and 45% with Xenopus gp41, mouse ZP3 and medaka L-SF, respectively. It included four potential N-linked glycosylation sites and 12 highly conserved cysteine residues of mammalian ZP3/ZPC molecules. This result suggests that CynopsZPC (cpZPC) has molecular features similar to those of mammalian ZP3/ZPCs. Messenger RNA for cpZPC was detected in the ovary and faintly in the testis. Two bands corresponding to 84 kDa and 70 kDa in the egg envelopes were detected by immunoblotting with an antiserum raised against a 9 amino acid peptide in the C-terminus domain of cpZPC. The molecular size of 84 kDa fits with the size of a putative sperm-binding protein reported by Nakai et al. (1999), suggesting that cpZPC may contribute to sperm binding to the egg envelope in C. pyrrhogaster. The results of immunohistochemistry suggest that cpZPC was localized in the inner surface of the egg envelope. Similar localization is seen only in fish, suggesting that cpZPC is a unique molecule which may allow us to investigate the functional evolution of the egg envelope in vertebrates.
Makabe-Kobayashi
Youko
Y
Department of Biology, Faculty of Science, Yamagata University, Koshirakawa, Yamagata, Japan.
Kudaira
Etsuko
E
Watanabe
Akihiko
A
Onitake
Kazuo
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary; D004527:Egg Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010053:Ovary / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012455:Salamandridae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015044:Zona Pellucida / Q000502:physiology*; D000072216:Zona Pellucida Glycoproteins
D017931:DNA Primers; D018076:DNA, Complementary; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse