PAWP, a Sperm-specific WW Domain-binding Protein, Promotes Meiotic Resumption and Pronuclear Development during Fertilization

doi:10.1074/jbc.M609132200

PAWP, a Sperm-specific WW Domain-binding Protein, Promotes Meiotic Resumption and Pronuclear Development during Fertilization^*^♦

^{^‡}Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada, the Departments of ^{^§}Animal Sciences and ^{^¶}Obstetrics and Gynecology, University of Missouri, Columbia, Missouri 65211-5300, and the ^{^∥}Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, 20 Marie-Curie, Ottawa, Ontario K1N 6N5, Canada

2 To whom correspondence should be addressed. Tel.: 613-533-2858; Fax: 613-533-2566; E-mail: ro3{at}post.queensu.ca.

Abstract

We report a novel alkaline extractable protein of the sperm head that exclusively resides in the post-acrosomal sheath region of the perinuclear theca (PT) and is expressed and assembled in elongating spermatids. It is a protein that shares sequence homology to the N-terminal half of WW domain-binding protein 2, while the C-terminal half is unique and rich in proline. A functional PPXY consensus binding site for group-I WW domain-containing proteins, and numerous unique repeating motifs, YGXPPXG, are identified in the proline-rich region. Considering these molecular characteristics, we designated this protein PAWP for postacrosomal sheath WW domain-binding protein. Microinjection of recombinant PAWP or alkaline PT extract into metaphase II-arrested porcine, bovine, macaque, and Xenopus oocytes induced a high rate of pronuclear formation, which was prevented by co-injection of a competitive PPXY motif containing peptide derived from PAWP but not by co-injection of the point-mutated peptide. Intracytoplasmic sperm injection (ICSI) of porcine oocytes combined with co-injection of the competitive PPXY peptide or an anti-recombinant PAWP antiserum prevented pronuclear formation and arrested fertilization. Conversely, co-injection of the modified PPXY peptide, when the tyrosine residue of PPXY was either phosphorylated or substituted with phenylalanine, did not prevent ICSI-induced fertilization. This study uncovers a group I WW domain module signal transduction event within the fertilized egg that appears compulsory for meiotic resumption and pronuclear development during egg activation and provides compelling evidence that a PPXY motif of sperm-contributed PAWP can trigger these events.

Footnotes

↵³ The abbreviations used are: PT, perinuclear theca; PAS, post-acrosomal sheath; DTT, dithiothreitol; PY, PPXY; PBS, phosphate-buffered saline; recPAWP, recombinant PAWP; GST, glutathione S-transferase; AsA, arylsulfatase A; IVF, in vitro fertilization; NCSU, North Carolina State University; BSA, bovine serum albumin; ICSI, intracytoplasmic sperm injection; TRITC, tetramethylrhodamine isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; DIG, digoxin; MII, metaphase II.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s) AF393575 (human) and AF322215 (bull).
↵^* This work was supported by Canadian Institutes of Health Research Grant MOP-62706 (to R. O.), National Research Initiative Competitive Grants 99-35203-7785 and 2002-35203-12237 from the United States Department of Agriculture Cooperative State Research, Education and Extension Service (to P. S.), by Food for the 21st Century Program of the University of Missouri-Columbia (to P. S.), by the Post-doctoral Fellowship Program of Korea Science and Engineering Foundation (KOSEF; to Y.-J. Y.), and by the National Institutes of Health R 21 Opportunities for Research at Regional Primate Research Centers (to R. O., P. S., and G. Schatten). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-5.
↵^♦ This article was selected as a Paper of the Week.
↵¹ These authors contributed equally to this work.
- Received September 26, 2006.
- Revision received February 2, 2007.