XB-ART-35318J Biol Chem April 20, 2007; 282 (16): 12164-75.
PAWP, a sperm-specific WW domain-binding protein, promotes meiotic resumption and pronuclear development during fertilization.
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.
PubMed ID: 17289678
Article link: J Biol Chem
Genes referenced: wbp2nl yes1
Article Images: [+] show captions
|FIGURE 8. Parthenogenetic pronuclear development in porcine (Sus scrofa) and frog (X. laevis) oocytes upon microinjection of recPAWP. A large female pronucleus (b) with a nuclear envelope that contains nuclear pore complexes (f) and an up-regulation of c-Yes tyrosine foci (d) were observed in the recPAWP-injected porcine oocytes. Sham-injected oocytes on the contrary remained at MII-arrested stage (a, c, e). Similarily sham or sham + BSA-injected Xenopus oocytes remained in the MII-arrested state with one polar body present (g). Injection of either alkaline PT extract or recPAWP induced a high degree of oocyte activation evident by the extrusion of the second polar body (h). The forming pronucleus of the activated oocyte, often difficult to observe due to the vast size and the non-transparent outer coat of the Xenopus egg, was caught sinking into the cytoplasm (PN and asterisk in inset) after activation with recPAWP. When the alkaline PT extract or recPAWP was co-injected with the PPXY peptide, the oocytes remained in the MII-arrested state as in the sham and BSA injected eggs (g), while co-injection of the mutated PPXF peptide was ineffective in blocking oocyte activation (see supplemental Fig. 4). DNA was labeled using DAPI in porcine oocytes and SYTOX Green in Xenopus oocytes. Bars = 10 μm.|