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 domainbinding 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 spermcontributed PAWP can trigger these events. The perinuclear theca (PT)3 of the mammalian sperm head is a condensed cytosolic structure layered between the sperm acrosome and nucleus and, continuing caudally, between the plasmalemma and nucleus. On a compositional basis, the PT can be subdivided into three structurally continuous regions, the subacrosomal layer, the outer periacrosomal layer on the outer aspect of the equatorial segment and the post-acrosomal sheath (PAS) (1, 2). Traditionally, PT has been considered as a cytoskeletal scaffold responsible for maintaining the overall architecture of the mature sperm head. However, recent studies indicate that the bulk of proteins making up the PT are not traditional cytoskeletal proteins but rather a variety of cytosolic proteins linked together and susceptible to extraction under different regimens (3). For example, alkaline extractable SubH2Bv, exclusive to the subacrosomal layer, is implicated in acrosome-nuclear docking during spermiogenesis (1). Salt-extractable non-nuclear somatic core histones, residing in the PAS, may be involved in stabilizing the chromatin of the decondensing sperm nucleus soon after oocyte entry (4, 5). The DTT salt or alkaline extractable calicin and cylicin II share a basic pI with the histones and bind to actin in vitro (6 -8). Detergentand salt-resistant fraction, "calyx fraction" of the PT, contain...

Mitochondrial distribution and microtubule organization were examined in porcine oocytes after parthenogenesis, fertilization and somatic cell nuclear transfer (SCNT). Our results revealed that mitochondria are translocated from the oocyte's cortex to the perinuclear area by microtubules that either constitute the sperm aster in in vitro-fertilized (IVF) oocytes or originate from the donor cell centrosomes in SCNT oocytes. The ability to translocate mitochondria to the perinuclear area was lower in SCNT oocytes than in IVF oocytes. Sperm-induced activation rather than electrical activation of SCNT oocytes as well as the presence of the oocyte spindle enhanced perinuclear mitochondrial association with reconstructed nuclei, while removal of the oocyte spindle prior to sperm penetration decreased mitochondrial association with male pronuclei without having an apparent effect on microtubules. We conclude that factors derived from spermatozoa and oocyte spindles may affect the ability of zygotic microtubules to translocate mitochondria after IVF and SCNT in porcine oocytes. Mitochondrial association with pronuclei was positively related with embryo development after IVF. The reduced mitochondrial association with nuclei in SCNT oocytes may be one of the reasons for the low cloning efficiency which could be corrected by adding yet to be identified, sperm-derived factors that are normally present during physiological fertilization.

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The concentration of free amino acids and the osmolalities in porcine oviductal (OF) and uterine fluids (UF) on Day 3 (D3) and Day 5 (D5) were measured by HPLC and Vapor Pressure Osmometer, respectively. Based on these measurements we designed new media based on PZM3 by modifying the amino acid composition and osmolality. The effectiveness of the modified PZM3 on the development of porcine IVF embryos was then investigated. A total of 24 free amino acids were measured, including 20 protein and 4 non-protein amino acids (β Alanine, Taurine, Ornithine and Citrulline). There was no significant difference in the total concentration of amino acids among D3OF (13.06±3.63 mmol/L), D3UF (10.54±5.16 mmol/L) or D5UF (10.23±6.69 mmol/L). But the total concentration of amino acids in D5OF (5.89±1.47 mmol/L) was significantly lower than the three fluids above. Some individual amino acids varied significantly depending on where they were collected from and the day. The blastocyst rates of porcine IVF embryos were not improved when embryos were cultured in PZM3 with amino acids at D3OF (PZM3-D3OF, 20.3±7.9%) or D5UF (PZM3-D5UF, 14.3±10.7%) concentrations or in PZM3-D3OF for the first 48 (20.5±15.1), 72 (25.6 ±10.4) and 96 (18.7±10.0) hours and then transferred into PZM3-D5UF compared with PZM3 with Sigma amino acid solution (PZM3-SAA) (30.8±9.1%). However, when IVF embryos were cultured in PZM3-D5UF, the average nuclear number per blastocyst (57.6±8.3) was increased compared to PZM3-SAA (40.5±3.5). The osmolalities in D3OF, D3UF, D5OF and D5UF were 318±8, 320±32, 321 and 293±8 mOsM, respectively. When the IVF embryos were cultured in PZM3-SAA and PZM3-D3OF at a variety of osmolalities (150-360mOsM), higher blastocyst rates were obtained at 270-300 mOsM in the PZM3-SAA group (24.6%-33.9%) and 270-290 mOsM in PZM3-D3OF group (22.4%-24.2%). The blastocyst rate gradually decreased when the osmolality was increased or decreased in both groups. When the embryos were cultured in PZM3-SAA at 330 mOsM for the first 72 hours and then transferred to 250 mOsM (33.3±3.4%), the blastocyst rate was higher than original PZM3 (21.2±2.2%)(288 mOsM).