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J. Biol. Chem., Vol. 282, Issue 16, 12164-12175, April 20, 2007
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1
¶12
From the
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
Received for publication, September 26, 2006 , and in revised form, February 2, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-actin and may interact with calicin and cylicin in situ (7-9). The DTT-cetyltrimethylammonium bromide-extractable Stat4, a transcription factor, presumably found throughout the PT (10), may contribute to the zygotic development after fertilization. Together, these data imply that the PT is an assembly of specialized proteins that serve multiple functions in both spermiogenesis and fertilization. Based on the relative ease of solubilization of the PAS in the oocyte cytoplasm and the signaling nature of some of its constituents (2), the PAS is the most likely region to harbor sperm proteins regulating oocyte activation and early stages of zygotic development after fertilization.
Several hypotheses have been proposed to account for how oocyte activation is achieved by the spermatozoon during mammalian fertilization. The currently favored cytosolic factor model postulates that upon sperm-oocyte fusion, signaling molecules are released into the egg cytoplasm that activate the dormant egg by eliciting intracellular Ca2+ oscillations, which serve as a secondary messenger for downstream effectors of zygotic development (reviewed in Refs. 11 and 12). Thus far, two sperm molecules, tr-kit (truncated c-Kit tyrosine kinase) and phospholipase C
, have been shown to parthenogenetically trigger oocyte activation leading to cleaved mouse embryos. tr-kit appears to activate the oocyte via a Fyn-phospholipase C
1-mediated signaling pathway (13, 14); a similar pathway has also been established in echinoderm and ascidian eggs (15). The discovery of the sperm-specific phospholipase C, on the other hand, offered the possibility that a phospholipase C protein of paternal origin was directly involved in triggering Ca2+ oscillations during oocyte activation (16, 17).
The role of sperm perinuclear theca during fertilization can be best appreciated from the observations that oocyte activation can be fully triggered by introducing a sperm head with only the nucleus and PT present via intracytoplasmic sperm injection (18). Furthermore, local solubilization of the PAS-PT is sufficient to elicit full oocyte activation in the absence of complete sperm incorporation into the ooplasm (2, 19). Kurokawa et al. (12) showed that three supernatants of successive sperm extractions (i.e. sonication, Triton X-100, and alkaline carbonate, pH 11.5) were all able to trigger Ca2+ oscillations comparable with those observed at natural fertilization. These investigators emphasized that only in the case of high pH treatment was the Ca2+ releasing activity fully released indicating that the factor was most likely a PT component according to our definition (4, 20). Perry et al. (21) proposed a so-called trans-complementation scheme of sperm-borne oocyte activating factors, detailing that full oocyte activation and embryo development can be achieved only with the combination of a DTT-soluble fraction obtained from Triton-demembranated mouse sperm head and the heat-stable component of the head after DTT extraction. Their observation indicated that at least two different sperm factors are involved during oocyte activation. Collectively, these observations corroborate our hypothesis that PAS-PT is the site housing sperm oocyte activating factor(s). In this context, we report the characterization a novel, sperm-specific PAS-PT protein, PAWP, which appears to promote meiotic resumption and pronuclear formation, mediated by an unprecedented WW domain-signaling pathway during fertilization.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Differential Sperm Head Treatments—Isolated bull sperm heads underwent two cycles of freeze-thaw followed by centrifugation (14,000 x g). The combined supernatants were then dialyzed and lyophilized for Western blot analysis along with the pellet. Isolated bull sperm heads were also treated with 0.1% Triton X-100 at 4 °C for 1 h and washed three times with PBS. The pellet was re-suspended in 15 mM DTT at 4 °C for 1 h. The suspension was then separated into supernatant and pellet by centrifugation for immunoblot analysis (20).
Molecular Cloning of cDNA Encoding PAWP—Anti-PT serum was used to screen a bull testicular Stratagene ZAP ExpressTM cDNA library as described (1). One of six clones was chosen for full-length sequencing (Cortech, Queen's University, Kingston, Ontario, Canada) and the sequence of both its nucleotide strands was determined.
For obtaining the human homologue of PAWP, primers were designed according to the draft sequence (GenBankTM accession number XM_001168) and used for standard reverse transcription-PCR followed by cDNA amplification (Qiagen, Mississauga, Ontario, Canada) with upstream primer (5'-ATG CCA TTT GAT CTG ATG-3') and downstream primer (5'-TAC CTC ATT GTC AGG TAG-3') at Tm 52 °C for 34 cycles. The human cDNA was inserted into pCR® II-TOPO® vector within an EcoRI restriction site (Invitrogen), sequenced, and deposited to GenBankTM (accession no. AF393575 [GenBank] ). The mouse version of PAWP was cloned and amplified in the same fashion, with upstream primer (5'-GAG CTC GAT GGC ACT GAA CCA G-3') and downstream primer (5'-GCG GCC GCC AAG GTT AAC ATC TTA GAG C-3' (the underlined sequences represent SacI and NotI restriction sites, respectively) at Tm 52 °C for 34 cycles.
Construction, Expression, and Purification of Bovine recPAWP—Full-length PAWP cDNA was subcloned into the pET28b vector (Novagen, Madison, WI) between the HindIII and XhoI sites and sequenced for verification. The construct was then transformed into Escherichia coli BL21 DE3[PlyS] cells (Novagen) for expression and the subsequent nickel column purification of His-tagged recPAWP, using the protocol provided by the vendor (Qiagen). The His tag was removed using thrombin (Sigma).
PAWP Sperm Equivalent Estimation—The amount of PAWP present in a single spermatozoon was calculated based upon densitometric comparison of known concentrations of recPAWP with known amounts of bovine spermatozoa by utilizing anti-recPAWP antiserum probed immunoblots. The reactive bands of the immunoblots were analyzed by using Scion Image Beta 4.02 Acquisition and Analysis software (Scion Corp., Frederick, MA). The standard curve generated from recPAWP (ranged from 0.025 to 0.2 µg) by using Microsoft Excel Data Analysis software was used for extrapolation of PAWP protein concentration present in a single spermatozoon.
Northern Blotting and Chemiluminescent Detection—Total RNA from various bovine and rat tissues was isolated by using RNeasy mini-preparation kit (Qiagen), separated by agarose gel electrophoresis and transferred to and UV-cross-linked to positively charged nylon membrane (0.45 µm pore size, Roche Applied Science). DIG-labeled sense and antisense riboprobes were synthesized and used for Northern blot hydridization according to the DIG System User's Guide for Filter Hybridization (Roche Applied Science).
Ultrastructural Immunocytochemistry—The LR-white-embedded bovine testicular and epididymal tissues were processed for immunogold labeling by using previously described procedures (5). Ultrathin sections were mounted on Formvar-coated nickel grids and blocked with 10% normal goat serum prior to overnight incubation of the primary antibodies at 4 °C. The sections were then washed and incubated with goat anti-rabbit secondary antibody conjugated to 10-nm gold particles (1:20; Sigma) followed by counterstaining with uranyl acetate and lead citrate. The final sections were analyzed by transmission electron microscope (Hitachi 7000).
Verification of Functional PPXY Motif(s) in PAWP—The binding the two putative PPXY motifs of recPAWP was examined by using GST-fused wild-type WW domains of YAP and Nedd4 (referred to as YAP and Nedd4). To show the specificity of the binding, two YAP WW domain mutants were used, YAPP202A and YAPH129F (all WW domain proteins, their respective mutants, and ligands were generous gifts from Dr. Marius Sudol) (23-25). Approximately equal amounts (5 µg) of purified GST-fused WW domains were separated by SDS-PAGE and transferred to nitrocellulose membrane for both positive binding and competitive inhibition assays. For positive binding assay, blots were blocked with 2% milk in PBS (pH 7.4, 0.05% Tween) and incubated with recPAWP (0.1 µg) in same buffer at 4 °C overnight followed by anti-recPAWP antibody detection. A synthetic peptide, Ac-PPVRYGSPPPGYEAPT-CONH2 (bold fonts indicate the PPXY motif overlapping with the underlined YGXPPXG motif) was designed as a competitive inhibitor to recPAWP by comparing bull, human, and mouse amino acid sequences. (Fig. 2b, inset). The above peptide was modified to demonstrate the specificity and the importance of the PPXY motif. In one modification, the tyrosine residue was constitutively phosphorlyated (PPGYP), while in the other the tyrosine residue was substituted with phenylalanine, PPGF. A synthetic peptide Ac-GTGKSPRRTL-CONH2, matching amino acid sequence of a common bovine sperm head protein, arylsulfatase A (AsA), was also used as a negative control. All peptides were synthesized, and purity was confirmed by HPLC (SynPep Corp., Dublin, CA). A PPXY (PY) ligand from WBP-1 (WW-binding protein 1) for YAP was also used as a competitive binding inhibitor (23, 26). For the competitive inhibition assay, PPXY peptide, its derivatives PPGYP, PPGF, and PY ligand, were preincubated with Western or dot blots loaded with both wild-type and mutant WW domains at 4 °C overnight followed by incubation of recPAWP and immunodetection. In another competitive inhibition assay, YAP and its mutant YAPP202A GST fusion constructs were preincubated with recPAWP prior to applying the latter onto the blots and immunodetection. In this study, GST tag was removed from all constructs, since it posed steric hindrance. Affinity-purified anti-recPAWP anti-serum was used to detect the presence of recPAWP on the blot. Control blots loaded with the equal amount of proteins were used to demonstrate that the anti-serum had no cross reactivity to the GST-fused WW domain proteins.
Porcine Oocyte Collection and in Vitro Fertilization—Ovaries were collected from pre-pubertal gilts at a local slaughterhouse, and oocytes were collected as described previously (27). Oocytes were washed twice and transferred into TCM-199 medium containing 0.1% polyvinyl alcohol, 10 ng/ml epidermal growth factor, 0.5 µg/ml FSH, 0.5 µg/ml LH, and 0.57 mM cysteine (mTCM-199) at 39 °C in atmosphere of 5% CO2 in humidified air. After 22-24 h, the oocyte-cumulus cell complexes were transferred to mTCM-199 without FSH and LH. They were cultured for additional 20 h under the same condition. Freshly ejaculated sperm-rich fraction was collected from a fertile boar and frozen as described previously (27). A sperm pellet was thawed in 2 ml of Dulbecco's PBS (calcium- and magnesium-free) supplemented with 0.1% polyvinyl alcohol at 39 °C and separated on a two layer (80 and 60%) Percoll gradient (Amersham Biosciences AB, Uppsala, Sweden). The sperm pellet was re-suspended in modified Tris-buffered medium. The final sperm concentration for IVF was 1 x 106 cells/ml. Fertilized oocytes were cultured at 39 °C in North Carolina State University (NCSU)-23 medium with the addition of 0.4% BSA in atmosphere of 5% CO2 in humidified air.
Intractyoplasmic Sperm Injection (ICSI) and Microinjection of recPAWP—For the manipulation, microdrops of 6 µl of 4% PVP and 20 µl of HEPES-buffered NCSU23 with adjusted osmolarity (HEPES-NCSU23) were placed on a Petri dish and covered with mineral oil. Affinity-purified anti-recPAWP (1 µl) or its preimmune serum (1 µl, diluted 1:200 first) were diluted into 1 µl of sperm suspension (4 x 106/ml) in modified Tris-buffered medium (1:1) as well as the PPXY peptides and AsA control peptide (1 µl each, both with initial concentration of 1 mg/ml in modified KHM injection buffer (78 mM KCl, 0.5 mM MgCl2, 50 mM HEPES, pH 7.0)). This suspension (2 µl) was thoroughly mixed in a 6 µl drop of 4% PVP (final dilution of anti-serum and peptides, 1:8). ICSI was performed using an Eppendorf Cell Tram microinjection system equipped with Prime Tech piezo drill. After the spermatozoon was immobilized with piezo pulses, it was aspirated into a micropipette with PVP solution, with or without experimental molecules, and injected into the oocyte within a microdrop of HEPES-NCSU23. After ICSI, oocytes were washed twice and cultured in a 10-µl drop of NCSU23 for 8 h at 39 °C in atmosphere of 5% CO2 in humidified air. The microinjection of recPAWP into mammalian oocytes was carried out as described in ICSI experiments, except for the absence of an injected spermatozoon. An estimated 10 pl of solution containing 0.1 to 1.0 pg of soluble recPAWP, control BSA, or AsA peptide (sham injection) was aspirated into the injection pipette. Pronuclear development was determined after processing of the oocytes for immunofluorescence.
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Bull, rhesus monkey, and boar spermatozoa were fixed and processed for immunofluorescence and DAPI staining as described previously (29). The primary antibodies used were anti-PAWP_N and anti-PAWP_C (1:200). Images were acquired with a Nikon Eclipse 1000 microscope with high numerical aperture objectives, and an RTE/CCD 1217 camera (Princeton Instruments, Inc., Trenton, NJ), operated by MetaMorph software. Digital images were edited by using Adobe Photoshop 6.0 software (Adobe Systems Inc., Mountain View, CA).
Microinjection of recPAWP into Xenopus Oocytes—The isolation and manipulation of oocytes were performed as described previously (30). Sexually mature Xenopus laevis females were primed with gonadotropin (pregnant mare serum gonadotropin, 50 IU/frog, Sigma) 3 days before operations. Ovarian fragments were removed surgically under hypothermia and subjected to collagenase A digestion (2 mg/ml, Roche Applied Science, Mississauga, Ontario, Canada) in calciumfree OR2 medium (83 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES, pH 7.8), with 1 mg/ml soybean trypsin inhibitor (Sigma) for 3 h to obtain follicle-cell free oocytes. Stage VI oocytes were then individually selected and grouped. Collagenase-treated oocytes were stimulated with progesterone (5 µM) and germinal vesicle breakdown was assessed by the appearance of a white spot (maturation spot) on the pigmented animal pole after 3-6 h. Three hours after germinal vesicle breakdown, the mature eggs were arrested at metaphase II with the first polar body present (PB). Injection of alkaline PT extract or recPAWP (0.02 ng/nl) in a total volume of 27 nl with or without PPXY peptides (0.04 ng/nl) was conducted in calcium-free OR2 medium. Artificial activation was achieved by incubating metaphase II-arrested oocytes in OR2 medium with 0.5 µg/ml calcium ionophore A23187 [GenBank] (Sigma) for 60 s. Twenty minutes after injection (or ionophore treatment), oocytes were fixed in 100% methanol for 30 min, rehydrated in 50% (v/v) methanol, and then transferred to Tris-buffered saline containing SYTOX Green (1:10,000; Molecular Probes) for detection of polar bodies as viewed under a Olympus IMT2-RFL inverted fluorescence microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Isolation of PAWP cDNA Clone and Sequence Analysis—Positive cDNA clones were obtained from immunoscreening of a bull testicular cDNA library by using anti-PT serum (first boost), and after appropriate processing the clones were sequenced and found to be identical. The isolated cDNA clone was a transcript of 1.4 kb (see supplemental Fig. 1; GenBankTM accession number AF322215 [GenBank] ) and the longest open reading frame was found to code for a 313 amino acid polypeptide (Fig. 2). Its calculated pI was 5.60 and molecular mass 31,966 Da.
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Cloning of Human and Mouse PAWP—We were able to identify both the human (GenBankTM accession number AK129656 [GenBank] ) and mouse (GenBankTM accession number AK015863 [GenBank] ) homologues of bovine PAWP in GenBankTM and amplify them by reverse transcription-PCR of total testicular RNA using appropriate primers. Sequence similarities and unique features of PAWP found in bull, human, and mouse are shown by multiple amino acid sequence alignment in Fig. 2. In all three species, the N-terminal region is highly similar while the C-terminal region is more variable. However, in the C-terminal region, the hallmark YGXPPXG repeats of PAWP appear in all three sequences examined, 12 in bull, 9 in human, and 5 in mouse. Furthermore, all three sequences contain PY motifs (shaded sequences in Fig. 2): 2 in bull, 1 in human, and 6 in mouse. Interestingly, at least one PY motif is always found overlapping with the YGXP-PXG motif.
Verification of the Identity of PAWP—Recombinant PAWP (recPAWP) was created in a bacterial system. Its authenticity was verified by immunoprobing with anti-PAWP_N and anti-PAWP_C antibodies. RecPAWP was then used for the production of a polyclonal anti-serum (anti-recPAWP) and for affinity purification of all anti-sera raised against PAWP. After affinity purification, all anti-sera, including the serum used to immunoscreen the PAWP clone from the cDNA expression library, labeled a single 32-kD band in Western blots of bull PT extracts and sperm, confirming the identity of the cloned and deduced sequence (see supplemental Fig. 3)
Expression Profile of PAWP—Northern blots of total RNA isolated from various bull tissues were probed with DIG-labeled antisense RNA transcribed from PAWP cDNA and a 1.6-kb transcript was detected in testicular tissue (Fig. 3a). This tissue specificity was also verified by PCR on selected tissues (data not shown). Species-comparative Northern blot of total testicular RNA from bull, human, and mouse (Fig. 3a, lower panel) showed the presence of two transcripts in bull and human and an additional transcript in the mouse. A 1.6-kb band was most prominent in the bull and human, while a 2.6-kb band dominated in the mouse. Tissue-comparative Western blots (Fig. 3b) immunolabeled with anti-recPAWP antibody confirmed the preferential expression of PAWP in the testis. This antibody was able to detect PAWP homologues in the sperm of different species (Fig. 3c).
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Localization of PAWP by Immunocytochemistry—Immunoperoxidase staining of the paraffin-embedded bull testicular tissue sections with anti-recPAWP antibody was restricted to the elongated spermatid population, and no immunostaining was evident in the round spermatids by this method. PAWP became prominent in the cytoplasmic lobe of step 11 spermatids, although it first became detectable in step 9 (Fig. 4a). Immunofluorescence labeling of mature spermatozoa of different species including the bull, rhesus monkey, and pig revealed its localization to the PAS-PT. In the bull, the labeling demonstrated an anterior-posterior gradation in the PAS-PT (Fig. 4b), while in the primate spermatozoa, it appeared as if PAWP delineated the anterior half of the PAS (Fig. 4c). In rabbit and porcine spermatozoa, PAWP also appeared as a distinct band in the PAS-PT region (Fig. 4, d and e). It is important to note that this immunoreactive region is posterior to the crescent-shaped equatorial segment region of the sperm acrosome (arrow, Fig. 4e). At the ultrastructural level of cauda epididymal sperm sections, immunogold labeling with anti-recPAWP was found predominantly over the post-acrosomal sheath of the mature bull sperm PT (Fig. 5, a and b). No immunoreactivity was found in any other sperm structures (Fig. 5c). A fortuitous oblique section through the caudal face of the sperm head (Fig. 5d) indicated that PAWP is distributed over the surface of the PAS-PT.
Distribution of PAWP during Natural Fertilization and ICSI—The localization of PAWP during fertilization was examined by using both ICSI and IVF techniques in porcine oocytes. Both experiments revealed similar PAWP distribution inside of the ooplasm. Oocytes were fixed and processed 6-8 h post-insemination/IVF (Fig. 6, a-d) and 2-4 h post-ICSI (Fig. 6, e-h) to analyze the dynamics of PAWP at the stage of sperm nuclear decondensation and pronuclear development. Initially, PAWP appeared as a distinct band covering the PAS region below the equatorial region of the acrosome in the intact sperm (Fig. 6b). Migration of PAWP to an anterior pole of the sperm nucleus was observed during the initial swelling thus marking the onset of sperm nucleus decondensation and male PN development (Fig. 6, c and e). As the PAS-PT region of the nucleus dissolved, PAWP moved toward the apical pole of the nucleus (Fig. 6f). At this stage, the apical pole of the sperm nucleus was still intact, due to the characteristic persistence of a complex of subacrosomal PT layer and inner acrosomal membrane (28). When the sperm nucleus swelled, PAWP was seen around and throughout the early male pronucleus (Fig. 6g). In the nascent large male pronucleus after ICSI, only trace amounts of PAWP were found in the nucleoplasm (Fig. 6d). In more advanced zygotes with two large, apposed pronuclei surrounded by the nuclear envelope, little PAWP was detected in the nucleoplasm (Fig. 6h).
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Pronuclear Formation in Porcine and Xenopus Oocytes Microinjected with recPAWP and Alkaline PT Extract—Microinjection of recPAWP consistently induced formation of a single pronucleus in MII-arrested porcine oocytes (Figs. 8, a and b, and 9) as well as in bovine (Fig. 9) and non-human primate (Fig. 9) oocytes and released Xenopus occytes from metaphase II arrest, as indicated by the second polar body extrusion and pronuclear formation (Figs. 8, g and h, and 10). Importantly, resumption of metaphase II induced by recPAWP could be prevented by the co-injection of a competitive PPXY motif containing peptide derived from PAWP but not by co-injection of the mutated PPXF peptide (Fig. 10), indicating that the interacting oocyte molecule was a group I WW domain containing protein. Also of note was that meiotic resumption induced by the sperm alkaline PT extract was also blocked by co-injection of PPXY peptide (Fig. 10). This inhibitory effect by the PPXY peptide indicates that egg activation elicited by the PT extract is PAWP-mediated, fortifying the PT egg-activating hypothesis put forward by Kimura et al. (18) and Sutovsky et al. (2). Recombinant PAWP-injected porcine oocytes displayed distinct cytoplasmic foci of tyrosine kinase c-Yes (Fig. 8, c and d), a proposed target for YAP and WBP-2, and a fully developed female pronucleus with a presumably functional nuclear envelope containing nuclear pore complexes (Fig. 8, e and f). Sham-injected control oocytes that were examined under differential image contrast microscopy and appeared to form a female pronucleus (counted as positive) had neither c-Yes foci in their cytoplasm nor nuclear pore complexes around their nuclei when examined by immunofluorescence.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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PY motifs are identified in molecules mediating protein-protein interactions, which bind WWI domain protein modules found in a variety of cellular signaling pathways (32, 33). One of the important goals of this study was to examine whether the PY motifs of PAWP are functional. Our in vitro data showed that PAWP is capable of binding to various WWI domains including YAP, Nedd4, and dystrophin through its PY motifs but not to groups II and III WW domains. PAWP-WWI interaction was highly specific as it was abolished by various point mutations in the WWI domains or PY motifs.
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It is uncertain at this point which of the two PY motifs present in bovine PAWP is responsible for the WWI interaction. However, it has been suggested in the case of dystrophin--dystroglycan complex, where -dystroglycan (ligand) contains two PY motifs, that the non-binding PY motif provides the stability of the complex (38). In the case of PAWP, the first PY motif that overlaps with the YGXPPXG motif of PAWP, is more likely to be participating in mediating the PAWP-WW I domain interactions since its synthetic peptide can successfully compete for this interaction.
Given the involvement YAP65 in transcriptional control and its obligatory co-activation by PY containing proteins, PEBP2 (39) and WBP-2 (40) there is a possibility that sperm-contributed PAWP could modulate some aspect of early zygotic transcription. A sequence comparison of WBP-2 and PAWP (see supplemental Fig. 2) reveals similar N-terminal halves suggesting conservation of a function that remains to be investigated. The only commonality in the C-terminal halves of these proteins is the shared PY motifs implying the involvement in WWI domain protein interactions. Unique to PAWP in this region is the presence of YGXPPXG repeating motifs, occasionally incorporating the PY motif, which may provide protein recognition specificity. In fact the repeating YGXPPXG motif along with the PY motif(s) appear to be the only conserved features of the C-terminal half of PAWP between species. The variability in the C-terminal region of PAWP may stem from interspecies differences in sperm head morphology and the molecular composition of the perinuclear theca. We suggest that it is the conserved N-terminal half of PAWP that dictates its function, while the C-terminal half ensures the binding specificity of PAWP through the YGXPPPGY motif, which we have shown blocks sperm induced egg activation.
It has been established in mouse that the bulk of early zygotic transcription occurs within the male pronucleus, but the reason for this gender bias is not known (41). One possible explanation is that transcriptional factors bind preferentially to paternal chromatin due to its differential histone modification (42). It is plausible that a transcriptional factor or co-activator could be carried in the sperm head PT and migrates inside the nascent male pronucleus at an early stage of pronuclear development. The signaling properties and timing of PAWP migration from PT to male pronucleus in ICSI and IVF zygotes (this study) fit this pattern. An unrelated transcription factor, Stat4, has also been localized to sperm PT (10) and could be released into ooplasm at fertilization. The combination of a functionally versatile PY motif in PAWP and its strategic localization in the PAS region of sperm, where oocyte-activating factors presumably reside (18), prompted an examination of the involvement of PAWP in fertilization.
In addition to the cortical reaction (data not shown) and second polar body formation, a high rate of parthenogenetic pronuclear development was achieved after recPAWP microinjection into MII-arrested oocytes of different species and taxa. Oocytes injected with recPAWP exhibited a fully developed female pronucleus surrounded by a nuclear envelope equipped with nuclear pore complexes, and c-Yes tyrosine foci were observed throughout the ooplasm. These signs of activation were comparable with eggs fertilized in vitro indicating a resemblance to normal fertilization. In cases of spontaneous activation none of these indicators of activation were found. Interestingly, in ionomycin-activated rat eggs, it was demonstrated that resumption of meiosis and PN development were significantly hindered by Src family tyrosine kinase inhibitors, while cortical granule exocytosis was not (43). Another study in mouse demonstrated that during sequential fractionation of freeze-thaw sperm extracts, the ability to support pronuclear formation was lost before Ca2+-releasing ability (44). Drawing from these studies, PAWP may function through its interaction with a WW-domain protein-Src family tyrosine kinase-protein complex (a precedent of interaction set by YAP65).
To confirm and examine the role of PAWP in pronuclear formation, ICSI was chosen for our inhibition experiments over IVF for several reasons. First, the co-injection of PAWP antagonists with the spermatozoa assures their sufficient concentration near the sperm head at the time of PAS-PT solubilization. Second, we avoided polyspermy, which occurs at high frequency during porcine IVF (27). Third, both the full compliment of paternal and maternal influences were taken into account thus enabling us to test the redundancy of any particular signaling molecule. As demonstrated, the anti-recPAWP antibody inhibited the formation of both male and female pronuclei, supporting the role of PAWP in PN development. Since our far-Western experiments showed that the PY ligands of PAWP specifically interact with WWI domain containing proteins, we hypothesized that this interaction, presumably with an oocyte WWI domain containing protein, is essential for egg activation. To test this hypothesis in ICSI, we substituted the antibody with our PAWP-PY peptide. Our hypothesis was confirmed by the prevention of pronuclear development and further validated by the fact that phosphorylation of the tyrosine residue or its substitution with phenylalanine rendered the PY peptide ineffective, as was predicted from previous PY-WWI domain interactions (31), from our far-Western analysis and from our parthenogenic trials.
The precise molecular mechanism by which PAWP contributes to meiotic resumption and pronuclear formation at present is not clear. Although there could be several alternative explanations, our data indicate that no indigenous sperm borne oocyte-activating factor introduced into the egg with the sperm was able to compensate for the PY peptide or anti-PAWP antibody-imposed blockade of fertilization after ICSI. Not ignoring the evidence for sperm-borne phospholipase involvement, a possibility remains that the PAWP-mediated signaling pathway acts upstream of calcium signaling. Most research points to a calcium increase as the sole requirement for resumption of the oocyte cell cycle (45, 46). However, we do not know yet if the PY peptide-induced block of oocyte activation affects sperm-induced calcium oscillations. Alternatively, the PAWP-mediated signaling pathway may act downstream of calcium-induced signaling during fertilization. In any case, the disruption of PAWP signaling prevents metaphase-anaphase transition of oocyte chromosomes and male pronuclear formation, leading to developmental arrest. Our data suggest that PAWP most likely imposes its effects on pronuclear development by interacting with an oocyte-derived WWI domain-containing protein. It is possible that the PAWP-WWI pathway targets tyrosine kinase regulation of the oocyte meiotic spindle and/or male chromatin remodelling factors. Future endeavors will focus on resolving how the PAWP-mediated pathway fits in with calcium oscillations during mammalian egg activation and on the identification of the interacting partner of PAWP in the oocyte.
In summary, we have uncovered a novel sperm-specific WWI domain-binding protein that exclusively resides in the PAS-PT, the region of the sperm believed to harbor the egg activating factors. This potential signaling molecule, PAWP, first appears in elongating spermatids coincident with the time frame when spermatids acquire their egg-activating ability. We provide evidence that PAWP promotes meiotic resumption and pronuclear development by specifically interacting through its PY motif(s) with the WWI domain of an unidentified oocyte protein.
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FOOTNOTES |
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* 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.
2 To whom correspondence should be addressed. Tel.: 613-533-2858; Fax: 613-533-2566; E-mail: ro3{at}post.queensu.ca.
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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, male pronucleus; 
, female pronucleus; scale bars:10 µm; negative controls for immunostaining are shown in 
2 test. The difference between columns a and b are significant (p < 0.01).
