Human Sperm Do Not Bind to Rat Zonae Pellucidae Despite the Presence of Four Homologous Glycoproteins
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m413569200
ISSN1083-351X
AutoresTanya Hoodbhoy, Saurabh Joshi, Emily S. Boja, Suzannah A. Williams, Pamela Stanley, Jurrien Dean,
Tópico(s)Animal Genetics and Reproduction
ResumoThe specificity of sperm-egg recognition in mammals is mediated primarily by the zona pellucida surrounding ovulated eggs. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona pellucida contains three glycoproteins, ZP1, ZP2, and ZP3, which are conserved in rat and human. The recent observation that human zonae pellucidae contain a fourth protein raises the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62–70% amino acid identity with human ZP1, ZP2, ZP3, and ZP4/ZPB were detected in rat zonae pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither, and the presence of human follicular fluid did not alter the specificity of sperm binding. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals. The specificity of sperm-egg recognition in mammals is mediated primarily by the zona pellucida surrounding ovulated eggs. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona pellucida contains three glycoproteins, ZP1, ZP2, and ZP3, which are conserved in rat and human. The recent observation that human zonae pellucidae contain a fourth protein raises the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62–70% amino acid identity with human ZP1, ZP2, ZP3, and ZP4/ZPB were detected in rat zonae pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither, and the presence of human follicular fluid did not alter the specificity of sperm binding. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals. After passage through the lower female reproductive tract, mammalian spermatozoa fertilize ovulated eggs in the ampulla of the oviduct. A key event in successful fertilization is sperm binding to the surface of the extracellular zona pellucida that surrounds the egg. Following zona penetration and fusion with the egg plasma membrane, peripherally located cortical granules within the egg exocytose their contents, which modify the zona matrix such that sperm no longer bind. These events are carefully orchestrated to ensure that a single sperm fertilizes a single egg (1.Yanagimachi R. Knobil E. Neil J. The Physiology of Reproduction. Raven Press, New York1994: 189-317Google Scholar). Despite decades of investigation, the molecular basis of mammalian sperm-egg recognition remains controversial. Human sperm are particularly fastidious and bind to old world primate eggs but not to eggs of other species. In contrast, mouse sperm are quite promiscuous, binding with near universality to eggs from other mammalian orders (2.Bedford J.M. Anat. Rec. 1977; 188: 477-488Crossref PubMed Scopus (123) Google Scholar). The mouse zona pellucida is composed of three major glycoproteins, ZP1, ZP2, and ZP3, one of which, ZP2, is proteolytically cleaved following fertilization (3.Wassarman P.M. Annu. Rev. Biochem. 1988; 57: 415-442Crossref PubMed Scopus (483) Google Scholar). Mouse lines have been established that lack each of the zona proteins as well as lines in which human ZP2 and/or human ZP3 replace endogenous mouse proteins (4.Hoodbhoy T. Dean J. Reproduction. 2004; 127: 417-422Crossref PubMed Scopus (123) Google Scholar). Mice without ZP1 form a zona pellucida matrix to which mouse sperm bind and Zp1 null females are fertile, albeit with decreased fecundity (5.Rankin T. Talbot P. Lee E. Dean J. Development. 1999; 126: 3847-3855PubMed Google Scholar). Mice in which endogenous proteins are replaced with human ZP2, human ZP3, or both are also fertile but do not support human sperm binding (6.Rankin T.L. Tong Z.-B. Castle P.E. Lee E. Gore-Langton R. Nelson L.M. Dean J. Development. 1998; 125: 2415-2424PubMed Google Scholar, 7.Rankin T.L. Coleman J.S. Epifano O. Hoodbhoy T. Turner S.G. Castle P.E. Lee E. Gore-Langton R. Dean J. Dev. Cell. 2003; 5: 33-43Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Thus, mouse ZP1 is not required for sperm-egg recognition, and human ZP2 and ZP3 are not sufficient to support human sperm binding. These results suggest that "humanized" zona matrices either lack a factor or contain a factor that prevents the binding of human sperm. Several possibilities can be entertained: 1) human sperm binding requires post-translational modification(s) of ZP2 and ZP3 by glycosylation pathways peculiar to human oocytes (8.Dell A. Chalabi S. Easton R.L. Haslam S.M. Sutton-Smith M. Patankar M.S. Lattanzio F. Panico M. Morris H.R. Clark G.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15631-15636Crossref PubMed Scopus (85) Google Scholar); 2) conversely, post-translational glycosylation(s) of ZP2 and ZP3 (either mouse or human) by mouse oocytes actively prevents human sperm binding; 3) additional components not provided in the mouse zona matrix are required to support human sperm binding (9.Lefièvre L. Conner S.J. Salpekar A. Olufowobi O. Ashton P. Pavlovic B. Lenton W. Afnan M. Brewis I.A. Monk M. Hughes D.C. Barratt C.L.R. Hum. Reprod. 2004; 19: 1580-1586Crossref PubMed Scopus (263) Google Scholar); or 4) supramolecular architecture involving more than one determinant plays a critical role (10.Dean J. Bioessays. 2004; 26: 29-38Crossref PubMed Scopus (74) Google Scholar). During intracellular processing in growing oocytes, both N- and O-glycans are added to the zona pellucida proteins prior to their secretion. N-Glycans are preassembled as a dolichol-linked high mannose oligosaccharide and cotranslationally attached to canonical sites (Asn-Xaa-Ser/Thr; where Xaa is any amino acid except proline). Subsequent modifications by glycosyltransferases and glycosidases result in hybrid and complex N-glycans. This progression beyond the initial multiantennary high mannose structures requires N-acetylglucosaminyltransferase I (GlcNAcT I) 1The abbreviations used are: GlcNAcT, β1–6 N-acetylhexosaminehexose; HPLC, high performance liquid chromatography; MS, mass spectrometry; CID, collision-induced dissociation; MgatI, N-acetylglucosaminyltransferase I; C2, Core 2. encoded by MgatI (11.Kumar R. Yang J. Larsen R.D. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9948-9952Crossref PubMed Scopus (142) Google Scholar). Of the 16 potential N-glycosylation sites on mouse ZP1 (four), ZP2 (six), and ZP3 (six), only one (ZP3-Asn227) remains free of glycan (12.Boja E.S. Hoodbhoy T. Fales H.M. Dean J. J. Biol. Chem. 2003; 278: 34189-34202Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), and the remainder are high mannose or biantennary complex N-glycans (13.Easton R.L. Patankar M.S. Lattanzio F.A. Leaven T.H. Morris H.R. Clark G.F. Dell A. J. Biol. Chem. 2000; 275: 7731-7742Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Mucin O-glycans are formed by the sequential addition of sugars on either serine or threonine residues after the initial folding of the protein during intracellular processing. Presumably peptide sequences or three-dimensional structures of the folded protein dictate those residues that will be modified, but the governing rules remain obscure. Mucin O-glycans initiate with the transfer of N-acetylgalactosamine to serine or threonine followed by subsequent elongation by a distinct glycosyltransferase. Core 2 O-glycans are generated by the action of a Core 2 (C2) β1–6 N-acetylglucosaminyltransferase (GlcNAcT), of which there are three isoforms (I–III) (14.Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar, 15.Yeh J.C. Ong E. Fukuda M. J. Biol. Chem. 1999; 274: 3215-3221Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 16.Schwientek T. Yeh J.C. Levery S.B. Keck B. Merkx G. van Kessel A.G. Fukuda M. Clausen H. J. Biol. Chem. 2000; 275: 11106-11113Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Biochemical and mass spectrometry analyses detect few or no O-glycans on mouse ZP2 (12.Boja E.S. Hoodbhoy T. Fales H.M. Dean J. J. Biol. Chem. 2003; 278: 34189-34202Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 17.Araki Y. Orgebin-Crist M.C. Tulsiani D.R. Biol. Reprod. 1992; 46: 912-919Crossref PubMed Scopus (21) Google Scholar, 18.Nagdas S.K. Araki Y. Chayko C.A. Orgebin-Crist M.-C. Tulsiani D.R.P. Biol. Reprod. 1994; 51: 262-272Crossref PubMed Scopus (60) Google Scholar), and of the O-glycans on ZP1 and ZP3, the most common are Core 2 glycans containing 2–6 monosaccharides, although Core 1 glycans are present as well (13.Easton R.L. Patankar M.S. Lattanzio F.A. Leaven T.H. Morris H.R. Clark G.F. Dell A. J. Biol. Chem. 2000; 275: 7731-7742Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Albeit widely proposed, recent biochemical and genetic studies have not substantiated a role for a specific sugar or class of glycans as the sole mediators of sperm binding. In extant glycan recognition models, the inability of sperm to bind to the early embryo has been attributed to the release from cortical granules of glycosidases that remove terminal glycans following fertilization. However, the persistence of sperm binding to the zona pellucida despite cortical granule exocytosis in genetically altered mice in which ZP2 is not cleaved is not consistent with either N- or O-glycans acting as primary "sperm receptors" and then being clipped off (7.Rankin T.L. Coleman J.S. Epifano O. Hoodbhoy T. Turner S.G. Castle P.E. Lee E. Gore-Langton R. Dean J. Dev. Cell. 2003; 5: 33-43Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Of note, cleavage of ZP2 could alter the architecture of the zona matrix and mask the accessibility of specific glycans after fertilization rather than releasing them. However, mice with mutations of specific glycan attachment sites (19.Liu C. Litscher S. Wassarman P.M. Mol. Biol. Cell. 1995; 6: 577-585Crossref PubMed Scopus (33) Google Scholar) or lacking an oocyte glycosyltransferases required for specific sugars proposed as recognition determinants (20.Thall A.D. Maly P. Lowe J.B. J. Biol. 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Thus, the unequivocal identification of a particular glycan as the mediator of sperm-egg recognition has not been achieved to date. Initially, three glycoproteins were detected in the human zona pellucida (27.Shabanowitz R.B. O'Rand M.G. J. Reprod. Fertil. 1988; 82: 151-161Crossref PubMed Scopus (91) Google Scholar, 28.Bauskin A.R. Franken D.R. Eberspaecher U. Donner P. Mol. Reprod. Dev. 1999; 5: 534-540Google Scholar), and their primary structures were deduced from cDNA (29.Chamberlin M.E. Dean J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6014-6018Crossref PubMed Scopus (157) Google Scholar, 30.Liang L.-F. Dean J. Dev. Biol. 1993; 156: 399-408Crossref PubMed Scopus (97) Google Scholar, 31.Harris J.D. Hibler D.W. Fontenot G.K. Hsu K.T. Yurewicz E.C. Sacco A.G. DNA Seq. 1994; 4: 361-393Crossref PubMed Scopus (318) Google Scholar). More recent reports raised the possibility of an additional protein in the human zona pellucida (32.Hughes D.C. Barratt C.L. Biochim. Biophys. Acta. 1999; 1447: 303-306Crossref PubMed Scopus (99) Google Scholar), the existence of which has been confirmed experimentally (9.Lefièvre L. Conner S.J. Salpekar A. Olufowobi O. Ashton P. Pavlovic B. Lenton W. Afnan M. Brewis I.A. Monk M. Hughes D.C. Barratt C.L.R. Hum. Reprod. 2004; 19: 1580-1586Crossref PubMed Scopus (263) Google Scholar). As noted, mouse zonae pellucidae contain three glycoproteins, ZP1, ZP2, and ZP3, and their primary structures also were deduced from cDNA (33.Ringuette M.J. Chamberlin M.E. Baur A.W. Sobieski D.A. Dean J. Dev. Biol. 1988; 127: 287-295Crossref PubMed Scopus (141) Google Scholar, 34.Liang L.-F. Chamow S.M. Dean J. Mol. Cell. Biol. 1990; 10: 1507-1515Crossref PubMed Scopus (149) Google Scholar, 35.Epifano O. Liang L.-F. Familari M. Moos Jr., M.C. Dean J. Development. 1995; 121: 1947-1956PubMed Google Scholar). Despite close examination, no additional zona proteins were detected in native mouse zonae pellucidae by mass spectrometry (12.Boja E.S. Hoodbhoy T. Fales H.M. Dean J. J. Biol. Chem. 2003; 278: 34189-34202Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Mouse and rat evolutionarily diverged only 12–24 million years ago (36.Mullins L.J. Mullins J.J. Genome Biol. 2004; 5: 221Crossref PubMed Scopus (25) Google Scholar), and the primary protein structures of rat ZP1, ZP2, and ZP3 are 88–92% identical to their murine homologues (37.Akatsuka K. Yoshida-Komiya H. Tulsiani D.R. Orgebin-Crist M.C. Hiroi M. Araki Y. Mol. Reprod. Dev. 1998; 51: 454-467Crossref PubMed Scopus (22) Google Scholar). However, the existence of an additional zona transcript deduced from cDNA (GenBank™ accession number NM_172330) raised the possibility of a fourth protein in the rat zona pellucida. Here we determine the protein composition of native rat zonae pellucidae by microscale mass spectrometry and identify ZP4 as a component. We also investigate the ability of human sperm to bind to normal and mutant rodent eggs as models for gain- and loss-of-function assays. Reagents—Molecular biology grade stock salt solutions for mass spectrometric analyses were purchased from Quality Biological Inc. (Gaithersburg, MD) (MgCl2 and CaCl2); Digene (Beltsville, MD) (NaCl); and Fisher (triethanolamine). Bovine pancreatic DNase I, phenylmethylsulfonyl fluoride, Complete Mini protease inhibitor mixture tablets, and the endoproteinases Asp-N and Glu-C were from Roche Applied Science, whereas sequencing grade modified porcine trypsin was from Promega (Madison, WI). Urea, dithiothreitol, iodoacetamide, ammonium bicarbonate, Hoechst, bovine testicular hyaluronidase, and turkey egg white trypsin inhibitor were obtained from Sigma-Aldrich, and Percoll was from Amersham Biosciences. The Glyko deglycosylation kit was purchased from Prozyme Inc. (San Leandro, CA). All of the HPLC grade solvents were of the highest grade commercially available from J. T. Baker (Phillipsburg, NJ). EM grade paraformaldehyde was obtained from Electron Microscopy Sciences (Fort Washington, PA). Egg and embryo collection media (M2 and M16) were from Cell and Molecular Technologies Inc. (Phillipsburg, NJ). Isolation of Rat Zonae Pellucidae—Rat zonae pellucidae were isolated from ovarian homogenates on Percoll gradients as described (38.Bleil J.D. Wassarman P.M. Dev. Biol. 1980; 76: 185-202Crossref PubMed Scopus (463) Google Scholar) with minor modifications. All glassware and plasticware were siliconized (Sigmacote; Sigma-Aldrich). Rat ovaries (4 weeks old; Sprague-Dawley) were purchased from Harlan Bioproducts for Science, Inc. (Indianapolis, IN), and extraneous tissue was mechanically removed. "Complete" TEA buffer (25 mm triethanolamine, pH 8.5, 150 mm NaCl, 1 mm MgCl2, 1 mm CaCl2) was supplemented immediately before use with turkey egg white trypsin inhibitor (2 mg/ml), phenylmethylsulfonyl fluoride (0.05 mm), 1 protease inhibitor mixture tablet/10 ml buffer, DNase I (11 mg/ml), and hyaluronidase (11 mg/ml). "Incomplete" TEA lacked DNase I, hyaluronidase, and protease inhibitors. The ovaries were homogenized (Dounce; 30 strokes) at a concentration of 1 ovary/ml of Complete TEA buffer in 3-ml batches. Nonidet P-40 (300 μl, 10% solution) and sodium deoxycholate (300 μl, 10% solution) in Incomplete TEA were added consecutively (30 strokes each), and the mixture was transferred to a 50-ml conical tube after passing through a 100-μm nylon mesh (BD Falcon, Bedford, MA). The homogenizer was rinsed with a volume of 100% Percoll equal to that of the ovarian homogenate, and the "rinsate" was then passed through the nylon mesh and added to the ovarian homogenate. Quick-Seal centrifuge tubes (16 × 76 mm; Beckman, Palo Alto, CA) were filled with the homogenate, 50% Percoll mix, and the volume was brought up to the capacity of the tube (∼14 ml) with 50% Percoll in Incomplete TEA. 50 μl of blue density marker beads (specific gravity, 1.018 gm/ml) (Amersham Biosciences) were added to a balance tube consisting of 50% Percoll in Incomplete TEA to identify the position of the zona band, and the tubes were heat-sealed and subjected to ultracentrifugation (50 Ti rotor at 25,000 rpm, 4 °C, 45 min). The isolated zonae in a distinct white band were removed with an 18-gauge, 1.5-inch needle attached to a 3-ml syringe and washed by rehomogenizing and rebanding (50% Percoll-Incomplete TEA, twice). Finally, the zonae were washed in Incomplete TEA (three times for 15 min) and HPLC grade water (three times for 15 min) by centrifugation (13,000 rpm, 4 °C). Isolated zonae were reconstituted in 50 μl of water and examined microscopically to estimate the number of zonae/μl prior to lyophilization and storage at –80 °C. Liquid Chromatography and Mass Spectrometry Analysis of Protein Digests—Each zona pellucida sample processed for mass spectrometric analysis was obtained from 1–5 rat ovaries. Zonae were denatured (8 m urea), reduced (5 mm dithiothreitol) and alkylated (0.5 m iodoacetamide), had buffer exchanged to remove excess reagents (50 mm NH4HCO3, pH 7.8), and de-N-glycosylated (peptide N-glycosidase) with or without de-exo-O-(sialidase A, β(1–4)-galactosidase and β-N-acetylglucosaminidase) or de-exo- and endo-O-glycosylated as described previously (12.Boja E.S. Hoodbhoy T. Fales H.M. Dean J. J. Biol. Chem. 2003; 278: 34189-34202Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Trypsin, Asp-N, trypsin + Asp-N, and Glu-C digests of rat zonae pellucidae were analyzed on a Micromass QTOF Ultima Global (Micromass, Manchester, UK) in electrospray mode (12.Boja E.S. Hoodbhoy T. Fales H.M. Dean J. J. Biol. Chem. 2003; 278: 34189-34202Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Briefly, 4–5 μl of each digest was loaded onto a Vydec C18 MS column (100 × 0.15 mm; Grace Vydec, Hesperia, CA), and chromatographic separation was performed at 1 μl/min using the following gradient: 0–5% B over 5 min; 5–40% B over 80 min; 40–95% B over 10 min; and 95% B held over 5 min (solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile). The top three most abundant ions in the preceding MS scan (m/z 300–1990 with a threshold of >10 counts/s) were subjected to CID. Data processing was accomplished by MassLynx 4.0 and submitted to Mascot search (biospec.nih.gov). Further in-depth analysis of the MS data was performed manually. Sperm Binding Assays—Mouse eggs were obtained from normal (FVB) and genetic mutant (huZP3 rescue (6.Rankin T.L. Tong Z.-B. Castle P.E. Lee E. Gore-Langton R. Nelson L.M. Dean J. Development. 1998; 125: 2415-2424PubMed Google Scholar), huZP2/3 double rescue (7.Rankin T.L. Coleman J.S. Epifano O. Hoodbhoy T. Turner S.G. Castle P.E. Lee E. Gore-Langton R. Dean J. Dev. Cell. 2003; 5: 33-43Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), conditional MgatI null (26.Shi S. Williams S.A. Seppo A. Kurniawan H. Chen W. Zhengyi Y. Marth J.D. Stanley P. Mol. Cell. Biol. 2004; 24: 9920-9929Crossref PubMed Scopus (81) Google Scholar), and C2 GlcNAcT null mice (24.Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; 9: 881-890Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Rat eggs were obtained from Fisher females, and sperm binding assays were carried out as previously described (6.Rankin T.L. Tong Z.-B. Castle P.E. Lee E. Gore-Langton R. Nelson L.M. Dean J. Development. 1998; 125: 2415-2424PubMed Google Scholar). Briefly, superovulated eggs were obtained by injecting 4-week-old rodents with 5 (mice) or 20 (rats) international units of pregnant mare serum gonadotropin followed 46–48 h (mice) or 48–50 h (rat) later by the same amount of human chorionic gonadotropin. Unfertilized eggs were collected 15–16 h later in M2 medium supplemented with one protease inhibitor tablet/10 ml (M2+inhibitors), and the cumulus cells were removed by treatment with hyaluronidase (250 μg/ml) for 2–5 min in the same medium. Mouse and rat two-cell embryos were collected in M2 medium 40 h after human chorionic gonadotropin administration and mating (1:1) of superovulated females with males of proven fertility. Eggs (20–30/experimental group) and two-cell embryos (2–3/group) were washed once with M2+inhibitors and three times with M2 alone and transferred to a 20-μl insemination droplet of M16 pre-equilibrated under mineral oil in a 5% CO2, 37 °C incubator. Human sperm collected for research purposes only were obtained from Fairfax Cryobank (Fairfax, VA). To select for motile sperm, 1.0 ml of pre-equilibrated M16 was layered over 500 μl of human semen and sperm allowed to "swim up" (1 h, 5% CO2, 37 °C incubator). Sperm from the top 500 μl were selected for capacitation. Mouse or rat caudal sperm (6.Rankin T.L. Tong Z.-B. Castle P.E. Lee E. Gore-Langton R. Nelson L.M. Dean J. Development. 1998; 125: 2415-2424PubMed Google Scholar) or the aforementioned ejaculated human sperm were capacitated in pre-equilibrated M16 (1 h, 5% CO2/37 °C incubator) and added to the insemination droplet containing the mouse or rat eggs at a concentration of 1 × 106/ml. After 30 min of exposure to sperm, the eggs were washed with a wide bore glass pipette through a series of 5 × 50-μl droplets of M16 until no more than 2–5 sperm bound to control two-cell mouse or rat embryos as an arbitrary end point. The samples were then fixed in 2% paraformaldehyde and stained with Hoechst, and the sperm heads were quantified from 1-μm optical sections obtained on a 510 LSM confocal microscope (Carl Zeiss, Thornwood, NY). Sperm binding experiments were also carried out with eggs preincubated in superfluous human follicular fluid obtained from Suburban Hospital (Bethesda, MD). These experiments were carried out using normal mouse and rat eggs, as well as mouse eggs with humanized zonae in which cognate mouse zona proteins were replaced with human ZP3 alone or both human ZP2 and ZP3 (6.Rankin T.L. Tong Z.-B. Castle P.E. Lee E. Gore-Langton R. Nelson L.M. Dean J. Development. 1998; 125: 2415-2424PubMed Google Scholar, 7.Rankin T.L. Coleman J.S. Epifano O. Hoodbhoy T. Turner S.G. Castle P.E. Lee E. Gore-Langton R. Dean J. Dev. Cell. 2003; 5: 33-43Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The eggs were incubated with 0, 50, or 100% follicular fluid for 15 min and then inseminated in M16 containing 0 or 50% follicular fluid. Sperm binding experiments were performed with human and control mouse sperm, and the eggs were washed, fixed, and stained as described above. All of the experiments were conducted in compliance with the guidelines of the Animal Care and Use Committee of the National Institutes of Health under a Division of Intramural Research, NIDDK-approved animal study protocol. Human sperm and follicular fluid were used under an National Institutes of Health Institutional Review Board-approved protocol. Conservation of ZP4 Genes—Comparing the recently reported rat genome (39.Gibbs R.A. et al.Nature. 2004; 428: 493-521Crossref PubMed Scopus (1687) Google Scholar) with those of human (40.International Human Genome Sequencing ConsortiumNature. 2004; 431: 931-945Crossref PubMed Scopus (3567) Google Scholar) and mouse (41.Waterston R.H. et al.Nature. 2002; 420: 520-562Crossref PubMed Scopus (5442) Google Scholar), the genetic loci of four genes potentially encoding zona pellucida proteins were identified (Fig. 1A). The genes are scattered among somatic chromosomes, but each gene is syntenic among the three species with similar exon maps for ZP1 (12 exons), ZP2 (18–19 exons), and ZP3 (8 exons). Transcripts encoding three rat, human, and mouse zona proteins have previously been characterized, and a fourth human zona transcript has been reported recently (9.Lefièvre L. Conner S.J. Salpekar A. Olufowobi O. Ashton P. Pavlovic B. Lenton W. Afnan M. Brewis I.A. Monk M. Hughes D.C. Barratt C.L.R. Hum. Reprod. 2004; 19: 1580-1586Crossref PubMed Scopus (263) Google Scholar). Rat Zp4, human ZP4/ZPB and mouse Zp4 genes, each containing 12 exons, were detected on syntenic chromosomes 17q12.1, 1q43, and 13A1, respectively (Fig. 1A). The nucleic acid sequence within the conserved exons of rat Zp4 (Fig. 1B) is 74% identical to human and encodes a hypothetical protein of 540 amino acids. Within its 12 exons, mouse Zp4 has a potential transcript that is 88% identical to the rat gene and expressed sequence tags (e.g. Riken, BY366037) corresponding to short aberrantly spliced portions of genomic sequences present in 8-cell mouse embryos. A similar "wash through" of abundant oocyte transcripts into the early embryo has been previously observed for other mouse zona transcripts (e.g. NCBI, AU043141). However, each potential open reading frame of the hypothetical mouse ZP4 transcript contains multiple stop codons (9.Lefièvre L. Conner S.J. Salpekar A. Olufowobi O. Ashton P. Pavlovic B. Lenton W. Afnan M. Brewis I.A. Monk M. Hughes D.C. Barratt C.L.R. Hum. Reprod. 2004; 19: 1580-1586Crossref PubMed Scopus (263) Google Scholar), and after re-examination of earlier mass spectra (12.Boja E.S. Hoodbhoy T. Fales H.M. Dean J. J. Biol. Chem. 2003; 278: 34189-34202Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), no tryptic peptides encoding ZP4 were detected in mouse zonae pellucidae. To determine whether three or four zona proteins are present in rat, zonae pellucidae were isolated from 200 rat ovaries, deglycosylated, digested with endoproteinases, and partially characterized by microscale liquid chromatography to separate peptides for mass spectrometry analysis. As expected, native rat ZP1, ZP2, and ZP3 proteins were readily detected with 52, 82, and 91% coverage, respectively, of their secreted polypeptide backbones (complete analysis to be published elsewhere). In addition, rat ZP4 was detected with 70% coverage of its polypeptide chain (Table I). Thus, it was concluded that although individual zona proteins are well conserved with a high percentage of amino acid identity, the mouse zona pellucida contains three proteins (ZP1–3), and rat and human contain four zona proteins.Table ILC-MS and MS/MS analysis of rat ZP4 Open table in a new tab Mass Spectrometry Analysis of Rat ZP4—The N terminus of rat ZP4 is predicted at Gln29 (42.Von Heijne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3693) Google Scholar), and the mass of an Asp-N cleaved peptide appearing as the monoisotopic +2 and +3 charged ions at m/z 1184.10 and 789.72 (Fig. 2A) represents the N-terminal carbamidomethylated peptide 29QHVTELPGVLHCGLQSFQFAV49 without cyclization of Gln29. Similarly, the C terminus of ZP4 was determined after a Glu-C digest by a tetracarbamidomethylated peptide (440KQVLGGQVYLHC*SASVC*QPAG(M·Ox)PSC*TVIC*PAS RR473, where C* indicates carbamidomethylated Cys) at m/z 1264.293+ and 948.474+ (Fig. 2B). This result indicates that the C terminus ends at Arg473 preceding a dibasic motif as has been observed in mouse ZP1, ZP2, and ZP3 (12.Boja E.S. Hoodbhoy T. Fales H.M. Dean J. J. Biol. Chem. 2003; 278: 34189-34202Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and recombinant human ZP3 (43.Zhao M. Boja E. Hoodbhoy T. Nawrocki J. Kaufman J.B. Kresge N. Ghirlando R. Shiloach J. Pannell L. Levine R. Fales H. Dean J. Biochemistry. 2004; 43: 12090-12104Crossref PubMed Scopus (32) Google Scholar). The resultant core 445-amino acid ZP4 polypeptide chain (Gln29–Arg473) has a predicted molecular mass of 48,878 daltons. Rat ZP4 contains four potential N-glycosylation sites (Asn-Xaa-Ser/Thr; where Xaa is not proline), and each is glycosylated in native rat zonae pellucidae. Peptide N-glycosidase F endoglycosidase releases protein-bound N-linked glycans and, by converting the involved asparagine residue to an aspartic acid, provides a signature increase in mass (0.98 Da). Subsequent Asp-N digested peptides 50NLSLEAESPVLTTW63, 228NITTGC233, and 336NYSSYYGT343 all demonstrate a mass increase of 0.98 Da compared with t
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