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Originally published In Press as doi:10.1074/jbc.M413569200 on January 27, 2005

J. Biol. Chem., Vol. 280, Issue 13, 12721-12731, April 1, 2005
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Human Sperm Do Not Bind to Rat Zonae Pellucidae Despite the Presence of Four Homologous Glycoproteins*

Tanya Hoodbhoy{ddagger}§, Saurabh Joshi{ddagger}, Emily S. Boja¶, Suzannah A. Williams||, Pamela Stanley||, and Jurrien Dean{ddagger}

From the {ddagger}Laboratory of Cellular and Developmental Biology, NIDDK and the Laboratory of Biophysical Chemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892 and the ||Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461

Received for publication, December 2, 2004 , and in revised form, January 27, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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).

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). The mouse zona pellucida is composed of three major glycoproteins, ZP1, ZP2, and ZP3, one of which, ZP2, is proteolytically cleaved following fertilization (3). 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). Mice without ZP1 form a zona pellucida matrix to which mouse sperm bind and Zp1 null females are fertile, albeit with decreased fecundity (5). 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, 7). 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); 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); or 4) supramolecular architecture involving more than one determinant plays a critical role (10).

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)1 encoded by MgatI (11). 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), and the remainder are high mannose or biantennary complex N-glycans (13).

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) {beta}1–6 N-acetylglucosaminyltransferase (GlcNAcT), of which there are three isoforms (I–III) (14–16). Biochemical and mass spectrometry analyses detect few or no O-glycans on mouse ZP2 (12, 17, 18), 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).

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). 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) or lacking an oocyte glycosyltransferases required for specific sugars proposed as recognition determinants (20–26) remain fertile. 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, 28), and their primary structures were deduced from cDNA (29–31). More recent reports raised the possibility of an additional protein in the human zona pellucida (32), the existence of which has been confirmed experimentally (9). As noted, mouse zonae pellucidae contain three glycoproteins, ZP1, ZP2, and ZP3, and their primary structures also were deduced from cDNA (33–35). Despite close examination, no additional zona proteins were detected in native mouse zonae pellucidae by mass spectrometry (12). Mouse and rat evolutionarily diverged only 12–24 million years ago (36), and the primary protein structures of rat ZP1, ZP2, and ZP3 are 88–92% identical to their murine homologues (37). However, the existence of an additional zona transcript deduced from cDNA (GenBankTM accession number NM_172330 [GenBank] ) 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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) 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 x 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, {beta}(1–4)-galactosidase and {beta}-N-acetylglucosaminidase) or de-exo- and endo-O-glycosylated as described previously (12).

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). Briefly, 4–5 µl of each digest was loaded onto a Vydec C18 MS column (100 x 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), huZP2/3 double rescue (7), conditional MgatI null (26), and C2 GlcNAcT null mice (24). Rat eggs were obtained from Fisher females, and sperm binding assays were carried out as previously described (6). 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) 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 x 106/ml. After 30 min of exposure to sperm, the eggs were washed with a wide bore glass pipette through a series of 5 x 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, 7). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Conservation of ZP4 Genes—Comparing the recently reported rat genome (39) with those of human (40) and mouse (41), 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). 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 [GenBank] ) 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 [GenBank] ). However, each potential open reading frame of the hypothetical mouse ZP4 transcript contains multiple stop codons (9), and after re-examination of earlier mass spectra (12), no tryptic peptides encoding ZP4 were detected in mouse zonae pellucidae.



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  		FIG. 1.
Genetic loci of four zona pellucida genes are conserved among mouse, rat and human. A, conserved exon maps of syntenic loci of Zp1, Zp2, Zp3, and Zp4/ZPB zona pellucida genes located on somatic chromosomes of rat, human, and mouse. B, conservation of the twelve exons (E1–E12) of the rat Zp4 gene compared with the mouse and human homologues. The numbers represent the size (bp) of each exon.

 
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.


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  		TABLE I
LC-MS and MS/MS analysis of rat ZP4

The residue numbers are based on the amino acid sequence of full-length ZP4. C* indicates carbamidomethylated cysteine; C—C indicates cysteines involved in disulfide bonding (a solid line between two cysteines indicates precise S—S pairings, whereas the absence of lines represents undetermined pairing among four cysteines). M* indicates methionine sulfoxide. N* indicates N-linked asparagine converted to aspartate after peptide N-glycosidase treatment (+0.985 Da). ¥ represents N-linked peptides. {Lambda} represents O-linked peptides.

 
Mass Spectrometry Analysis of Rat ZP4—The N terminus of rat ZP4 is predicted at Gln29 (42), 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) and recombinant human ZP3 (43). The resultant core 445-amino acid ZP4 polypeptide chain (Gln29–Arg473) has a predicted molecular mass of 48,878 daltons.



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  		FIG. 2.
Determination of N and C termini of rat ZP4. A, an Asp-N cleaved carbamidomethylated N-terminal peptide 29QHVTELPGVLHC*GLQSFQFAV49 (where C* indicates carbamidomethylated Cys) without cyclization of Gln29 is shown by the +2 and +3 charged ions at m/z 1184.10 and 789.72. B, the C terminus ends at Arg473 as detected by the Glu-C digested peptide 440KQVLGGQVYLHC*SASVC*QPAG(M·Ox)PSC*TVIC*PASRR473 as shown by the +3 and +4 charge states at m/z 1264.29 and 948.47.

 
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 their predicted masses, indicating that Asn50, Asn228, and Asn336 are glycosylated (Table I). For instance, the CID spectrum of a +2 charged ion at m/z 780.89 (Fig. 3A, inset) representing 50NLSLEAESPVLTTW63 unequivocally assigned the peptide sequence by y1–3 and y6, with b2–4 and a7 ions confirming the change from Asn to Asp (Fig. 3A). Although no sequence coverage was obtained between Asn74 and Ala117, the observation that Asp-N cleaved after Lys73 implies that Asn74 was converted to an Asp by peptide N-glycosidase F, thereby creating a site that otherwise would not have been available to Asp-N. This result indirectly shows that Asn74 was originally N-glycosylated. A tryptic peptide 293VSC*TYSIHSIMSPVNMQVWTLPPPLPK319 with an attachment of one N-acetylhexosamin-hexose was observed only in the N- and exo-O-deglycosylated sample. As shown in Fig. 3B, the +4 and +5 charged ions at m/z 862.69 and 690.36 represent this peptide, presumably with sugar linkage at one of the five potential attachment sites (Thr296, Ser298, Ser301, Ser304, and Ser312). No CID spectrum was available to confirm the attachment site.



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  		FIG. 3.
Localization of N- and O-glycosylation sites in rat ZP4. A, an example of a +2 charged Asp-N cleaved peptide 50N*LSLEAESPVLTTW63 (where N* indicates N converted to D) at m/z 780.89 (inset) and its CID spectrum clearly indicating a mass increase of 0.98 Da after peptide N-glycosidase F digestion and thus N-glycosylation occurring at Asn50. B, O-glycosylation at one of the six potential Ser/Thr sites (predicted at Thr312) in a carbamidomethylated tryptic peptide 293VSC*TYSIHSIMSPVNMQVWTLPPPLPK319 was observed with a mass increase of 365.15 Da (one N-acetylhexosamin-hexose) as shown by its +4 and +5 charge states at m/z 862.69 and 690.36.

 
Using the same nomenclature reported previously, priming the ions from peptide P2 (12), three disulfide bridges were identified of 20 cysteines present in mature rat ZP4. One of the disulfide bridges is formed between Cys233/Cys252 as illustrated by the +3 and +4 charged ions at m/z 1104.18 and 828.38 from a trypsin/Asp-N digest (Fig. 4A). The CID spectrum of m/z 1104.17 (Fig. 4B) included the y1–5, y9*, and b2–5 ions from 228NITTGCDPVMK238 (peptide P1; NIT converted to DIT), as well as y'1–5 and b'2–5 ions from 239TSTFVLFQFPLTSCGTTQR257 (peptide P2). Two more disulfide pairs were found among four cysteine residues near the C terminus of ZP4. The +3 charged ion at m/z 1086.82 from both trypsin and trypsin/Asp-N digests represent the peptide 441QVLGGQVYLHCSASVCQPAGMPSCTVICPASR472 with the loss of 4 Da because of the formation of two disulfide linkages (data not shown). No precise linkages could be assigned as a result of their close proximity and the lack of appropriate cleaving reagents, similar to mouse ZP3 (12). These results are summarized (Fig. 5) and compared with the homologous human protein (31).



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  		FIG. 4.
Disulfide bond linkage in rat ZP4. A, a disulfide bond formed between Cys233 and Cys252 is shown by the +3 and +4 charged ions at m/z 1104.17 and 828.38 representing 228NITTGCDPVMK238 (peptide P1; NIT converted to DIT) linked to 239TSTFVLFQFPLTSCGTTQR257 (peptide P2) from trypsin/Asp-N digestion. B, the CID spectrum of m/z 1104.17 included the y1–5, y9*, and b2–5 ions from P1, as well as y'1–5 and b'2–5 ions from P2. Primes refer to peptide P2.

 



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  		FIG. 5.
Summary of rat ZP4. The primary amino acid sequence (single-letter code) of rat ZP4 is compared with human ZP4/ZPB, and amino acid identities are indicated by dashes in the human sequence. Native rat ZP4 extends from an N-terminal glutamine (Gln29) to a C-terminal arginine (Arg473) immediately upstream of a potential dibasic cleavage site. There are 20 cysteine residues (yellow on a blue background). Three are in a trefoil domain (tan background; residues 149–191), and 10 are in the zona domain (yellow background; residues 197–471) of which eight are conserved (Cys198, Cys233, Cys252, Cys295, Cys377, Cys398, Cys451, and Cys464) among all of the zona domains (54). One disulfide bond was experimentally determined, Cys233/Cys252 (solid line), and two disulfide bonds were present in the peptide containing four cysteine residues (Cys451, Cys456, Cys464, and Cys468). The linkage of the latter (dotted lines) was indeterminate because of clustering of cysteine residues and the absence of appropriate cleavage sites. All four of the potential N-linked sites (white on green background) were glycosylated (shown in red; Asn50, Asn74, Asn228, and Asn336), and a single O-glycan was detected on a peptide containing 5 potential glycosylation sites (red asterisks; Thr296, Ser298, Ser301, Ser304, and Thr312). Peptides representing ~30% of mature rat ZP4 were not identified (white on gray backgrounds) because of the paucity of biological material.

 
Sperm Binding to Heterologous Eggs—To determine whether the presence of a fourth protein altered the specificity of sperm-egg interactions, sperm binding assays with mouse and rat eggs were conducted in parallel with capacitated mouse, rat, and human spermatozoa. After washing with wide bore pipettes until 1–5 sperm bound to two-cell embryos, the eggs were fixed, stained with Hoechst, and examined by confocal microscopy to quantify total sperm binding (Fig. 6). Each assay was repeated in triplicate with 20–30 eggs. Mouse sperm bound well to both mouse (85.0 ± 5.7 sperm/egg) and rat (81.9 ± 3.6 sperm/egg) eggs, but rat sperm bound considerably less well to rat (7.7 ± 1.2 sperm/egg) than to mouse (62.5 ± 2.7 sperm/egg) eggs. In marked contrast, capacitated human sperm bound to neither mouse (containing moZP1–3) nor rat (containing ratZP1–4) eggs.



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  		FIG. 6.
Heterologous sperm binding to mouse and rat eggs. Capacitated mouse, rat, or human sperm (1 x 106/ml) were incubated with 20–30 ovulated mouse or rat eggs for 30 min and then washed with a wide bore pipette until no more than 1–5 sperm bound to control mouse or rat two-cell embryos (insets), respectively. Eggs/sperm were fixed and stained with Hoechst. A, mouse sperm binding to mouse eggs. B, rat sperm binding to mouse eggs. C, human sperm binding to mouse eggs. D, mouse sperm binding to rat eggs. E, rat sperm binding to rat eggs. F, human sperm binding to rat eggs. Scale bar, 10 µm.

 
These data suggest that rat ZP1–4, homologous to the four human zona proteins, are not sufficient to support human sperm binding. The role of follicular fluid in mediating human sperm-egg interactions has been controversial (44, 45). To ascertain whether follicular fluid facilitates human sperm binding to zonae pellucidae, normal mouse eggs, normal rat eggs, huZP3 rescue eggs, or huZP2/3 double rescue eggs were preincubated with human follicular fluid (Fig. 7). The specificity of sperm binding was assayed as before, and in the presence of human follicular fluid, mouse sperm bound to mouse eggs (80.4 ± 3.8), rat eggs (68.3 ± 5.4), huZP3 rescue eggs (71.1 ± 3.2), or huZP2/3 double rescue eggs (127.9 ± 8.2). However, even in the presence of human follicular fluid, human sperm did not bind to any of the normal rodent or transgenic mouse eggs with chimeric mouse-human zona pellucida matrices (Fig. 7).



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  		FIG. 7.
Sperm binding to ovulated eggs in the presence of follicular fluid. Experimental procedures were as for Fig. 6 except that eggs were preincubated with human follicular fluid (50% in M16). A, mouse sperm binding to mouse eggs. B, human sperm binding to mouse eggs. C, mouse sperm binding to rat eggs. D, human sperm binding to rat eggs. E, mouse sperm binding to huZP3 rescue eggs. F, human sperm binding to huZP3 rescue eggs. G, mouse sperm binding to huZP2/3 double rescue eggs. H, human sperm binding to huZP2/3 double rescue eggs. Scale bar, 10 µm.

 
Sperm Binding to Glycosylation-deficient Eggs—Mouse sperm bind to a zona pellucida matrix composed of only mouse ZP2 and ZP3 (5), and yet human sperm will not bind to a zona matrix in which human ZP2 and ZP3 replace the endogenous mouse proteins (7). Earlier biochemical data (18, 46, 47), and more recent mass spectrometry analyses (8, 12), indicate that most of the carbohydrate on mouse ZP2 and ZP3 is N-linked with few if any O-linked sugars on ZP2 and only two variably occupied clusters on ZP3. Overall glycosylation of the zona proteins is quite heterogeneous, and although high mannose and biantennary complex N-glycans predominate, the majority of the less abundant O-glycans are mucin Core 2 type (13). To determine whether complex N-glycans or Core 2 O-glycans were capable of masking access or otherwise preventing human sperm binding, ovulated eggs lacking either 1) N-acetylglucosaminyltransferase I (MgatI), which initiates hybrid and complex N-glycan synthesis (26) or 2) C2 GlcNAcT I required for the formation of Core 2 O-glycans (24) were examined for human sperm binding. The structure of zonae pellucidae surrounding C2 GlcNAcT I null eggs appeared normal morphologically, but those of the MgatI conditional mutant was significantly thinner with more adherent cumulus cells. Nevertheless mouse sperm bound to zonae pellucidae deficient in Core 2 O-glycans (51.8 ± 3.0) or lacking hybrid or complex N-glycans (16.0 ± 4.4), but human sperm did not (Fig. 8).



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  		FIG. 8.
Sperm binding to ovulated eggs lacking specific N-or O-glycans. Experimental procedures were as for Fig. 6. A, mouse sperm binding to C2 GlcNAcT I null eggs deficient in core 2 O-glycans. B, same as A but with human sperm. C, mouse sperm binding to F/F:ZP3Cre MgatI null eggs lacking hybrid or complex N-glycans. D, same as C but with human sperm. Scale bar, 20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Successful sperm-egg recognition at the surface of the zona pellucida is a prerequisite for normal fertilization and the onset of embryonic development. Although tens of millions of spermatozoa are deposited in the lower female reproductive tract, relatively few encounter ovulated eggs emphasizing the imperative of productive sperm binding to the zona pellucida. Zona matrices surrounding rodent and human eggs are seemingly simple structures composed of either three or four glycoproteins. However, despite intense investigations, the molecular basis for sperm binding has remained enigmatic.

Internal fertilization offers no compelling reason for taxon-specific sperm-egg recognition among mammals, and yet human sperm are fastidious and will not bind to mouse eggs. The recent observations that human zonae pellucidae contain four zona proteins (ZP1, ZP2, ZP3, and ZP4/ZPB) and mouse eggs but three (ZP1, ZP2, and ZP3) raised the possibility that the additional zona protein is required for human sperm binding (9, 12). Human ZP2 and ZP3 are not sufficient to support human sperm binding in chimeric mouse-human zonae (7), and establishing mouse lines expressing four human zona proteins in lieu of the endogenous mouse genes is a daunting task. Therefore, other mammalian species containing four zona proteins were sought. In rat, the three transcripts encoding ZP1, ZP2, and ZP3 initially reported (37) have been augmented by a fourth that encodes a hypothetical protein of 540 amino acids that is 68% identical to human ZP4/ZPB with 20 conserved cysteine residues and both a trefoil and zona domain (Fig. 5). Microscale mass spectrometry was used to analyze the protein composition of native rat zonae pellucidae. Rat ZP4 (along with rat ZP1–3) was readily identified with 70% peptide coverage, and definition of N and C termini establishes a molecular mass of 48.9 kDa for the core polypeptide.

However, sperm binding assays to ovulated eggs indicate that rat ZP1–4 (62–70% identical to the secreted homologous human zona proteins) are not sufficient to support human sperm binding, and using human follicular fluid to preincubate (50% or 100% v/v) rodent eggs or supplement (50% v/v), the sperm binding assay media does not alter the outcome. In these assays, human sperm do not bind, even transiently, to mouse (containing moZP1–3), rat (ratZP1–4), huZP3 rescue (moZP1/2, huZP3), or huZP2/3 double rescue (moZP1 and huZP2/3) eggs in the presence or the absence of human follicular fluid. Seemingly, the structures of these four different zona matrices are not permissive for human sperm binding, and additional determinants must be sought.

Carbohydrate side chains have long been implicated as receptors in sperm-egg interactions in mice, although efforts to identity specific sugar moieties (or their attachment sites) as recognition determinants have been inconclusive. Most models are predicated on the release of the "adhesive" glycan by cortical granule glycosidases to account for the inability of sperm to bind to the zona matrix after fertilization. Zp1 null mice are fertile, albeit with decreased fecundity, indicating that if mouse sperm adhesion is mediated by a single glycan, it must be present on either ZP2 or ZP3 (5). N-Glycans occupy 11 of 12 potential sites on mouse ZP2 and ZP3 (12), and the majority are high mannose or biantennary complex (13, 18, 46). Mice lacking N-acetylglucosaminyltransferase I (MgatI null) required to initiate hybrid and complex N-glycan synthesis do not survive as embryos (48, 49). However, using the Zp3 promoter to drive Cre recombinase in mice in which the MgatI gene is flanked by loxP sites, mouse lines incapable of expressing N-acetylglucosaminyltransferase I in oocytes have been established. High mannose but not hybrid and complex N-glycans are detected in zona pellucidae of these conditionally mutant females, and the mice remain viable and fertile, albeit with morphologically abnormal zonae and decreased fecundity (26).

Few, if any O-glycans have been detected on ZP2 (8, 12, 18), and the majority present on ZP3 are reported to have Core-2 structures (13). However, mice lacking Core 2 {beta}1–6-N-acetylglucosaminyltransferase (C2 GlcNAcT I null) involved in formation of mucin Core 2 O-glycans remain fertile (24). Although two other human isoforms, C2 GlcNAcT II and C2 GlcNAcT III, have been identified, the first is not detected in ovarian tissues by Northern blot analysis (15), and the second is reported to be expressed exclusively in the thymus (16). Taken together, these data suggest that, if sperm adhesion is glycan mediated, it must be via high mannose N-glycans on ZP2 or ZP3 or O-glycans on ZP3 other than mucin Core 2 structures. Alternatively, rather than forming receptor sites, carbohydrates could function to block human sperm from binding to rodent eggs either sterically or through electrostatic interactions. However, human sperm do not bind to ovulated eggs from the MgatI conditional null mice or to eggs deficient in Core 2 O-glycans. These data imply that the restriction of N-glycans to high mannose or deficiency of Core 2 O-glycans neither precludes mouse sperm binding nor supports human sperm binding to the zona pellucida surrounding ovulated mouse eggs.

Thus, it has been difficult to ascribe sperm binding to a single protein or carbohydrate determinant in the zona pellucida, which raises the possibility that supramolecular structures play important roles in sperm-egg recognition. This formulation is consistent with genetic data implicating the cleavage status of ZP2 as primary in determining whether or not the three-dimensional structure of the zona matrix is permissive for sperm binding (7). ZP3 is essential for formation of the zona pellucida matrix (6, 50) and can partner with either ZP1 (Zp2 null), ZP2 (Zp1 null), or both (normal), although the width and stability of the ZP1/ZP3 matrix are considerably thinner than the ZP2/ZP3 matrix because of limiting amounts of ZP1 (5, 51). Each zona protein has a conserved "zona domain" implicated in the polymerization of the zona pellucida matrix (52), and disulfide bonds mapped by microscale mass spectrometry suggest two zona domain isoforms (12). Zona domain I (e.g. ZP3) contains eight conserved cysteine residues followed by a 45–50-amino acid C-terminal tail, and zona domain II (e.g. ZP1 and ZP2) contains 10 conserved cysteine residues ending 3–5 amino acids shy of the C terminus (12). Although the data set is incomplete, it appears that the first four cysteine residues in each isoform have similar linkages: Cys1–Cys4; Cys2–Cys3 (loop within loop). However, the linkages of the remaining cysteines differ. In zona domain I it is Cys5–Cys7 and Cys6–Cys8 (cross-over motif), whereas in zona domain II it is Cys5–Cys6, Cys7–Cys8, and Cys9–Cys10 (sequential motif). Thus, it appears that formation of a zona pellucida matrix requires two different zona domain isoforms, at least one type I, and one or more type II. The presence of 10 conserved cysteine residues and the presence of two disulfide bonds among the last four cysteine residues suggests that ZP4 contains a zona domain II isoform. These observations predict that, if present in sufficient quantities, ZP3/ZP4 proteins could form a zona pellucida matrix, as observed with the ZP3/ZP1 and ZP3/ZP2 matrices. A further prediction would be that if sufficiently robust zona matrices lacking ZP2 could be formed, they, in the absence of a cleavable protein with zona domain II isoform, would retain the ability to bind sperm even after fertilization.

Two variables beyond the composition of the zona pellucida may have import: the geometry of the zona matrix and the sperm surface. One of the most evolutionarily dynamic aspects of sperm-egg recognition among mammals is morphology of the sperm head (53). Mouse sperm (125 µm long), like that of all rodents, have a curved surface on the anterior head that resembles a hook and the length of the head (8 µm) is comparable with the thickness of the zona pellucidae (7–8 µm) that surrounds the 80-µm-diameter egg. Human sperm (60 µm) are more compact and morphologically distinct from mice with an anterior head resembling a flattened spade the length of which (4 µm) is significantly less than the width of the human zona pellucidae (10–15 µm) surrounding the larger, 120-µm-diameter human egg. Whether these distinct morphological differences between anterior sperm surfaces and zona matrices affect three-dimensional molecular structures important for taxon-specific sperm-egg recognition remains to be determined.


   FOOTNOTES
 
* 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. Back

§ To whom correspondence should be addressed: Laboratory of Cellular and Developmental Biology, NIDDK, Bldg. 50, Rm. 3128, National Institutes of Health, Bethesda, MD 20892-8028. Tel.: 301-594-1405; Fax: 301-496-5239; E-mail: tanyah{at}intra.niddk.nih.gov.

1 The abbreviations used are: GlcNAcT, {beta}1–6 N-acetylhexosaminehexose; HPLC, high performance liquid chromatography; MS, mass spectrometry; CID, collision-induced dissociation; MgatI, N-acetylglucosaminyltransferase I; C2, Core 2. Back



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