Sperm receptors are located in the mammalian egg extracellular coat, or zona pellucida. Mouse and hamster sperm receptor glycoproteins, mZP3 (83 × 103Mr) and hZP3 (56 × 103Mr), respectively, have very similar polypeptides (44×103Mr; 81% identical) that are glycosylated to different extents. Purified mZP3 and hZP3 can bind to mouse sperm, prevent them from binding to eggs and induce them to undergo exocytosis, the acrosome reaction, in vitro. A DNA construct that placed the hZP3 gene under the control of mZP3 gene 5 -flanking sequence was used in this report to produce two mouse lines that harbored the foreign sperm receptor transgene. In both lines, the transgene was expressed only by growing oocytes, at a level comparable to that of the endogenous mZP3 gene, and the developmental pattern of transgene expression resembled that of the mZP3 gene. In addition to mZP3, transgenic mouse oocytes synthesized and secreted a glycoprotein indistinguishable from hZP3, and incorporated both glycoproteins into a mosaic zona pellucida. Importantly, hZP3 purified from such zonae pellucidae exhibited both sperm receptor and acrosome reaction-inducing activities in vitro and, following fertilization of transgenic mouse eggs, was inactivated. These results demonstrate that a biologically active foreign sperm receptor can be synthesized and secreted by transgenic mouse oocytes, assembled into a mosaic zona pellucida, and inactivated following fertilization as part of the secondary block to polyspermy.

Fertilization in mammals is initiated by binding of free- swimming sperm to the egg zona pellucida, a thick extra- cellular coat surrounding mammalian eggs (Gwatkin, 1977; Yanagimachi, 1988; Dietl, 1989). This event is mediated by egg-binding proteins located on the sperm head and sperm receptors located in the egg zona pellucida (Wassarman, 1987a,b, 1990).

In mice, the sperm receptor is mZP3 (83×103Mr), one of three zona pellucida glycoproteins (Bleil and Wassarman, 1980a; Wassarman, 1987a,b, 1988, 1990; Kinloch et al., 1991). mZP3 consists of a 44×103Mr polypeptide, 3 or 4 asparagine- (N-) linked oligosaccharides and an un- determined number of serine/threonine- (O-) linked oligosaccharides (Wassarman, 1988, 1990). A specific class of O-linked oligosaccharides is responsible for mZP3 sperm receptor activity (Florman and Wassarman, 1985; Wassarman, 1987a,b, 1988, 1989, 1990, 1991). The mZP3 gene has been cloned and its primary structure determined (Kinloch et al., 1988; Ringuette et al., 1988; Kinloch and Wassarman, 1989a). Evidence suggests that mZP3 gene expression is restricted to growing oocytes within ovaries of juvenile and adult female mice (Bleil and Wassarman, 1980b; Salzmann et al., 1983; Philpott et al., 1987; Roller et al., 1989; Kinloch and Wassarman, 1989a). Cis-acting sequences located in the mZP3 gene 5′-flanking region, together with oocyte-specific proteins, apparently are responsible for the pattern of expression during development (Lira et al., 1990; Schickler et al., 1992).

In hamsters, the sperm receptor is hZP3 (56×103Mr), one of three zona pellucida glycoproteins (Moller et al., 1990). The hZP3 gene has been cloned and its primary structure determined (Kinloch et al., 1990). mZP3 and hZP3 polypeptides are very similar to each other (44×103Mr; 81% identical), yet are glycosylated to different extents. Consequently, mature mZP3 (83×103Mr) and hZP3 (56 × 103Mr) have significantly different molecular weights. However, either purified mZP3 or purified hZP3 can bind to mouse sperm and induce them to undergo the acrosome reaction in vitro (Moller et al., 1990; Kinloch et al., 1991), suggesting that the two glycoproteins have at least certain O-linked oligosaccharide determinants in common.

To determine whether biologically active foreign sperm receptors could be introduced into the mouse zona pellu-cida, we produced transgenic mice harboring the hZP3 gene placed under control of the mZP3 gene promoter. These mice enabled us to address several important issues, including the following. (i) Whether expression of the hZP3 gene is regulated like the endogenous mZP3 gene during mouse development. (ii) Whether mouse oocytes can discriminate between mZP3 and hZP3 nascent polypeptides, and process the latter properly into a biologically active glycoprotein that resembles the authentic sperm receptor. (iii) Whether hZP3 can associate with mouse zona pellucida glycoproteins and be accommodated together with mZP3 in a mosaic zona pellucida. (iv) Whether, like mZP3, hZP3 is inactivated as part of the secondary block to polyspermy following fertilization of transgenic mouse eggs. Results of experiments reported here provide evidence bearing on these, as well as other aspects of mammalian development.

Plasmid construction

The plasmid used to generate transgenic mice is depicted schematically in Fig. 1. Briefly, p6.5-5′m/hZP3 contains the entire hamster ZP3 (hZP3) gene coding region (nts +28 to +8200; Kinloch et al., 1990), including the polyadenylation and termination signals, placed under control of the mouse ZP3 (mZP3) gene promoter (6.5 kb of the mZP3 gene 5′-flanking sequence and nucleotides 1-10 of mZP3 mRNA; Lira et al., 1990). p6.5- 5′m/hZP3 was constucted by cloning the mZP3 promoter region, comprising a 6.5 kb SstII fragment, into the SstII site of pPGK/hZP3 (Kinloch et al., 1991). The plasmid was digested with ApaI and fractionated over a 10-40% sucrose gradient. The ∼11.3 kb m/hZP3 fragment (Fig. 1) was recovered from appropriately pooled fractions by precipitation with ethanol and resuspended at 2.5 μg/ml in injection buffer (10 mM Tris-HCl, pH 7.5; 0.15 mM EDTA).

Fig. 1.

Schematic diagram of m/hZP3 transgene and PCR analysis of transgenic mice. Top: Schematic diagram of m/hZP3 transgene. The 5′- flanking region of mZP3 is denoted by a closed box and the hZP3 coding region is denoted by an open box. The numbering represents the nt position of the mZP3 and hZP3 loci with respect to their transcriptional start sites (+1). The m/hZP3 transgene was excised from plasmid p6.5- 5′m/hZP3 using restriction endonuclease Apa I (A). Bottom: PCR analysis of tail DNA. PCR analysis was performed on tail DNA from all mice born following the microinjection procedures.Shown is a 1.5% agarose gel containing amplified DNA from representative samples. The 511 bp and 406 bp DNA fragments represent the endogenous mZP3 gene and m/hZP3 transgene, respectively. Lanes M and H contain positive control plasmid templates for the mZP3 and hZP3 primer sets, respectively (pPGK/mZP3 and pPGK/hZP3; Kinloch et al., 1991). B, blank lane (H2O control). Sd, size standards (1 kb DNA ladder; BRL).

Fig. 1.

Schematic diagram of m/hZP3 transgene and PCR analysis of transgenic mice. Top: Schematic diagram of m/hZP3 transgene. The 5′- flanking region of mZP3 is denoted by a closed box and the hZP3 coding region is denoted by an open box. The numbering represents the nt position of the mZP3 and hZP3 loci with respect to their transcriptional start sites (+1). The m/hZP3 transgene was excised from plasmid p6.5- 5′m/hZP3 using restriction endonuclease Apa I (A). Bottom: PCR analysis of tail DNA. PCR analysis was performed on tail DNA from all mice born following the microinjection procedures.Shown is a 1.5% agarose gel containing amplified DNA from representative samples. The 511 bp and 406 bp DNA fragments represent the endogenous mZP3 gene and m/hZP3 transgene, respectively. Lanes M and H contain positive control plasmid templates for the mZP3 and hZP3 primer sets, respectively (pPGK/mZP3 and pPGK/hZP3; Kinloch et al., 1991). B, blank lane (H2O control). Sd, size standards (1 kb DNA ladder; BRL).

Production and characterization of transgenic mice

Purified DNA fragment (described above) was microinjected into the male pronucleus of fertilized mouse [(C57BL/6J × DBA/2J)F2; The Jackson Laboratory] eggs. Microinjected eggs were then transferred into oviducts of [(C57BL/6J × CBA/J)F1; The Jackson Laboratory] foster mothers according to published procedures (Hogan et al., 1986).

Identification of transgenic founders was carried out by polymerase chain reaction (PCR) analysis. A small piece of mouse tail was digested in 50 μl of lysis buffer (50 mM KCl, 10 mM Tris- HCl, pH 8.3, 2 mM MgCl2, 0.45% NP-40, 0.45% Tween-20, and 60 μg/ml Proteinase K) and incubated overnight at 55°C, followed by 30 minutes at 95°C. Two pairs of oligonucleotide primers were used for PCR analysis: 5′-CTCAGCCTGCTCATGGCTCTA -3′ and 5′-GAATAGATGGCTCGAGCCTGT -3′, specific for the m/hZP3 transgene, and 5′-CAGCTCTACATCACCTGCCA-3′ and 5′-CACTGGGAAGAGACACTCAG -3′, as an internal control specific for the endogenous mZP3 gene. These primers amplify fragments of 406 nt and 511 nt from the hZP3 and mZP3 genes, respectively. A 25 μl PCR reaction mix [50% lysis buffer, 50% PCR buffer (16.6 mM ammonium sulphate, 67 mM Tris-HCl pH8.8, 0.1 mg/ml BSA), 0.2 mM NTPs, 1 unit Taq polymerase (Perkin-Elmer/Cetus, Norwalk, CT), 0.2 μg of each oligonucleotide primer, and 4 μl of DNA sample] (F. Koentgen, personal communication) was overlaid with 25 μl of mineral oil and run in a Perkin Elmer/Cetus DNA Thermal Cycler for 40 cycles of 94°C, 20 sec; 60°C, 30 sec; and 72°C, 30 sec. Ten μl of each sample was subjected to electrophoresis on a 1.5% agarose gel. Positive founder mice were mated to generate transgenic pedigrees.

Reverse-PCR analysis

Total RNA was isolated from tissues using RNAzol B, according to the manufacturer’s instructions (Cinna/Biotecx Labs, Friendswood, TX). First strand cDNA was prepared in a 20 μl reaction mix (5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1 mM dNTPs, 1 U/ μl RNase inhibitor, 2.5 U/ μl reverse tran - scriptase, 2.5 μM random hexamers) as described by Perkin Elmer/Cetus, and run in a Perkin Elmer/Cetus Thermal Cycler for 1 cycle of 20°C, 10 minutes; 42°C, 20 minutes; 99°C, 5 minutes; 5°C, 5 minutes. Three pairs of oligonucleotide primers were used for PCR analyses: 5′-CACCTTTGCCAGACCCGAACT-3′ and 5′-CTGCGGTTTCGAGAAACTAGC-3′, specific for mZP3 transcripts; 5′-CCGTGCTGCTGCACCAACCCC-3′ and 5′-CTGCG-GCGTCTAGATGCCGAC-3′, specific for hZP3 transcripts; 5′- GTGGGCCGCTCTAGGCACCAA-3′ and 5′-CTCTTT- GATGTCACGCACGATTTC-3′, specific for mouse β-actin transcripts (Clontech Labs, Inc., Palo Alto, CA); these primers amplify fragments of 395 nt, 715 nt, and 540 nt, respectively. A 25 μl PCR reaction mix (5 μl cDNA mix, 2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 0.625 U Taq Polymerase (Perkin Elmer/Cetus, Norwalk, CT), 0.2 μg of appropriate oligonucleotide primer set) was overlaid with mineral oil and run in a Perkin Elmer/Cetus Thermal Cycler for 40 cycles of 94°C, 20 sec; 60°C, 30 sec; 72°C, 30 sec. Ten μl of each sample was subjected to electrophoresis on 2% agarose gels.

RNase protection analysis

RNase protection assays were performed essentially as described by Ambion, Inc. (Austin, TX). Total RNA was isolated from oocytes or ovulated eggs using RNAzol B, as described by the supplier (Cinna/Biotecx Labs, Friendswood, TX). High specific activity RNA probes were transcribed, using SP6 RNA polymerase, from either EcoRI linearized pGEM- G9/S-A (mZP3 specific probe; Kinloch et al., 1988) or HindIII linearized pGEM- hBR2 (hZP3 specific probe; Kinloch et al., 1990). RNA was mixed

with 1 × 105 counts/minute of probe in 20 μl hybridization buffer (80% formamide, 40 mM PIPES, pH 6.4, 400 mM sodium acetate, pH 6.4, 1 mM EDTA) and incubated overnight at 42°C. Following incubation in the presence of RNase A (0.5 U/ml) and RNase T1 (100 U/ml), protected fragments were analyzed on 6% denaturing polyacrylamide gels.

Western gel immunoblot analysis

Ovaries were sonicated in 0.5 ml of lysis buffer (1% Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 1 mM iodoacetamide, 0.2 U/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 mM Tris-Cl, pH 8, 140 mM NaCl, 0.025% sodium azide), and the lysate was cleared by centrifugation at 30,000 g for 30 minutes at 4°C. The supernatant was recovered and adjusted to 2% SDS/10% glycerol/50 mM DTT/0.05% Bromphenol blue (protein sample buffer). Alternatively, isolated ZP were dissolved in protein sample buffer. Aliquots were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membrane. The membrane was processed for western gel immunoblotting by first incubating it in a blocking buffer, then in the presence of a rabbit polyclonal antiserum (Pocono Rabbit Farms, Canadensis, PA) directed against either mZP3 or hZP3 peptides (1:750 dilution), and finally in the presence of a goat anti-rabbit IgG-alkaline phosphatase conjugate (1:3,000 dilution; Bio-Rad, Rockville Centre, NY), essentially according to published procedures (Ausubel, 1989). Blocking buffer contained 3% BSA, 0.5 M NaCl, 20 mM Tris-Cl, pH 7.5; washing buffer contained 0.2% Tween-20, 0.5 M NaCl, 20 mM Tris-Cl, pH 7.5; and antibody solutions contained 1% BSA, 0.5 M NaCl, 20 mM Tris-Cl, pH 7.5, 0.2% Tween-20.

Glycoprotein purification

Purification of mZP3 and hZP3 from ovaries has been described (Bleil et al., 1988; Moller et al., 1990). hZP3 was purified from transgenic mouse ovaries essentially as follows. Zonae pellucidae were isolated from ovaries of 50 transgenic mice as described previously (Bleil et al., 1988). Zonae pellucidae were dissolved in protein sample buffer and glycoproteins were separated on a 10% preparative SDS-PAGE gel. hZP3 was recovered from gel slices using a Centrilutor micro-electroeluter, as described by the manufacturer (Amicon, Beverly, MA). The sample was extensively dialyzed against 8 M urea, then against distilled water, and aliquots were analysed by SDS-PAGE, followed by western immunoblotting and silver-staining to determine purity and concentration.

Sperm receptor assay

Assays for sperm receptor activity (“competition assays”) were carried out in vitro essentially as previously described (Bleil and Wassarman, 1980a; Florman and Wassarman, 1985; Moller et al., 1990), using gametes and embryos obtained from randomly bred, Swiss albino mice (CD-1; Charles River Breeding Labs, Wilmington, MA). Assays were carried out in Earle’s modified medium 199 (Gibco B.R.L., Grand Island, NY) containing 25 mM Hepes, pH 7.3, 30 μg/ml pyruvate, and 4 mg/ml BSA (M199-M), at 37°C in a humidified atmosphere of 5% CO2 in air. Capacitated (M- 199M, 4 mM EGTA, 37°C, 1 hour) mouse sperm in 50 μl of medium (5 × 105 sperm/ml, final concentration) were incubated in the presence of either M199-M alone or M199-M containing a purified glycoprotein to be tested. Then 12-15 ovulated mouse eggs and 3-4 two-cell mouse embryos, obtained as previously described (Bleil and Wassarman, 1980a; Florman and Wassarman, 1985; Moller et al., 1990), were added to the cultures and the incubation continued for an additional 30 minutes. Ovulated eggs and two-cell embryos were washed by mouth-pipetting with fresh M-199M, until no sperm remained associated with embryos, and cells were fixed in 1% glutaraldehyde and the number of bound sperm per egg determined by light microscopy. In all competition assays, at least 70% of sperm remained highly motile up to the time of fixation, or the experiment was discarded.

Acrosome reaction assay

Capacitated mouse sperm in 25 μl of M199-M (1 × 106 sperm/ml, final concentration) were mixed with 25 μl samples containing M199-M alone, M199-M containing a purified glycoprotein to be tested, or ionophore A23187 (10 μM, final concentration; Sigma, St. Louis, MO), incubated for 1 hour at 37°C, and then fixed and processed, essentially as previously described (Moller et al., 1990). Briefly, treated sperm were fixed with 2% formaldehyde for 30 minutes, washed by centrifugation at 10,000 revs/minute for 2 minutes in 0.15 M ammonium acetate, pH 9, and dried onto gelatin-coated (0.25% gelatin, 0.5% chromium sulphate) glass slides. Sperm were stained with 0.04% Coomassie G-250 (in 3.5% perchloric acid) for 2.5 minutes, washed with distilled water, and scored for the presence or absence of an acrosome by bright field microscopy.

Immunohistological analysis

Ovarian paraffin sections were dewaxed, hydrated and incubated first in a blocking buffer (5% goat serum, 5% fetal calf serum, 0.1% gelatin 4 mg/ml polyvinylpyrrolidone in PBS), then in the presence of a rabbit polyclonal antiserum directed against either mZP3 or hZP3 peptides (1: 500 dilution in blocking buffer), then in the presence of goat anti-rabbit IgG-horseradish peroxidase conjugate (1:2,000 dilution in blocking buffer; Bio-Rad, Rockville Centre, NY), and finally processed for silver enhancement using the DAB enhancement kit (Amersham, Arlington Heights, IL), according to manufacturer’s instructions.

Cryosections of ovaries excised from transgenic and control mice were prepared and processed for immunohistological examination using rabbit antisera directed specifically against either mZP3 or hZP3 peptides and goat anti-rabbit IgG coupled to 5 nm colloidal gold (Jansen Biotec, Piscataway, NJ). Sections were prepared and immunostained essentially as previously described (Griffiths et al., 1983; Polak and Varndell, 1984), except that all solutions contained polyvinylpyrrolidone (5 mg/ml; 40 × 103Mr), all incubations were carried out in a 40°C water bath, and sections were re-embeded in epon prior to examination by transmission electron microscopy (Mortillo and Wassarman, 1991). Sections mounted on copper grids were examined with a Philips 201 electron microscope at 60 kV.

Production of transgenic animals

Seventy-one fertilized mouse eggs were microinjected with the m/hZP3 fragment (Fig. 1) and 50 microinjected eggs were transplanted into oviducts of pseudopregnant recipients. PCR analysis of tail DNA was performed to determine which of the resulting progeny contained the m/hZP3 transgene. Three of 29 newborn animals exhibited a 406 nt DNA fragment that corresponded to amplification of a segment of the m/hZP3 transgene (Fig. 1). In addition, all samples contained a 511 nt fragment that corresponded to amplification of a segment of the endogenous mZP3 gene. Transgenic lines were established from two of the positive animals (designated m/h-6 and m/h-11; a third animal, designated m/h-29, died), more than 180 progeny were analyzed by PCR, and the pattern of amplification was found to be reproducible. Founder animals m/h-6 and m/h-11 distributed the transgene to 47% and 46% of their progeny, respectively. Mice homozygous for the transgene were established for line m/h-11.

Expression of m/hZP3 by transgenic mice

We reported previously that the first 470 nt of mZP3 gene 5′-flanking sequence is sufficient to target expression of the firefly luciferase gene specifically to growing oocytes (Schickler et al., 1992). To determine the tissue specificity of m/hZP3 expression, a combination of reverse transcription followed by PCR amplification was employed. PCR primers, specific for hZP3 mRNA, were used to amplify a region of cDNA that had been reverse transcribed from ovarian RNA samples obtained from littermates of transgenic lines m/h-6 and m/h-11. For both lines, a 720 nt fragment, specific for m/hZP3 mRNA, was amplified from ovarian cDNA prepared from mice shown to carry the transgene. This fragment was absent when littermates not carrying the transgene were analyzed. In all cases, a 395 nt fragment specific for mZP3 mRNA was amplified (Fig. 2). To confirm that the 720 nt fragment did, indeed, correspond to an m/hZP3-specific sequence, aliquots of PCR reactions were digested with SstI, and the 542 nt and 172 nt restriction fragments produced (data not shown) are consistent with SstI digestion of this portion of m/hZP3 cDNA (Kinloch et al., 1990). Reverse-PCR was also carried out with ovarian RNA obtained from founder m/h-29 immediately after it died, and a 720 nt fragment, indicative of m/hZP3 expression, was amplified.

Fig. 2.

Reverse PCR analysis of transgenic progeny. Top: PCR analysis was performed on ovarian cDNA samples from progeny of transgenic lines m/h-6 and m/h-11. Amplified DNA fragments were separated on 1.5% agarose gels. Mice harboring the m/hZP3 transgene are denoted + and mice lacking the transgene are denoted –. The 395 bp and 720 bp DNA fragments represent mZP3 and hZP3 mRNAs, respectively. Bottom: PCR analysis of tissue cDNA samples. B, brain; E, epididymis; I, intestine; H, heart; L, liver; M, muscle; S, spleen; T, testis; U, uterus; Ot, ovary; all from a mouse harboring the m/hZP3 transgene. Ot, ovary from a control (non-transgenic) littermate. Amplified DNA fragments were separated on 1.5% agarose gels. The 540 bp and 720 bp DNA fragments represent β-actin and hZP3 mRNAs, respectively. Sd, size standards (1 kb DNA ladder; BRL).

Fig. 2.

Reverse PCR analysis of transgenic progeny. Top: PCR analysis was performed on ovarian cDNA samples from progeny of transgenic lines m/h-6 and m/h-11. Amplified DNA fragments were separated on 1.5% agarose gels. Mice harboring the m/hZP3 transgene are denoted + and mice lacking the transgene are denoted –. The 395 bp and 720 bp DNA fragments represent mZP3 and hZP3 mRNAs, respectively. Bottom: PCR analysis of tissue cDNA samples. B, brain; E, epididymis; I, intestine; H, heart; L, liver; M, muscle; S, spleen; T, testis; U, uterus; Ot, ovary; all from a mouse harboring the m/hZP3 transgene. Ot, ovary from a control (non-transgenic) littermate. Amplified DNA fragments were separated on 1.5% agarose gels. The 540 bp and 720 bp DNA fragments represent β-actin and hZP3 mRNAs, respectively. Sd, size standards (1 kb DNA ladder; BRL).

PCR primers, specific for either hZP3 or β-actin mRNA, were used to amplify regions of cDNA reverse transcribed from RNA obtained from a variety of tissues excised from transgenic mice. While a 540 nt DNA fragment, representative of β-actin mRNA, was amplified using cDNA from each tissue tested, the 720 nt fragment, representative of hZP3 mRNA, was amplified only from brain and ovary cDNA (Fig. 2). Expression of the transgene in brain was detected only with line m/h-11 and presumably reflects a position effect.

Results of reverse-PCR analyses indicated that m/hZP3 was expressed only in ovaries. To demonstrate that ovarian expression of m/hZP3 was restricted to growing oocytes, as is the case for mZP3 (Roller et al., 1989; Kinloch and Wassarman, 1989a; Lira et al., 1990; Schickler et al., 1992), and to assess the level of expression, RNase protection assays were carried out. RNA was prepared using oocytes isolated from positive (determined by PCR of tail DNA) littermates of transgenic lines m/h-6, m/h-11, and a negative control littermate, and from ovulated eggs isolated from line m/h-11. One-half of each RNA sample was hybridized with probes corresponding to antisense transcripts of either m/hZP3 or mZP3. A 226 nt m/hZP3specific fragment was protected in oocyte samples from positive transgenic mice of both lines, but not in the oocyte sample from a negative, control littermate (Fig. 3). In all samples a 233 nt mZP3-specific fragment was protected. Densitometry of the gel revealed that m/hZP3 mRNA accumulated to 48% and 30% of the level of mZP3 mRNA for lines m/h-6 and m/h-11, respectively.

Fig. 3.

RNase protection analysis of oocytes and ovulated eggs. RNase protection analysis was performed with oocyte and ovulated egg RNA from transgenic progeny and with yeast tRNA. The 233 nt fragment represents mZP3 mRNA protected by the mZP3-specific antisense probe. The 226 nt fragment represents hZP3 mRNA protected by the hZP3-specific antisense probe. Top: Oocyte RNA from control mice (non-transgenic littermates; 75 oocytes, lanes A and B), oocyte RNA from mice harboring the m/hZP3 transgene (60 oocytes, line m/h-6, lanes C and D; 75 oocytes, line m/h-11, lanes E and F), and yeast tRNA (10 μg, lanes G and H), protected with either mZP3-specific probe (lanes A, C, E, and G) or hZP3-specific probe (lanes B, D, F, and H). Sd, size standards (123 bp DNA ladder; BRL) with appropriate sizes indicated. Bl, blank lane. Bottom: Oocyte RNA (100 oocytes, lanes A and C), ovulated egg RNA (200 eggs, lanes B and D) from mice harboring the m/hZP3 transgene, and yeast tRNA (10 μg, lanes E and F) protected with either mZP3-specific probe (lanes A, B, and E) or hZP3-specific probe (lanes C, D, and F).

Fig. 3.

RNase protection analysis of oocytes and ovulated eggs. RNase protection analysis was performed with oocyte and ovulated egg RNA from transgenic progeny and with yeast tRNA. The 233 nt fragment represents mZP3 mRNA protected by the mZP3-specific antisense probe. The 226 nt fragment represents hZP3 mRNA protected by the hZP3-specific antisense probe. Top: Oocyte RNA from control mice (non-transgenic littermates; 75 oocytes, lanes A and B), oocyte RNA from mice harboring the m/hZP3 transgene (60 oocytes, line m/h-6, lanes C and D; 75 oocytes, line m/h-11, lanes E and F), and yeast tRNA (10 μg, lanes G and H), protected with either mZP3-specific probe (lanes A, C, E, and G) or hZP3-specific probe (lanes B, D, F, and H). Sd, size standards (123 bp DNA ladder; BRL) with appropriate sizes indicated. Bl, blank lane. Bottom: Oocyte RNA (100 oocytes, lanes A and C), ovulated egg RNA (200 eggs, lanes B and D) from mice harboring the m/hZP3 transgene, and yeast tRNA (10 μg, lanes E and F) protected with either mZP3-specific probe (lanes A, B, and E) or hZP3-specific probe (lanes C, D, and F).

RNase protection experiments were also carried out using RNA prepared from oocytes and eggs isolated from line m/h-11. These experiments revealed that the level of m/hZP3 mRNA fell dramatically in ovulated eggs (Fig. 3), consistent with the behavior of mZP3 mRNA during meiotic maturation of oocytes (Roller et al., 1989; Kinloch and Wassarman, 1989a). The m/hZP3 (226 nt) and mZP3 (233 nt) protected fragments were only detected in oocytes, not in ovulated eggs.

Synthesis of hZP3 glycoprotein by transgenic mice

RNA analyses revealed that the m/hZP3 transgene was expressed by oocytes in mice of both transgenic lines. To demonstrate that hZP3 glycoprotein was present in transgenic mouse ovaries, ovarian lysates were prepared and analyzed on western gel immunoblots using antisera raised against either mZP3 (anti-mZP3p) or hZP3 (anti-hZP3p) peptides (Kinloch et al., 1991). Immunoblots of ovarian lysates of transgenic lines harboring the m/hZP3 gene revealed that, in addition to mZP3 (83 × 103Mr), the transgenic mice synthesized hZP3 (Fig. 4). hZP3 from transgenic mice migrated as a broad band at 56 × 103Mr, similar to the migration of authentic, purified hamster hZP3 on SDS-PAGE. Lysates of control littermates, lacking the m/hZP3 transgene, exhibited only mZP3 (83 ×103Mr) on immunoblots.

Fig. 4.

Western gel immunoblot analysis of transgenic progeny. Ovarian lysates from transgenic progeny were subjected to SDS- PAGE, transferred to nitrocellulose membrane, and processed for western immunoblotting using polyclonal antisera directed against either mZP3 or hZP3. Lysates from mice harboring the m/hZP3 transgene (m/h-11, lanes A and D; m/h-6, lanes B and E) and control mice (non-transgenic littermates; lanes C and F), probed with either anti-mZP3p (lanes A-C) or anti-hZP3p (lanes D-F). The positions of mZP3 and hZP3 are indicated.

Fig. 4.

Western gel immunoblot analysis of transgenic progeny. Ovarian lysates from transgenic progeny were subjected to SDS- PAGE, transferred to nitrocellulose membrane, and processed for western immunoblotting using polyclonal antisera directed against either mZP3 or hZP3. Lysates from mice harboring the m/hZP3 transgene (m/h-11, lanes A and D; m/h-6, lanes B and E) and control mice (non-transgenic littermates; lanes C and F), probed with either anti-mZP3p (lanes A-C) or anti-hZP3p (lanes D-F). The positions of mZP3 and hZP3 are indicated.

Immunohistochemical analyses provided further evidence for hZP3 synthesis by transgenic mouse oocytes. Paraffin sections were prepared from ovaries excised from mice containing the m/hZP3 transgene, as well as from negative control littermates, and processed for immunohistochemistry using anti-mZP3p and anti-hZP3p. As seen in Figure 5, anti-hZP3p detected hZP3 in zonae pellucidae around oocytes within transgenic mouse ovaries, but not in zonae pellucidae of control mice. As expected, anti-mZP3p detected mZP3 in zonae pellucidae of all mice examined.

Fig. 5.

Immunohistochemistry of ovarian sections from transgenic progeny. Ovarian paraffin sections were prepared and processed for immunohistochemistry using rahhit polyclonal antisera directed against either mZP3 or hZP3. and goat anti-rabbit IgG coupled to horseradish peroxidase, as described in Experimental Procedures. Shown are representative sections from mice harboring the mlhZP3 transgene (panels B, D and F) and control mice (non-transgenic littermates; panels A, C:, and E), prohed with either anti mZP3p (panels C and D) or anti-hZP3p (panels A, B, E, and F). o, oocyte; zp, zona pellucida; fc, follicle cell.

Fig. 5.

Immunohistochemistry of ovarian sections from transgenic progeny. Ovarian paraffin sections were prepared and processed for immunohistochemistry using rahhit polyclonal antisera directed against either mZP3 or hZP3. and goat anti-rabbit IgG coupled to horseradish peroxidase, as described in Experimental Procedures. Shown are representative sections from mice harboring the mlhZP3 transgene (panels B, D and F) and control mice (non-transgenic littermates; panels A, C:, and E), prohed with either anti mZP3p (panels C and D) or anti-hZP3p (panels A, B, E, and F). o, oocyte; zp, zona pellucida; fc, follicle cell.

Secretion of hZP3 by transgenic mice

The immunohistochemical analyses described above strongly suggest that hZP3 is secreted by oocytes and incorporated into the zona pellucida. To provide additional evidence, two experimental approaches were employed. First, zonae pellucidae were isolated from ovaries of both transgenic lines, as well as from littermates negative for the m/hZP3 transgene, and were probed on immunoblots using anti-hZP3p and anti-mZP3p. The immunoblots revealed that mice from lines m/h-6 and m/h-11 had hZP3 in their zonae pellucidae, but that control littermates did not (Fig. 6, top panel). Quantitation of immunoblots by densitometry indicated that, for lines m/h-6 and m/h-11, hZP3 represented 36% and 26%, respectively, of total ZP3 present in zonae pellucidae.

Fig. 6.

ZP3 composition of ZP from transgenic mouse oocytes. Top: western gel immunoblot analysis of ZP from transgenic progeny. Purified mZP3 and hZP3, ZP from mice harboring the m/hZP3 transgene, and ZP from control mice (non- transgenic littermates) were subjected to SDS- PAGE, transfered to nitrocellulose membrane, and processed for western immunoblotting using polyclonal antisera directed against mZP3 and hZP3, as described in Experimental Procedures. Lanes A-C, purified hZP3 (50, 150, and 250 ng, respectively). Lanes D and E, 150 ZPs from mice harboring the m/hZP3 transgene (m/h-6 and m/h-11, respectively). Lane F, 150 ZP from control mice. Lanes G-I, purified mZP3 (50, 150, and 250 ng, respectively). Bottom: Immunohistochemistry of ZP from mice harboring the m/hZP3 transgene. Ovarian semi-thin cryosections were prepared and processed for immunohistochemistry using a rabbit polyclonal antiserum directed against hZP3 and goat anti-rabbit IgG coupled to 5 nm colloidal gold, as described in Experimental Procedures. Shown is a representative transmission electron micrograph of a ZP section. Gold particles, indicative of the presence of hZP3, appear as black dots on the ZP filaments. Some gold particles are indicated by arrows. The dark patches seen in the electron micrograph are microvilli that traverse the ZP.

Fig. 6.

ZP3 composition of ZP from transgenic mouse oocytes. Top: western gel immunoblot analysis of ZP from transgenic progeny. Purified mZP3 and hZP3, ZP from mice harboring the m/hZP3 transgene, and ZP from control mice (non- transgenic littermates) were subjected to SDS- PAGE, transfered to nitrocellulose membrane, and processed for western immunoblotting using polyclonal antisera directed against mZP3 and hZP3, as described in Experimental Procedures. Lanes A-C, purified hZP3 (50, 150, and 250 ng, respectively). Lanes D and E, 150 ZPs from mice harboring the m/hZP3 transgene (m/h-6 and m/h-11, respectively). Lane F, 150 ZP from control mice. Lanes G-I, purified mZP3 (50, 150, and 250 ng, respectively). Bottom: Immunohistochemistry of ZP from mice harboring the m/hZP3 transgene. Ovarian semi-thin cryosections were prepared and processed for immunohistochemistry using a rabbit polyclonal antiserum directed against hZP3 and goat anti-rabbit IgG coupled to 5 nm colloidal gold, as described in Experimental Procedures. Shown is a representative transmission electron micrograph of a ZP section. Gold particles, indicative of the presence of hZP3, appear as black dots on the ZP filaments. Some gold particles are indicated by arrows. The dark patches seen in the electron micrograph are microvilli that traverse the ZP.

In a second approach, semi-thin cryosections of ovaries excised from transgenic mice and control littermates were prepared and processed for immunohistology using colloidal gold. As seen in Figure 6 (bottom panel), zonae pellucidae of transgenic mice contained material that was recognized by anti-hZP3p. Zonae pellucidae of control littermates contained only mZP3 (data not shown). Quantitation of gold particles revealed that hZP3 constituted 48% and 14% of total ZP3 contained in zonae pellucidae of transgenic lines m/h-6 and m/h-11, respectively (Table 1). The total amount of ZP3 in zonae pellucidae of transgenic mice and in zonae pellucidae of control littermates was very similar. These observations strongly suggest that hZP3 is secreted by transgenic mouse oocytes and incorporated into the zona pellucida.

Table 1.

Quantification of antibody binding to transgenic mouse oocyte ZP

Quantification of antibody binding to transgenic mouse oocyte ZP
Quantification of antibody binding to transgenic mouse oocyte ZP

Biological activities of hZP3 synthesized by transgenic mice

hZP3 was purified from transgenic mouse oocyte zonae pellucidae and analyzed on western gel immunoblots using anti-hZP3p and anti-mZP3p. This allowed both an assessment of the effectiveness of the purification procedure and quantitation of hZP3 by comparison to known amounts of authentic, purified hamster egg hZP3.

Two experimental approaches were used to determine whether hZP3 purified from transgenic mouse oocyte zonae pellucidae exhibited biological activity. In the first approach, an in vitro “competition assay” was employed (Bleil and Wassarman, 1980a; Florman and Wassarman, 1985) using mouse gametes. Results of previous experiments demonstrated that purified hZP3 was as effective as mZP3 in such an assay (Moller et al., 1990; Kinloch et al., 1991). Capacitated mouse sperm were exposed to culture medium alone or to culture medium containing either purified mouse oocyte mZP3 or hZP3 purified from transgenic mouse oocyte zonae pellucidae. Sperm were then incubated with ovulated eggs and two-cell embryos, and the extent of binding of sperm to eggs was determined. Exposure of sperm to either mZP3 or hZP3 resulted in about 75% inhibition of sperm binding to eggs, as compared with sperm incubated in medium alone (Table 2). These results suggest that hZP3 secreted by transgenic mouse oocytes exhibits sperm receptor activity in vitro.

Table 2.

Sperm receptor and acrosome reaction-inducing activities of transgenic mouse oocyte hZP3

Sperm receptor and acrosome reaction-inducing activities of transgenic mouse oocyte hZP3
Sperm receptor and acrosome reaction-inducing activities of transgenic mouse oocyte hZP3

In a second approach, acrosome reaction-inducing activity of transgenic mouse oocyte hZP3 was assessed. Capacitated mouse sperm were incubated in culture medium alone, or in the presence of ionophore A23187, egg mZP3, or hZP3 purified from transgenic mouse oocyte zonae pellucidae, and were then scored for the presence or absence of an intact acrosome (Bleil et al., 1988). Sperm incubated in the presence of medium alone and in the presence of ionophore provided “background” and “maximal” values, respectively, for the percentage of sperm that acrosome-reacted during a 1 hour incubation (Table 2). Sperm incubated in the presence of either mZP3 or hZP3 underwent the acrosome reaction to similar extents (∼66%). Therefore, hZP3 purified from transgenic mouse oocytes exhibits both sperm receptor and acrosome reaction-inducing activity in vitro.

Following fertilization, sperm receptors are modified such that they are no longer recognized by sperm (Gwatkin, 1977; Bleil and Wassarman, 1980a). Since it was possible that hZP3 present in zonae pellucidae of transgenic mouse eggs might escape this modification, two-cell embryos were isolated from both transgenic and non-transgenic mice (littermates), and were incubated with mouse sperm in vitro. Under conditions in which sperm remained bound to ovulated eggs (29 ± 4 sperm bound/egg), sperm did not bind to two-cell embryos from either transgenic (2 ± 1 sperm bound/embryo) or non-transgenic (2 ± 1 sperm bound/embryo) mice. Therefore, like mZP3, hZP3 present in zonae pellucidae of transgenic mouse eggs is inactivated following fertilization.

Previously, we reported that either 6.5 kb or 0.47 kb of mZP3 gene 5′-flanking sequence can target expression of the firefly luciferase gene exclusively to growing oocytes in transgenic mice (Lira et al., 1990; Schickler et al., 1992). However, reporter mRNA steady-state levels were extremely low in these experiments. In experiments reported here, instead of comparing expression of a cDNA reporter gene with expression of the mZP3 gene, we used a genomic fragment containing all exons and introns of the hZP3 gene (a gene bearing high homology to the mZP3 gene) and a small portion of 3′-flanking sequence (nt +28 to +8,900; Kinloch et al., 1990, 1991) linked to 3.3 kb of mZP3 promoter. Thus, the organization of this transgene (m/hZP3) is very similar to the endogenous mZP3 gene. In this context, it has been shown that genomic constructs, or constructs containing at least the first intron, are expressed far more efficiently than identical constructs lacking introns (Brinster et al., 1988; Palmiter et al., 1991).

m/hZP3 was expressed exclusively in growing mouse oocytes, in agreement with previous results that employed reporter gene constructs (Lira et al., 1990; Schickler et al., 1992). Furthermore, m/hZP3 was expressed at levels that compare favorably with the high levels of endogenous mZP3 expression (Table 3; Roller et al., 1989; Kinloch and Wassarman, 1990). This suggests that all information required for correct mZP3 expression is contained within the proximal 3.3 kb of mZP3 gene 5′-flanking sequence and, perhaps, within one or more mZP3 introns. Based on Southern blot analysis, mouse lines m/h-6 and m/h-11 contain ∼7 and ∼3 copies of the m/hZP3 transgene, respectively, at a single unique locus. Therefore, differences in levels of expression of m/hZP3 and mZP3 by transgenic mouse oocytes probably reflect differences in copy number and/or position effects. For instance, locus control regions (LCRs) are required for position-independent, high level expression of transgenes containing regions of the human β-globin complex (Townes and Behringer, 1990). Alternatively, differences in levels of expression could be due to differences in the 5′-untranslated region. m/hZP3 contains the first 10 nt of the 29 nt, 5′-untranslated region of the mZP3 gene; the other 19 nt are vector sequence. For certain genes, sequences around the promoter region, including the 5′-untranslated region, can influence positioning of nucleosomes (Gross and Garrard, 1988; Felsenfeld, 1992). Thus, subtle changes in these sequences may affect transcriptional complex assembly and transcriptional activity.

Table 3.

Estimates of mZP3 and hZP3 mRNA and glycoprotein levels in transgenic mouse oocytes

Estimates of mZP3 and hZP3 mRNA and glycoprotein levels in transgenic mouse oocytes
Estimates of mZP3 and hZP3 mRNA and glycoprotein levels in transgenic mouse oocytes

The fact that m/hZP3 mRNA is present at high steadystate levels in transgenic mouse oocytes permitted assessment of the fate of these transcripts during ovulation. In this respect, once again, the behavior of m/hZP3 mRNA during mouse development mimics that of mZP3 mRNA (Roller et al., 1989; Kinloch and Wassarman, 1989a). m/hZP3 mRNA accumulates to very high steady-state levels in fully grown oocytes, is degraded during ovulation, and falls to very low levels in unfertilized eggs. mRNA degradation can depend upon recognition of certain internal structural features, such as stem loops, of individual messages (Brawerman, 1989). For some mRNAs (e.g., transferrin receptor, GM-CSF, and c-fos), sequences responsible for instability have been identified in the 3′- untranslated region (Shaw and Kamen, 1986; Fritz et al., 1991; Koeller et al., 1991). In this context, it is unlikely that sequences in the unusually short 3′-untranslated region of mZP3 and hZP3 mRNAs are involved in their degradation during ovulation. The two mRNAs do not share any sequence homology, except for the polyadenylation signal. Perhaps, like c-fos (Kabnick and Housman, 1988), sequences responsible for degradation are located within the highly homologous coding regions of mZP3 and hZP3 mRNAs. Stability of mRNAs can also be influenced by their 3′-poly(A) tract (Bernstein and Ross, 1989; Laird-Offringa et al., 1990; Atwater et al., 1990). It is well documented that certain maternal mRNAs are either adenylated or deadenylated during ovulation in mice, and that the modification profoundly affects their stability (Huarte et al., 1987; Kinloch and Wassarman, 1992).

Comparison of results presented here with those obtained with firefly luciferase (Lira et al., 1990; Schickler et al., 1992) reveals a major difference with respect to protein accumulation during oocyte growth in transgenic mice. Each fully grown oocyte, isolated from mice harboring the firefly luciferase transgene, possessed approximately 2 pg of luciferase. On the other hand, the zona pellucida of each fully grown oocyte, isolated from mice harboring the m/hZP3 transgene, possessed as much as 700 pg of hZP3; a value consistent with the amount of mZP3 in each zona pellucida (Table 3). Since the steady-state level of luciferase mRNA in transgenic mice was not determined, the presence of relatively low amounts of luciferase could be due to low rates of transcription and/or translation, as well as to high rates of mRNA and/or protein degradation. In the case of oocytes from mice carrying m/hZP3, the amount of hZP3 present is consistent with the levels of hZP3 mRNA present (Table 3). This suggests that hZP3 and mZP3 mRNAs are translated at comparable rates in oocytes from transgenic mice and that both proteins are stable. Thus, transgenic mice have the potential to serve as a source of relatively large amounts of foreign sperm receptors.

Some of the most significant observations reported here concern glycosylation of nascent sperm receptor polypeptides and its relationship to biological activity. For example, the results strongly suggest that the glycosylation machinery of mouse oocytes distinguishes between nascent mZP3 and hZP3 polypeptides and processes both properly. As a result, hZP3 secreted by oocytes of transgenic mice resembles hamster egg hZP3 (56× 103Mr), not mouse egg mZP3 (83 × 103Mr), and exhibits biological activity in vitro. This occurs despite the fact that mZP3 and hZP3 polypeptides have very similar primary structures (81% identity; Kinloch et al., 1990). These findings are of interest in view of results of recent experiments with embryonal carcinoma (EC) cell lines stably transfected with either the mZP3 or hZP3 gene (Kinloch et al., 1991). Although cells transfected with mZP3 or hZP3 genes synthesized and secreted glycoproteins that had molecular weights similar to mouse or hamster sperm receptors (EC-mZP3, 83× 103Mr; EC-hZP3, 49× 103Mr, respectively, only the former glycoprotein exhibited biological activity in vitro. Since O-linked oligosaccharides, not polypeptide, account for sperm receptor activity (Florman and Wassarman, 1985; Wassarman, 1989) and, since the EC cells used were derived from mice, it was possible that faulty oligosaccharide processing of glycoproteins from mammals other than mice could account for the functional differences between EC-mZP3 and EC- hZP3. A similar explanation has been used to account for faulty oligosaccharide processing of other glycoproteins in heterologous systems (Kornfeld and Kornfeld, 1985). Results reported here strongly suggest that this may not be the correct interpretation of the EC cell transfection experiments. Mouse oocytes synthesize hZP3 that is active as a sperm receptor and acrosome reaction-inducer.

The mouse zona pellucida consists of long, interconnected filaments (Greve and Wassarman, 1985; Wassarman, 1988; Wassarman and Mortillo, 1991). Each filament is a polymer constructed of dimers, consisting of mZP2 (120× 103Mr) and mZP3, located every 15 nm or so along the filament. The filaments are interconnected by another zona pellucida glycoprotein, mZP1 (200× 103Mr). Therefore, in addition to its other roles, mZP3 serves as a structural component of the zona pellucida as it is assembled during oogenesis. In this context, it is of considerable interest that a foreign sperm receptor glycoprotein, hZP3, can interact properly with mZP2 and be incorporated into a mouse zona pellucida. Electron micrographs revealed that hZP3 is an integral part of the zona pellucida and is not simply trapped within the filamentous network. Furthermore, since the amount of hZP3 present in zona pellucida simply reflects the amount synthesized by oocytes of transgenic mice, apparently hZP3 combines with mZP2 to form dimers as readily as does the native glycoprotein, mZP3. These results suggest that the protein and/or carbohydrate determinants on mZP3 that support its interactions with mZP2, are conserved on hZP3. Clearly, the difference in extent of glycosylation of mZP3 and hZP3 polypeptides, does not significantly affect the ability of hZP3 to combine with mZP2 to form zona pellucida filaments. Since the majority of mZP3 oligosaccharides are located on the carboxy-terminal half of the polypeptide (T.K. Rosiere and P.M. Wassarman, unpublished results), it is tempting to speculate that the amino-terminal half of mZP3 (or hZP3) polypeptide interacts with mZP2 to form dimers.

Following fertilization of mouse eggs, mZP3 is inactivated as sperm receptor and acrosome reaction-inducer by enzymes released from egg cortical granules during the cortical reaction (Bleil and Wassarman, 1980a, 1983; Wassarman, 1987a,b, 1990; Ducibella, 1991). There is evidence to suggest that such enzymes modify those mZP3 O-linked oligosaccharides that mediate sperm binding to eggs (J. Bleil and P. Wassarman, unpublished results). Results described here indicate that hZP3 present in mouse egg zonae pellucidae is also inactivated following fertilization. This suggests that mouse egg cortical granule enzymes, which recognize and modify mZP3 O-linked oligosaccharides, also recognize and modify hZP3 O-linked oligosaccharides. This is consistent with our previous finding that the mouse cortical granule protease that specifically cuts mZP2 following fertilization, also cuts hZP2 (Moller et al., 1990).

By combining the experimental approach described here with site-directed mutagenesis of sperm receptor genes, it should be possible now to identify regions of sperm receptors essential for both zona pellucida assembly and biological activity. Furthermore, this experimental approach could be used to produce eggs with mosaic zona pellucida containing sperm receptors of other mammals. In addition to providing a source for larger amounts of certain sperm receptors (e.g., human sperm receptor) than are currently available, such eggs could provide valuable information about the molecular basis of species-specificity during mammalian fertilization.

We are grateful to Dr Jeff Mann for advice and assistance in producing transgenic mice and Ms. Alice O’Connor for assistance in manuscript preparation. We thank our colleagues for critically reviewing the manuscript.

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