Conserved Furin Cleavage Site Not Essential for Secretion and Integration of ZP3 into the Extracellular Egg Coat of Transgenic Mice

Immunocytochemical analysis of zona-free oocytes and eggs.Growing oocytes and eggs were isolated from NIH Swiss mice, and after their zonae were removed by exposure to acidified Tyrode's medium (12), they were processed for imaging by Nomarski optics and confocal microscopy (30) with monoclonal antibodies specific to mouse ZP1 (33), ZP2 (8), and ZP3 (9) as primary antibodies and rhodamine-lissamine-conjugated goat anti-rat immunoglobulin G (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) as a secondary antibody.

EGFP-mouse ZP3 (MoZP3) expression vectors.The 5′ region (bp 1 to 149) of pZP3.5 (34), including the N-terminal signal peptide sequence (amino acids 1 to 40), was amplified by PCR with two primers, each containing either an NheI or an AgeI recognition site, respectively, i.e., 5′-GGCTAGCTGAGCCCAGCTGTACTCCA-3′ and 5′-AACCGGTGATGAGGACCCCACTGGGG-3′. The resultant PCR product was cloned into the NheI-AgeI site of pEGFP-C2 (Clontech Laboratories, Palo Alto, Calif.) upstream and in frame with enhanced green fluorescent protein (EGFP). The remainder of ZP3 was assembled from a second PCR product (bp 93 to 435) by using primers 5′-TCAGATCTCCCAGACTCTGTGGCTT-3′ and 5′-CAATGGGTACCTCCACAC-3′, each with a BglII or a KpnI recognition site, respectively. This PCR fragment was cloned into the BglII and KpnI sites downstream of and in frame with EGFP. The rest of ZP3 (bp 179 to 1189) was isolated from pZP3.5 after digestion with SpeI and SacII and cloned into the corresponding sites in ZP3 and the multicloning site of pEGFP-C2. The resultant plasmid was designated pSEGFP-MoZP3.

PCR mutagenesis was used to change the furin recognition site (amino acids 350 to 353) in mouse pSEGFP-MoZP3 from Arg-Asn-Arg-Arg-COOH to Ala-Asn-Ala-Ala-COOH. With pZP3.5 as a template, fragments from PCR amplifications with either primer set 5′-CCAAGCTAGTTTCTGCCAACGCAGCTCACGTGACCG-3′ and 5′-TAATACGACTCACTATAGGG-3′ or primer set 5′-TACATCACCTGCCATCTCAA-3′ and 5′-CGGTCACGTGAGCTGCGTTGGCAGAAACTAGCTTGG-3′ were isolated by agarose gel electrophoresis and reamplified without additional primers or templates. The SapI-ApaI fragment of the resultant PCR product (bp 1031 to 1123) containing the mutated ZP3 furin site was substituted for the corresponding region of pSEGFP-MoZP3, and the result was designated pSEGFP-FurX. Taq DNA polymerase was used for amplification, and all PCRs were performed for 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s with a Perkin-Elmer GeneAmp PCR System (Perkin-Elmer, Norwalk, Conn.). The sequences of PCR fragments were confirmed by dideoxy sequencing (36).

Transient expression of pSEGFP-MoZP3 and pSEGFP-FurX.Transient-transfection assays were performed with Lipofectamine (Life Technologies, Rockville, Md.) in accordance with the manufacturer's protocol. On the day before transfection, 1.5 × 10⁵ mouse 10T[1/2] embryonic fibroblast cells in 2 ml of Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) were seeded in each well of six-well (35-mm-diameter) tissue culture plates. The cells were incubated overnight until they were 50 to 80% confluent. For each transfection, 6 μl of PLUS reagents was added to 100 μl of DMEM (without FBS) predissolved with 1 μg of either pSEGFP-MoZP3 or pSEGFP-FurX and incubated at room temperature (RT) for 15 min. Lipofectamine (4 μl in 100 μl of DMEM) was then added and mixed. After 15 min of incubation at RT, the mixture was diluted with 0.8 ml of DMEM and overlaid on the growing cells. After 3 h at 37°C, the medium was replaced and incubated for an additional 48 h with one refeeding prior to harvest. Alternatively, 1 day after transfection, the cells were changed to the same medium containing 0.2 μg of brefeldin A (BFA) per ml and cultured for an additional 12 h. The cells were then washed with phosphate-buffered saline (PBS) and changed to DMEM with 10% FBS (lacking BFA). After 30 min in culture, the cells were fixed with 2% paraformaldehyde for 40 min at RT.

The fixed 10T[1/2] cells were blocked and permeabilized with blocking solution (PBS supplemented with 1.5 mg of glycine per ml, 1 mg of saponin [S-4521; Sigma, St. Louis, Mo.] per ml, 10% donkey serum [Jackson ImmunoResearch Laboratories]) at RT for 1 h. Rabbit anti-bovine protein disulfide isomerase (PDI; 1:200; StressGen Biotechnologies, Victoria, British Columbia, Canada) or rabbit anti-rat α-mannosidase II (1:100; gift of Kelley Moremen [University of Geogia] and Marilyn G. Farquhar [University of California, San Diego]) diluted with blocking solution was added, and the mixture was incubated at RT for 1 h. After being washed with the blocking solution, the cells were further incubated with Cy5-labeled donkey anti-rabbit antibodies (1:500; Jackson ImmunoResearch Laboratories) at RT for 1 h. Following additional washes with the blocking solution and then PBS, the slides were plated with mounting medium (ProLong Antifade Kit; Molecular Probes, Eugene, Oreg.) and examined on a 510 LSM confocal microscope (Carl Zeiss, Thornwood, N.Y.). EGFP was excited at 488 nm with an argon laser light, and fluorescence images were collected at 515 to 530 nm. Cy5 was excited with a HeNe laser light at 633 nm, and fluorescence images were collected at ≥670 nm.

Western blot analyses.Cell supernatants from transient transfections were concentrated by Microcon 10 (Millipore, Bedford, Mass.), and cell pellets were washed twice with PBS and lysed with 50 mM Tris-HCl (pH 7.5)-1% NP-40-10 mM EDTA containing 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (1 mM), aprotinin (5 μg/ml), leupeptin (1 μg/ml), benzamimidine hydrochloride (0.1 mM), and pepstatin (1 μg/ml). Protein samples (pellets or supernatants from 10⁴ cells) were treated with or without endoglycosidase H (endo-H) or peptide-N-glycosidase F (PNGase F), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and assayed by Western blotting (4). For endo-H or PNGase F treatment, samples were denatured at 100°C for 10 min and then incubated with 100 U of enzyme per sample at 37°C for 1 h in accordance with the manufacturer's (New England Biolabs, Beverly, Mass.) instructions.

Expression of EGFP-MoZP3 and EGFP-FurX in microinjected oocytes.Oocytes isolated from 11- to 13-day-old mouse ovaries (23) were incubated in M199 medium supplemented with 0.28 mM sodium pyruvate, 25 mM HEPES (pH 7.4), and 2 mg of BSA per ml at 37°C prior to microinjection. About 10 pl of solution, containing 50 ng of plasmid DNA per ml, was injected into the nucleus of each oocyte. The injected oocytes were cultured (37°C, 5% CO₂) for 24 h in the same supplemented M199 medium. The oocytes were then stained with 1 μg of the lipophilic dye PM-R18 (octadecyl rhodamine B Cl⁻; Molecular Probes) per ml for 20 min and separated into two groups. One group was fixed with 2% paraformaldehyde for 1 h at RT. The other group was transferred into 20 mM Tris-HCl, pH 7.4, containing 1% NP-40 and 0.5 M NaCl and freeze-thawed 10 times on ethanol-dry ice to isolate zona ghosts (37). The fixed oocytes and treated zona ghosts were washed three times with PBS and put on a slide chambered with Gene-Frame (20-μl cavity volume; Advanced Biotechnologies, Leatherhead, United Kingdom). Images were obtained by confocal microscopy with an argon laser light (488 nm) to visualize EGFP at 515 to 530 nm and a HeNe 543 laser light to detect PM-R18 at 570 nm.

EGFP-MoZP3 and EGFP-FurX transgenic mice. NheI-EcoRV fragments isolated from pSEGFP-MoZP3 and pSEGFP-FurX were inserted into the SpeI-EcoRV sites of a plasmid containing 6 kbp of the mouse Zp3 promoter, which was previously shown to direct oocyte-specific expression in transgenic mice (33). A bovine growth hormone polyadenylation signal was cloned into EcoRV-NotI sites downstream of the ZP3-EGFP sequences, and the 8.1-kbp Zp3-MoZP3-EGFP fragment was purified by agarose electrophoresis after digestion with I-SceI meganuclease and NotI. Following pronuclear injection, founders were identified and transmission of the transgene in their progeny was followed by PCR and Southern analyses (33). The primers used for the PCR were specific to EGFP (5′-GTCGCCACCATGGTGAGCAA-3′ and 5′-ACTTGTACAGCTCGTCCATG-3′), and the conditions are described above. The presence of the normal and mutant furin sites was confirmed by sequencing of PCR products of the transgenes with genomic tail DNA as a template, flanking primers (5′-GTCGCCACCATGGTGAGCAA-3′ at the 5′ end of EGFP and 5′-CCTCGAGTTTCTTCTTTTATTGCGG-3′ at the 3′ end of ZP3), and the above-described PCR conditions. The nucleic acid sequence was obtained with ABI PRISM Fluorescent DNA Analysis Kits and an ABI Prism 310 Genetic Analyzer in accordance with the manufacturer's instructions. All experiments with mice were conducted under protocols approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases-National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee.

Detection of zona proteins at the plasma membrane.To confirm biochemically the presence of the zona proteins at the growing-oocyte surface, zonae pellucidae were removed from growing oocytes or eggs by brief exposure to acidified Tyrode's medium (Fig. 1A and B). After being washed, the germ cells were incubated with monoclonal antibodies to either ZP1, ZP2, or ZP3. With a rhodamine secondary antibody and confocal microscopy, each zona protein was detected on the growing-oocyte surface (Fig. 1C, E, and G). All of the zona proteins were also detected within growing oocytes, where they are actively synthesized, but were not present (or were present in very diminished amounts) in ovulated eggs, in which zona protein synthesis has stopped (Fig. 1D, F, and H).

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FIG. 1.

Immunocytochemical analysis of oocytes and eggs. Nomarski images of a growing oocyte from a 3-week-old NIH Swiss mouse before (A) and after (B) treatment with acidified Tyrode's medium to remove the zona pellucida. Confocal microscopy of zona-free oocytes (C, E, and G) and eggs (D, F, and H) stained with antibodies specific to ZP1 (C and D), ZP2 (E and F), and ZP3 (G and H) after permeabilization with saponin. Each zona protein was present within and at the cell surface of growing oocytes (C, E, and G) but not of ovulated eggs (D, F, and H). Bar, 25 μm. MAb, monoclonal antibody.

ZP1, ZP2, and ZP3 each have a signal peptide that directs them into a secretory pathway, a 260-amino-acid zona box of unknown function, and a transmembrane domain near the carboxyl terminus. At 31 to 47 amino acids upstream of the transmembrane domain are short regions with basic residues implicated as the furin cleavage site involved in zona protein secretion (Fig. 2A). The 424-amino-acid ZP3 protein was selected as a model with which to determine if the furin recognition site is required for zona protein incorporation into the egg coat. The identification of a monoclonal antibody binding site (22) and its ability to bind to the extracellular zona pellucida (9) suggested that cleavage occurs downstream of the antibody binding site (amino acids 336 to 342) and presumably upstream of the transmembrane domain (amino acids 385 to 410), in a region that includes the potential furin cleavage site (amino acids 350 to 353).

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FIG. 2.

Conserved zona pellucida protein motifs and construct design. (A) Each of the three mouse zona pellucida proteins, ZP1, ZP2, and ZP3, has a signal peptide that directs it into a secretory pathway, a signature zona box, and a transmembrane domain near the carboxyl terminus, upstream of which (30 to 47 residues) is a potential furin cleavage site. A monoclonal antibody binds a linear epitope on ZP3 (amino acids [aa] 336 to 342). (B) cDNA encoding ZP3 with a normal (RNRR) or a mutant (ANAA) furin site was cloned in frame with EGFP into expression plasmids under the control of a cytomegalovirus immediate-early (CMV IE) promoter. Constructs were transfected into 10T[1/2] cells or injected into the nuclei of growing mouse oocytes for expression studies. (C) The normal and furin site mutant EGFP-ZP3 cDNAs were inserted downstream of ∼6.0 kbp of the mouse ZP3 promoter and just upstream of a bovine growth hormone (BGH) poly(A) signal. After isolation, the DNA fragment was injected into the nuclei of one-cell zygotes to establish transgenic mouse lines expressing normal and furin site mutant EGFP-ZP3 proteins.

Expression of EGFP-ZP3 fusion proteins in 10T[1/2] cells.ZP3 cDNA was cloned into the expression vector pEGFP-C2, which encodes EGFP and is well suited for analysis of intracellular trafficking by confocal microscopy. The 5′ region of the ZP3 cDNA that included the N-terminal signal sequence (amino acids 1 to 22) was cloned upstream, and the reminder of the ZP3 cDNA was cloned downstream of EGFP to establish an EGFP-ZP3 fusion protein driven by the cytomegalovirus promoter (Fig. 2B). PCR mutagenesis was used to change the potential furin recognition site, RNRR, to ANAA, and the SapI-ApaI fragment from a correctly mutated clone was substituted for the same region of EGFP-MoZP3. The fidelity of the normal (pSEGFP-MoZP3) and mutant (pSEGFP-FurX) constructs was verified by DNA sequencing.

Because of variable levels of expression in clonal cell lines after stable transfection, transient-expression assays with populations of mouse 10T[1/2] embryonic fibroblasts were used to examine the expression, processing, and secretion of the normal (ZP3) and mutant (FurX) proteins. Both constructs were expressed in 10T[1/2] embryonic fibroblast cells, and EGFP could be detected by confocal microscopy (data not shown). BFA dissociates Golgi structures, mixes them with the endoplasmic reticulum, and blocks protein translocation to the trans-Golgi. To investigate the processing of the EGFP-fused proteins, transfected 10T[1/2] cells were cultured in the presence of 0.2 μg of BFA per ml for 12 h, at which time the Golgi structures were disrupted and the synthesized EGFP fusion proteins were distributed in endoplasmic reticulum networks (data not shown). The cells were then incubated in the absence of BFA. Enhancement of the EGFP-ZP3 signal occurs concomitantly with reassembly of the Golgi structure (near the nucleus, often in contact with the nuclear membrane). After 30 min, the EGFP signals were localized in the Golgi region (Fig. 3) and no significant differences in synthesis and translocation were observed between the normal ZP3 (Fig. 3B and H) and FurX (Fig. 3E and K) proteins.

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FIG. 3.

Confocal microscopy of EGFP-ZP3 in 10T[1/2] cells. Cells transfected with plasmid expression vectors were incubated with BFA overnight, allowed to recover for 30 min, and fixed for imaging. Cells expressing normal EGFP-ZP3 (A to C and G to I) or EGFP-ZP3 mutated at the furin site, FurX (D to F and J to L), were incubated with antibodies to PDI (A and D) or α-mannosidase II (ManII) (G and J) and imaged to detect antibody binding with a Cy-5-conjugated secondary antibody (A, D, G, and J), EGFP-ZP3 (B, E, H, and K), or both (C, F, I, and L). Bar, 20 μm.

To identify whether the EGFP-fused proteins correctly traffic from the endoplasmic reticulum to the Golgi, two antibodies, one to an endoplasmic reticulum residential enzyme, PDI, and the other to a Golgi marker, α-mannosidase II, were used to colocalize the expressed EGFP-ZP3 fused proteins. Although some EGFP-ZP3 signal was observed in the endoplasmic reticulum at 30 min after release from the BFA block, most had progressed to the Golgi (Fig. 3B, E, H, and K). In some cells, the EGFP signal in the endoplasmic reticulum seemed more pronounced with the furin mutant ZP3 (Fig. 3B and E) but this observation was inconsistent (Fig. 3H and K). More consistent was the colocalization of the EGFP signal with antibodies specific to α-mannosidase II in the Golgi (Fig. 3I and L) and the absence of colocalization with antibodies to PDI in the endoplasmic reticulum (Fig. 3C and F). This progression to the Golgi occurred in the presence (Fig. 3J through L) or absence (Fig. 3G through I) of the mutated furin site. These observations suggested that processing of the recombinant ZP3 protein in the Golgi apparatus did not require the integrity of the furin site, although differences in the efficiency of transport of normal and mutant ZP3 in heterologous cells would not be detected in these studies.

Secretion of EGFP-MoZP3 fusion proteins from 10T[1/2] cells.The transiently transfected 10T[1/2] cells and the culture supernatants were analyzed by Western blotting for expression and secretion of the EGFP-ZP3 proteins. Because bovine serum in the culture medium distorted the electrophoretic separation, the transfected cells were cultured in the absence of serum. NP-40 extracts of the cell pellets were separated by SDS-PAGE and probed with a monoclonal antibody specific to ZP3. Both normal ZP3 and FurX proteins were detected (Fig. 4A) as major bands migrating with an apparent molecular mass of 75 kDa. After treatment with endo-H, the bands shifted to 70 kDa, which is consistent with the removal of three or four N-linked, high-mannose oligosaccharides normally attached to the core protein during transit through the endoplasmic reticulum (35). The remaining 70 kDa is accounted for by EGFP (27 kDa) and mouse ZP3 polypeptide lacking the signal peptide (44 kDa). The faint higher-molecular-weight bands in Fig. 4A may represent further posttranslational modification in the Golgi (∼100 kDa) or aggregation (>200 kDa).

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FIG. 4.

Western blot assay of EGFP-ZP3. Three days after transfection with plasmid vectors encoding either EGFP-ZP3 (normal) or EGFP-ZP3 mutated at the furin site (FurX), 10T[1/2] cell pellets (A) and supernatants (B) were harvested. Pellets were lysed with detergent and incubated with (+) or without (−) endo-H, which cleaves high-mannose, but not complex, N-linked oligosaccharide side chains. Cell supernatants were incubated with (+) or without (−) PNGase F, which cleaves high-mannose, hybrid, and complex N-linked oligosaccharides. Proteins in the pellets and supernatants were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with a monoclonal antibody specific to mouse ZP3.

Supernatants (0.6 ml) from transfected cells were collected and concentrated prior to Western blot analysis. Both normal ZP3 and FurX proteins were secreted with apparent molecular masses of ∼105 kDa and were indistinguishable from one another (Fig. 4B). The mature form of native mouse ZP3 has a polypeptide chain of ∼37 kDa and migrates at ∼83 kDa on SDS-PAGE after posttranslational modifications. The addition of EGFP (27 kDa) would account for the observed molecular masses of the secreted recombinant proteins. Normally, high-mannose, N-linked oligosaccharides attached in the endoplasmic reticulum are further matured into complex side chains as a glycoprotein passes through the Golgi apparatus. Consistent with such modifications, neither normal ZP3 nor FurX secreted protein was appreciably affected by treatment with endo-H, which does not cleave complex oligosaccharides (data not shown). However, after treatment with PNGase F, which cleaves complex mannose N-linked oligosaccharides, the molecular mass of both normal ZP3 and FurX decreased to ∼80 kDa (consistent with the removal of three or four N-linked sugar side chains). Thus, mutation of the furin site did not appear to affect the intracellular trafficking, the posttranslational modification, or the secretion of EGFP-ZP3 protein from 10T[1/2] cells. Similar results were obtained when the blots were probed with antibodies to EGFP (data not shown).

Expression of EGFP-ZP3 in growing oocytes and incorporation into the zona pellucida.To confirm these unexpected results with cells that normally express zona proteins, pSEGFP-MoZP3 and pSEGFP-FurX were microinjected into the nuclei of growing oocytes isolated from 11- to 13-day-old mice (Fig. 5). Signals obtained by confocal microscopy were optimized by injecting ∼0.5 fg of plasmid/oocyte and incubating the oocytes for 20 to 40 h. Under the experimental conditions described, the lipophilic dye PM-R18 primarily stained the plasma membrane of the oocytes although weak staining of the microvilli between oocytes and cumulus cells was observed in some instances (Fig. 5A and D).

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FIG. 5.

Localization of EGFP-ZP3 within the growing-oocyte zona pellucida. Plasmid vectors expressing either normal EGFP-ZP3 or EGFP-ZP3 mutated at the furin site (FurX) were injected into the nuclei of growing oocytes, which were cultured for 20 to 40 h. Oocytes were incubated with a lipid membrane stain (PM-R18) before (A and D) or after (G and J) freeze-thawing in the presence of 0.5 M NaCl and 1% NP-40. PM-18 (A, D, G, and J) and EGFP-ZP3 (B, E, H, and K) were viewed individually and together (C, F, I, and L) after superimposition on a light microscopic image. Bar, 20 μm.

Both constructs, pSEGFP-MoZP3 and pSEGFP-FurX, were successfully transcribed by the oocytes, and recombinant fusion proteins were directed into secretory pathways prior to detection at the periphery (Fig. 5B and E). Because there is close contact between the zona pellucida and the plasma membrane in growing oocytes, it was difficult to determine if the colocalization at the periphery was limited to the membrane or also reflected incorporation into the extracellular zona pellucida (Fig. 5C and F). Therefore, zona ghosts were prepared by freeze-thawing in the presence of detergent (1% NP-40) and salt (0.5 M NaCl) to remove the plasma membrane. Solubilization of the plasma membrane was confirmed by absent staining with PM-R18 (Fig. 5G and J), and the persistence of an EGFP signal at the periphery (Fig. 5H, I, K, and L) indicated incorporation of ZP3 into the inner aspect of the zona pellucida matrix. No significant difference was observed between normal ZP3 and ZP3 with the mutated furin site.

EGFP-MoZP3 is incorporated into the zona pellucida of transgenic mice.These in vitro tissue culture studies were extended in transgenic mice. An 8.0-kbp fragment containing the 6.0-kbp oocyte-specific mouse ZP3 promoter (33) upstream of EGFP-ZP3 with the normal or the mutated furin site, followed by the bovine growth hormone poly(A) signal, was assembled (Fig. 2C). After its injection into the male pronuclei of one-cell zygotes, mouse lines with normal EGFP-ZP3 or with EGFP-FurX were established and they passed the transgene through their germ line. Mice expressing either EGFP-ZP3 or EGFP-FurX protein had normal-appearing zonae pellucidae (Fig. 6H, K, M, and Q). Hemizygous EGFP-ZP3 and EGFP-FurX females were fertile when mated with normal males and produced litters (11.4 ± 0.8 and 10.6 ± 0.7 [standard error of the mean] pups, respectively), the sizes of which were not statistically significantly different (unpaired t test, P > 0.05) from those of the litters of normal mice (8.8 ± 0.6 [standard error of the mean] pups) or from one another. Thus, the presence of EGFP (27 kDa) at the N terminus of mouse ZP3 did not adversely affect formation of the zona pellucida matrix or fecundity.

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FIG. 6.

Transgenic mice incorporate EGFP-ZP3 into the zona pellucida. Mouse lines with transgenes expressing either EGFP-ZP3 or EGFP-ZP3 mutated at the furin site were established. The morphology of growing oocytes (H and N) and ovulated eggs (K and Q) from normal (H and K) and furin mutant (N and Q) EGFP-ZP3 transgenic mice was indistinguishable from that of oocytes (B) and eggs (E) isolated from nontransgenic mice. EGFP signals were observed throughout the width of the zona pellucida surrounding oocytes (G, I, M, and O) and eggs (J, L, P, and R) from normal (G to L) and furin mutant (M to R) EGFP-ZP3 transgenic mice. No signal was observed in oocytes (A to C) and eggs (D to F) isolated from nontransgenic mice. Bar, 20 μm.

Oocytes and eggs were collected from transgenic animals, fixed in 2% paraformaldehyde, and examined by confocal microscopy. The EGFP signal was not observed in nontransgenic oocytes and eggs (Fig. 6A through F) but was readily detected in the zona pellucida surrounding growing oocytes (Fig. 6G, I, M, and O) and ovulated eggs (Fig. 6J, L, P, and R) isolated from transgenic mice expressing either EGFP-ZP3 or EGFP-FurX. As anticipated, no EGFP expression was observed in surrounding somatic cells (Fig. 6I and O). The EGFP-ZP3 fusion proteins were present throughout the width of the zona matrix in mice expressing either the normal or the mutant form of FurX. The mutation of the furin site in the genome of the transgenic mice was confirmed by nucleic acid sequencing of PCR products specific to the transgene with tail DNA as the template (Fig. 7), and the phenotype was the same in three independently derived ZP3-FurX mutant lines. Thus, the integrity of the furin site does not appear to be required for ZP3 synthesis, intracellular trafficking, secretion, or incorporation into the extracellular zona pellucida matrix of transgenic mice.

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FIG. 7.

The furin site in transgenic mice. A schematic representation of mouse ZP3 (424 amino acids) with the N-terminal signal peptide, the monoclonal antibody (MAb) binding site, and the transmembrane domain is at the top. The nucleic acid sequence of a PCR product obtained with primers that flank the potential furin cleavage site in the transgene confirmed the mutation (RNRR → ANAA) in genomic tail DNA isolated from EGFP-FurX transgenic mice. The nucleic acid sequence of FurX is color coded (A, green; C, blue; G, black; T, red) and shown directly above the traces. The single-letter amino acid sequences (amino acids 336 to 360) of normal ZP3 and FurX are at the top, and dashes indicate identical residues.