Development 121, 1947-1956 (1995) 1947 Printed in Great Britain © The Company of Biologists Limited 1995 Coordinate expression of the three zona pellucida genes during mouse oogenesis Olga Epifano1,*, Li-fang Liang1, Mary Familari1, Malcolm C. Moos, Jr2 and Jurrien Dean1 1Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA 2Laboratory of Developmental Biology, Center for Biologics Evaluation and Research, Bethesda, MD 20892, USA *Author for correspondence SUMMARY The mammalian zona pellucida is an extracellular matrix restricted to the oocyte. ZP2 transcripts, but not ZP1 or that surrounds growing oocytes, ovulated eggs and early ZP3, are detected in resting (15 µm diameter) oocytes, and embryos. The mouse zona is composed of three sulfated all three zona transcripts coordinately accumulate as glycoproteins: ZP1, ZP2 and ZP3. Each is critically oocytes begin to grow. Together they represent approxi- involved in fertilization, the postfertilization block to mately 1.5% of the total poly(A)+ RNA in 50-60 µm oocytes. polyspermy and protection of the preimplantation embryo. In the latter stages of oogenesis, their abundance declines We have previously isolated cDNAs encoding mouse ZP2 and each zona transcript is present in ovulated eggs at less and ZP3 and now report the isolation of a full-length cDNA than 5% of its maximal level. No zona transcripts were encoding ZP1. Mouse ZP1 is composed of a 623 amino acid detected above background signal in granulosa cells. We polypeptide chain with a signal peptide and a carboxyl conclude that, in mice, the three zona pellucida genes are terminal transmembrane domain, typical of all zona expressed in a coordinate, oocyte-specific manner during proteins. Sequence comparison demonstrate that mouse the growth phase of oogenesis. Our data support the ZP1 is an orthologue of a rabbit zona protein, R55. The hypothesis that the transcription of the zona genes is con- expression of R55 has been reported previously in both trolled, in part, by shared regulatory element(s). oocytes and granulosa cells. However, by northern analysis and in situ hybridization with 33P-labelled antisense probes to each of the three mouse zona mRNAs, we have deter- Key words: zona pellucida, ZP1, ZP2, ZP3, oocyte-specific gene mined that the expression of each mouse zona gene is expression, in situ hybridization, RNase protection assay, mouse INTRODUCTION secreted, native ZP2 and ZP3 proteins are heterogeneous with 3 average Mr of 120-140 and 83×10 , respectively. No primary The zona pellucida surrounds growing oocytes, ovulated eggs structural information is available for ZP1 and little is known and preimplantation embryos in mammals. It plays a critical about the protein. After staining or metabolic labeling, it role in the species-specificity of fertilization, the postfertiliza- appears as the least abundant of the three mouse zona glyco- tion block to polyspermy and protects the early embryo as it proteins on SDS-PAGE, where it has an apparent Mr of 185- passes down the oviduct (Yanagimachi, 1994). While primor- 200×103. Under reducing conditions, ZP1 co-migrates with 3 dial oocytes do not have a zona pellucida, the zona glycopro- ZP2 at 120×10 Mr suggesting that it is present in the zona as teins represent a major secretory product of growing oocytes. a disulfide-bond-linked dimer (Bleil and Wassarman, 1980b; The zona matrix first appears as amorphous material deposited Shimizu et al., 1983). in the space between the oocyte and the surrounding granulosa A current model proposes specific biological functions for cells. This material is subsequently assembled into long each mouse zona protein (Greve and Wassarman, 1985). Con- filaments forming a highly porous matrix that increases in siderable in vitro data suggest that mouse sperm initially bind thickness to 7 µm in fully grown mouse oocytes (Phillips and to O-linked oligosaccharides attached to ZP3 (Florman and Shalgi, 1980). Wassarman, 1985; Kinloch et al., 1995), although the identifi- The mouse zona is composed of three sulfated glycoproteins cation of the corresponding sperm receptor remains controver- designated ZP1, ZP2 and ZP3 (Bleil and Wassarman, 1980b; sial (Youakim et al., 1994; Leyton et al., 1992; Cheng et al., Shimizu et al., 1983). The primary structures of ZP2 and ZP3 1994). The primary interaction with ZP3 triggers the sperm have been deduced from full-length cDNAs that encode acrosome reaction (Saling, 1991), releasing lytic enzymes polypeptides of 80,217 and 46,307 Mr, respectively (Ringuette (considered important for sperm penetration of the zona et al., 1988; Liang et al., 1990). However, due to post-transla- matrix) and exposing additional macromolecules, which tional modifications, including glycosylation, the sizes of the appear to be involved in secondary binding of sperm to ZP2 1948 O. Epifano and others (Bleil et al., 1988). ZP1 has been proposed as a crosslinker of (NIH Swiss) after homogenization and centrifugation through a filaments composed of ZP2/ZP3 dimers. Selective proteolytic Percoll gradient (Bleil et al., 1988). Following solubilization (60°C, degradation of ZP1 or reduction of disulfide bonds results in 1 hour in 200 µl 50 mM Tris, pH 8), the zona proteins were separated disruption of interconnections between zona filaments in the by preparative non-reducing SDS-PAGE (7%). Gel purified ZP1 µ mouse (Greve and Wassarman, 1985). Thus, ZP1 appears to (approximately 50 g) was used to immunize a 6-week-old male rat provide structural integrity to the mouse zona matrix to (Sprague Dawley) by intraperitoneal injection of a 1:1 suspension of acrylamide:Freund’s complete adjuvant (Difco) containing 20 µg of maintain its biologic activity. mouse ZP1 protein followed by 2 further injections (15 µg in Freund’s Additional observations suggest that the role of ZP1 may not incomplete adjuvant) at 20-day intervals. The specificity of the be the same in all species. Zonae pellucidae have been bio- resultant antisera was confirmed by probing a western blot (Burnette, chemically characterized in other mammals, including rabbit 1981) of mouse zona pellucida proteins separated by SDS-PAGE. (Dunbar et al., 1981), pig (Hedrick and Wardrip, 1987) and human (Shabanowitz and O’Rand, 1988). Although all zonae Protein sequencing are composed of three glycoproteins, variations in nomencla- ZP1 protein from 500 mice was purified by SDS-PAGE (Moos et al., ture have confused the correspondence of particular zona 1988) using a 6% Long Ranger (AT Biochem) gel and 4 M urea in proteins among mammals. The recent cloning of zona genes in the sample buffer. After transfer to Immobilon-P PVDF membrane different species has improved our understanding of the struc- (Matsudaira, 1987), the N-terminal sequence of ZP1 was determined on an Applied Biosystems Sequencer (Model 477A with 120A PTH tural homology of zona proteins among mammals. However, amino acid analyzer). there remain discrepant data on the biologic function of par- To obtain internal sequence, zona proteins were separated by ticular zona proteins. For example, although no in vitro sperm SDS-PAGE, stained with 0.01% 3, 3′-dipentyloxacarbocyanine iodide binding activity was detected with mouse ZP1 (Bleil and (Molecular Probes) in 2% MeOH, 0.2% SDS, 50 mM NaHCO3 Wassarman, 1980a), the porcine homologue to mouse ZP1 has (20°C, 20 minutes) and destained in water (M. Moos, details to be been reported to have sperm-binding activity (Sacco et al., published elsewhere). The ZP1 band was excised (302 nm transillu- × 1989; Yurewicz et al., 1991). Whether these observations mination), washed (2 1 ml H2O, 15 minutes) and dried in vacuo to represent biological differences among mammals or differ- 20% of its original volume. After rehydration (1 ml, 50 mM Tris-HCl ences in experimental design remains to be determined. pH 8.3, 2 mM CaCl2, 4 M urea), ZP1 was incubated (20°C, 16 hours) with 1 g of sequencing grade modified trypsin (Promega). Zona gene expression provides a potential paradigm for Tryptic peptides were extracted sequentially with 0.5 ml 4 M urea, studying mechanisms of oocyte-specific gene expression and a 1 ml 1% trifluoroacetic acid (TFA), and 1 ml 0.1% TFA, 80% aceto- marker of oocyte growth and differentiation. Previously, we nitrile for 1 hour each with vigorous agitation at 20°C. The extracts reported the characterization of mouse Zp2 and Zp3 genes were combined, filtered (0.22 µm) and injected onto a 2.1×250 mm (Liang and Dean, 1993; Chamberlin and Dean, 1990) and Vydac 214TP52 C4 column equilibrated at 60°C with 0.1% TFA, 5 showed that both transcripts were detected only in oocytes % acetonitrile (Solvent A) at a flow rate of 150 L/minutes in a (Liang et al., 1990; Ringuette et al., 1988). In additional exper- Hewlett-Packard model 1090 chromatograph. After a 15 minute iments, in situ hybridization of ovarian sections with a ZP3 isocratic hold, peptides were eluted by increasing the concentration antisense probe confirmed the presence of zona transcripts in of Solvent B (0.085% TFA in 80% acetonitrile) to 35% over 60 oocytes and did not detect transcripts in granulosa cells minutes, from 35% to 75% over 30 minutes, and from 75% to 100% over 15 minutes (Stone and Williams, 1986). Peptides detected by (Philpott et al., 1987). These data led to the hypothesis that the absorption at 215 nm were collected by hand and analyzed as above zona genes are expressed in an oocyte-specific manner. While with modification (Tempst and Riviere, 1989) using Applied Biosys- there is general agreement that the zona genes are expressed in tems Model 610A software (Matsudaira, 1987). oocytes, others have reported that zona genes are also expressed in granulosa cells, where their protein products can be detected cDNA library construction and screening (Wolgemuth et al., 1984; Lee and Dunbar, 1993).