Development 121, 1947-1956 (1995) 1947 Printed in Great Britain © The Company of Biologists Limited 1995

Coordinate expression of the three during mouse

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 restricted to the . ZP2 transcripts, but not ZP1 or that surrounds growing , 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 : 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 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 . Sequence comparison demonstrate that mouse the growth phase of oogenesis. Our data support the ZP1 is an orthologue of a rabbit zona , 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 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 . 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 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 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 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). These later 5 µg of ovarian poly(A)+ RNA from 250 10-day-old mice were studies were conducted in the rabbit, using rc55, a cDNA that isolated on an Oligotex-dT column (Qiagen) and used to create an encodes a zona protein distinct from mouse ZP2 or ZP3, raising oligo(dT)-primed, directionally-cloned cDNA expression library in the possibility that it is the homologue of mouse ZP1. lambda ZAP (Stratagene). The library was packaged with Gigapack Having a complete set of the mouse zona cDNAs would II Gold extract (Stratagene); a single reaction (50 ng of cDNA) resulted in 1-2×106 pfu. Using T3 and T7 oligonucleotides in a PCR make possible the simultaneous determination of the analysis of 34 clones, the library was estimated to be 97% recombi- expression of the three mouse genes. Additionally, these nant with an average insert size of 1.2 kb. reagents would be of particular value in further establishing the 1×106 pfu of the primary library were screened with rat anti-mouse role of each zona protein in forming the zona matrix and in ZP1 antiserum (1:400) as described (Sambrook et al., 1989), except defining domains important for specific biological functions. the blocking solution was 1% BSA and the washes were 20 mM Tris- We now report the isolation of a mouse ZP1 cDNA clone and HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20. Filter-bound primary establish that it is a homologue of rabbit rc55. Using a sensitive antibodies were detected with alkaline-phosphatase-conjugated, goat RNase protection assay and in situ hybridization, we examine anti-rat antibodies (BIO-RAD) and a BCIP/NBT color development the abundance of the three zona transcripts during oocyte solution (Sigma) per the manufacturer’s instructions. Immunopositive growth and demonstrate that the expression of these genes is clones were plaque-purified, and plasmids were excision rescued following the manufacturer’s instructions (Stratagene). coordinate and oocyte-specific. Polymerase chain reaction MATERIALS AND METHODS After heating (99¡C, 5 minutes), 1×107 pfu of the amplified library were used in PCR with a vector-specific, oligo-primer 5′ AACAGC- Purification of ZP1 protein and production of antisera TATGACCATGATTACGCC 3′ and a ZP1-specific oligo-primer Zonae pellucidae were isolated from 3000, 3-week-old mouse (396-419 nt, Fig. 1), 5′ TGTGCTATATCCACACGGCCATTG 3′, in Coordinate expression of the zona genes 1949 a Perkin Elmer GeneAmp PCR System 9600. The following con- serum albumin and 0.4 mM sodium pyruvate (Sigma) (Hogan et al., ditions were used: 95¡C for 2 minutes; 35 cycles of 95¡C for 15 1986). Oocytes were size-selected by micrometer lens, and groups of seconds, 65¡C for 45 seconds, 72¡C for 1 minute; 72¡C for 7 minutes. 50 were solubilized in 10 µl 5 M guanidine thiocyanate. The PCR products were gel purified and subcloned into the TA Fragments derived from ZP1 (95-611 nt), ZP2 (22-486 nt) and ZP3 cloning vector (Invitrogen). (1-233 nt) cDNAs were subcloned into Bluescript-KS. After lin- earization at the 3′-end, sense-strand RNAs were transcribed in the DNA sequencing presence of excess cold NTPs and trace amount of [5-3H]CTP (NEN). The sequence of the cDNA inserts was determined by a DMSO- The synthetic mRNAs were quantified by incorporation measurement modified dideoxy chain termination method (Seto, 1990) using [α- and diluted for use in standard curves. ZP1 (95-611 nt), ZP2 (34-481 35S]dATP (Amersham), the Sequenase Sequencing Kit (US Bio- nt) and ZP3 (28-233 nt) cDNA fragments subcloned in Bluescript-KS chemicals, Ver. 2) and separation on gels (6%) made from Sequagel-6 were linearized at the 5′-end and antisense probes were prepared by acrylamide solution (National Diagnostics). Both strands were incorporation of [α32-P]UTP (Amersham) into the transcripts, using sequenced using T3 and T7 primers as well as specific internal the MAXIscript kit (Ambion). Unincorporated nucleotides were oligonucleotide primers. Sequence comparison and translation were removed by G-50 Sephadex minicolumns (5 PRIME→3 PRIME). performed using the Genetic Computer Group (1991) and PCGene The RNase protection assay was performed using the RPA II kit (IntelliGentics) computer software, respectively. The accession (Ambion) protocol, except the mixture containing the probes was number of the cDNA sequence in GenBank is U20448. added directly to the sample lysate in 5 M guanidinium thiocyanate (Haines and Gillespie, 1992). Standard curves for each zona transcript Northern analysis were constructed using known amounts (0.8-120 attomole) of Internal organs were dissected from 10-day-old mice, and total RNA synthetic sense-strand RNAs. Yeast RNA, oocyte lysates or standards was isolated by the RNAzol B method (Tel-Tec, Inc.). Poly(A)+ RNA containing increasing amounts of the three synthetic RNAs were was purified using Oligotex-dt columns (Qiagen), electrophoresed in simultaneously hybridized with 1.2 fmole of ZP1 (562 nt), ZP2 (406 a 1% agarose/formaldehyde gel and transferred to a Nytran nylon nt) and ZP3 (259 nt) 32P-labelled antisense probes. The protected membrane (Schleicher & Schuell) (Derman et al., 1981). A ZP1 fragments were separated on a 4% acrylamide-8 M urea gel and cDNA fragment (150-1963 bp) was 32P-labelled by random-priming detected by autoradiography. End-labelled DNA fragments of pUC19 (Boehringer Mannheim) according to the manufacturer’s instructions digested with Sau3AI were used as molecular weight markers and do and used to probe the northern blot in aqueous hybridization solution not correspond exactly to the size of the RNA probes. The intensity (6 × SSC) at 65¡C. Final washes were at 65¡C with 0.1×SSC, 0.1% of each zona band was determined by PhosphorImager (Molecular SDS (Sambrook et al., 1989). The filter was stripped by boiling and Dynamics) using ImageQuant software. Results represent the average rehybridized with a hamster actin probe (Ringuette et al., 1986). of three independent experiments (± s.e.m.), each conducted with a standard curve. In situ hybridization Ovaries from 13-day old neonates were fixed in 4% paraformalde- hyde in PBS (1 hour, room temperature), rinsed in PBS and dehy- drated. Tissues were embedded in paraffin and 4 µm sections were RESULTS placed on silanated slides (Manova et al., 1990). cDNA fragments of ZP1 (1647 bp), ZP2 (1723 bp) and ZP3 (944 bp) were subcloned into Cloning and characterization of mouse ZP1 cDNA Bluescript (Stratagene). Sense and antisense labelled transcripts were Despite extensive screening of previously reported mouse generated using a MAXIscript T7/T3 kit (Ambion) according to the ovarian cDNA expression libraries with immunologic probes manufacturer’s specifications. Reactions (20 µl) were optimized (22¡C, 2 hours) for full-length transcripts using 80 pmoles of [α- reactive with ZP1, no clones encoding mouse ZP1 were iden- 33P]UTP (Amersham) and 1 µg of the appropriate linearized template. tified. Therefore, a primary cDNA library was constructed + The probes were purified on G-50 Sephadex mini-columns (5 using poly(A) RNA from 10-day-old mouse ovaries and PRIME→3 PRIME). screened with antisera specific to gel-purified ZP1. Seven Shorter probe fragments (200-400 bp) were derived by alkaline immunoreactive clones with inserts ranging from 1.5 to 1.9 kbp hydrolysis (1/10 volume 1M sodium carbonate, pH 10.2 at 65¡C, 10 were plaque purified and their inserts isolated as plasmids. Five minutes). The RNA fragments were precipitated and resuspended in had sequences with open reading frames containing amino acid hybridization buffer at a concentration of 5×104 cps/minute/µl. In situ sequence that exactly matched that of an internal peptide hybridization was performed as described (Manova et al., 1990) obtained from native ZP1. The longest of these clones (150- except that the slides were prehybridized and hybridized at 60¡C 1963 nt, Fig. 1) was missing the 5′ end of the ZP1 cDNA. overnight using 50% formamide and 0.4 M NaCl. After dipping in × 7 Kodak NTB-2 emulsion, the slides were exposed for 4 (ZP2 and ZP3) Therefore, 1 10 pfu of the amplified cDNA library were used or 7 (ZP1) days. They were then developed in Kodak Developer D- as a substrate in a PCR using synthetic oligonucleotide primers 19 and Kodak Fixer and stained in hematoxylin (Fisher) and eosin specific to ZP1 (396-419 nt, Fig. 1) and to the 5′ phage multi- (Polyscience, Inc.) according to standard procedures. Multiple slides cloning site. Each of two separate PCR reactions resulted in of each of two hybridizations were examined. 384 bp products containing the identical DNA sequence (36- 419 nt, Fig. 1). Taking advantage of a unique restriction site Oocyte collection and RNase protection assay (Sph I) present in the overlap of the 5′ PCR fragment and the 15 µm (resting), 40 µm, 50 µm, 60 µm and 80 µm diameter oocytes 3′ fragment obtained from screening the library, a single near were isolated from the ovaries of 3-, 8-, 10-, 14-day-old and 3-week- full-length clone (MoZP1.2) was constructed and used in sub- old female NIH Swiss mice, respectively (Huarte et al., 1987). sequent analysis. The remaining 5′ sequence (35 nt) of the Ovulated eggs (metaphase II) were isolated from the ampullae of 5- week-old, superovulated mice 16 hours after hCG injection and freed untranslated region was obtained from a genomic clone after of cumulus cells by 300 µg/ml digestion (Sigma) the transcription start was determined by an RNase protection (Hogan et al., 1986). Oocytes were isolated in incomplete Brinster assay (data to be published elsewhere). medium (Ca2+/Mg2+-free) containing 0.4 mM sodium pyruvate and The 1963 nt full-length mouse ZP1 mRNA (Fig. 1) has a then transferred into complete Brinster medium with 3 mg/ml bovine single open reading frame of 1869 nt flanked by relatively short 1950 O. Epifano and others

5′ and 3′ untranslated regions of 57 and 37 nt, respectively. cross-hybridization. Mouse ZP1 (623 amino acids) is signifi- Similar short untranslated regions are characteristic of the two cantly longer than rabbit R55 (540 amino acids), a difference other mouse zona transcripts (Ringuette et al., 1988; Liang et al., that is mostly accounted for by a 77 amino acid region in the 1990). The AUG initiation codon lies within the context of the amino-terminal third of the mouse protein that is not present in ANNAUG motif associated with vertebrate initiator codons the rabbit protein (Fig. 3). Overall similarity of the 427 aligned (Kozak, 1991), and an AAUAAA polyadenylation signal is residues in the two polypeptide chains is 51% with a central located just 9 nt downstream of the termination codon. The open core of 225 amino acids that have 66% similarity (53% reading frame encodes a protein of 623 amino acids with a cal- identity). Fifteen cysteine residues present in the mouse ZP1 are culated relative molecular mass of 69,679 (8.7% acidic, 10.6% precisely aligned in rabbit R55 with an additional three non- basic, 9.6% aromatic, 45.9% hydrophobic residues). The identity aligned cysteine residues being present near the N terminus of of the ZP1 clone was confirmed by matches between the the mouse protein (Fig. 3). This degree of conservation of predicted amino acid sequence deduced from the cDNA with sequence and placement of cysteine residues suggests that the that determined by microsequencing an N-terminal and three three-dimensional structure of mouse ZP1 and rabbit R55 is internal tryptic peptides derived from native mouse ZP1 (Fig. 1). conserved as well. Whether or not the two proteins have the A 20 amino acid N-terminal signal peptide is predicted from same biological role in fertilization remains an area for future the deduced amino acid sequence (Von Heijne, 1985, 1986) investigations. and the signal peptidase cleavage site at amino acid position 21 was confirmed by N-terminal sequencing of ZP1 (Figs 1,2). Tissue-specific expression of Zp1, Zp2 and Zp3 Like other zona proteins, ZP1 has a carboxyl terminal trans- genes membrane domain (amino acids 590-615) that may be Poly (A)+ RNA was isolated from , brain, heart, liver, important for its intracellular traf- ficking and/or assembly into the zona matrix (Fig. 2). 41 residues upstream of this hydrophobic domain is a potential prote- olytic cleavage site (Arg-Arg-Arg- Arg) (Hosaka et al., 1991). This motif, first noted in a porcine zona protein (Yurewicz et al., 1993), is also present in mouse ZP2 and ZP3 (Fig. 2). The next 277 amino acids upstream of the potential furin cleavage site (residues 268-544) are 47% similar (34% identical) to a similar region of mouse ZP2 (residues 363-635), but not to ZP3, as indicated by the horizontal line in Fig. 2. Like ZP2 and ZP3, secreted ZP1 is heavily glycosy- lated. Its predicted polypeptide chain contains 6 potential N-glyco- sylation sites and more than 93 (serine and threonine) potential O- glycosylation sites (Fig. 1). Relationship of mouse ZP1 and rabbit rc55 Rabbit rc55 encodes R55, a major component of the rabbit zona pellucida (Schwoebel et al., 1991). However, preliminary hybridization studies with rc55 cDNA and mouse Fig. 1. Nucleotide and deduced amino acid sequence of mouse ZP1 cDNA. The nucleotides are genomic DNA did not detect cross- numbered on the right. The initiation and the termination codon are boxed and the polyadenylation hybridizations between the two signal is overlined. The single 1869 nt open reading frame is translated into the ZP1 polypeptide in species, even under low-stringency the second line, and the amino acids are numbered on the left. A 21 amino acid signal peptide is wash conditions. Now, however, indicated by a dashed line, and the signal peptidase cleavage site by an inverted triangle. Amino acid the independent cloning of mouse sequences that were experimentally determined by sequencing the N terminus of the secreted protein (X-L-X-L-E-P-G-F-E-Y, where X is indeterminable) and three internal tryptic peptides (V-F-I-Q-A- ZP1 confirms that rabbit rc55 and V-L-P-N; F-S-S-Y-Y-Q-G-S-D-Y; L-L-R-E-P-V-Y-V-E-V-R) are underlined with a solid line. Six mouse ZP1 are homologues, and the potential N-linked glycosylation sites (Asn-X-Thr/Ser) are underlined with a double line. A potential relatively low nucleic acid identity furin processing site is underlined with a triple line, and the putative transmembrane domain is may have accounted for the lack of underlined with a dotted line. Coordinate expression of the zona genes 1951 kidney, spleen and testis. Using a 32P-labelled ZP1 cDNA As a control, the northern blot was reprobed with a labelled probe in a northern blot analysis, a single 2.15 kb signal was actin cDNA and actin transcripts were detected in all tissues detected only in the ovary (Fig. 4). Assuming a poly(A) tail of (data not shown). Earlier studies had determined that the 150-200 nt, the size of the mouse ZP1 transcript corresponds expression of mouse ZP2 and ZP3 genes is similarly restricted to the size of the 1963 nt full-length cDNA described above. to the ovary (Ringuette et al., 1986; Liang et al., 1990). To localize zona gene expression further to a specific cell type, ovaries from 13-day-old mice were fixed, sectioned and hybridized in situ with sense and antisense 33P-labelled RNA probes specific to mouse ZP1, ZP2 and ZP3 (Figs 5,6). Mouse ZP1 transcripts were readily detected in growing oocytes where they are present diffusely in the cytoplasm. Although present in the earliest growing oocytes, their abundance appears to increase as the oocytes grow. The hybridization signal over granulosa cells and resting oocytes was no greater than background. ZP2 transcripts were also present in growing oocytes, where they appear as an abundant mRNA; again, no hybridization signal above background was observed in

Fig. 2. Secondary structure of mouse ZP1, ZP2 and ZP3 proteins. Hydropathy profiles of mouse ZP1 (623 amino acids), ZP2 (713 amino acids) and ZP3 (424 amino acids) were determined using a window size of 7 residues (Kyte and Doolittle, 1982). ZP1, like ZP2 and ZP3, has an N-terminal hydrophobic peak representing its 21 amino acid signal peptide (the signal peptides of ZP2 and ZP3 are 34 and 22 residues in length, respectively). ZP1 also has a transmembrane domain (25 amino acids long) near its carboxyl terminus, as has been previously observed in the ZP2 and ZP3 proteins. The arrows indicates putative furin proteolytic cleavage sites 41, 47 and 34 residues upstream of the transmembrane domains of ZP1, ZP2 and ZP3, respectively. The thick horizontal line in the ZP1 and ZP2 profiles (not present in ZP3) represents a 277 amino acid domain ending at the potential furin site in which the two polypeptides are 47% similar (34% identical).

Fig. 3. Alignment of mouse ZP1 and rabbit R55 proteins. When mouse ZP1 (MoZP1) and rabbit R55 (RabR55) are compared (Needleman and Wunsch, 1970), 427 residues of the two proteins align. Identical amino acids and conserved substitutions are shaded in black and grey, respectively. Aligned but non-similar and non-aligned residues are indicated by lower case letters and dots, respectively. Asterisks indicate cysteine residues conserved between the two homologous proteins. Amino acids of each protein are numbered on the left. 1952 O. Epifano and others granulosa cells. Additionally, hybridization signals were detection using 33P-antisense RNA probes in an in situ hybrid- detected in resting oocytes probed for ZP2 (arrows, Fig. 6D,E), ization assay, none of the three zona transcripts were detected but not in resting oocytes probed for ZP1 or ZP3 (Fig. in granulosa cells. Only ZP2 transcripts were detected in 6A,B,G,H). In confirmation of an earlier report (Philpott et al., resting oocytes in amounts greater than background. Control 1987), ZP3 transcripts hybridized exclusively to growing ZP1, ZP2 and ZP3 sense probes did not demonstrate any oocytes and not to granulosa cells. Thus, at the limit of specific hybridization to growing oocytes (Fig. 6C,F,I). Zona transcript accumulation during oogenesis The temporal accumulation of the three zona transcripts during oogenesis was determined by a RNase protection assay using a mixture of zona-specific probes (Fig. 7A). Fifty oocytes at different stages of growth and maturation were isolated, analyzed and the protected probe fragments were separated by denaturing-PAGE. Although no zona transcripts were apparent in RNA isolated from 50 or 800 resting oocytes shown in Fig. 7B (lane 2,3), using prolonged exposure times for autoradio- graphy, low levels of ZP2 (but not ZP1 or ZP3) were detected in RNase protection assay samples containing 800 resting oocytes (data not shown). This is in accord with the in situ hybridization data (Fig. 6D,E), where the signal was present in the primordial oocytes. No protection was observed with yeast RNA, confirming the specificity of the hybridization (Fig. 7B, lane 1). As the oocyte begins to grow, ZP1, ZP2 and ZP3 transcripts accumulate coordinately and reach maximum levels in mid- Fig. 4. Tissue-specific expression of the Zp1 gene. After gel sized oocytes (50-60 µm in diameter), before declining in the electrophoresis, RNA isolated from seven mouse tissues was later stages of oocyte growth (Fig. 7B). By comparing the transferred to a nylon membrane and probed with 32P-labelled insert from mouse ZP1 cDNA. Each lane contains 1 µg of poly(A)+ RNA. intensity of the RNAse protection signal in oocyte samples After final washes with 0.1× SSC at 65¡C, the blot was exposed at with that obtained from known amounts of synthetic zona tran- −70¡C for 2 days. No additional signals were detected in non-ovarian scripts, the accumulation of zona transcripts can be quantified tissue even after 6 days of exposure. Numbers to the left represent (Fig. 7C). At their maximum, each oocyte contains approxi- RNA molecular size markers (Kb). mately 0.24±0.016 (s.e.m.), 0.82±0.10, and 1.0±0.11 attomoles

Fig. 5. In situ hybridization of mouse ovaries. 33P-labelled anti-sense RNA probes derived from ZP1 (A,B), ZP2 (C,D) and ZP3 (E,F) cDNAs were hybridized to formaldehyde-fixed, paraffin embedded ovarian sections from 13-day-old mice. Sections hybridized with ZP1 probes were exposed for 7 days; those hybridized with ZP2 and ZP3 probes were exposed for 4 days. For each probe, dark-field (A,C,F) and bright-field (B,D,F) images are provided. Oocytes, within growing follicles scattered throughout the ovary, are labelled with all three probes (A,C,E). Primordial oocytes, abundant in the periphery of the ovary, are labelled with the ZP2 probe (C) but not ZP1 (A) or ZP3 (E). Scale bar, 100 µm. Coordinate expression of the zona genes 1953 of ZP1, ZP2 and ZP3 transcript, respectively. Low levels (less and protein in granulosa cells. Since the initial cloning of than 5% of peak values) of each transcript can still be detected cDNAs encoding mouse ZP3 (Ringuette et al., 1986, 1988), in metaphase II ovulated eggs. These data indicate that the sto- mouse ZP2 (Liang et al., 1990) and rabbit rc55 (Schwoebel et ichiometry of ZP1, ZP2 and ZP3 transcripts is approximately al., 1991), it has been proposed that these three cDNAs encode 1:4:4. The overall time course suggests that expression of the prototypes of each of the three zona proteins. Thus, the zona pellucida genes is coordinately regulated in an oocyte- different results described above might reflect differences specific manner to ensure the availability of zona transcripts among mammals or might reflect differences among the three during oogenesis. zona proteins. To resolve this issue in the mouse, we have char- acterized ZP1 and demonstrated that it is the mouse homologue of rabbit rc55. Using the three mouse zona probes, we simul- DISCUSSION taneously assessed the temporal, spatial and quantitative aspects of zona gene expression during mouse oogenesis. The site of zona pellucida biosynthesis has been controversial By northern blot analysis, the three zona transcripts were almost since the zona matrix was first described more than 100 detected in ovary and not in six other tissues, including testis years ago. Some investigators have identified oocytes as the (this paper and Ringuette et al., 1986; Liang et al., 1990). Using source (Kang, 1974; Haddad and Nagai, 1977; Bousquet et al., in situ hybridization and 33P-labelled probes, the expression of 1981; Flechon et al., 1984; Leveille et al., 1987), whereas all three zona transcripts were further localized to oocytes. No others have localized zona biosynthesis to the surrounding signals above background were detected in granulosa cells. To granulosa cells (Chiquoine, 1954; Hadek, 1965). In metabolic our knowledge, there are no published accounts of the sensi- studies, isolated oocytes devoid of granulosa cells, when tivity of 33P-labelled probes used in in situ hybridization. cultured in the presence of radioactive precursors, synthesize However, it is reported that the signal-to-noise ratio is 10-50 de novo ZP1, ZP2 and ZP3 (Bleil and Wassarman, 1980c); times better than with 35S-labelled probes (McLaughlin and morphologic studies demonstrate that mouse oocytes mis- Margolskee, 1993), which can detect as few as 10-14 tran- directed to the adrenal gland (and lacking granulosa cells) nev- scripts per cell (Arcellana-Panlilio and Schultz, 1994). Thus, ertheless produce a zona pellucida (Zamboni and Upadhyay, we conclude that in vivo expression of the mouse zona tran- 1983). Although these studies demonstrate the ability of the scripts is restricted to oocytes. The brief report of rabbit rc55 oocyte to synthesize zona proteins by itself, they do not address whether or not granulosa cells produce zona proteins as well. The cloning of cDNAs encoding zona proteins and the development of immunologic probes to specific zona compo- nents have permitted more molecular investigations of this question. However, these more recent studies have examined only a single zona protein or transcripts under different experimental con- ditions, making direct compar- isons difficult. For example, in situ hybridization studies of mouse (Philpott et al., 1987) and marmoset (Koothan-Thillai et al., 1993) ovaries detected ZP3 tran- scripts in oocytes, but not in granulosa cells. Immunohisto- chemical studies with a mono- clonal antibody to mouse ZP2 (East and Dean, 1984) and poly- clonal antisera to a mouse ZP3 peptide (Millar et al., 1989) detected zona proteins only within the zona matrix. However, other immunohistochemical studies Fig. 6. In situ hybridization of mouse ovaries. Higher magnification photomicrographs of ovarian (Wolgemuth et al., 1984) and sections after hybridization with 33P-labelled antisense probes specific to ZP1 (A,B), ZP2 (D,E) and northern blot analysis of granulosa ZP3 (G,H). For each probe, dark-field (A,D,G) and bright-field (B,E,H)) images are provided. Control cells grown in culture for 5 days hybridization with 33P-labelled sense probes are shown in panels C,F,I. The arrows indicate the (Lee and Dunbar, 1993) report the primordial follicles, in which oocytes are labelled by the ZP2 probe (D,E) but not the ZP1 (A,B) or presence of rabbit rc55 transcript ZP3 probes (G,H). Scale bar, 40 µm. 1954 O. Epifano and others

Fig. 7. Developmental expression of zona pellucida mRNAs during oogenesis. RNase protection assay for detection of mouse ZP1, ZP2 and ZP3 messages in oocyte lysates. (A) 32P-labelled antisense ZP1 (lane 1, 562 nt), ZP2 (lane 2, 406 nt) and ZP3 (lane 3, 259 nt) probes. (B) Protected ZP1 (516 nt), ZP2 (338 nt) and ZP3 (205 nt) fragments detected after RNase A/T1 digestion and autoradiography. Lane 1, yeast RNA (10 µg); lane 2, 800 resting oocytes; lane 3, 50 resting oocytes; lane 4, 50 oocytes of 40 µm diameter; lane 5, 50 oocytes of 50 µm diameter; lane 6, 50 oocytes of 60 µm diameter; lane 7, 50 fully grown oocytes (80 µm diameter); lane 8, 50 ovulated eggs. Sau3A1 fragments of pUC19 used as molecular weight markers (DNA does not correspond exactly to the size of the RNA probes) are indicated on the left of panels A and B. (C) Quantification of the abundance of ZP1(᭿), ZP2 (᭹) and ZP3 (᭡) transcripts during oogenesis. The molar amount for each ZP message was determined by PhosphorImager analysis based on the hybridization signals in B and the hybridization signals obtained by using increasing amount of synthetic ZP1, ZP2 and ZP3 transcripts. The values in the ordinate represent the average of three experiments (± s.e.m.). Abbreviations: R, resting oocytes; OV, ovulated eggs. in growing oocytes, but not in granulosa cells, by in situ fully formed and appears to be quite stable (Shimizu et al., hybridization of rabbit ovary (Dunbar et al., 1995) suggests 1983). After ovulation, eggs have less than 5% of the peak that this may be true in the rabbit as well. A possible explana- levels of zona transcripts. ZP1 is the least abundant of the three tion of the paradoxical detection of rc55 by northern blot in transcripts, representing approximately 25% of ZP2 or ZP3 granulosa cells grown in vitro (Lee and Dunbar, 1993), but not RNAs, which are present in roughly equimolar amounts. These by in situ hybridization of ovarian sections, is that the rc55 data suggest that the three zona genes may have transcriptional gene might be derepressed in granulosa cells grown in the regulatory elements in common. Analysis of the promoter absence of oocytes. region of the Zp2 and Zp3 genes has identified a binding site Our results also indicate that the three zona pellucida genes for a putative transcription factor Zona Activating Protein exhibit an ordered pattern of expression during oogenesis. ZP2 (ZAP-1) implicated in the regulation of zona gene expression transcripts are detected in oocytes before birth, well before the (Millar et al., 1991, 1993). It remains to be determined if ZAP- growth phase of oogenesis and perhaps as early as 16 days 1 is also involved in the regulation of Zp1. gestation (Millar et al., 1993). In the current study, ZP2 tran- The primary structure of ZP1 has been deduced from a full- scripts were detected by in situ hybridization in roughly 90% length cDNA of mouse ZP1. Comparison of its amino acid of the resting oocytes whereas ZP1 and ZP3 transcripts were sequence with that of rabbit R55 confirms that the two proteins detected only after the oocytes begin to grow. Whether the are orthologues. Gel electrophoretic analysis of mouse ZP1 early detection of the ZP2 transcript reflects a relative (Bleil and Wassarman, 1980b; Shimizu et al., 1983) and rabbit abundance (e.g., ZP1 and ZP3 are present but at levels below R55 (Dunbar et al., 1981) suggests that each protein is heavily the sensitivity of the assay) or an earlier onset of Zp2 gene glycosylated. Mouse ZP1 is present in the extracellular matrix expression (e.g., Zp1 and Zp3 genes are not yet activated) as a high molecular weight protein (180-200×103) which, remains to be determined. As the diameter of the oocyte under reducing conditions, has an apparent mass of 120×103 3 increases, all three zona transcripts accumulate, and in mid- Mr. In contrast, rabbit R55 appears as a broad band (90×10 sized oocytes (50-60 µm diameter) represent approximately Mr) resolved only by 2-dimensional electrophoresis whose 1.5% of the total poly(A)+ RNA. The accumulation of the ZP1, mobility changes little after reduction of disulfide bonds. These ZP2 and ZP3 RNAs appears coordinate, with a dramatic data suggest that mouse ZP1 (but not rabbit R55) is dimerized increase during the early stages of oogenesis and then a decline via disulfide bonds. The detection of a single N-terminal as the oocyte matures. In mature oocytes, the zona matrix is sequence after microsequencing of the secreted protein further Coordinate expression of the zona genes 1955 suggests that ZP1 exists as a homo- rather than a heterodimer. interaction: Identification of a in mouse egg zonae pellucidae It is tempting to speculate that the three cysteine residues possessing receptor activity for sperm. Cell 20, 873-882. present in native mouse ZP1 (but not rabbit R55) are involved Bleil, J. D. and Wassarman, P. M. (1980b). Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse in ZP1-ZP1 dimerization. Presumably, the remaining 15 oocyte’s zona pellucida. Dev. Biol. 76, 185-202. conserved cysteine residues present in both proteins are Bleil, J. D. and Wassarman, P. M. (1980c). Synthesis of zona pellucida involved in maintaining the three-dimensional structure of ZP1 proteins by denuded and follicle-enclosed mouse oocytes during culture in (and R55) in the zona matrix. vitro. Proc. Natl. Acad. Sci. USA 77, 1029-1033. Although distinct from mouse ZP2 and ZP3, ZP1 shares Bousquet, D., Leveille, M. C., Roberts, K. D., Chapdelaine, A. and Bleau, G. (1981). The cellular origin of the zona pellucida antigen in the human and certain motifs with the other two zona proteins. Each of the hamster. J. Exp. Zool. 215, 215-218. three proteins has a signal peptide that directs it into a secretory Burnette, W. N. (1981). ‘Western blotting:: electrophoretic transfer of proteins pathway and is cleaved from the mature polypeptide chain. from sodium dodecyl sulfate--polyacrylamide gels to unmodified Each zona protein has a transmembrane domain near its nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195-203. carboxyl terminus about 40 amino acids downstream of a Chamberlin, M. E. and Dean, J. (1990). Human homolog of the mouse sperm potential furin cleavage site. The proteolytic processing of the receptor. Proc. Natl. Acad. Sci. USA 87, 6014-6018. transmembrane domains may be an intermediate step in the Cheng, A., Le, T., Palacios, M., Bookbinder, L. H., Wassarman, P. M., secretion of the zona proteins. Accumulating evidence suggests Suzuki, F. and Bleil, J. D. (1994). Sperm-egg recognition in the mouse: that specific protein domains have been conserved among zona characterization of sp56, a sperm protein having specific affinity for ZP3. J. Cell Biol. 125, 867-878. classes of each and among widely divergent species. Chiquoine, A. D.(1954). The identification, origin and migration of the Ending at the aforementioned potential furin cleavage site, primordial germ cells in the mouse embryo. Anat. Rec. 118, 135-146. mouse ZP1 shares a 277 amino acid domain with mouse ZP2. Derman, E., Krauter, K., Walling, L., Weinberger, C., Ray, M. and This domain, first noted between rabbit rc55 and mouse ZP2 Darnell, J. E.,Jr. (1981). Transcriptional control in the production of liver-specific mRNAs. Cell 23, 731-739. (Schwoebel et al., 1991), is also present in a protein Dunbar, B. S., Liu, C. and Sammons, D. W. (1981). Identification of the three surrounding teleost eggs that is synthesized in and secreted major proteins of porcine and rabbit zonae pellucidae by high resolution from the liver after estrogen induction (Lyons et al., 1993). two-dimensional gel electrophoresis: comparison with serum, follicular, Thus, it appears that this motif has persisted over approxi- fluid, and ovarian cell proteins. Biol. Reprod. 24, 1111-1124. mately 650 million years of evolution in egg envelope proteins. Dunbar, B. 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