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The Major Chicken Egg Envelope Protein ZP1 Is Different from ZPB and Is Synthesized in the Liver

2000; Elsevier BV; Volume: 275; Issue: 37 Linguagem: Inglês

10.1074/jbc.275.37.28866

1083-351X

Nina Bausek, Marianne Waclawek, Wolfgang J. Schneider, Franz Wohlrab,

Proteins in Food Systems

The extracellular matrix surrounding vertebrate oocytes is called the zona pellucida in mammals and perivitelline membrane (pvm) in birds. We have analyzed this structure in chicken follicles and laid eggs and have identified a 95-kDa component of the pvm, which, by protein sequencing, shows homology to mammalian zona pellucida proteins. Surprisingly, we could not detect this protein in ovarian granulosa cells or oocytes but instead found high levels in the liver of the laying hen. In contrast, it is absent in rooster liver but can be efficiently induced by estrogen treatment of the animal. An immunoscreen of a liver λ-ZAP library yielded a cDNA coding for a protein of 934 amino acids. It displayed significant homology to members of the ZP1/ZPB family from other species, notably to mouse and rat ZP1, and was therefore designated chkZP1. It is clearly different from a protein designated chkZPB that had been deposited in the data base previously. Alignment of the known members of the ZP1/ZPB family demonstrated the existence of at least three subgroups, with representatives of both the ZP1 and the ZPB sequence homology group occurring in vertebrates. Northern blot analysis of liver extracts revealed the presence of a single 3.2-kilobase mRNA coding for chkZP1, distinct from the chkZPB transcript detectable in follicles. Immunohistochemical analysis of follicle sections demonstrates that chkZP1 can be found in the blood vessels of the theca cell layer as well as in the pvm surrounding the oocyte. Thus, in the chicken, at least one of the major pvm components is synthesized in the liver and is transported via the bloodstream to the follicle. The extracellular matrix surrounding vertebrate oocytes is called the zona pellucida in mammals and perivitelline membrane (pvm) in birds. We have analyzed this structure in chicken follicles and laid eggs and have identified a 95-kDa component of the pvm, which, by protein sequencing, shows homology to mammalian zona pellucida proteins. Surprisingly, we could not detect this protein in ovarian granulosa cells or oocytes but instead found high levels in the liver of the laying hen. In contrast, it is absent in rooster liver but can be efficiently induced by estrogen treatment of the animal. An immunoscreen of a liver λ-ZAP library yielded a cDNA coding for a protein of 934 amino acids. It displayed significant homology to members of the ZP1/ZPB family from other species, notably to mouse and rat ZP1, and was therefore designated chkZP1. It is clearly different from a protein designated chkZPB that had been deposited in the data base previously. Alignment of the known members of the ZP1/ZPB family demonstrated the existence of at least three subgroups, with representatives of both the ZP1 and the ZPB sequence homology group occurring in vertebrates. Northern blot analysis of liver extracts revealed the presence of a single 3.2-kilobase mRNA coding for chkZP1, distinct from the chkZPB transcript detectable in follicles. Immunohistochemical analysis of follicle sections demonstrates that chkZP1 can be found in the blood vessels of the theca cell layer as well as in the pvm surrounding the oocyte. Thus, in the chicken, at least one of the major pvm components is synthesized in the liver and is transported via the bloodstream to the follicle. perivitelline membrane polyacrylamide gel electrophoresis group of overlapping clones polymerase chain reaction phosphate-buffered saline Vertebrate eggs are surrounded by an insoluble extracellular matrix, which is called zona pellucida in mammals, chorion in fish, and perivitelline membrane or vitelline envelope in amphibians and birds (1Dumont J.N. Brummett A.R. Dev. Biol. 1985; 1: 235-288Google Scholar, 2Bellairs R. Harkness M. Harkness R. J. Ultrastruct. Res. 1963; 8: 339-359Crossref Scopus (93) Google Scholar, 3Cotelli F. Andronico F. Brivio M.F. Lora Lamina C. J. Ultrastruct. Mol. Struct. Res. 1988; 99: 70-78Crossref Scopus (46) Google Scholar, 4Wassarman P. Chen J. Cohen N. Litscher E. Liu C. Qi H. Williams Z. J. Exp. Zool. 1999; 285: 251-258Crossref PubMed Scopus (69) Google Scholar). This structure represents the initial sperm-binding site, participates in the induction of the acrosome reaction, and mediates the prevention of polyspermy. In mammals, the zona pellucida is composed of three component glycoproteins, called ZP1, ZP2, and ZP3, (also known as ZPB, ZPA, and ZPC, respectively) (5Harris J.D. Hibler D.W. Fontenot G.K. Hsu K.T. Yurewicz E.C. Sacco A.G. DNA Seq. 1994; 4: 361-393Crossref PubMed Scopus (318) Google Scholar), that show significant conservation across all species studied. The classification of these proteins often has been difficult because of their extensive heterogeneity, which is due to multiple posttranslational modifications. In many cases it has been possible to make unambiguous assignments to particular gene families only after isolation of the cDNAs. Although the different polypeptides are highly conserved, they show large differences in their functional properties (6Prasad S.V. Skinner S.M. Dunbar B.S. Coufaris C. Mastroianni L. New Horizons in Reproductive Medicine. Parthenon Publishing, New York1997: 129-144Google Scholar). Thus, the primary sperm receptor in mouse appears to be ZP3 through its O-linked oligosaccharides (7Wassarman P.M. J. Reprod. Fertil. Suppl. 1990; 42: 79-87PubMed Google Scholar), whereas in the rabbit the ZP1 homologue (8Prasad S.V. Wilkins B. Skinner S.M. Dunbar B.S. Mol. Reprod. Dev. 1996; 43: 519-529Crossref PubMed Scopus (63) Google Scholar), and in the pig, a heterodimer between ZPB (ZP1) and ZPC (ZP3), is thought to possess sperm binding activity (9Yurewicz E.C. Sacco A.G. Gupta S.K. Xu N. Gage D.A. J. Biol. Chem. 1998; 273: 7488-7494Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Furthermore, sperm binding in the pig appears to be mediated not by O-linked but rather byN-linked carbohydrates (10Yonezawa N. Mitsui S. Kudo K. Nakano M. Eur. J. Biochem. 1997; 248: 86-92Crossref PubMed Scopus (42) Google Scholar, 11Yonezawa N. Aoki H. Hatanaka Y. Nakano M. Eur. J. Biochem. 1995; 233: 35-41Crossref PubMed Scopus (75) Google Scholar). Oocytes of oviparous species are large when compared with those of mammals. In the chicken, the pvm1 is composed of two layers, an inner layer, deposited in the preovulatory phase, and an outer layer, added during passage through the oviduct; these layers are separated by a thin membrane (2Bellairs R. Harkness M. Harkness R. J. Ultrastruct. Res. 1963; 8: 339-359Crossref Scopus (93) Google Scholar). This membrane and the outer layer are added to the inner membrane only after ovulation, i.e.during migration of the oocyte through the oviduct (12Back J.F. Bain J.M. Vadehra D.V. Burley R.W. Biochim. Biophys. Acta. 1982; 705: 12-19Crossref PubMed Scopus (61) Google Scholar, 13Kido S. Morimoto A. Kim F. Doi Y. Biochem. J. 1992; 286: 17-22Crossref PubMed Scopus (37) Google Scholar). For successful fertilization to occur, sperm first has to bind to the pvm, a process that is species-specific (14O'Rand M.G. Gamete Res. 1988; 19: 315-328Crossref PubMed Scopus (88) Google Scholar), and then penetrate it (15Liu D.Y. Baker H.W. Biol. Reprod. 1993; 48: 340-348Crossref PubMed Scopus (78) Google Scholar). The outer layer appears to be involved in a block to polyspermy via the acrosome reaction (16Barros C. Crosby J.A. Moreno R.D. Cell Biol. Int. 1996; 20: 33-39Crossref PubMed Scopus (57) Google Scholar). Despite the long history of ultrastructural studies on the chicken follicle (2Bellairs R. Harkness M. Harkness R. J. Ultrastruct. Res. 1963; 8: 339-359Crossref Scopus (93) Google Scholar), little is known about the molecular details of the composition of the pvm. Of the major bands obtained on SDS-polyacrylamide gels after electrophoresis of laid egg pvm under reducing conditions, all but two are outer layer components. Of these two, one protein of 34 kDa has been characterized (17Waclawek M. Foisner R. Nimpf J. Schneider W.J. Biol. Reprod. 1998; 59: 1230-1239Crossref PubMed Scopus (98) Google Scholar, 18Takeuchi Y. Nishimura K. Aoki N. Adachi T. Sato C. Kitajima K. Matsuda T. Eur. J. Biochem. 1999; 260: 736-742Crossref PubMed Scopus (84) Google Scholar). This protein is a homologue of the mammalian ZP3/ZPC; it has been demonstrated to be synthesized exclusively by the granulosa cells surrounding the oocyte and is secreted in a polarized fashion (17Waclawek M. Foisner R. Nimpf J. Schneider W.J. Biol. Reprod. 1998; 59: 1230-1239Crossref PubMed Scopus (98) Google Scholar). The nature of the other band had been unclear. In this paper, we report the characterization, molecular cloning, site of expression, and localization of this protein and its identification as an avian ZP1 homologue. We also show that it is clearly different from a related protein designated chkZPB. 30–40-week-old Derco-brown laying hens and roosters (Heindl Co., Vienna, Austria) were used as a source for eggs, follicles, and tissues. Antibodies were raised in adult female New Zealand White rabbits (see below). Perivitelline membranes from chicken eggs or ovarian follicles were obtained as described (17Waclawek M. Foisner R. Nimpf J. Schneider W.J. Biol. Reprod. 1998; 59: 1230-1239Crossref PubMed Scopus (98) Google Scholar). Briefly, for isolation of pvm from freshly laid eggs, yolk was drained from oocytes by puncturing the membranes after removal of egg white. The membranes were washed in Tris-buffered saline (137 mm NaCl, 2.5 mm KCl, 2.5 mmTris·HCl, pH 7.6) and incubated for 1 h at 4 °C in 200 mm Tris·maleate (pH 6.5), 2 mmCaCl2, 0.5 mm phenylmethylsulfonyl fluoride, 2.5 mm leupeptin, and 1.4% Triton X-100. After centrifugation for 40 min at 4 °C at 300,000 × g, the detergent-pretreated membranes were solubilized in the equivalent of 0.2 ml/egg of Tris-buffered saline containing 2% SDS and 50 mm dithiothreitol. Insoluble constituents were removed by centrifugation at 12,000 rpm for 20 min in an Eppendorf centrifuge. For isolation of pvm from follicles, ovaries were dissected from mature laying hens immediately after decapitation. Granulosa cell sheets containing the inner pvm as well as granulosa cells and basement membranes were prepared from follicles larger than 2 cm in diameter as described (19Gilbert A.B. Evans A.J. Perry M.M. Davidson M.H. J. Reprod. Fertil. 1977; 50: 179-181Crossref PubMed Scopus (278) Google Scholar). The protocol for subsequent solubilization was identical to the one for pvm from laid eggs. Chicken pvm proteins were separated by preparative SDS-PAGE (4.5–18%) under reducing conditions. The 95-kDa band was cut out and eluted electrophoretically at 180 V for 8 h in 25 mm Tris, 250 mm glycine, pH 8.3, containing 0.1% SDS. Microsequencing of tryptic digests was performed essentially as described (20Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). Three sequence fragments were characterized, designated 95.1 (the N-terminal peptide, LLQYHYDCRDFGMQLLAYP), t2 (TQLVPVGPATLQLPF), and t3.1 (PGLXXPGLPSXPGLVS), respectively. A 190-kDa band present under nonreducing conditions (see Fig. 2) had the same N-terminal sequence as the 95-kDa species and is presumably a dimeric form of this protein. Subsequently, synthetic peptides were obtained corresponding to fragments 95.1 and t2, coupled to maleimide-activated keyhole limpet hemocyanin (Pierce), and used to raise antisera in rabbits. Three series of intradermal injections of 500 μg of each of the antigens in a total volume of 400 μl, mixed with an equal volume of Freund's complete (day 0) or incomplete (days 14 and 35) adjuvant were administered to the animals. Additionally, an antiserum directed against the entire gel-purified 95-kDa protein (anti-p95 antiserum) was raised according to the same protocol. Preimmune and immune sera were stored at −80 °C. A laying hen liver λ ZAPII cDNA library (Takara) was grown and induced with isopropyl-1-thio-β-d-galactopyranoside-soaked filters. 1 × 106 plaques were screened with polyclonal anti-p95 antiserum (see above) according to standard methods (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995Google Scholar) as modified by the picoBlue™ immunoscreening kit (Stratagene). Four positive clones were picked, and the released phage particles were grown in XL1-Blue MRF′ cells that were coinfected with ExAssist™ helper phage for in vivo excision using the ExAssist/SOLR system (Stratagene). All clones obtained were sequenced on both strands. The phage were digested with several restriction enzymes and recloned into BlueScript vectors, and the contig sequences were determined on an ABI sequencer. Contigs were assembled with the help of Assembly Line (Oxford Molecular) and alignments were determined by ClustalW (MacVector 6.5.3). Chickens were sacrificed by decapitation, and tissues were frozen immediately in liquid nitrogen. For estrogen treatment, roosters were injected intramuscularly with 10 mg/kg 17-β-estradiol (dissolved in 1,2-propanediol at 20 mg/ml) 48 h prior to removal of tissues; control roosters received vehicle only. Total RNA was extracted using TriReagent (Molecular Research Center, Inc.) and was subjected to electrophoresis on a 1.2% agarose gel in the presence of glyoxal (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. 2 nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), followed by blotting onto positively charged nylon membranes (Roche Molecular Biochemical). RNA was covalently bound to the dried membrane by UV cross-linking. A 2921-base pair fragment of chkZP1 was used as hybridization probe and labeled with [α-32P]dCTP by random priming. Hybridization was performed at a probe concentration of 2 × 106 cpm/ml in a buffer containing 1% bovine serum albumin, 7% SDS, 0.5m sodium phosphate (pH 6.8), and 1 mm EDTA at 65 °C for 16 h. Washes were performed at 65 °C, first in 40 mm sodium phosphate buffer (pH 6.8), 0.5% bovine serum albumin, 5% SDS, and 1 mm EDTA and then in 1% SDS, 40 mm sodium phosphate (pH 6.8), and 1 mm EDTA. The blot was subsequently exposed to X-Omat Blue XB-1 (Kodak) film with intensifying screen at −80 °C. To obtain a chicken ZPB (accession number AB025428)-specific probe, total RNA was isolated from small follicles using TriReagent (Molecular Research Center, Inc.), according to the manufacturer's instructions. 5 μg of this RNA was then reverse transcribed using the SuperscriptTM preamplification system (Life Technologies, Inc.), and an aliquot of the obtained cDNA was used as a template for PCR. Primers were chosen to amplify a 510-base pair region of the chicken ZPB cDNA (forward primer, 5′-TTGGAGCTGTGTTCTTCTTGG-3′; reverse primer, 5′-GGTTGTAACAACAGCCTCGC-3′). PCR was performed for 30 cycles of denaturation for 1 min at 95 °C, annealing was performed for 1 min at 58 °C, and primer extension was performed for 1.5 min at 72 °C. The PCR product was subcloned into pCR2.1 (Invitrogen), and its identity was verified by sequencing. It was then labeled with [α-32P]dCTP by random priming and used as a hybridization probe for Northern blotting as described above. Freshly obtained chicken tissues were homogenized in 5 ml/g of buffer A (25 mm Tris·HCl, pH 8.0, 1 mm CaCl2, 1 mmphenylmethylsulfonyl fluoride, and 1 μm leupeptin) using an Ultra Turrax T25 and then centrifuged at 1,500 × gfor 10 min. The supernatant was passed through cheesecloth and spun at 100,000 × g for 1 h. The resulting pellet was resuspended in buffer A and aspirated through 18- and 22-gauge needles. After recentrifugation, cells were resuspended in extraction buffer (125 mm Tris·maleate, pH 6.0, 1 mmCaCl2, 0.5 mm phenylmethylsulfonyl fluoride, 1 μm leupetin, 160 mm NaCl, and 1% Triton X-100). Serum was delipidated prior to gel electrophoresis by treatment with a 20-fold excess of precooled chloroform/methanol (2:1). After 30 min at 4 °C, the mixture was spun for 15 min at 4000 rpm in a tabletop centrifuge, followed by dissolution of the pellet in the original volume of sample buffer. Protein extracts and sera were separated by one-dimensional SDS-PAGE (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). Proteins were transferred to nitrocellulose (Hybond-ECL, Amersham Pharmacia Biotech) for immunoblotting. Transfers were performed in 25 mm Tris, 192 mm glycine, and 20% methanol for 1 h at 17 V at room temperature or overnight at 6 V and 4 °C. The membranes were blocked in 80 mmNa2HPO4, 20 mmNaH2PO4, 100 mm NaCl (PBS), 0.1% Tween, 5% nonfat dry milk for 1 h, followed by incubation with antiserum in PBS-t (PBS with 0.1% Tween). After three washes in PBS-t, the membrane was incubated with protein A-horseradish peroxidase (1:5000) for 1 h. Bands were visualized by the enhanced chemoluminescence procedure as suggested by the manufacturer (Amersham Pharmacia Biotech). The positions of migration of molecular weight standards (Bio-Rad) were determined by staining with Ponceau S (0.5% in 1% acetic acid). Perivitelline membranes from follicles were obtained as described above, washed in Tris-buffered saline, and taken up in 20 mm sodium phosphate (pH 6.8), 10 mm EDTA, 0.2% SDS. After addition of 1 milliunit ofN-glycosidase F (Roche Molecular Biochemicals; fromFlavobacterium meningosepticum)/100 μl of suspension and subsequent incubation at 37 °C for 16 h, the samples were centrifuged at 15,000 × g for 2 min, and the supernatant was analyzed by SDS-PAGE as described. For differential interference contrast microscopical analysis of tissue sections after immunohistochemistry, procedures were as described (24Stockinger W. Hengstschlager-Ottnad E. Novak S. Matus A. Huettinger M. Bauer J. Lassmann H. Schneider W.J. Nimpf J. J. Biol. Chem. 1998; 273: 32213-32221Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Briefly, chickens were anesthetized with 2 ml of nembutal and perfused via the left ventricle with 300 ml of PBS, followed by 300 ml of a solution containing 75 mml-lysine, 75 mm sodium phosphate (pH 7.3), 2% paraformaldehyde, and 2.4 mg/ml sodium meta-periodate. Specimens were embedded in paraffine using a Tissue-Tek VIP (Miles Scientific) embedding machine, and 3-μm slices were cut on a Microm HM335E microtome. Slices were deparaffinized in xylol exchange medium XEM-200 (Vogel) and rehydrated by consecutive washes in 96, 70, and 50% ethanol and pure water. Endogenous peroxidase was blocked by incubating the slices in 3% H2O2 for 5 min. Unspecific binding of antibodies was inhibited by blocking with a solution of 1% milk powder and 3% total goat serum in PBS for 1 h at room temperature. Polyclonal anti-p95 antiserum was applied at a dilution of 1: 800 in PBS for 1 h. After three washes in PBS, the following incubations were performed at room temperature: goat anti-rabbit biotinylated IgG (Sigma) diluted 1:500 in blocking solution for 1 h, five washes with PBS, peroxidase-labeled avidin (Sigma; 1:200 in 1% milk in PBS) for 1 h, and three final washes with PBS. For the color reaction, slices were incubated in 0.1 msodium acetate (pH 5.1) containing 150 μl of 30% H2O2 and 20 mg of 3-amino 9-ethylcarbazole/100 ml of buffer. The staining process was followed under the microscope and stopped by immersing the slides in water. Results were observed on a Zeiss Axiovert 135 microscope. In a previous communication, we had demonstrated that SDS-dissolved chicken perivitelline membranes from laid eggs, when subjected to SDS-PAGE under reducing conditions, show four major protein bands with apparent molecular weights of 5,000, 13,000, 34,000, and 95,000, respectively (17Waclawek M. Foisner R. Nimpf J. Schneider W.J. Biol. Reprod. 1998; 59: 1230-1239Crossref PubMed Scopus (98) Google Scholar). Sequencing of the N termini and of tryptic fragments of the proteins indicated that the 5- and 13-kDa species represent chicken vitelline membrane outer protein I and lysozyme, respectively, whereas the 34-kDa protein is an avian homologue of the mammalian zona pellucida component known as ZP3 or ZPC (17Waclawek M. Foisner R. Nimpf J. Schneider W.J. Biol. Reprod. 1998; 59: 1230-1239Crossref PubMed Scopus (98) Google Scholar). The nature of the 95-kDa protein (p95) was not immediately clear. Although BLAST searches of public data bases revealed that the N-terminal sequence had homology to proteins of the ZP1/ZPB family, an internal tryptic fragment (t3.1) showed no similarity to known zona pellucida components of other species. Rather, it resembles glutamine-, proline-, and glycine-rich proteins such as fibroin or glutenin, so that an unambiguous assignment to the zona pellucida protein family was not directly possible. Furthermore, during the course of this work, a chicken ZPB sequence had been deposited in the public data bases (accession number AB025428); however, it did not contain any of the fragments we had sequenced. To characterize the 95-kDa band, we first raised polyclonal antisera against the entire protein as well as against synthetic oligopeptides corresponding to the sequences we had obtained (see “Materials and Methods”). Fig. 1 shows identical samples run on the same gel probed with three different antibodies. As is evident in panel A, the antiserum against the entire protein (lane 1) as well as the one against the N-terminal peptide 95.1 (lane 2) recognize only a single band of the expected size on immunoblots of chicken pvm. Although the same polypeptide is recognized by the antiserum against the internal peptide t2, we consistently also observed reactivity toward two polypeptides of 43 and 48 kDa, respectively (lane 3). The nature of these bands is unclear; however, Coomassie Blue-stained SDS-polyacrylamide gels of pvm extracts show the 95-kDa protein as well as the 34-kDa ZP3 but do not exhibit major proteins migrating at these positions (17Waclawek M. Foisner R. Nimpf J. Schneider W.J. Biol. Reprod. 1998; 59: 1230-1239Crossref PubMed Scopus (98) Google Scholar). To test for the possibility that p95 is a glycoprotein, the pvm was treated with N-glycosidase F and subsequently subjected to SDS-PAGE. As can be seen in Fig. 1 B, the band detected by the antibody directed against the entire protein was shifted to an apparent molecular mass of approximately 93 kDa (lane 2). This increase in mobility indicates the presence ofN-linked sugars on p95. Surprisingly, the pvm is not the only tissue where p95 can be detected. As can be seen in Fig.2, it is also present at high levels in the liver and serum of laying hens but is lacking from the liver and serum of roosters. Because for this immunoblot, SDS-PAGE had been performed under nonreducing conditions, an additional band migrating at approximately 190 kDa representing a dimeric form of p95 (17Waclawek M. Foisner R. Nimpf J. Schneider W.J. Biol. Reprod. 1998; 59: 1230-1239Crossref PubMed Scopus (98) Google Scholar) was observed. In mammals and most other species studied to date, zona proteins are synthesized either in the oocyte itself or in the follicle cells surrounding it. In some species of fish, however, the liver has been reported to be the site of synthesis for components of the piscine equivalent of the zona pellucida. These proteins are then transported to the oocytes via the blood stream (25Oppen-Berntsen D.O. Hyllner S.J. Haux C. Helvik J.V. Walther B.T. Int. J. Dev. Biol. 1992; 36: 247-254PubMed Google Scholar, 26Oppen-Berntsen D.O. Gram-Jensen E. Walther B.T. J. Endocrinol. 1992; 135: 293-302Crossref PubMed Scopus (87) Google Scholar, 27Lyons C.E. Payette K.L. Price J.L. Huang R.C. J. Biol. Chem. 1993; 268: 21351-21358Abstract Full Text PDF PubMed Google Scholar, 28Del Giacco L. Vanoni C. Bonsignorio D. Duga S. Mosconi G. Santucci A. Cotelli F. Mol. Reprod. Dev. 1998; 49: 58-69Crossref PubMed Scopus (41) Google Scholar, 29Sugiyama H. Yasumasu S. Murata K. Iuchi I. Yamagami K. Dev. Growth Differ. 1998; 40: 35-45Crossref PubMed Scopus (48) Google Scholar). The results of Fig. 2suggest that a similar situation exists in the chicken, a finding that is unexpected in light of the site of synthesis of chicken ZP3, namely ovarian granulosa cells (17Waclawek M. Foisner R. Nimpf J. Schneider W.J. Biol. Reprod. 1998; 59: 1230-1239Crossref PubMed Scopus (98) Google Scholar). Fig. 2 also demonstrates that p95 expression is sex-specific, because it is largely absent from rooster liver and rooster serum. These data strongly suggest that the liver is the site of synthesis for p95 and raise the possibility that the gene is under estrogen control. Indeed, estrogen treatment of roosters resulted in a dramatic induction of p95 synthesis in liver and serum to levels comparable with those in the laying hen (Fig. 2). The notion that p95 might be synthesized by hepatic tissues prompted us to screen a λ-ZAP laying hen liver cDNA library with our antiserum raised against purified p95. We obtained several candidate clones, which were sequenced on both strands (see “Materials and Methods”). All clones were overlapping, apparently representing different parts of a single transcript of 2932 nucleotides in length. The sequence has been deposited in the public data bases under accession number AJ289697. It contains a long open reading frame coding for a polypeptide of 934 amino acids with a predicted molecular mass of 100 kDa (Fig.3 A). Comparison of the sequence of this protein with the sequence of the N-terminal peptide (95.1; see above) indicates that Leu-25 of the precursor protein represents the N terminus of the mature protein found in the laid egg. In fact, theoretical calculations using the algorithm of von Heijne and co-workers (30Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4942) Google Scholar) predict a signal peptide with the most likely cleavage site at the sequence GLA↓LL at exactly this position. For this reason and on the grounds of sequence alignments, we believe that the first ATG in the cDNA (position 9) indeed represents the initiator codon. In addition, we find a consensus furin cleavage site (RX(K/R)R↓) at position 900–903. The protein contains 21 cysteines, as well as three consensus sites forN-linked glycosylation (Asn in positions 65, 121, and 723). The putative mature protein lacking the signal sequence and the C terminus would have a molecular mass of 94 kDa, in excellent agreement with the results obtained by immunoblotting (Fig. 1). Comparative analysis of the protein sequence with the data bases using the BLAST algorithm revealed high scoring matches with the ZP1/ZPB family of proteins. Alignment of the chicken sequence with ZP1/ZPB proteins from other organisms shows a highly conserved hydrophobic region, called the zona pellucida domain (31Bork P. Sander C. FEBS Lett. 1992; 300: 237-240Crossref PubMed Scopus (292) Google Scholar), in the C-terminal part of the molecule. Further similarities are the presence of a so-called trefoil domain (32Hoffmann W. Hauser F. Trends Biochem. Sci. 1993; 18: 239-243Abstract Full Text PDF PubMed Scopus (112) Google Scholar) and the above-mentioned furin cleavage site. In addition, the extreme N terminus also has significant homology to some other ZP1/ZPB family members, notably to those found in mouse and rat. In contrast, the central part of the p95 sequence, which contains one of the tryptic fragments (t3.1) that we had sequenced initially, shows no apparent similarity to any of the mammalian zona pellucida components. Taken together, the alignment demonstrates that the chicken isolate is highly homologous to ZP1/ZPB proteins in both the N- and the C-terminal domains but contains an additional central region with some resemblance to several other extracellular proteins like fibroin or wheat glutenin. The similarity of p95 to other zona proteins and the fact that we had originally isolated it as one of two major components of the inner pvm support its assignment to the family of zona pellucida proteins. The sequence data also clearly distinguish this protein from a sequence deposited in the data base called chicken ZPB. In fact, the dendrogram in Fig. 3 Bshows that the sequence obtained here appears to be more closely related to the mouse, rat, and human ZP1 proteins than to the chicken or human ZPB polypeptides. This indicates that it represents a distinct avian gene related to the ZP1 family, and we therefore designate it chicken zona pellucida protein 1 or chkZP1. The dendrogram further suggests that, according to sequence homologies, we can distinguish between several distinct subgroups in the ZP1/ZPB gene family. One group comprises the mouse, rat, human, and chicken ZP1 proteins (ZP1 group), one contains the human, rabbit, chicken, cat, andXenopus ZPB proteins, as well as the marmoset and macaque ZP1 orthologues (ZPB group), and a third group encompasses the fish homologues from winter flounder, medaka, and Atlantic salmon (Fig.3 B). Using a probe spanning the entire coding region of the cDNA, we then performed Northern blotting experiments on various chicken tissues (Fig. 4). It is obvious that the only major site of synthesis of chkZP1 mRNA is the liver. Some transcripts are also detectable in the adrenal glands, but at low levels when compared with hepatic tissues (Fig.4 A). The probe hybridizes to a single transcript of approximately 3.2 kilobases, suggesting that major splice variants are not expressed in the tissues studied. In accordance with the results obtained by immunoblotting (Fig. 2), the chkZP1 transcript is absent in rooster liver but can be induced by estrogen treatment of the animal to levels comparable with those detected in mature females (Fig.4 B). It is noteworthy that we could not detect significant expression in gonadal tissues, such as follicles (Fig. 4 B) or