Proc. Nati. Acad. Sci. USA Vol. 87, pp. 6014-6018, August 1990 Developmental Biology Human homolog of the mouse sperm receptor (human zona pellucida/ZP3/oocyte-specific gene expression/cis-acting elements/human sperm receptor) MARGARET E. CHAMBERLIN AND JURRIEN DEAN Laboratory of Cellular and Developmental Biology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Christian B. Anfinsen, May 31, 1990 ABSTRACT The human zona pellucida, composed of see ref. 7). The recent cloning of mouse ZP2 and ZP3 cDNAs three glycoproteins (ZP1, ZP2, and ZP3), forms an extracel- (8-10) and the characterization of their genomic loci (8, 11, lular matrix that surrounds ovulated eggs and mediates species- 12) has provided a wealth of molecular detail on the primary specific fertilization. The genes that code for at least two of the structure of the zona proteins and their developmentally zona proteins (ZP2 and ZP3) cross-hybridize with other mam- regulated expression during oogenesis (8, 13). malian DNA. The recently characterized mouse sperm receptor Less is known about the primary structure and the func- gene (Zp-3) was used to isolate its human homolog. The human tions of the zona pellucida proteins from other species, homolog spans =18.3 kilobase pairs (kbp) (compared to 8.6 including that of human. The human zona pellucida is com- kbp for the mouse gene) and contains eight exons, the sizes of posed of three glycoproteins, ZP1 (90-110 kDa), ZP2 (64-76 which are strictly conserved between the two species. Four kDa), and ZP3 (57-73 kDa) (14, 15), at least one of which short (8-15 bp) sequences within the first 250 bp of the 5' appears to be modified following fertilization (15). However, flanking region in the human Zp-3 homolog are also present more detailed biochemical studies of individual human zona upstream ofmouse Zp-3. These elements may modulate oocyte- pellucida proteins are difficult because of the paucity of specific gene expression. By using the polymerase chain reac- biological material. An alternate approach to learning more tion, a full-length cDNA of human ZP3 was isolated from about the human zona pellucida is to deduce the structure of human ovarian poly(A)I RNA and used to deduce the structure individual proteins from their cognate genes. We have dem- of human ZP3 mRNA. Certain features of the human and onstrated (9, 10) that the genes that code for the zona mouse ZP3 transcripts are conserved. Both have unusually pellucida proteins are conserved among mammals. Taking short 5' and 3' untranslated regions, both contain a single open advantage of the cross-hybridization of a mouse cDNA with reading frame that is 74% identical, and both code for 424 human DNA, we now report the isolation of full-length amino acid polypeptides that are 67% the same. The similarity cDNA clones of human ZP3* and the characterization of the between the two proteins may define domains that are impor- genomic locus of human ZP3, the homologue to the mouse tant in maintaining the structural integrity of the zona pellu- sperm receptor. cida, while the differences may play a role in mediating the species-specific events of mammalian fertilization. MATERIALS AND METHODS Human Genomic DNA. A human genomic library in Charon Many details ofmammalian fertilization have been elucidated 4A (16) was screened with pZP3.2 (10). A recombinant, from studies in the mouse. Fertilization begins when a AHuZP3.14, was isolated and plaque-purified. The 14.0- capacitated mouse spermatozoon attaches to the zona pel- kilobase-pair (kbp) insert (containing exons 1-5) was digested lucida, an extracellular matrix that surrounds the ovulated with BamHI and EcoRI, the resultant fragments were sub- egg of mammals. After tight binding to the zona, the dehis- cloned into pBluescript, and the nucleic acid sequence of 11.7 cence of the sperm acrosome results in the release of lytic kbp was determined (17). enzymes that, coupled with the forward motility of the The 3' end of the gene including exons 6-8 was obtained sperm, results in passage through the zona pellucida. The by the polymerase chain reaction (PCR) (GeneAmp; Perkin- sperm then traverses the perivitelline space, fuses with the Elmer/Cetus). One microgram of human genomic DNA from egg's plasma membrane, and is absorbed into the cytoplasm, K562 cells (gift of A. Dean) was primed with oligonucleotide where its nucleus undergoes decondensation and repackag- H78 (exon 6) and H77 (exon 8) for 25 cycles (94°C for 1 min, ing into the male pronucleus of the one-cell zygote (1). 60°C for 2 min, and 72° for 3 min) and a final extension at 72°C The sperm-egg interaction that takes place in the oviduct for 15 min. The 1.8-kbp fragment was blunt end-ligated into after ovulation of the egg and insemination of sperm is pBluescript (Stratagene) and sequenced (17). relatively species-specific (2-5). In part, this specificity is The 3' flanking sequences were determined by PCR of a dependent on the affinity of the spermatozoon for the zona circularized Xba I fragment encompassing exon 8 (Fig. 1). pellucida of the homologous species (for review, see ref. 6). One microgram of human genomic DNA was digested with The mouse zona pellucida is composed of three sulfated Xba I, extracted, precipitated with ETOH, resuspended (1 glycoproteins, ZP1, ZP2, and ZP3, and specific functions ,ug/ml), and cyclized overnight with 10 units ofT4 ligase. The have been ascribed to each. ZP3 induces the sperm acrosome Xba I fragment containing exon 8 and the 3' flanking region reaction and mediates the initial binding of sperm to the egg were amplified by using exon 8 oligonucleotide primers H101 via O-linked oligosaccharide side chains. ZP2 acts as a and H86 for 25 cycles of PCR. The resultant 570-bp fragment secondary sperm receptor and it, along with ZP3, is biochem- was blunt end-ligated into pBluescript and sequenced (17). ically modified after fertilization to provide the postfertiliza- Southern Blot Analysis. Exon-specific DNA probes were tion block to polyspermy. ZP2 and ZP3 exist as dimers in long amplified by PCR with human cDNA as a substrate (see filaments that appear to be cross-linked by ZP1 (for review, below) and the following oligonucleotide primer sets: H36 The publication costs of this article were defrayed in part by page charge Abbreviations: PCR, polymerase chain reaction; nt, nucleotides. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M35109). 6014 Downloaded by guest on October 3, 2021 Developmental Biology: Chamberlin and Dean Proc. Natl. Acad. Sci. USA 87 (1990) 6015 B xB Bg x B Bg X X 111 I I 1VI * 1 A 51 =I I I11 11 0* - 4 - - - 1 1 l 2 3 4 5 67 8 I kb -44+- B Bg Bg B x x Bg I1 I I Ism I I in . I I B I IN I in l 3 l 2 34 5 67 8 FIG. 1. Genomic locusST-of the human homolog of mouse Zp-3. (A) Exon map of human Zp-3 homolog with eight exons (1-8) and restriction enzyme sites for BamHI (B), Bgl II (Bg), and Xba I (X). Horizontal arrows indicate Alu repeat sequences. Solid lines indicate regions for which the nucleic acid sequence was determined. Distance of the dashed line was estimated from Southern blot analysis. (B) Exon map of mouse Zp-3 (12) for comparison. and H75 (exons 4 and 5); H78 and H81 (exons 6 and 7); and was determined (23) as was its hydropathicity (24) and H80 and H77 (exon 8). Labeled probes (18) were used in hydrophilicity (25). Signal peptide cleavage sites were de- Southern blots of human K562 DNA after digestion with a fined by a sliding window-weighted matrix algorithm (26). variety of restriction endonucleases as described (12). Human ZP3 cDNA. Poly(A)+ RNA (19) was purified from RESULTS total RNA (20) isolated from a human ovary (supplied by the National Disease Research Interchange) and used as a tem- Genomic Locus of Human Zp-3 Homolog. A clone contain- plate for first-strand synthesis with oligonucleotide primer ing a 14.0-kbp insert was isolated from a human genomic A2T15 according to the manufacturer's instruction (Promega library (16) screened with a mouse ZP3 cDNA probe (10). Of Riboclone cDNA synthesis system). The first strand was the 14.0-kbp insert, 11.7 kbp were sequenced and contained amplified by PCR with A2T15 and oligonucleotide H36 from the first five exons ofthe human Zp-3 homolog as well as 1.1 human exon 4 (940C for 5 min, 500C for 2 min, and 720C for kbp of the 5' flanking region and 1.78 kbp of intron 5 (27). 40 min, followed by 40 cycles of 940C for 2 min, 580C for 2 Despite repeated screening of the library with probes from min, and 720C for 3 min) (21). The resultant 800-bp band was mouse cDNA specific for exons 6-8, a genomic fragment reamplified with A2 [lacking poly (dT)] and H74 from exon 5 containing the balance of the human exons was not isolated. (25 cycles of 94°C for 1 minm 55°C for 2 min, and 72°C for 3 Instead, the remaining exons of the human Zp-3 homolog min and a final extension at 72°C for 15 min). The 670-bp were characterized by amplifying genomic DNA by the PCR insert was blunt-end ligated into pBluescript and sequenced with oligonucleotides from exons 6 and 8 (based on the cDNA (17).