At fertilization, mouse sperm bind to the zona pellucida (which consists of glycoproteins ZP1, ZP2, and ZP3) that surrounds eggs. A ZP2 cleavage model of gamete recognition requires intact ZP2, and a glycan release model postulates that zona glycans are ligands for sperm. These two models were tested by replacing endogenous protein with ZP2 that cannot be cleaved (Zp2Mut) or with ZP3 lacking implicated O glycans (Zp3Mut). Sperm bound to two-cell Zp2Mut embryos despite fertilization and cortical granule exocytosis. Contrary to prediction, sperm fertilized Zp3Mut eggs. Sperm at the surface of the zona pellucida remained acrosome-intact for more than 2 hours and were displaced by additional sperm. These data indicate that sperm-egg recognition depends on the cleavage status of ZP2 and that binding at the surface of the zona is not sufficient to induce sperm acrosome exocytosis.

The zona pellucida surrounding ovulated mouse eggs contains three glycoproteins, two of which (ZP2 and ZP3) are reported sperm receptors. After fertilization, the zona pellucida is modified ad minimus by cleavage of ZP2, and sperm no longer bind. Crosstaxa sperm binding is limited among mammals, and human sperm do not bind to mouse eggs. Using transgenesis to replace mouse ZP2 and/or ZP3 with human homologs, mouse lines with human-mouse chimeric zonae pellucidae have been established. Unexpectedly, mouse, but not human, sperm bind to huZP2 and huZP2/huZP3 rescue eggs, eggs fertilized in vitro with mouse sperm progress to two-cell embryos, and rescue mice are fertile. Also unanticipated, human ZP2 remains uncleaved after fertilization, and mouse sperm continue to bind early rescue embryos. These observations are consistent with a model in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.

Adeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) and on expression of the rep gene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR-AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest.

Zp1 encodes one of the three major glycoproteins of the zona pellucida, an extracellular matrix that surrounds growing oocytes, ovulated eggs, and preimplantation embryos. The mouse gene is composed of 12 exons ranging in size from 82 to 364 base pairs and spans 6.5 kilobase pairs on chromosome 19 (2.13 ؎ 1.5 centimorgans distal to D19Bir1). The Zp1 exon map is similar to ZPB, a human orthologue, and an E-box (CANNTG), implicated in oocyte-specific gene expression of mouse Zp2 and Zp3, is similarly located upstream of the transcription start site. The single copy Zp1 gene encodes a 623-amino acid protein, the carboxyl-terminal half of which is significantly similar to a corresponding region of mouse ZP2. The conservation of this same region in a fish egg envelope protein suggests that not only has this protein domain been duplicated in mammals but that it has been conserved and used as an egg envelope protein in species that diverged 650 million years ago.Among vertebrates, different reproductive strategies have evolved based on mating behavior, gamete structures, and the specificity of recognition molecules on the surface of sperm and eggs. In all vertebrates, however, a prerequisite to successful fertilization is penetration of sperm through an acellular envelope surrounding ovulated eggs. In mammals, capacitated sperm bind in a seemingly non-site-directed manner to the zona pellucida. Following the induction of the acrosome reaction and release of lytic enzymes, sperm penetrate the zona and fuse with the egg's plasma membrane, triggering the postfertilization block to polyspermy (1). In contrast, most fish sperm lack an acrosome and penetrate the vitelline envelope surrounding fish eggs via a discrete micropyle (2). Most commonly, the micropylar channel is sufficiently narrow to permit the passage of a single sperm, and subsequent fusion with the plasma membrane induces the cortical granule reaction, resulting in a block to polyspermy (3). It has become increasingly clear that the proteins of the zona pellucida are conserved among eutherian mammals and that the proteins of the vitelline envelope are conserved among teleostean fish. More recently, it has become apparent that, although critical for speciation, the proteins from the mammalian egg envelope are distinctly related to those of the teleostean envelope.The mouse zona pellucida contains three major glycoproteins: ZP1, ZP2, ZP3. Genes encoding the latter two zona proteins have been characterized. Zp2 is composed of 18 exons (4), of which six encode a 241-amino acid domain reported as 28% identical with the wf& protein of the white flounder teleost (5). Zp3 contains eight exons (6, 7), of which the first six encode a 261-amino acid domain that is 33% identical with ZI-3, a major component of the inner layer of the egg envelope of a second teleost, Oryzias latipes (8). Although similar structural domains are present in egg envelope proteins of teleosts and eutherian mammals, the site of synthesis is quite different in these two classes of vertebrates. In ...