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The zona pellucida surrounds all mammalian oocytes and plays a vital role at fertilization and in early development. The genes that code for two of the mouse zona proteins (ZP2 and ZP3) represent a developmentally regulated set of genes whose expression serves as markers of mouse oocyte growth and differentiation. We previously characterized the single-copy Zp-3 gene and showed that its expression is oocyte specific and restricted to a narrow window of oocyte development. We now define the Zp-2 gene transcript and show that it is coordinately expressed with Zp-3 only during the 2-week growth phase of oogenesis that occurs prior to ovulation. Like Zp-3, the expression of Zp-2 is restricted to oocytes, and, although not detectable in resting oocytes, both ZP2 and ZP3 transcripts accumulate to become very abundant messengers in 50-,um-diameter oocytes. Ovulated eggs contain ZP2 and ZP3 transcripts which are 200 nucleotides shorter than those found in growing oocytes and have an abundance of less than 5% of the peak levels. In an attempt to understand the molecular details associated with the developmentally regulated, tissue-specific gene expression of the zona genes, the Zp-2 genetic locus has been characterized and its 5' flanking sequences have been compared with those of Zp-3. Both genes contain three short (8-to 12-base-pair) DNA sequences of 80 to 88% identity located within 250 base pairs of their transcription start sites.At birth the mouse ovary contains 10,000 to 15,000 primordial oocytes (29), the vast majority of which are in the prophase of the first meiotic division. During the reproductive life of the female, cohorts of these resting oocytes (10 to 15 p.m in diameter) enter into a 2-week growth phase which culminates in meiotic maturation and subsequent ovulation (3, 11). Virtually nothing is known about the signals that induce this growth and differentiation, but one potential marker of these phenomena is the expression of the zona pellucida genes.The zona pellucida surrounds all mammalian oocytes; in the mouse it is composed of three sulfated glycoproteins, ZP1, ZP2, and ZP3 (7,45). The mouse zona proteins are coordinately synthesized in the growing oocyte (8,45) and are secreted to form a filamentous zona matrix (39) in which ZP2 and ZP3 complex into copolymers cross-linked by ZP1 (24). At fertilization, sperm initially bind to ZP3 via 0-linked oligosaccharide side chains (9, 10, 21). Following the induction of the sperm acrosome reaction on the surface of the zona, ZP2 acts as a secondary sperm receptor that is necessary for the maintenance of sperm binding to the egg (6). ZP2 is proteolytically cleaved after fertilization (5, 36), and this modification, along with presumed changes in ZP3, are postulated to play an important role in the postfertilization block to polyspermy.The genes that code for the mouse zona pellucida represent a remarkable set of developmentally regulated, oocytespecific genes which specifiy products vital to fertilization and early mammalian development. We have previously ...

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The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR 3 ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.Extracellular matrices provide assorted roles in biology, and the mechanisms of their formation appear quite disparate. The matrix that surrounds vertebrate eggs and early embryos is variously known as the vitelline envelope, the perivitelline membrane, or the zona pellucida. The component parts are most commonly synthesized and secreted from female germ cells, but in some vertebrates they are produced in the somatic compartment of the ovary or in the liver, which requires both transport to the egg and assembly on its surface. Although the molecular biology of the mouse egg coat has been well studied, little is known about the intracellular protein trafficking of individual components or the mechanisms by which they are secreted to form the insoluble extracellular matrix required for fertilization and embryonic development.The mouse zona pellucida consists of three major sulfated glycoproteins (ZP1, ZP2, and ZP3), each of which is encoded by a single-copy gene in the mouse genome. Although the primary structures of ZP1 (623 amino acids, 68 kDa), ZP2 (713 amino acids, 80 kDa), and ZP3 (424 amino acids, 46 kDa) are distinct, they share certain motifs among themselves, as well as with homologues in other mammals (29). Each zona protein has an N-terminal signal peptide, a signature zona box (260 amino acids with eight conserved cysteine residues, (3), and a transmembrane domain near its carboxyl terminus. The conservation of zona proteins among mammals suggests that the three-dimensional structures of the proteins in each class of zona proteins are similar and that the interactions among classes that effect the supramolecular structure of the zona pellucida may be preserved as well. This hypothesis ha...