Conserved furin cleavage site not essential for secretion and integration of ZP3 into the extracellular egg coat of transgenic mice.

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 has been substantiated by the ability of transgenic mice expressing human ZP3 to restore the zona matrix and fertility in Zp3-null mice that are otherwise unable to form a zona pellucida (33). Additionally, although Xenopus laevis and mammals evolutionarily diverged 350 to 370 million years ago (16), microinjected mRNAs encoding mouse ZP1, ZP2, and ZP3 are translated and incorporated into the extracellular vitelline envelop surrounding Xenopus eggs (7). The zona pellucida matrix is assembled during oogenesis as the oocyte grows from ∼15 to 80 μm prior to ovulation into the oviduct. Targeted mutagenesis of each mouse zona gene indicates that either ZP1 and ZP3 or ZP2 and ZP3 are sufficient for zona matrix formation, although the ZP1-and-ZP3 matrix is not sustained through the latter stages of oogenesis (18, 30-32). The detection of zona proteins (ZP1and ZP2) at the zona-free oocyte surface from Zp3-null mice (30) is consistent with tethering of the zona proteins to a plasma membrane. However, the presence of the perivitelline space between the plasma membrane of the mature eggs and the extracellular zona matrix indicates that the zona proteins are subsequently released from the membrane for incorporation into the zona pellucida. A conserved furin cleavage site (RXK/RR) upstream of the carboxyl-terminal transmembrane domain in most, but not all, mammalian zona proteins has been suggested as the processing site for cleavage and release of zona proteins (17, 42, 44). In this study, by using site-specific mutagenesis, we examined the involvement of the furin recognition site in the secretion of mouse ZP3 in mouse embryonic fibroblasts and its incorporation into the zona pellucida surrounding mouse oocytes. Unexpectedly, our data indicate that cleavage at the conserved furin site is not required for ZP3 secretion or its participation in the extracellular egg coat.