ZP2 and ZP3 Traffic Independently within Oocytes prior to Assembly into the Extracellular Zona Pellucida

Abstract

The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1-to 5-m post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.

A Structural View of Egg Coat Architecture and Function in Fertilization1

“…On the other hand, consistent with the finding that ZP subunits traffic independently within oocytes before incorporation into the ZP [49], the position of the EHP does not support the idea that the patch might allow the precursors of ZP subunits to form complexes inside the oocyte [30]; rather, EHP appears to lock the ZP-N and ZP-C domains of the ZP3 precursor in a conformation that is incompatible with polymerization [25,43]. So what could happen when the CTP, which contains the EHP and the membrane anchor, is cleaved and leaves the dimer?…”

“…Moreover, a conserved N-glycosylation site (N159 in chicken ZP3), which was inactivated in the protein used for crystallography, is located in a disordered region of the interdomain loop that precedes T168 by a few residues. As in the case of the Oglycan attached to T168, this N-glycan is not required for secretion in either chicken ZP3 [43] or mouse ZP3 [49,57], and it might also contribute to sperm binding, at least in some species [41,42]. Notably, the structure suggests that this and all other Nglycans of mouse and human ZP3 are exposed on the extracellular side of the molecule.…”

Immunoglobulin to zona pellucida 3 mediates ovarian damage and infertility after contraceptive vaccination in mice

“…The thin, irregular appearance of the zona pellucida from rMCMV-mZP3 infected immunocompetent mice suggests that ZP formation has been disrupted by the presence of ZP-specific antibody. Antibodies generated by inoculating specific ZP3 B cell epitope peptides in adjuvant were reported to disrupt the ZP structure in the ovaries of inoculated mice, presumably due to the inability of ZP2 and ZP3 to co-localize during ZP synthesis [22,23]. Our data support these results.…”

Influence of the zona pellucida of the mouse egg on folliculogenesis and fertility

“…However, mZP2 and mZP3 are dependent on each other for incorporation into the ZP and apparently do so by a stochastic process (Tong et al, 1995;Qi and Wassarman, 1999;Hoodbhoy et al, 2006). In this context, isolated ZPDs polymerize into long fibrils on their own and it is the N-terminal sub-domain that is responsible for polymerization (Jovine et al, 2002).…”