Subcellular Distribution of ZP1, ZP2, and ZP3 Glycoproteins During Folliculogenesis and Demonstration of Their Topographical Disposition Within the Zona Matrix of Mouse Ovarian Oocytes1

Abstract

The zona pellucida (ZP) is an extracellular coat synthesized and secreted by the oocyte during follicular development and surrounding the plasma membrane of mammalian eggs. To date, the mechanism of synthesis and secretion, mode of assembly, and intracellular trafficking of the ZP glycoproteins have not been fully elucidated. Using antibodies against mouse ZP1, ZP2, and ZP3 in conjunction with the protein A-gold technique, we have shown an association of immunolabeling with the Golgi apparatus, secretory granules, and a complex structure called vesicular aggregate, respectively, in mouse ovarian follicles. In contrast, the neighboring granulosa cells were not reactive to any of the three antibodies used. Immunolabeling of ZP1, ZP2, and ZP3 was detected throughout the entire thickness of the ZP, irrespective of the developmental stage of ovarian follicles. Double and triple immunolocalization studies, using antibodies tagged directly to different sizes of gold particles, revealed an asymmetric spatial distribution of the three ZP glycoproteins in the zona matrix at various stages of follicular development. All three glycoproteins were specifically localized over small patches of darkly stained flocculent substance dispersed throughout the zona matrix. Very often, ZP1, ZP2, and ZP3 were found in close association. These results confirm findings from previous studies demonstrating that ovarian oocytes and not granulosa cells are the only source for mouse ZP glycoproteins. In addition, results from our morphological and immunocytochemical experiments suggest that the vesicular aggregates in the ooplasm are likely to serve as an intermediary in the synthesis and secretion of ZP glycoproteins. The stoichiometric disposition of ZP1, ZP2, and ZP3 in the zona matrix as revealed by double and triple immunolocalization studies provide further insight into some of the unanswered questions pertinent to the current model of mouse ZP structure proposed by the Wassarman group.

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

“…At the ultrastructural level, the pleiomorphic structures appeared as MVA, consisting of vesicles (50 to 300 nm) embedded in an amorphous material which included ZP3 (Fig. 1F) and ZP2 as reported in earlier studies (1,12).…”

Sperm arylsulfatase A binds to mZP2 and mZP3 glycoproteins in a nonenzymatic manner

“…proacrosin, sp56, zonadhesin, sp38), and proacrosin, like ASA, has the affinity for both mZP2 and mZP3. Electron microscopy immunogold labeling also indicates that mZP2 and mZP3 are localized next to each other on the ZP (El Mestrah et al 2002), and both of these ZP glycoproteins have the propensity to form fibrils (Greve & Wassarman 1985, Litscher et al 2008, Jimenez-Movilla & Dean 2011. The ability of sperm proteins, including ASA (this report) and proacrosin (Howes et al 2001), to bind to both mZP2 and mZP3 should give enhanced interaction between sperm and the ZP.…”

ZP2 and ZP3 cytoplasmic tails prevent premature interactions and ensure incorporation into the zona pellucida

“…Together these post-translational modifications account for roughly one-half of the mass of ZP2 and ZP3 on SDS-PAGE gels (Nagdas et al, 1994;Easton et al, 2000;Boja et al, 2003). The two proteins are observed in peripherally located multivesicular aggregates (MVAs) consisting of 1-5 m vesicles embedded in an amorphous matrix before transit to the plasma membrane and ultimate incorporation into the extracellular zona matrix (Merchant and Chang, 1971;El Mestrah et al, 2002;Hoodbhoy et al, 2006). ZP2 and ZP3 with intact transmembrane domains co-expressed in heterologous cells and oocytes are localized in vesicle-like structures, but seem more diffuse when transmembrane domains are absent (TM).…”