Secretion and assembly of zona pellucida glycoproteins by growing mouse oocytes microinjected with epitope-tagged cDNAs for mZP2 and mZP3.
The zona pellucida (ZP) is a highly organized extracellular coat that surrounds all mammalian eggs. The mouse egg ZP is composed of three glycoproteins, called mZP1-3, that are synthesized, secreted, and assembled into a ZP exclusively by growing oocytes. Here, we microinjected epitope-tagged (Myc and Flag) cDNAs for mZP2 and mZP3 into the germinal vesicle (nucleus) of growing oocytes isolated from juvenile mice. Specific antibodies and laser scanning confocal microscopy were used to follow nascent, recombinant ZP glycoproteins in both permeabilized and nonpermeabilized oocytes. When such cDNAs were injected, epitope-tagged mZP2 (Myc-mZP2) and mZP3 (Flag-mZP3) were synthesized, packaged into large intracellular vesicles, and secreted by the vast majority of oocytes. Secreted glycoproteins were incorporated into only the innermost layer of the thickening ZP, and the amount of nascent glycoprotein in this region increased with increasing time of oocyte culture. Consistent with prior observations, the putative transmembrane domain at the C terminus of mZP2 and mZP3 was missing from nascent glycoprotein incorporated into the ZP. When the consensus furin cleavage site near the C terminus of mZP3 was mutated, such that it should not be cleaved by furin, secretion and assembly of mZP3 was reduced. On the other hand, mZP3 incorporated into the ZP lacked the transmembrane domain downstream of the mutated furin cleavage site, suggesting that some other protease(s) excised the domain. These results strongly suggest that nascent mZP2 and mZP3 are incorporated into only the innermost layer of the ZP and that excision of the C-terminal region of the glycoproteins is required for assembly into the oocyte ZP.
Qi
Huayu
H
Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
Williams
Zev
Z
Wassarman
Paul M
PM
D000818:Animals; D018076:DNA, Complementary; D004527:Egg Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes / Q000235:genetics; D005260:Female; D016259:Genes, myc; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008845:Microinjections; D009842:Oligopeptides; D009865:Oocytes / Q000502:physiology* / Q000648:ultrastructure; D010455:Peptides / Q000235:genetics; D011956:Receptors, Cell Surface; D015044:Zona Pellucida / Q000502:physiology*; D000072216:Zona Pellucida Glycoproteins
D018076:DNA, Complementary; D004527:Egg Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D010455:Peptides; D011956:Receptors, Cell Surface; D000072216:Zona Pellucida Glycoproteins; C085706:FLAG peptide