Mouse cortical granules: Biochemical heterogeneity, migration, pre-fertilization release, and identification of a novel cortical granule protein

Cortical granules are specialized secretory vesicles that are positioned near the plasma membrane of oocytes. Following gamete membrane fusion, cortical granules undergo exocytosis, and their contents contribute to formation of the cortical granule envelope and to establishment of the block to polyspermy. Relatively little is known about the biochemical composition, distribution, and pre-fertilization release of mammalian cortical granules. In this study, we have demonstrated that mouse oocytes contain at least two biochemically distinct populations of cortical granules, of which one binds to both the lectin LCA and the ABL2 antibody and the other binds only to LCA. Both populations of cortical granules were evenly distributed in the cortex of germinal vesicle intact oocytes. However, a cortical granule free domain, which was devoid of both types of granules, formed over the spindle as the oocytes matured into metaphase I. During first polar body extrusion, a subpopulation of LCA-binding cortical granules became concentrated around the cleavage furrow, where they underwent exocytosis. As the oocytes entered metaphase II, a second cortical granule free domain developed as both types of granules over the spindle were redistributed toward the equator.;The ABL2 antibody recognizes a protein with a 75 kDa protein (p75) in mouse cortical granules. To identify the mouse cortical granule protein, p75 was isolated and subjected to amino acid sequencing using tandem mass spectrometry. p75 was identified as peptidylarginine deiminase (PAD) based on a partial amino acid sequence (VLIGGSFY). When a PAD antibody was used to probe western blots of p75, a single protein band was detected with a molecular weight of 75 kDa, confirming the identification of p75. PAD was demonstrated cytochemically in mouse cortical granules, and the protein was exocytosed extracellularly following fertilization or activation. After the cortical reaction, PAD remained associated with oolemma and behaved as a peripheral membrane protein. PAD remained on the blastomeres' plasma membranes until the blastocyst stage. In vitro treatment of 2-cell mouse embryos with various PAD antibodies and with the ABL2 antibody inhibited preimplantation embryo development, suggesting that the cortical granule PAD following its release functions in regulating early embryogenesis.