Abstract
BackgroundWhile mammalian cortical granules are important in fertilization, their biochemical composition and functions are not fully understood. We previously showed that the ABL2 antibody, made against zona free mouse blastocysts, binds to a 75-kDa cortical granule protein (p75) present in a subpopulation of mouse cortical granules. The purpose of this study was to identify and characterize p75, examine its distribution in unfertilized oocytes and preimplantation embryos, and investigate its biological role in fertilization.ResultsTo identify p75, the protein was immunoprecipitated from ovarian lysates with the ABL2 antibody and analyzed by tandem mass spectrometry (MS/MS). A partial amino acid sequence (VLIGGSFY) was obtained, searched against the NCBI nonredundant database using two independent programs, and matched to mouse peptidylarginine deiminase (PAD). When PAD antibody was used to probe western blots of p75, the antibody detected a single protein band with a molecular weight of 75 kDa, confirming our mass spectrometric identification of p75. Immunohistochemistry demonstrated that PAD was present in the cortical granules of unfertilized oocytes and was released from activated and in vivo fertilized oocytes. After its release, PAD was observed in the perivitelline space, and some PAD remained associated with the oolemma and blastomeres' plasma membranes as a peripheral membrane protein until the blastocyst stage of development. In vitro treatment of 2-cell embryos with the ABL2 antibody or a PAD specific antibody retarded preimplantation development, suggesting that cortical granule PAD plays a role after its release in preimplantation cleavage and early embryonic development.ConclusionOur data showed that PAD is present in the cortical granules of mouse oocytes, is released extracellularly during the cortical reaction, and remains associated with the blastomeres' surfaces as a peripheral membrane protein until the blastocyst stage of development. Our in vitro study supports the idea that extracellular PAD functions in preimplantation development.
Highlights
While mammalian cortical granules are important in fertilization, their biochemical composition and functions are not fully understood
The ABL2 antibody recognizes a 75-kDa ovarian protein that is present in cortical granules of mouse oocytes The ABL2 antibody precipitates a 75 kDa protein (p75) from mouse oocytes [20]
Other bands in the gel are from the antibody used for immunoprecipitation. (B) Confocal scanning laser micrographs of germinal vesicle intact mouse oocytes double labeled with the lectin Lens culinaris agglutinin (LCA) (LCA) and the ABL2 antibody (ABL2)
Summary
While mammalian cortical granules are important in fertilization, their biochemical composition and functions are not fully understood. Mammalian cortical granules are membrane-bound organelles located in the cortex of unfertilized oocytes [1,2]. While the biological significance of this pre-fertilization release is not yet known, it likely plays a role in fertilization since it occurs at a specific time and place and involves a specific population of cortical granules. These prior studies show that mammalian cortical granules are released both before and after fertilization and that their functions are probably more complex than previously realized
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