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ePAD; oocyte PAD OKDB#: 1750
 Symbols: ePAD; oocyte PAD Species: mouse
 Synonyms:  Locus:


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General Comment Wright PW, et al 2003 reported that ePAD is an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets. Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.
General function
Comment
Cellular localization Cytoplasmic
Comment
Ovarian function Oogenesis
Comment Peptidylarginine deiminase (PAD) is a mouse cortical granule protein that plays a role in preimplantation embryonic development Liu M, et al . BACKGROUND: While 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. RESULTS: To 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. CONCLUSION: Our 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. Wright PW, et al 2003 reported that ePAD is an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets. Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.
Expression regulated by
Comment
Ovarian localization Oocyte
Comment
Follicle stages Primordial, Primary, Secondary, Antral, Preovulatory
Comment
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created: March 30, 2003, 10:03 a.m. by: hsueh   email:
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last update: July 2, 2007, 10:28 a.m. by: hsueh    email:



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