Characterization of Metastasis Associated Protein 3 (MTA3) in Mouse Oocytes and Pre-Implantation Embryos.

Abstract

Metastasis associated protein 3 (MTA3) is a constituent of the Mi-2/nucleosome remodeling and deacetylase (Mi-2/NuRD) complex that regulates gene expression by altering chromatin structure, e.g., histone deacetylation. The biological function of MTA3 within the Mi-2/NuRD complex is unknown. An initial computational analysis of expressed sequence tags in the GenBank database revealed very high relative expression of MTA3 in mouse oocytes, fertilized eggs and blastocysts when compared to all other screened tissues. Because global chromatin reorganization is a critical aspect of preimplantation embryogenesis, we initiated studies to characterize MTA3 expression at this time of development. Immunohistochemical analysis of postnatal ovaries revealed that MTA3 is highly expressed in the germinal vesicle (GV) region of oocytes throughout postnatal development and in the nucleus of granulosa cells at all ovarian follicle stages. Surprisingly, we also found very high MTA3 expression in the cortical region of oocytes at all stages of follicular development. Confocal microscopic analysis of oocytes confirmed that MTA3 was present in the GV but that the perinuclear and cortical localization was much greater. During the progression of preimplantation development we observed nuclear localization that was most pronounced in the morula and blastocyst stages; however, the majority of MTA3 was in the cytoplasm where it localized to clusters of increasing size. Western blot analysis demonstrated that MTA3 protein expression in whole ovarian extracts gradually increased and reached its maximal level on postnatal day 19. MTA3 was also detected by immunoblotting of isolated GV oocytes and ovulated eggs. Although the amount of protein did not change, there was a shift in apparent size of MTA3 that suggested a post-translational protein modification occurs during oocyte maturation. Sequence analysis of isolated mRNA revealed that the predominant MTA3 variant in granulosa cells has 1679 nucleotides, whereas the predominant variant present in eggs and blastocysts has 1682 nucleotides because there is a 3 nucleotide insertion encoding a serine residue. This difference in the MTA3 protein sequence and its predominant expression in the cytoplasmic compartment suggest that it has functions in oocytes and preimplantation embryos in addition to supporting Mi-2/NuRD chromatin remodeling. (poster)