Dynamics and regulation of lysine-acetylation during one-cell stage mouse embryos

Previous studies show that treatment of zygotes with trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), impacts the subsequent development to a blastocyst as well as full-term development. To reveal the dynamics of protein acetylation, with and without TSA treatment during one-cell stage, we examined oocytes and zygotes by immunofluorescence and Western Blot analyses using anti-acetylated lysine and acetylated alpha-tubulin antibodies. In unfertilized oocytes, lysine acetylation level was extremely low over all but faintly detected in the spindle. Once oocyte activation occurs, a dramatic increase of lysine acetylation signal was observed mostly in the pronuclei and a fiber-like structure, the so called midbody, suggesting activation coupled up-regulation of lysine acetylation presumably in histones and alpha-tubulin. TSA treatment resulted in significantly more hyperacetylation not only in the midbody structure and pronuclei but also in the whole cytoplasm. Consistently, Western Blot analysis revealed that acetylation of proteins about 53 kDa and 11 kDa in size, corresponding to alpha-tubulin and histone H4 sizes respectively, were increased mainly after oocyte activation and exclusively enhanced by TSA treatment in zygotes. To confirm this behavior of acetylated nonhistone proteins, acetylated alpha-tubulin was examined and found to be faintly detected in the spindle of MII oocytes but later in whole in the cell of zygotes including the midbody, which was enhanced by TSA treatment. To elucidate the mechanism underlying up-regulation of lysine acetylation following oocyte activation, we assayed the HDAC activity, and found significant reduction of HDAC activity from MII to zygotic stages. Taken together, our data indicate that HDACs play an important role in maintaining low acetylated status in a MII oocyte. However, once an oocyte has been activated, histone and nonhistone proteins including alpha-tubulin are hyperacetylated partly due to a reduction of HDAC activity. TSA treatment of zygotes enhances their acetylation, which could affect subsequent embryonic development. (C) 2013 Elsevier Inc. All rights reserved.