We have shown okadaic acid (OA) and calyculin-A (CLA) inhibition of mouse oocyte phosphoprotein phosphatase 1 (PPP1C) and/or phosphoprotein phosphatase 2A (PPP2CA) results in aberrant chromatin condensation, as evidenced by the inability to resolve bivalents. Phosphorylation of histone H3 at specific residues is thought to regulate chromatin condensation. Therefore, we examined changes in histone H3 phosphorylation during oocyte meiosis and the potential regulation by protein PPPs. Western blot and immunocytochemical analysis revealed histone H3 phosphorylation changed during mouse oocyte meiosis, with changes in chromatin condensation. Germinal vesicle-intact (GV-intact; 0 h) oocytes had no phospho-Ser10 but did have phospho-Ser28 histone H3. Oocytes that had undergone germinal vesicle breakdown (GVBD; 2 h) and progressed to metaphase I (MI; 7 h) and MII (16 h) had phosphorylated Ser10 and Ser28 histone H3 associated with condensed chromatin. To determine whether OA-induced aberrations in chromatin condensation were due to alterations in levels of histone H3 phosphorylation, we assessed phosphorylation of Ser10 and Ser28 residues following PPP inhibition. Oocytes treated with OA (1 μM) displayed increased phosphorylation of histone H3 at both Ser10 and Ser28 compared with controls. To begin to elucidate which OA-sensitive PPP is responsible for regulating chromatin condensation and histone H3 phosphorylation, we examined spatial and temporal localization of OA-sensitive PPPs, PPP1C, and PPP2CA. PPPC2A did not localize to condensed chromatin, whereas PPP1beta (PPP1CB) associated with condensing chromatin in GVBD, MI, and MII oocytes. Additionally, Western blot and immunocytochemistry confirmed presence of the PPP1C regulatory inhibitor subunit 2 (PPP1R2) in oocytes at condensed chromatin during meiosis and indicated a change in PPP1R2 phosphorylation. Inhibition of oocyte glycogen synthase kinase 3 (GSK3) appeared to regulate phosphorylation of PPP1R2. Furthermore, inhibition of GSK3 resulted in aberrant oocyte bivalent formation similar to that observed following PPP inhibition. These data suggest that PPP1CB is the OA/CLA-sensitive PPP that regulates oocyte chromatin condensation through regulation of histone H3 phosphorylation. Furthermore, GSK3 inhibition results in aberrant chromatin condensation and appears to regulate phosphorylation of PPP1R2.
You have requested a machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Neither BioOne nor the owners and publishers of the content make, and they explicitly disclaim, any express or implied representations or warranties of any kind, including, without limitation, representations and warranties as to the functionality of the translation feature or the accuracy or completeness of the translations.
Translations are not retained in our system. Your use of this feature and the translations is subject to all use restrictions contained in the Terms and Conditions of Use of the BioOne website.
Vol. 76 • No. 4