Specific inhibition of mouse oocyte nuclear protein phosphatase-1 stimulates germinal vesicle breakdown

Abstract

Okadaic acid (OA)-induced germinal vesicle breakdown (GVBD) and localization of protein phosphatase-1 (PP1) in oocyte nuclei are suggestive of PP1's role in regulating oocyte GVBD. To explore this possibility, we microinjected protein phosphatase (PP) inhibitors OA, anti-PP1 antibody (anti-PP1), PP1 inhibitor I2, and anti-PP2A antibody (anti-PP2A) into nuclei of roscovitine (ROSC)-arrested mouse oocytes. Oocytes were also injected with recombinant PP1 in the absence of ROSC. Oocytes were assessed for GVBD and metaphase II (MII) development at 2 and 18 hr post-injection. Data were analyzed using Cochran-Mantel-Haenszel Statistics adjusted for time. Microinjection of OA significantly enhanced GVBD in comparison to controls at 2 and 18 hr ( P < 0.01), yet had no effect on MII development. Similarly, microinjection of anti-PP1 resulted in significantly higher levels of GVBD compared to controls at 2 and 18 hr ( P < 0.01). Interestingly, anti-PP1 microinjection also tended to enhance MII development at 18 hr in comparison to controls ( P < 0.09). Microinjection of I2, anti-PP2A, and PP1 had no effect on GVBD or MII development. If reduction of PP1 activity was important for GVBD, one would anticipate an endogenous means of regulating PP1 activity at this developmental stage. In somatic cells, phosphorylation of PP1 at Thr320 causes PP1 inactivation. Germinal vesicle-intact oocytes did not contain phosphorylated PP1, as determined using a specific Thr320-Phospho-PP1 antibody, Western blot analysis, and confocal immunocytochemistry. At or around the time of GVBD, oocyte PP1 became phosphorylated at Thr320, which remained phosphorylated through MII development. These data indicate that inhibition of intra-nuclear PP1, through specific antibody neutralization, mimics OA-stimulated GVBD, providing the first direct evidence that nuclear PP1 is involved in regulation of oocyte nuclear membrane integrity. In addition, phosphorylation of PP1 occurs at/or around GVBD indicating that inactivation of PP1 is an important intracellular event in regulation of nuclear envelope dissolution at GVBD. Mol. Reprod. Dev. 65: 96–103, 2003. © 2003 Wiley-Liss, Inc.