FOXO3 Expression and Localization During Pig Oocyte Meiosis Maturation and Embryo Early Development.

Forkhead box O3 (FOXO3), a member of the FOXO subfamily of Forkhead transcription factors, has been shown to play critical roles in apoptosis, oxidative stress, cell cycle and DNA repair. A role for ovarian FOXO3 in controlling the rate of primordial follicle activation into the growing follicular pool has been determined. FOXO3-deficient mice had global activation of the primordial follicle pool, while oocyte-specific over-expression of FOXO3 prevented activation of primordial follicles. In both cases, altered FOXO3 was associated with impaired fertility. FOXO3 protein activity is regulated via phosphorylation by kinases, such as the serine/threonine protein kinase (AKT), extracellular signal-regulated kinase (ERK) and I B kinase (IKK) and can be activated by Doxorubicin (DOX), an anti-neoplastic agent. The objective of this study was to characterize FOXO3 expression during oocyte maturation and early embryo development in the pig. We utilized pools of 25 germinal vesicle stage (GV) oocytes, MII-arrested oocytes (MII) and 4/8-cell stage in vitro fertilized (IVF) embryos (n=4 per stage) for quantitative reverse-transcriptase PCR to determine FOXO3 mRNA level. FOXO3 protein was measured using Western blot analysis from pools of 40 GV, MII oocytes, 2-cell and 4/8-cell stage IVF embryos (n=4 per stage). Relative quantification of FOXO3 mRNA demonstrated abundance was greatest in MII-arrested oocytes although not statistically different compared to GV oocytes. FOXO3 mRNA expression was 9.1-fold (P = 0.046) less in 4/8-cell stage embryos compared to MII oocytes. Western blot analysis demonstrated FOXO3 protein was present in all samples and was not significantly different (P > 0.05). By immunocytochemistry, FOXO3 was located throughout the cytoplasm albeit slightly enriched in the nuclear area of GV oocytes. Following in vitro maturation, FOXO3 cytoplasmic staining was much less in MII arrested oocytes (P=0.003). Immunostaining of phosphorylated FOXO3 at Ser253 was strongly enriched in the nuclear area and slightly stained throughout the cytoplasm in GV oocytes, and by the MII oocyte stage, cytoplasmic staining was significantly increased (P=0.014). Co-culture with DOX (2 μM) during in vitro maturation significantly increased total FOXO3 staining (P=0.0007), and decreased the phosphorylated FOXO3 (Ser253) staining (P=0.049) in MII oocytes as compared to controls. IVM with DOX (2μM) numerically increased maturation rate (control = 54.3±6.5%; DOX = 64.8±7.0%; P=0.14, n=3). Collectively, these data demonstrate the dynamic FOXO3 expression and localization in the pig oocyte and early embryo and suggest a role for FOXO3 in oocyte maturation in the pig. This project was supported by National Research Initiative Competitive Grant no. 2008-35205-05309 and 2008-35205-18712 from the USDA National Institute of Food and Agriculture.