PLATFORM SESSION 4. GAMETE BIOLOGY AND GAMETOGENESIS I
Sunday, August 1, 2004
3:00 PM–5:00 PM
Chairs: Leslie Heckert
Co-Chair: Jason Swain
(30) DISTRIBUTION AND EXPRESSION OF AKT/PROTEIN KINASE B DURING OOCYTE MEIOTIC MATURATION AND PREIMPLANTATION DEVELOPMENT IN MICE.
Hoshino, Yumi1, Matsumoto, Hiromichi1, Sasada, Hiroshi1, Sato, Eimei1, 1 Laboratory of Animal Reproduction, Sendai, Japan
ABSTRACT- Previously, we reported that phosphatidylinositol 3-kinase (PI3K) participates in meiotic maturation in mice. Akt, known as protein kinase B, has been identified as a serine-threonine kinase, and the activation of Akt is thought to be a critical step in the PI3K pathway that regulates cell growth and differentiation. Akt becomes phosphorylated at two residues, Thr308 and Ser473, and both are required for full activation. Therefore, we examined the distribution of total Akt, Thr308-phosphorylated Akt and Ser473-phosphorylated Akt during meiotic maturation in mouse oocytes. Confocal scaning laser microscopy analysis showed immunoreactive Akt was localized in both the cytoplasm and germinal vesicle (GV) in oocytes, while it was concentrated within the chromatin in germinal vesicle breakdown (GVBD) oocytes. At metaphase I (MI) and metaphase II (MII), localization of Akt was distributed in the spindle. Western blot analysis showed that expression of total Akt protein was at the same level in GV and MII oocytes. The expression of Akt mRNA was also detected at the same level in GV and MII oocytes. Immunodetection of Thr308-phosphorylated Akt was localized in a punctate pattern around chromosomes and in the cytoplasm in PMI oocytes. At MI and MII, four intense dots were observed. Merged figures with staining of microtubules and nuclei showed that Thr308-phosphorylated Akt was present in the region of pericentriolar materials (PCM). In contrast, the distribution of Ser473-phosphorylated Akt was similar to that of microtubules at PMI and was localized in spindles at MI and MII, while the expression of Akt was detected at very low to undetectable levels in embryos during preimplantation development at any stages. Western blot analysis also showed very low to undetectable levels of expression. In conclusion, Akt was involved in mouse meiotic maturation, whereas Akt protein disappeared after the pronuclear stage. The activity of Akt would be related to spindle formation in mouse oocytes. Thr308-phosphorylated Akt and Ser473-phosphorylated Akt may have different functions because they showed different localization at metaphase during meiotic maturation.
KEY WORDS: akt/protein kinase b, metaphase, oocyte meiotic maturation, spindle formation