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Wolfgang Tomek and Tatjana Smiljakovic

In somatic cells, the serine/threonine kinase Akt (or protein kinase B) was shown to contribute to processes linked to cellular growth, cell survival and cell cycle regulation. In contrast to these findings, the function of Akt during the meiosis of mammalian oocytes remains to be investigated. We analysed the phosphorylation pattern and the activity of Akt during meiotic maturation (transition from prophase I to metaphase II) of bovine oocytes. The oocytes were matured in vitro (IVM) for 0, 10 and 24 h to reach the germinal vesicle (GV), metaphase I (M I) and metaphase II (M II) stages respectively. The abundance and phosphorylation pattern of Akt was revealed by Western blotting using total Akt or phosphoso-Akt-specific antibodies. The activity of this particular kinase was determined by an in vitro kinase assay. Furthermore, functional properties were analysed by cultivating oocytes in the presence of the Akt inhibitor SH6. The results showed that the overall abundance of Akt did not change significantly during IVM. On the other hand, Akt became phosphorylated at Thr 308 and Ser 473, reaching its maximum at the M I phase. In the GV and M II stages, only low basal phosphorylation levels were observed on both sides. This phosphorylation profile corresponded strictly to the activity of the kinase. The cultivation of oocytes in the presence of the phosphatidylinositol analogue SH6 for 24 h showed that, with higher concentrations, up to 65% of the oocytes were arrested in the M I stage. This result indicated that Akt is involved in the M I/M II transition during the meiotic maturation of bovine oocytes. The physiological aspects of the Akt function will be discussed.

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Helmut Torner, Nasser Ghanem, Christina Ambros, Michael Hölker, Wolfgang Tomek, Chirawath Phatsara, Hannelore Alm, Marc-André Sirard, Wilhelm Kanitz, Karl Schellander, and Dawit Tesfaye

Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here, we aim to identify molecular and functional markers associated with oocyte developmental potential when selected based on G6PDH activity. Immature compact cumulus–oocyte complexes were stained with brilliant cresyl blue (BCB) for 90 min. Based on their colouration, oocytes were divided into BCB (colourless cytoplasm, high G6PDH activity) and BCB+ (coloured cytoplasm, low G6PDH activity). The chromatin configuration of the nucleus and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement. The abundance and phosphorylation pattern of protein kinases Akt and MAP were estimated by Western blot analysis. A bovine cDNA microarray was used to analyse the gene expression profiles of BCB+ and BCB oocytes. Consequently, marked differences were found in blastocyst rate at day 8 between BCB+ (33.1±3.1%) and BCB (12.1±1.5%) oocytes. Moreover, BCB+ oocytes were found to show higher phosphorylation levels of Akt and MAP kinases and are enriched with genes regulating transcription (SMARCA5), cell cycle (nuclear autoantigenic sperm protein, NASP) and protein biosynthesis (RPS274A and mRNA for elongation factor 1α, EF1A). BCB oocytes, which revealed higher mitochondrial activity and still nucleoli in their germinal vesicles, were enriched with genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10) and growth factor activity (bone morphogenetic protein 15, BMP15). This study has evidenced molecular and subcellular organisational differences of oocytes with different G6PDH activity.