Molecular and subcellular characterisation of oocytes screened for their developmental competence based on glucose-6-phosphate dehydrogenase activity.
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 1alpha, 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.
Torner
Helmut
H
Department of Animal Breeding and Husbandry, Institute of Animal Science, University of Bonn, Endenicher allee 15, 53115 Bonn, Germany.
Ghanem
Nasser
N
Ambros
Christina
C
Hölker
Michael
M
Tomek
Wolfgang
W
Phatsara
Chirawath
C
Alm
Hannelore
H
Sirard
Marc-André
MA
Kanitz
Wilhelm
W
Schellander
Karl
K
Tesfaye
Dawit
D
D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D004396:Coloring Agents; D048049:Extracellular Signal-Regulated MAP Kinases / Q000032:analysis / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D051058:Oncogene Protein v-akt / Q000032:analysis / Q000378:metabolism; D009865:Oocytes / Q000201:enzymology* / Q000378:metabolism; D009866:Oogenesis / Q000235:genetics / Q000502:physiology*; D010078:Oxazines; D010766:Phosphorylation; D013194:Staining and Labeling / Q000379:methods
D004396:Coloring Agents; D010078:Oxazines; C006797:Brilliant Cresyl Blue; D005954:Glucosephosphate Dehydrogenase; D051058:Oncogene Protein v-akt; D048049:Extracellular Signal-Regulated MAP Kinases