276 PHOSPHOINOSITIDE 3-KINASE REGULATES EXPRESSION OF KEY ENZYMES AND CELLULAR TRANSPORTERS OF GLUCOSE METABOLISM IN CUMULUS CELLS OF BOVINE COCs CULTURED IN VITRO
D. Kaiser de Souza A D , L. P. Salles A B , R. Camargo A C , B. Dolabela de Lima A C , F. A. G. Torres A B and A. A. M. Rosa e Silva A DA University of Brasilia, Brasilia, DF, Brazil;
B Laboratory of Molecular Biology, Department of Cellular Biology, Brasilia, DF, Brazil;
C Laboratory of Microbiology, Department of Cellular Biology, Brasilia, DF, Brazil;
D Laboratory of Biotechnology of Reproduction, Brasilia, DF, Brazil
Reproduction, Fertility and Development 27(1) 227-227 https://doi.org/10.1071/RDv27n1Ab276
Published: 4 December 2014
Abstract
The aim was to analyse the function of the PI3K pathway during oocyte maturation in bovine by use of the specific inhibitor, LY294002. Genes studied in cumulus cells (CC) were PDH, G6PDH, GLUT1, and GLUT4. PDH is an important enzyme for oxidative metabolism, G6PDH is related to resumption and progression of oocyte meiosis, and GLUT1 and GLUT4 are glucose transporters. This study was performed in defined medium (MIV B) in absence or presence of 10 ng mL–1 of FSH. Polar body extrusion was analysed after culture and correlated with gene expression. Experimental methods were bovine COC (n = 35–40/well, n = 3 replicates) collected from ovaries obtained from abattoirs (DF, Brazil) and cultivated in either 400 µL of medium MIV B, or MIV B + 100 µM of LY294002, or MIV B + 10 ng mL–1 of FSH; or MIV B + 10 ng mL–1 of FSH + 100 µM of LY294002 during 22 to 24 h. After culture, COC were mechanically denuded and CC from 20 COC/group were isolated. Gene expression of GLUT1, GLUT4, G6PDH, and PDH were measured by real time PCR. The CC of immature COC were also collected and analysed as the calibrator group. Student-Newman-Keuls was performed as a statistical test. The percentage of oocytes that extruded the polar body was determined. Two-way ANOVA, followed by Bonferroni test, and t-test were performed to determine statistical significance. In MIV B, the polar body extrusion rate was 25.48 ± 7.64%, while FSH increased it up to 74.52 ± 10.58% (P < 0.05). The extrusion of polar body was inhibited by LY294002 in the absence or presence of FSH (67.51 ± 6.13 and 31.02 ± 16.97%, respectively, P < 0.05). Gene expression of PDH was not altered by any culture medium in contrast to GLUT1, GLUT4, and G6PDH expression (Table 1). Only G6PDH expression showed the same pattern as the polar body extrusion, in absence or presence of FSH. In conclusion, PI3-K inhibition affects polar body extrusion and expression of genes related to glucose metabolism in CC. Lower G6PDH expression in CC may be related to low rates of polar body extrusion in treated oocytes.
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The authors thank FAP-DF (193.000.577/2009), CNPq, CAPES and Ponte Alta abattoir, Brasilia.
