168 The Effect of Prematuration Culture Using Epidermal Growth Factor Receptor Kinase Inhibitor on Nuclear Maturation of Bovine Oocytes and Cumulus Cell Gene Expression
Authors: Mingoti G. , Dall'Acqua P. , Nunes G. , Silva C. , Fontes P. , Nogueira M. 
Published: November 2018

Epidermal growth factor receptor (EGFR) activation is an essential step in triggering LH-induced meiotic resumption. Here, we used an EGFR kinase inhibitor (AG1478) to assess the blockage and reversal of meiosis block in bovine oocytes, the competence of such oocytes for the embryonic development, and the gene expression of cumulus cells (CC). Cumulus-oocyte complexes (COC, n = 583) were pre-matured (PM) during 8 h in TCM-199 with 1 μM AG1478 (EGFR-group). Next, COC were washed from meiotic inhibitor and cultured for 22 h in in vitro maturation (IVM) medium (TCM-199 with bicarbonate, 0.5 mg mL−1 FSH, 100 IU mL−1 hCG, and 10% FCS). The control group was only cultured for 22 h in IVM medium. Meiosis progression in oocytes was evaluated after PM (blocked_GV) and after IVM of PM oocytes (blocked_MII) or immediately after follicle aspiration (immature control = control_GV) and IVM (matured control = control_MII); oocytes were classified as immature (germinal vesicle; GV) or mature (metaphase II; MII). Another sample of matured oocytes was fertilized and cultured to the blastocyst stage. Abundance of 81 transcripts was evaluated by qPCR using a microfluidic platform (BioMark HD System™, Fluidigm®, South San Francisco, CA, USA) in CC collected at the same time as oocyte evaluation. Relative mRNA abundance was calculated by ΔCt (target genes were normalized by 2 reference genes: PPIA and RPL15). Data were analysed by ANOVA followed by Tukey’s test (P ≤ 0.05). The GV rates after 8-h PM differed (P ≤ 0.05) between 0-h oocytes (96.4 ± 2.2%) and EGFR- (59.8 ± 2.2%). After 22-h IVM, meiosis block was fully reversed and there was no difference (P ≥ 0.05) in MII rates between treatments (84.8%, averaged). Blastocyst rates (38.2%, averaged) were unaffected by treatment (P ≥ 0.05). Abundance of several transcripts was modulated by PM culture (blocked_GV v. control_GV), including up-regulation of genes that control the expansion of CC/COC competence markers (GREM1 and VCAN), cell proliferation/survival control (IGF1R and EGFR) and maintenance of cellular structural integrity/prediction of embryo quality (KRT8 and GATM), and down-regulation of genes associated with failures in pregnancy establishment/glucose metabolism (AKR1B1), endoplasmic reticulum stress (ATF4), meiotic arrest (ADCY3 and NOS2) and cell survival(HSPA1A). After IVM (blocked_MII v. control_MII), it was found an up-regulation in the relative abundance of PTX3 and PTGS2 (expansion of CC), RGS2 (regulator of G-protein signalling), LUM (embryo quality) and FOXO3 (apoptosis and oxidative stress resistance), whereas relative abundance of PFKP (glucose metabolism) was down-regulated. Our results indicate that blockade of meiosis with the EGFR kinase inhibitor before IVM is reversible and does not affect subsequent embryonic development. The gene expression profile of CC indicates a possible improvement in the quality of COC; however, more studies will be necessary to evaluate whether these improvements will be maintained until the embryonic stage.
Financial support provided by FAPESP (#2015/06733-5 and #2012/50533-2) and CNPq (#307416/2015-1).