Oocyte competence is acquired during the period of oocyte growth and maturation and is associated with follicular size, estrous cycle stage, level of atresia and can be related to the abundance of specific transcripts during oocyte growth and the final steps of folliculogenesis. The difference between competent and incompetent oocytes may depend on differential gene expression patterns. The cumulus cells that surround the oocyte play an essential role in controlling oocyte growth, maturation, fertilization and the ability to support further embryonic development. Cumulus cells maintain metabolic cooperation with oocytes, a crucial interaction for the regulation of maturation and chromatin remodeling of bovine oocytes and for the full competence of both cell types to support early embryogenesis. Because of this important connection during follicular development and maturation, analysis of these cells may provide important information regarding the acquisition of competence by oocytes. Thus, the aim of this study is to establish a methodology for gene silencing by lipofection in cumulus cells to evaluate the communication of cumulus cells and oocyte, by investigating the functional implications of these genes during in vitro maturation on oocyte competence, in order to provide further knowledge which may help to improve in vitro embryo production systems. For this purpose, the expression profiles of mRNAs coding for BMPRII and EGFR will be firstly investigated in cumulus cells throughout in vitro maturation culture, to determine their temporal expression. Gene silencing will be performed initially in isolated cumulus cells cultured in vitro to determine the best conditions for lipofecction (concentration and exposure time to the interference RNA). The best conditions will be next evaluated in cumulus-oocyte complexes (COCs). To confirm gene silencing in COCs the gene expression and the detection of BMPRII and EGFR proteins will be analyzed. The effects of gene silencing will be studied in terms of finctional aspects of cumulus cells, evaluating the expression of genes related to expansion (HAS2 and PTG2), proliferation (CCND2) and steroidogenesis (STAR), which are downstream of the BMPRII and EGFR signaling pathway. Besides, phenotypical effects on cumulus expansion, MAPK activation and their reflexes on oocyte maturation and embryo development will be assessed. Therefore, the silencing of the genes in study, present in the cumulus cells, will negatively influence cumulus expansion, decreasing the oocyte competence for maturation and, consequently, reducing embryonic development, thus demonstrating the importance of proper function of these genes during IVM for subsequent embryonic development.
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