Gene expression and in vitro developmental potential of sheep cumulus-oocyte complexes treated with roscovitine

Abstract

The majority of COCs for in vitro embryo production (PIV) has not yet completed its development and, therefore, does not have adequate mRNAs and proteins stock essential to support the later development stages. In this context, the temporary maintenance of the oocyte at germinal vesicle stage with meiosis inhibitors is a strategy to increase the in vitro development potential of oocytes and embryos. Thus, this study aims to evaluate the expression pattern of genes: GLUT1, SOD, MATER1, ZAR1, BMP15, GDF9 in oocytes, and GREM1, PTX3, PTGS2, ALK5, ALK6, BMPRII in cumulus cells, considered fundamental for the acquisition of oocyte competence and cumulus cells functionality, of ovine cumulus-oocyte complexes (COCs) treated with roscovitine (specific inhibitor of cyclin-dependent kinases) ; and evaluate the potential of these COCs to develop into viable embryos after the reversibility of roscovitine inhibitory action. For this, follicles with diameter between 2 and 5mm of ovine ovaries, obtained from slaughterhouses, will be aspirated and COCs grade 1and 2 will be cultivated, for 20 hours, in an specific medium (Control Group) with 75µM roscovitine (Treatment Group) added, at 38,5ºC in 5% CO2 atmosphere. For the reversibility of the roscovitine inhibitory action and meiosis progression, COCs will be cultivated for additional 18hours in inhibitor-free medium, under the same conditions described above. Before and after 20 and 38hours of in vitro culture, COCs from both experimental groups will be evaluated according to cumulus cells expansion, under stereomicroscope, to nuclear maturation, under fluorescence microscope and to gene expression in oocyte and cumulus cells by qRT-PCR. Part of COCs from Control and Treatment Groups, after in vitro culture for 20 and 38 hours, respectively, will be assigned for fertilization and in vitro culture embryonic and the embryos obtained will be evaluated according to development stage and quality, under stereomicroscope, and to feasibility, under fluorescence microscope. (AU)