Action of the butyrolactone I and roscovitine in maturation of bovine oocytes and its implications in resistence to cryopreservation

Abstract

This study aims to investigate the effect of butyrolactone I and roscovitine to temporarily block the resumption of meiosis, acting on the factors that control the meiotic cell cycle of bovine oocytes and cumulus cell expansion oophoros. Thus, this project starts from the hypothesis that the addition of these drugs during oocyte maturation will positively influence the production of embryos and that this effect will induce a better response to vitrification. The different groups of oocytes are evaluated for: 1) oocyte maturation and expansion of cumulus cells, 2) protein expression of the MPF (p3cdc2 and cyclin B1) and MAPK in oocytes, 2) the ability of oocytes treated or not blocking pre-meiotic maturation develop into viable embryos and 3) resistance to vitrification of embryos obtained. In Part I all slaughterhouse oocytes are matured in vitro in groups: G1: control group, G2: Group treated with butyrolactone I for 12 hours; G3: Group treated with BLI for 24 hours; G4: Group treated with roscovitine for 12 hours; G5: Group treated with roscovitine for 24 hours and G6: group treated with the combination of BL I (100 mM) and ROS (25 mM) for 12 hours followed by 22 hours of IVM and G7: Group treated withthe association of BL I (100 mM) and ROS (25 mM) for 24 hours followed by 22 hours of IVM. Embryos will be produced in vitro from oocytes of all groups, using means plus SOFA 5mg/mL by BSA and 2.5% FCS, fertilized and cultured in vitro. D7 will be evaluated in the rate of blastocyst formation, the rate of apoptosis. Then, the embryos will be vitrified D7 produced by the method known as "Cryotop" (Glass-Inga ®, Ingamed). After heating the embryos are then washed and cultured in SOFA 5mg/mL increased by BSA and 2.5% FBS. The index of apoptosis will be evaluated before and after the vitrification process / heating. In Part II will assess the effect of the addition of the drugs on in vitro maturation (IVM) of oocytes according to the groups described in Part I, and the production of proteins linked to the MPF of the oocytes and cumulus cell expansion.