Studies regarding melatonin on the function and quality of bovine cumulus-oocyte complexes

Abstract

In vitro production (IVP) of bovine embryos is an important tool for animal production and for genetic improvement of herds, which has great importance to the Brazilian economy due to the weight of the bovine herd for internal and external market. Besides, Brazil is the world´s leading producer of bovine embryos by IVP. However, IVP still faces several hurdles to overcome, as production is still lower than desired and IVP embryos show lower quality and capacity to support cryopreservation when compared to in vivo produced. Therefore, improvements to the process are still needed and the better comprehension of factors involved in the efficiency of the technique is important for its improvement. Among several factors that can interfere with IVP outcomes, the in vitro maturation (IVM) step plays an important role. Oocyte maturation process impacts the quality and developmental competence of the gamete. Melatonin is a hormone with diverse functions, which, besides acting as an antioxidant and antiapoptotic agent, also is capable of activating different cell signaling pathways. Recently (Grant# 2010/18023-9), we observed that melatonin is present in the bovine follicular fluid (Coelho et al, 2013) and that during IVM it stimulates meiosis resumption and cytoplasmic maturation (blastocyst production) at levels similar to FSH stimulation. Besides, it influenced the expression of antioxidant enzymes and reduced apoptosis in cumulus cells (CC), showing effects not only on the oocyte, but also on its CC (Valerino da Cunha, 2014). As these cells are essential for oocyte development, we propose to study the influence of melatonin receptor (MT1) on their functions. To investigate the role o MT1 on melatonin effects, the receptor will be silenced by lipofection of small interfering RNA (siRNA). Parameters assesses in this study are: 1) temporal expression profile for MT1 in CC during IVM; 2) lipofecction conditions (concentration and exposure period to lipofector); 3) effect of loss of function of MT1 in CC from treated cumulus-oocyte complexes (COCs) on CC expansion, expression of antioxidant enzymes (SOD1 and SOD2) and apoptosis rates and 4) consequences on oocytes regarding maturation rates, levels of reactive oxygen species (ROS) and developmental competence after in vitro fertilization and culture. (AU)