Functional study of the melatonin receptor MT1 in bovine cumulus cells using RNA interference (RNAi) by lipofection

• Grant number: 14/17181-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: November 01, 2014
• End date: May 21, 2018
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Cláudia Lima Verde Leal
• Grantee: Fernanda Cavallari de Castro
• Host Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil
• Associated scholarship(s): 17/04376-6 - Functional study of the melatonin receptor MT1 in bovine cumulus-oocyte complexes and its importance on oocyte maturation and embryo development in vitro under normal and heat stress culture conditions, BE.EP.DR

Abstract

Cumulus cells surrounding the oocyte play an important role for the oocyte´s growth, capacitation, maturation, fertilization and ability to support embryo development. These cells maintain their metabolism in cooperation with oocytes, and such interaction is important for the proper development of the gamete. The study of these cells can provide important information on oocyte competence. Melatonin is a molecule that mediates important reproductive functions and has also been shown to be important for cell functions such as oocyte maturation. The melatonin receptor MT1 has recently been detected in bovine cumulus cells. The aim of this study is to establish conditions for the silencing by lipofection of this gene in bovine cumulus cells and to use this strategy to evaluate the interaction between these cells and the oocyte and to investigate the functional implications of this gene silencing on oocyte competence, to promote better results in the production in vitro. Firstly the temporal expression profile of MT1 in cumulus cells will be determined during in vitro maturation (IVM). Gene silencing will be initially used to establish the best conditions for lipofection (concentration and exposure period to the RNAi - MT1). Best conditions will be assessed in cumulus oocyte-complexes (COCs) to determine the effect of loss of function by gene silencing in COCs on cumulus functions during IVM such as expansion, antioxidants genes expression (SOD1, SOD2, and GPx) and apoptotic cell rates (TUNEL) as well as its consequences for oocyte maturation rates, levels of reactive oxygen species (ROS) and developmental competence after in vitro fertilization and culture. (AU)