Intra and intercellular routes during foliculogenesis and in vivo and in vitro oocyte maturation

Abstract

In vitro production (IVP) of bovine embryos is a widely used biotechnology in Brazil. However, the in vitro embryo production system, although technically viable, leads to cellular and molecular alteration in oocytes and embryos, producing undesirable outcomes and embryo developmental rates ranging from 30% to 50%. Therefore, generate methodologies the lead to higher conversion of oocytes to embryos, and comprehend the mechanism that determine the oocyte competence is strategic for the Brazilian industry of embryo in vitro production. The follicular microenvironment and the oocyte metabolism are fundamental to determine the competence acquisition of the oocyte. Experiments evaluating intra and intercellular routes during the oocyte maturation and embryo development will be conducted aiming to investigate the biological process associated with the acquisition of competence and to generate increments on embryo in vitro production rates. The first research strategy will focus on lipid oxidative metabolism and mitochondrial activity. Previous studies conducted by our group showed a deficient migration and total quantity of active mitochondria and lipid droplets in in vitro maturation (IVM) compared to in vivo matured oocytes. Since that oocytes' mitochondria are responsible for the energetic supply until the blastocyst stage, and that fatty acids oxidation is the main cellular mechanism of energetic supply for oocyte and embryo development, the activity of both mitochondria and lipid droplets are fundamental for IVP. Previous reports show that modulator of beta-oxidation, such as L-carnitine and bezafibrate, can stimulate the oxidative process and lead to an increase of in vitro embryo production rates. Thus, this line of investigation aim to compare the mitochondrial activity and the lipid metabolism between in vivo and in vitro embryo production, as well as assess the effect of oxidative modulators supplementation (L-carnitine and bezafibrate) during IVM on embryo IVP rates. Both the lipid droplets and mitochondrial behavior will be evaluated, as well as the cellular oxidative stress during in vivo and in vitro maturation and development. The second and third research strategies will focus on extracellular routs of communication within the follicular microenvironment. The hypothesis that will be investigated is that proteins enclosed on cell-secreted vesicles (CSV) in the follicular fluid (FF) and miRNAs from cumulus cells modulate oocyte quality. Proteins in CSV (microvesicles and exosomes) from the FF associated with different developmental competences will be identified and quantified; and miRNA profile of cumulus cell correlated with oocyte competence will be determined. To validate miRNAs associated with oocyte development competence, functional tests will be performed mimicking (miRNA mimics) or inhibiting (miRNA inhibitors) target miRNAs previously related to the presence of absence of oocyte competence, and the effects on embryo IVP will be assessed. Results of these experiments will help to elucidate the effects of in vitro systems on beta-oxidation and lipid-mitochondrial metabolism, as well as help to clarify the mechanisms of intercellular communication within the ovarian follicle, characterize miRNA profile of cumulus cells, and identify potential miRNAs and protein biomarkers for oocyte quality. Moreover, the results will enable the development of new tools to improve embryo IVP through supplementation of modulators of mitochondrial oxidation that prevent cellular oxidative stress or through the mimicking of miRNA profiles associated with oocyte competence. (AU)