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Semi-quantitative assessment of the lipid content of bovine oocytes in vitro matured under the influence of the cGMP pathway

Grant number: 15/19888-7
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): November 01, 2015
Effective date (End): October 31, 2016
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Kátia Regina Lancellotti Schwarz
Grantee:Letícia Schefer
Home Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil

Abstract

Bovine oocytes and blastocysts produced in vitro have darker cytoplasm as a result of higher lipid accumulation in relation to embryos produced in vivo. This characteristic has been associated with a lower quality of the in vitro produced embryo and consequently to higher sensitivity to cryopreservation. Increasing knowledge on the control of lipid metabolism in oocytes may cause changes to the oocyte itself, in order to generate more freezable embryos, contributing to the dissemination of in vitro production (IVP) technique. In human adipocytes, besides the classical pathway mediated by cAMP on cell lipolysis, the cGMP/cGMP-dependent protein kinase (PKG) pathway activates enzymes important in the process. Therefore, intracellular levels of both nucleotides are involved in the regulation of lipolysis rate. As several enzymes for cGMP synthesis and degradation have been detected in the bovine cumulus oocytes complex (COCs) it is possible that the manipulation of this signaling pathway during maturation can exert influence on the lipid metabolism of oocytes in the same manner as reported in adipocytes. The aim of this study is to intervene in the cGMP pathways by different stimuli, seeking the increase this nucleotide during oocyte maturation and to assess its interference on lipid metabolism. Different components of cGMP pathway for synthesis and degradation of the nucleotide will be stimulated by drugs added to the culture media, such as Protoporphirin IX and atrial natriuretic peptide (NPPA), which stimulate enzymes for cGMP synthesis, such as guanylate cyclase (GC), as well as ODQ, a GC inhibitor. Interference on degradation will be given by sildenafil, a selective inhibitor of PDE5 and PDE6 (cGMP-specific degradation enzymes), associated with KT5823, a selective PKG inhibitor which also inhibits phosphorylation and activity of PDE5. For lipid assessment a comparative study of the most widely used dyes for detection of intracellular lipids, Nile red, BODIPY and Sudan Black B, will be conducted. The most effective technique will be selected for measuring the lipid content of oocytes derived from COCs submitted to the treatments. Cumulus cells of such COCs will be used for analysis of gene expression for PLIN2, protein that is part of the proteome of lipid droplets, measured by RT-qPCR techniques.