Influence of the second messenger cGMP and its signaling pathways on the lipid content of bovine complexes cumulus oocytes matured in vitro

Abstract

Although obtaining in vitro produced embryos (IVP) is an animal biotechnology widely used in research and for commercial purposes in Brazil and the world, such embryos are still of inferior quality compared to those produced in vivo. The greater accumulation of lipids in embryos produced in vitro has been suggested as one of the causes of this lower quality. Classically, cell lipolysis is mediated through cAMP/PKA, however, in human adipocytes it has been observed that the cGMP/PKG pathway may also be involved in this process. To better understand the interference of this pathway on lipolysis in bovine oocytes, we have previously investigated the interference of cGMP through the PKG-dependent signaling pathway (Scientific Initiation, Fapesp grant 2015/19888-7). The results suggested that although cGMP and PKG may indeed be involved in oocyte lipolysis, this action appears to be distinct depending on the type of stimulus used to raise cGMP levels (SCHEFER et al., 2017, in the final stage of preparation for submission). Cyclic GMP can be produced by different stimuli which include activation of membrane (mGC or NPR1 and 2) or soluble guanylate cyclase (sGC). Therefore, the objective of this study is to investigate the interference of the second messenger cGMP, whose synthesis will be stimulated by different pathways (sGC or mGS), on the lipid content of bovine COCs in continuity and complementation to the previous research. In addition, we propose to investigate whether such cGMP effects of different stimuli occur through PKG, on different parameters related to lipolysis. In experiment 1, COCs will undergo in vitro maturation (IVM) with different concentrations of NPPB (NPR1 stimulator); oocytes will then be evaluated in relation to lipid content and cumulus cells to the expression of NPR1 transcripts. In experiment 2, COCs will be matured in vitro with NPPB (concentration selected in the previous experiment) and Protoporphyrin IX (GC stimulator, used in the previous work) to compare the effect of different stimuli to cGMP synthesis on the lipid content of oocytes and cumulus cells. In the third experiment, the participation of PKG will be evaluated in the pathways stimulated by cGMP generated from different stimuli (NPPB and Protoporphyrin IX) associated or not to the inhibition of PKG with KT5823 during the IVM of COCs. This experiment will be developed in three stages to evaluate different parameters related to lipolysis. In the first, the lipid content of the cells (oocytes and cumulus cells) will be analyzed. In the second, quantification of the phosphorylated HSL protein (active PKG target effector protein) will be performed and, finally, in the third stage, gene expression of PLIN1 and PLIN2 in cumulus cells and oocytes will be evaluated. In addition, the maturation medium used in the 3rd experiment will be collected for quantification of glycerol resulting from the lipolysis of COCs. Therefore, we seek to obtain new knowledge regarding the lipolysis process in bovine COCs, which could support future studies to improve the culture conditions to generate IVP embryos with lower lipid accumulation. (AU)