Antioxidant roles of melatonin on bovine cumulus-oocyte complexes and embryos produced in vitro

Abstract

Melatonin (MLT) is an indoleamine that can act on reproductive seasonality in some species, has antioxidant and antiapoptotic activities and influences different cell signaling pathways. In vitro studies have shown beneficial effects of MLT use on in vitro maturation (IVM) and culture (IVC) of bovine oocytes and embryos, respectively. In this study, we propose to investigate the effects of MLT during IVM on oocyte maturation and during IVC on embryo development and quality. Antioxidant and mitochondrial function effects on oocytes and embryos will be assessed as well. In experiment 1, cumulus-oocyte complexes (COCs) will be cultured for 24 h for IVM with MLT and/or associations with gonadotropins: 1) NC (negative control; no gonadotrophins); 2) FSH; 3) FSH+MLT; 4) FSH+LH and 5) FSH+LH+MLT. Oocytes will be evaluated for maturation rate (metaphase II - MII), Mitochondrial Membrane Potential (MMP), ATP concentration, ROS levels and mitochondrial DNA copy number (mtDNA). The best combination of MLT and gonadotropins will be used in experiment 2 in which matured COCs will be submitted to in vitro fertilization (IVF) followed by IVC with MLT (0, 10-11, 10-9 and 10-7M). The embryos will be evaluated for cleavage rate, blastocyst development and quality (ICM/TE), mtDNA copy number and transcript abundance for genes related to oxidative stress (TXNRD1, GLRX2); NRF2 pathway (NFR2 and GCLM); glutathione pathway (GSTT1 and GCLC) and embryo quality markers (OCT4, BMP15, PLAC8, GLUT1). In the third experiment, COCs will be in vitro matured in three treatments: no antioxidant (control), MLT or with a mitochondrial antioxidant (mtAOx). After 24 h IVM, oocytes will be evaluated for MII, MMP, ATP, ROS and mtDNA. In experiment 4, IVC will include three treatments: no antioxidant, MLT or mtAOx. Embryos will be evaluated for cleavage rate, blastocysts development and quality (ICM/TE), mtDNA and transcript abundance for the same genes used in experiment 2. In experiment 5, treatments that obtained the best IVM and IVC rates in experiments 4 and 5, respectively, will be used. Four treatments will be tested: control (no antioxidant during IVM and IVC), best IVM and no IVC antioxidant, no IVM and best IVC antioxidant and, best IVM and best IVC antioxidant. Embryos will be evaluated for cleavage rate, blastocyst development and quality (ICM/TE), mtDNA, nuclear fragmentation (TUNEL), transcripts abundance as in experiment 2 and cryotolerance to vitrification.