Metabolic Activity During Bovine Oocyte Maturation In Vivo and In Vitro

Oocyte metabolism during maturation is vital for establishing developmental competence and embryo quality. In the first set of experiments, follicular fluid metabolomics and glucose concentrations were investigated after induced ovulation. Glucose levels remained constant while 74 metabolites were enriched 6-hours, and 252 metabolites enriched 22-hours after induced ovulation. Six hours after induced ovulation, pathways associated with histidine metabolism, methylhistidine metabolism, and β- alanine metabolism were enriched. After 22 hours, enriched pathways included histidine metabolism, arginine biosynthesis, and arginine and proline metabolism. In the second set of experiments, transcriptomics was performed on in vitro matured (IVM) oocytes resulting from conventional conditions or after C-type natriuretic peptide (CNP) treatment. Single oocyte RNA sequencing revealed significant differences in gene expression between germinal vesicle (GV) and mature oocytes from both conditions. While there were fewer differences between the two groups of mature oocytes, transcripts derived from the mitochondrial genome were significantly downregulated after CNP treatment. In the third set of experiments, molecular and lipid profiling were performed on conventional or CNP IVM oocytes and in vivo matured (IVO) oocytes. Molecular profiling with Raman microspectroscopy revealed similarities between CNP-IVM and IVO oocytes and identified reduced fatty acid concentrations compared to GV and conventional IVM oocytes. Lipid profiling with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) revealed similarities in lipid species between immature oocytes collected after CNP treatment and IVO oocytes. Oocytes allowed to mature after CNP treatment contained similar lipid species to those from GV and conventional IVM sample groups. In xii the last set of experiments, CNP treatment was combined with L-carnitine to improve mitochondrial function during IVM. While there were no differences in maturation rates or ATP concentrations, CNP treatment alone improved mitochondrial membrane potential the most while L-carnitine supplementation significantly increased oxidative stress. Molecular profiling of oocytes treated with or without etomoxir revealed that the significantly altered spectra observed after CNP treatment may be attributed to increased fatty acid oxidation, which was not observed with L-carnitine treatment. Overall, these results provide mechanistic insight to metabolic regulation of oocyte maturation and illustrate the beneficial effects of CNP treatment on cytoplasmic maturation during IVM.