Deregulation of oxidative phosphorylation pathways in embryos derived in vitro from prepubertal and pubertal heifers based on whole-transcriptome sequencing.

BACKGROUND: Although, oocytes from prepubertal donors are known to be less developmentally competent than those from adult donors it does not restrain their ability to produce full-term pregnancies. The transcriptomic profile of embryos could be used as a predictor for embryo's individual developmental competence. The aim of the study was to compare transcriptomic profile of blastocysts derived from prepubertal and pubertal heifers oocytes. Bovine cumulus-oocyte complexes (COCs) were obtained by ovum pick- up method from prepubertal and pubertal heifers. After in vitro maturation COCs were fertilized and cultured to the blastocyst stage. Total RNA was isolated from both groups of blastocysts and RNA-seq was performed. Gene ontology analysis was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery). RESULTS: A higher average blastocyst rate was obtained in the pubertal than in the pre-pubertal group. There were no differences in the quality of blastocysts between the examined groups. We identified 436 differentially expressed genes (DEGs) between blastocysts derived from researched groups, of which 247 DEGs were downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes, and 189 DEGs were upregulated. The genes involved in mitochondrial function, including oxidative phosphorylation (OXPHOS) were found to be different in studied groups using Kyoto Encyclopedia of Genes (KEGG) pathway analysis and 8 of those DEGs were upregulated and 1 was downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes. DEGs associated with mitochondrial function were found: ATP synthases (ATP5MF-ATP synthase membrane subunit f, ATP5PD- ATP synthase peripheral stalk subunit d, ATP12A- ATPase H+/K + transporting non-gastric alpha2 subunit), NADH dehydrogenases (NDUFS3- NADH: ubiquinone oxidoreductase subunit core subunit S3, NDUFA13- NADH: ubiquinone oxidoreductase subunit A13, NDUFA3- NADH: ubiquinone oxidoreductase subunit A3), cytochrome c oxidase (COX17), cytochrome c somatic (CYCS) and ubiquinol cytochrome c reductase core protein 1 (UQCRC1). We found lower number of apoptotic cells in blastocysts derived from oocytes collected from prepubertal than those obtained from pubertal donors. CONCLUSIONS: Despite decreased expression of genes associated with OXPHOS pathway in blastocysts from prepubertal heifers oocytes, the increased level of ATP12A together with the lower number of apoptotic cells in these blastocysts might support their survival after transfer.

Mitochondrial DNA content and developmental competence of blastocysts derived from pre-pubertal heifer oocytes.

In the cattle-breeding industry, there is an increasing demand for in vitro embryo production from pre-pubertal heifers. In this study, we evaluated the differences in mitochondrial DNA content, oxidative stress, and developmental competence in blastocysts derived from pre-pubertal and pubertal heifers. We found higher mitochondrial DNA copy numbers in blastocysts produced from pre-pubertal heifers than from pubertal heifers. In the group of pre-pubertal animals, there was a significantly lower number of blastocysts produced in vitro from the same number of collected oocytes, and these blastocysts did not differ from those obtained from pubertal oocytes in terms of their morphological quality. The morphologically appropriate blastocysts derived from pre-pubertal heifers had higher concentrations of reactive oxygen species and glutathione. In blastocysts derived from pre-pubertal heifers, we found alterations in the expression of gene markers for developmental competence, which correlated with higher mitochondrial DNA content, suggesting a lower quality of blastocysts derived from pre-pubertal animals than from pubertal animals. The inadequate redox balance in blastocysts obtained from pre-pubertal females, along with higher mitochondrial DNA copy number, as well as differential gene expression of markers of developmental competence, elucidate the low quality of blastocysts derived from pre-pubertal animals, despite their unaltered morphology.