Deregulation of oxidative phosphorylation pathways in embryos derived in vitro from prepubertal and pubertal heifers based on whole-transcriptome sequencing
Purpose: The purpose of this study was to examine whether different developmental competence of the embryos derived from prepubertal and pubertal animals is affected by the heterogeneity of their transcriptomic profiles. Moreover, the expression of molecular markers associated with mitochondrial function, expressed by the genes involved in OXPHOS, might be applied as a predictable marker for embryonic developmental capability. Overall design: Methods: Bovine cumulus-oocyte complexes were obtained by ovum pick- up method from pre- pubertal and pubertal heifers. After 24 hours, the mature cumulus-oocyte complexes (COCs) were fertilized in vitro using commercial bull semen. 18 hours after fertilization, the embryos were transferred to culture medium and grown for another 7 days to the blastocyst stage. Total RNA was isolated from two groups of blastocysts. The cDNA libraries were prepared according to the SMARTer Stranded Total RNA Seq Kit v3- Pico Input Mammalian (Takara Bio, USA, Inc). The quality and quantity of the obtained libraries were measured with Qubit (Invitrogen, ThermoFisher Scientific), TapeStation2200 (Agilent, Santa Clara, CA, USA) and High Sensitivity D1000 ScreenTape RNA ScreenTape (Agilent, Santa Clara, CA, USA) according to the manufacturer's instructions. The concentrations of the libraries obtained were normalized to 10 nm, and the next samples were pooled. NGS sequencing was performed on a NextSeq500 Illumina platform in 75 single-end cycles according to the manufacturer's instructions.  Analysis of the transcriptome profile were determined by the Database for Annotation, Visualization and Integrated Discovery (DAVID). We identified 436 differentially expressed genes (DEGs) between embryos derived from pre-pubertal and pubertal heifers. Results: We identified 436 differentially expressed genes (DEGs) between embryos derived from pre-pubertal and pubertal heifers. Kyoto Encyclopedia of Genes (KEGG) pathway analysis suggested that the genes involved in mitochondrial function including oxidative phosphorylation were different in two researched groups. Differences of the level of expression of genes 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). This study indicated that genes involved in OXPHOS could be predictive indicators for the embryo capacity, developmental competence and implantation ability. Conclusions: Accurate knowledge of the gene expression profile in blastocysts will allow researchers to better understand the molecular mechanisms controlling early embryo development. Moreover, taking into consideration, using the in vitro embryo production provides the increase of the production efficiency and is essential for the selection and breeding of the animals with genetic potential. Additionally, the possibility of using blastocysts derived from prepubertal heifers in embryo transfer programmes will directly provide possibilities to enhance genetic gain in domestic livestock via the reduction of generation interval. Moreover, our results could be beneficial for searching for molecular indicators that could be used to develop good-quality oocytes of greater developmental ability. Taken together, these results suggest that genes involved in OXPHOS could be predictive molecular markers and indicators for embryo capacity, developmental competence and implantation ability, which in turn should result in pregnancy. To unravel the gene expression pathways important for mitochondrial function, the transcriptomic profiles of genes involved in oxidative phosphorylation from blastocysts derived from prepubertal and pubertal heifers were compared.