Effects And Mechanisms Of Low Oxygen Concentration On In Vitro Maturation And Developmental Competence Of Yak Oocytes

Yak(Bos grunniens)is one of a few domestic animals that can well adapt to life at high altitudes of Tibetan Plateau and adjacent areas.Owing to its pivotal role in providing basic resources for Tibetans and other high-altitude nomadic pastoralists throughout history,the yak is dubbed “Boat of Plateau”.Yak has a seasonal reproduction activity and low reproduction performance,which hinders the development of yak industry.One way to improving yak industry efficiency is applying modern reproduction technologies such as in vitro production(IVP)of embryos to promote the reproduction potentials of yak.In IVP,oocyte is one of the most fundamental material and the quality of oocytes has a significant effect on the quality of the subsequent embryos.Oxygen plays an important role in oocyte maturation process,which has been confirmed in human,bovine,goat,and mouse et al.;however,there were few researches as to the effects of oxygen concentrations on the quality of yak oocytes.In the present study,we investigate the effects and mechanisms of different oxygen concentrations on quality of yak oocytes matured in vitro.The main results of this study were as follows:1.Effects of different oxygen concentrations on maturation and developmental competence of yak oocytes matured in vitroEffects of 5% and 20% oxygen concentrations on maturation rate,ROS level and GSH content of oocytes,parthenogenetic and IVF embryo development were studied.Results showed that 5% oxygen concentration increased maturation rate(P < 0.05)and GSH content(P < 0.05)of oocytes,decreased ROS levels of oocytes(P < 0.01),and improved blastocyst quality of parthenogenetic and IVF embryos.2.Effects of different antioxidants on maturation and developmental competence of yak oocytes matured in vitroEffects of supplementation with cysteamine,melatonin,resveratrol and vitamin C on maturation rate,ROS level and GSH content,mitochondrion distribution of oocytes and IVF embryo development were studied.Results showed that 1)cysteamine increased GSH content of oocytes;2)melatonin increased maturation rate(P < 0.05)and GSH content(P < 0.05)of oocytes,rate of oocytes with evenly distributed mitochondria(P < 0.05),cleavage rate of IVF embryos(P < 0.05),and decreased ROS levels of oocytes(P < 0.05);and 3)both of resveratrol and vitamin C increased GSH content(P < 0.05)and ROS levels(P < 0.05)of oocytes.3.Effects of different oxygen concentrations on transcriptome profiles of yak oocytesSingle cell transcriptome sequencing was performed to identify the differentially expressed genes(DEGs)in yak oocytes matured at 5% and 20% oxygen concentrations.120 DEGs were identified,among which 37 genes were upregulated and 83 genes downregulated in the 5% oxygen concentration group.The up-regulated DEGs were mainly involved in hypoxia,cell cycle/cell division,chromatin configuration and remodeling,and cytoskeleton,whereas the down-regulated DEGs were mainly involved in energy metabolism,protein synthesis,re-dox equilibrium and cumulus cell regulation.GO analysis showed the up-regulated DEGs were enriched in 7 terms(nucleoplasm,positive regulation of transcription DNA-templated,ERK1 and ERK2 cascade et al.)and the down regulated DEGs were enriched in 7 terms(mitochondria,mitochondrial respiratory chain complex I,cytosolic large ribosomal subunit and extracellular exosome et al.).KEGG analysis showed the upregulated genes were enriched in 11 pathways(HIF-1 signaling,neurotrophin signaling,focal adhesion,regulation of actin cytoskeleton,and PI3K-Akt signaling et al.),and the downregulated genes were enriched in 4 pathways(Oxidative phosphorylation et al.).4.Effects of different oxygen concentrations on transcriptome profiles of yak cumulus cellsTranscriptome sequencing was performed to identify the DEGs in yak cumulus cells matured at 5% and 20% oxygen concentrations.270 DEGs were identified,among which 229 genes were upregulated and 41 genes downregulated in the 5% oxygen concentration group.The up-regulated DEGs were mainly involved in hypoxia response,glycolysis,immune/inflammatory response and apoptosis,whereas the downregulated DEGs were mainly involved in anti-apoptosis and anti-inflammatory.GO analysis showed the up-regulated DEGs were enriched in 26 terms(response to hypoxia,glycolytic process,ATP metabolic process and inflammatory response et al.)and the down-regulated DEGs were enriched in protein homodimerization and receptor binding.KEGG analysis showed the up-regulated DEGs were enriched in 9 pathways(Neuroactive ligand-receptor interaction,c AMP signaling pathway and Glycolysis/Gluconeogenesis et al.)and the down-regulated DEGs were not enriched.This study investigated the effects of different oxygen concentrations and antioxidants on maturation and developmental competence of yak oocytes matured in vitro as well as the effects of different oxygen concentrations on transcriptome profiles of yak oocytes and cumulus cells.The following conclusion was drawn: compared with20% oxygen concentration,5% oxygen concentration exerted less oxidative stress on yak oocytes,enhanced capacity of cumulus cells in providing more metabolic substrates,and thus promoted oocyte in vitro maturation and early embryo development of yak.These results provided valuable reference data for further investigating effects of oxygen concentrations on maturation,developmental competence and cumulus cell –oocyte interaction in yak oocytes.