Analysis Of Ovarian Differences In Cold And Warm Season And The Effects Of Melatonin On Oocyte Maturation And Embryo Development Of Yak In Vitro

(1)This study was aimed to investigate the ovary status changes of yak in warm（W-yak）and cold（C-yak）season using histology and transcriptome.The ovaries was collected for detecting the numbers of corpus luteum,follicle development and RNA-Seq analysis from W-yak and C-yak.The results showed that（1）yak ovulation focus in August and September because of the number of corpus luteum was significantly higher than the other months（P<0.05）.（2）The process of follicle development is disrupted in C-yak（November）ovary,this result from abnormal accumulation of antral follicles compare to W-yak（July）.（3）The phenomenon of polycystic bubble was represent functional disorder of granulosa cells within an antral follicle in C-yak ovary.（4）RNA-seq data showed that estrogen secretion and metabolism signaling pathway was disorder in C-yak ovary,such as FST,CYP1A1,GNAI2 and PI3K.Both the obtained ovarian morphologic changes and the differently expressed genes provided experimental data related to ovary status between W-yak and C-yak.This study also laid the foundation for understanding yak seasonal breeding physiology.（2）To study the effects of melatonin on oocyte maturation and embryo development of yak oocytes in mature or/and culture medium with different doses.When compared with the control group,the oocyte maturation rates were significantly increased（67.9%vs 82.4%,78.4%）,and the blastocyst rates were significantly increased（26.3%vs 36.8%,34.4%）in the IVC+10^-9 group and the IVC+10^-11 group.The IVM+10^-9 group showed the decreased ROS level,apoptotic rate,and spindle dislocation and chromosomal abnormalities rates,Moreover,the mitochondrial membrane potential was increased when compared to the untreated group.In Experiment 2,the blastocyst rates from IVC+10^-99 and IVC+10^-11 groups were significantly higher than the control group（36.9%vs 43.2%,47.5%）.Furthermore,the number and the percentage of apoptotic cells in blastocysts were markedly decreased in the IVC+10^-9 group（6.9±0.6;5.5%vs 3.5±0.4;3.0%）compared to the control group.In experiment 3,compared with the control group,IVC+10^-9 group reduced the number of apoptotic cells in blastocysts,increased the expression levels of antioxidant gene（SOD2）and heat shock protein（HSPB1）.The expression of proapoptotic genes,such as p53,Bax and capase-3 were decreased.The expression levels of anti-apoptotic genes BCL2L1 and Survivin were significantly higher than that of the control group.Compared with the control,the reactive oxygen species level in the IVM/IVC+10^-9 treatment group decreased.In conclusion,the addition of 10^-9 M concentration of melatonin directly affects embryo developmental capacity and quality in the in vitro production stage of yak embryos.（3）Melatonin plays a critical role in several types of cells as an antioxidant to protect intracellular molecules from oxidative stress.The anti-oxidation effect of melatonin in yak embryos is largely unknown.We report that melatonin can protect the development of yak preimplantation embryos against oxidative stress induced by hydrogen peroxide（H₂O₂）.Therefore,the quality of blastocysts developed from zygotes exposed to H₂O₂ was promoted.In addition,we observed that melatonin reduced H₂O₂-induced intracellular ROS levels and prevented mitochondrial dysfunction in zygotes.These phenomena revealed the effective antioxidant activity of melatonin to prevent oxidative stress in yak embryos.To determine the underlying mechanism,we further demonstrated that melatonin protected preimplantation embryos from oxidative damage by preserving antioxidative enzymes.Collectively,these results confirmed the anti-oxidation effect of melatonin in yak embryos,which significantly improved the quantity and quality of blastocysts in the in vitro production of embryos in yaks.（4）Melatonin decreased the apoptosis rate and ROS level in yak SCNT embryos.It also increased cell number,inner cell mass（ICM）cell numbers,and the ratio of ICM/total cells.Gene expression analysis showed that melatonin suppressed the expression of the pro-apoptotic genes p53 and Bax.Moreover,melatonin also increased the expression of the antioxidant genes SOD2,Gpx4,the anti-apoptotic gene BCL2L1,and the pluripotency-related gene SOX2 in SCNT blastocysts.The global H3K9ac level of melatonin-treated SCNT embryos at the blastocyst stage was higher than those of the untreated control.We concluded that exogenous melatonin regulates the expression of genes related to apoptosis,antioxidant function,and development.Moreover,melatonin reduced apoptosis and ROS in yak SCNT embryos and enhanced the blastocyst quality,thereby ultimately improving the cloning efficiency of yak oocyte.（5）The study was to explore the effects of melatonin on fibroblasts,which acts as the donor cell for in vitro development and quality of yak somatic embryos.At 10^-9 M,melatonin significantly enhanced the proliferation rate of yak fetal fibroblasts（PFF）,and the expressions of H3K9me3,H3K9me2 and H3K18ac in donor cells were significantly increased,and the expressions of pro-apoptotic genes p53 and Bax were significantly decreased,and the mRNA expression of the anti-apoptotic gene BCL2L1 was significantly increased.Furthermore,the blastocyst rate was significantly increased in the 10^-9 M melatonin-treated donor cell group,and the total number of cells,ICM cells and the ratio of ICM:TCN of blastocysts were significantly improved.TUNEL analysis showed that melatonin treatment significantly reduced the ratio of apoptotic nuclei,reduced the intracellular ROS level,and increased the genomic H3K9ac and H3K18ac acetylation levels of blastocysts;The mRNA levels of development-related genes OCT4,pluripotency-related gene SOX2,antioxidant genes SOD2,Gpx4 and heat stress protein HSPB1 were significantly up-regulated in SCNT-T group,However,the levels of proapoptotic genes p53and Bax were significantly lower than those in SCNT group.The expression levels of apoptosis genes BCL2L1 and Survivin were significantly higher than those of the control.This study confirmed that donor cells treated with melatonin can promote the development of cloned embryos.The optimal concentration of melatonin(10^-9M)reconstructed embryos,thus enhanced the formation of yak SCNT blastocysts and improved embryo quality.