Study On The Effect Of Low Oxygen Concentration On Developmental Competence Of Yak Oocyte Maturation,Pre-implantation Embryos And Its Mechanisms

Yak(Bos grunniens)is more adaptable to hypoxic and low-oxygen environment,but their oocyte maturation rate and embryo development ability in vitro are relatively low.This seriously affects in vitro fertilization,somatic cell cloning,and the advancement of other reproductive technologies.The gas-phase environment of in vitro culture system plays an important role in the development of oocytes and early embryos,and oxygen is a vital component of the gas-phase environment of in vitro culture systems.However,the effect of low oxygen concentration on the development of yak oocytes and pre-implantation embryos in yaks has not been reported yet.The present study aimed to explore the effects of different oxygen concentrations in yak oocyte maturation and in vitro fertilized(IVF)embryo development competence as well as study its mechanism,and provide theoretical basis to further optimize yak in vitro embryo production system.Accordingly,the following experiments were conducted and their corresponding results achieved:1.This experiment observed the expansion of mature Cumulus-oocyte complexes(COCs)and the discharge of the first polar body in different oxygen concentration groups(20%,10%,5%and 1%O₂).To judge the maturity state of oocytes,the cleavage rate and blastocyst rate of oocytes in different oxygen concentration groups after parthenogenetic activation(PA)were counted.Furthermore,Quantitative Real-Time PCR(q RT-PCR)and Western blot(WB)techniques were used to determine the expression of genes and proteins relating to glucose transport,development,and metabolism in yak oocytes under different oxygen concentrations.The results showed that when oxygen concentration was 5%,COCs spread best,and maturation rate of oocytes was significantly higher than in other groups(p<0.05);the cleavage rate of parthenogenetic embryos in the 5%O₂ concentration group was significantly higher than that of 20%O₂ and 1%O₂ concentration group(P<0.05),meanwhile the blastocyst rate of the 5%O₂ concentration group was significantly higher than in other groups(P<0.05).Also,there were significant differences in the expression of genes and proteins that relates to oocyte development,antioxidant,and metabolism between groups with different oxygen concentrations.These results showed that during the in vitro maturation of yak oocytes,5%oxygen concentration improved oocyte maturation and the development ability of subsequent parthenogenetic embryos.2.In this experiment,the localization of HIF-1?and VEGF protein in yak ovary was detected by immunohistochemistry,and the expression of HIF-1?,VEGF,Bax and Bcl-2,and the cell apoptotic rate of COCs in different oxygen concentrations(20%,10%,5%,and 1%O₂)were detected by q RT-PCR,Western blotting,TUNEL,and immunofluorescence staining methods.Results showed that HIF-1?protein was mainly located in the granular layer of follicles,follicular theca,zona pellucida,corona,and germinal epithelium of ovary,while VEGF protein was mainly distributed in the granular layer of follicles and follicular theca.With decreased oxygen concentration,HIF-1?and VEGF expressions first increased and then decreased,reaching the highest under 5%oxygen concentration and was significantly higher than in the other groups(P?0.05).Also,in the yak COCs matured under 5%oxygen,the expression of pro-apoptotic factor Bax(gene and protein)was significantly lower than that of the other groups(P<0.05),while the expression levels of anti-apoptotic factor Bcl-2 was significantly higher than in the other groups(P<0.05).These results mean that the apoptosis rate of mature COCs in the 5%oxygen concentration group was significantly lower than that of the other groups(P?0.05).The results showed that during in vitro maturation of yak oocytes,the 5%oxygen concentration may inhibit the apoptosis of COCs and promote the development of oocytes through HIF-1?-mediated VEGF pathway.3.The mature COCs in 5%oxygen concentration group were subjected to in vitro fertilization under different oxygen concentrations(20%,10%,5%,and 1%O₂),and the effects of different oxygen concentrations on the cleavage rate,blastocyst rate,blastocyst hatching rate,and total cell number of fertilization embryos in vitro were observed.In addition,we used q RT-PCR,immunofluorescence staining and TUNEL kit to detect the expression of genes related to embryonic development and the rate of apoptosis.The results showed that the cleavage rate of 5%O₂ concentration group was significantly lower than that of 20%O₂and 10%O₂concentration group(P<0.05),but was significantly higher than the 1%O₂ concentration group(P<0.05).The blastocyst rate,the total number of blastocysts,the number of cells in the inner cell mass,the number of trophoblast cells,and the ratio of ICM/TE and the apoptosis rate were significantly higher than those of other groups(P<0.05).Furthermore,the m RNA transcription levels of CDX2,POU5F1,SOX2 and NANOG genes in the blastocysts of the 5%O₂concentration group were significantly higher than those of the other groups(P<0.05),and the transcription levels of the pro-apoptotic gene Bax significantly reduced(P<0.05),while the transcription level of the anti-apoptotic factor Bcl-2significantly increased(P<0.05).The results showed that during the development of yak IVF embryos,5%oxygen concentration reduced the cleavage rate of embryo,but significantly increased the blastocyst rate of the embryos,thus enhanced the development ability of yak embryo before implantation and inhibited its cells from apoptosis.4.The expression and localization of p38 MAPK and HIF-1?at different stages of fertilization embryos under normoxia(20%O₂)and hypoxia(5%O₂)in vitro were detected by q RT-PCR and immunofluorescence staining.The results showed that under normal and low oxygen concentration,with the development of fertilized embryos in vitro,the expression of HIF-1?and p38 MAPK increased first and then decreased,which reaching the highest in 8 cells(P<0.05)and the lowest in the blastocyst stage(P<0.05).The expression of p38 MAPK at the 2-cell to 8-cell stage was not significantly different between normoxia and hypoxia concentration(P>0.05)but the difference was significant at the morula and blastocyst stages(P<0.05),while the expression of HIF-1?in the embryos at each stage was significantly different(P<0.05).Under normoxia,HIF-1?protein was mainly expressed in the cytoplasm of embryos from 2 cells to blastocyst stage and the expression in blastocysts was very weak,while p38 MAPK protein was expressed in cytoplasm and nucleus of embryos,with the expression being more obvious in the cytoplasm.Under hypoxia,HIF-1?protein is mainly expressed in the cytoplasm of the embryo from 2 cells to the morula stage,while in the blastocyst stage,HIF-1?is expressed in both the cytoplasm and the nucleus,and is obviously expressed in the nucleus.Also under hypoxic conditions,the p38 MAPK protein is expressed in the cytoplasm and nucleus of the embryo,from the 2 cell to the morula stage is more pronounced in the cytoplasm,and is more pronounced in the nucleus at the blastocyst stage.The results also showed that HIF-1?protein was transferred to the nucleus and expressed in blastocysts under 5%oxygen concentration.It was speculated that 5%oxygen concentration might regulate the expression of HIF-1?gene through p38 MAPK gene,which increased the blastocyst rate and further improved the development of the embryo before pre-implantation.This study showed that an oxygen concentration of 5%is beneficial to maturation of yak oocytes in vitro,and enhanced the development ability of yak embryos via in vitro fertilization before implantation,which provides a theoretical basis for further exploring the in vitro maturation environment of yak oocytes and establishing an in vitro embryo production system for yak.