IGF?EGF And LIF Regulate The Development Of Yak Embryos Produced By IVF And PA And Cryo-adaptation Of COCs

Yak(Bos grunniens)as an important domestic of the Qinghai-Tibet Plateau,has been challenged by the cold environment for a long time.The assisted reproductive technology(ART)can improve and promote the development of animal husbandry in the Qinghai-Tibet Plateau.The aims of this study were to improve the quality of embryonic development of yak in vitro,explore the mechanism of growth factor regulating embryonic apoptosis in vitro.We can reduce the damage thought improving yak embryo culture method after COCs vitrification.The experiments and results in this study as follows:(1).The objective of our present study was to determine the effects of insulin-like growth factor I(IGF-I)on the development of yak(Bos grunniens)embryos after cumulus-oocyte complexes(COCs)vitrification and warming followed by in vitro fertilization(IVF).The yak COCs underwent vitrification and then IVF.Embryos were incubated in synthetic oviductal fluid(SOF)supplemented with four concentrations(0,50,100,and 200 ng/ml)of IGF-I,while the yak COCs without vitrification or IGF-I supplementation acted as the control group;the Bax,Bcl-2,AQP3 mRNA and aquaporin 3(AQP3)protein expression levels in the five groups of blastocysts were evaluated using quantitative Real-time PCR and immunofluorescence analyses.The cleavage rate,blastocyst rate,total cell count per blastocyst and the rate of growth of the inner cell mass(ICM)and trophectoderm(TE)were evaluated.The results were as follows:(1)The AQP3 gene and protein expression in the control and 100 ng/ml IGFI treatment groups were the highest.(2)The Bax gene expression was the lowest and the Bcl-2 gene expression was the highest in the control and 100 ng/ml IGF-I treatment groups.(3)The rates of cleavage and blastocysts in the control and 100 ng/ml IGF-I groups were higher than those in the other three groups.The total cell count per blastocyst in the vitrified and warmed 100 ng/ml IGF-I group(106.7±4.9)and the control group(107.3±4.2)was higher than that in the vitrified and warmed 0 ng/ml IGFI(91.2±3.1),50 ng/ml IGF-I(92.3±3.7),and 200 ng/ml IGF-I(92.4±3.7)groups.Therefore,we conclude that IGF-I can improve yak blastocyst developmental ability,cytomembrane permeability and formation of the blastocyst cavity after COCs vitrification by improving the Bax,Bcl-2 and AQP3 expression levels.(2).The COCs of yak were collected and cultured then IVF.Four concentrations(0,50,100,200 ng/m L)of IGF-I was added to IVF embryos respectively.Real-time PCR and indirect immunofluorescence were used to detect the expression level of Glut-1,Bax and Bcl-2 genes in four blastocysts and the total cleavage rate,blastocyst rate and average number of cells in each blastocyst and the number of ICMs in order to assess the regulation of IGF-I on embryonic development of yak in vitro.The results were as follows: the Bax gene expression in the control and 100 ng/ml IGF-I treatment groups were the lowest.The 0 ng/ml and 50 ng/ml treatment group were the highest and the difference was not significant(P> 0.05).The 200 ng / m L IGF-I treatment group was slightly higher than 100 ng / mL treatment group.The Bcl-2 gene expression in the control and 100 ng/ml IGF-I treatment groups were the highest.The expression of Bcl-2 gene in 50 ng/ml and 200 ng/ml treatment group were higher than that in control group,and the difference was not significant(P> 0.05).The expression of Glut-1 gene in 100 ng/mL and 200 ng/mL IGF-I treatment group were higher than that in control group,and the difference was significant(P<0.05).The increase in the expression level of Glut-1 can effectively provide energy for the development of yak in vitro embryos.The Glut-1 gene and protein expression in the control and 50 ng/ml IGFI treatment group was significantly higher than that in the control group(P <0.05),and significantly lower than the other two groups(P <0.05).Therefore,IGF-I treatment could significantly increase the expression of Glut-1 gene.The cleavage rate and blastocyst rate of 100 ng / mL IGF-I group were significantly higher than those of the other three groups,and the percentage of total cells and ICM in blastocyst was also improved.The results showed that IGF-I was an important autocrine or paracrine factor in the maturation and embryonic development of yak oocytes,and increased the developmental ability of fertilization of yak in vitro though up-regulated the expression of Bax,Bcl-2 and Glut-1.(3).The COCs of yak were collected and cultured then PA.Four concentrations(0,50,100,200 ng/m L)of EGF was added to the immature COCs and PA embryos respectively.0 ng/m L EGF was an experimental control group.Real-time PCR and indirect immunofluorescence were used to detect the relative expression of Cx43 and HSP90 in four groups of blastocysts.The maturation rate of oocytes in four groups was counted according to the number of the first polar body excreted by parthenogenetic activation,and the cleavage rate and blastocyst rate were analyzed.The results were as follows: EGF could significantly improve the maturation rate of COCs and the blastocyst rate and developmental quality of PA embryos(P<0.01),which the best concentration was 100 ng/mL.EGF could significantly increase the expression of Cx43 and HSP90 genes in yak blastocysts.The optimal concentrations of EGF on Cx43 and HSP90 genes were respectively 50 ng/m L and 100 ng/mL,and 100 ng/m L EGF could significantly increase the expression of Cx43.The results showed that EGF can significantly improve the developmental capacity of yak goat embryos by regulating the expression of apoptosis and developmental genes such as HSP90 and Cx43.(4).The COCs of yak were collected and cultured then IVF.200 ng/m L of LIF was added to the immature COCs and IVF embryos,0 ng/m L LIF as control group.Realtime PCR was used to detect the expression of STAT3 gene in immature COCs,mature COCs,2-cell embryos,8-cell embryos,morula and blastocysts of yak in two groups.The expression of STAT3 protein in COCs was detected by indirect immunofluorescence staining.Embryos were expressed at various stages,and the developmental rates of embryos at different stages were counted.The results were as follows: LIF could significantly increase the STAT3 gene expression in embryonic blastocysts of yak IVF(P <0.01),and had no significant effect on the expression of STAT3 immature COCs,mature COCs,2-cell embryos,8-cell embryos and morula.LIF had no significant effect on yak pre-embryonic development,but it could significantly improve the development rate of yak from morula to blastocyst stage(P<0.01).In addition,the expression of STAT3 in mature COCs and blastocysts was significantly higher than that in immature COCs and pre-embryos.The results showed that LIF regulates the transcriptional gene STAT3 in yak embryos and significantly increases the blastocyst rate of yak embryos.This study provides some theoretical basis for promoting the development of yak embryos in vitro.This study from the yak COCs in vitro maturation and vitrification,the embryo in vitro development,growth factor for yak are investigated in biological processes,and the mechanism of IVF and PA embryonic development.The effect of IGF-I to yak mature COCs after vitrification in IVF development to related apoptosis genes Bax and Bcl-2 and AQP3 gene and protein expression,to enhance yak IVF embryo development,reducing the vitrification freezing damage of yak embryonic development..IGF-I regulate glucose metabolism related gene Glut-1 and apoptosis related genes expression in IVF embryos,inhibit embryo cell apoptosis at the same time.EGF regulate Cx43 gene expression in parthenogenetic activation yak embryos,improve the connection and communication between adjacent cells,regulating HSP90 gene at the same time,to adapt the stress environment in vitro.LIF regulate the STAT3 gene expression in yak IVF embryonic development,so as to improve the ability of embryonic development.