The Effect And Mechanism Of LIF In The Oocyte Maturation And Early Embryonic Development Of Yak

Yaks are seasonal estrus animals and their reproductive performance is low.The application of assisted reproduction techniques in yak populations is a good alternative.In vitro maturation of oocytes and in vitro developmental culture of embryos have been applied as important assisted reproduction techniques in a variety of mammals,including humans.Since the process of oocyte maturation and embryo development is complex and influenced by many external factors,it is particularly important to investigate the mechanisms of in vitro maturation of oocytes and in vitro development of early embryos in yaks.Leukemia inhibitory factor(LIF)was first identified to play an important function in the interaction between embryo and uterus during embryo attachment.As LIF was studied more deeply,it was found to play a key role in oocyte maturation and embryo development.However,the role of LIF in regulating oocyte maturation and pre-implantation embryonic development in yaks and its mechanism are not clear.In this study,we investigated the effects of LIF on oocytes and embryos by studying the role of LIF in the maturation of yak oocytes and the development of preimplantation embryos in vitro,so as to elucidate the mechanism of LIF on the maturation of yak oocytes and the development of preimplantation embryos.1.Different concentrations of LIF(0,25,50,100 ng/m L)were added to yak oocytes during in vitro maturation,and the oocyte early apoptosis levels and apoptosisrelated genes(Bax,Bcl-2,Bax/Bcl-2,Caspase-3,and Survivin)expression,and the first polar body rate of oocytes was counted to determine the effect of LIF on oocyte maturation ability.The results were as follows: the first polar body rate was upregulated and the apoptosis level was downregulated in the oocytes treated with different concentrations of LIF.Among them,compared with other experimental groups(control,25 and 100 ng/m L LIF-treated groups),the first polar body expulsion rate of oocytes in the 50 ng/m L LIF-treated group was significantly higher(P < 0.01);the apoptosis level of oocytes in the 50 ng/m L LIF-treated group was significantly lower than that in the control group(P < 0.01)and oocytes in other LIF-treated groups(P < 0.05).The expression of Bcl-2,Bax/Bcl-2,Caspase-3 and Survivin genes in oocytes was regulated by 50 ng/m L LIF,and the difference was significant(P < 0.05).We also found that the proliferation of oocytes in COCs was better in the 50 ng/m L LIF-treated group.These results suggested that LIF improved the first polar body rate and oocyte spreading of oocytes by regulating the apoptosis of yak oocytes,and 50 ng/m L LIF had the best effect.2.Different concentrations of LIF(0,25,50 and 100 ng/m L)were added to yak oocytes during in vitro maturation.The effects of LIF on the developmental capacity of yak oocytes were determined by examining the mitochondrial,oxidative stress and actin levels,the ability to bind sperm,and the apoptotic level and total cell number of blastocysts after the activation of solitary females in each test group.The results were as follows: 25 and 100 ng/m L LIF significantly increased oocyte mitochondrial and actin levels and the ability to bind sperm(P < 0.01)and decreased ROS(Reactive oxygen species)levels(P < 0.01)compared to the control group.Meanwhile,in the other three experimental groups(control,25,and 100 ng/m L LIF-treated groups),the50 ng/m L LIF-treated group significantly increased oocyte mitochondrial and actin levels and the ability to bind to sperm(P < 0.01)and significantly decreased oxidative stress levels(P < 0.01).In addition,the apoptosis rate of blastocysts was significantly down-regulated and the total cell number was significantly up-regulated in the 50ng/m L LIF-treated group after oocyte solitary activation(P < 0.01).These results suggested that LIF can improve the sperm binding ability and the quality of subsequent blastocysts by regulating the levels of mitochondria,oxidative stress and actin in oocytes,with 50 ng/m L LIF having the best effect.3.50 ng/m L of LIF and/or 5 μM JAK inhibitor was added to yak oocyte maturation fluid,and three experimental groups were set up(control group: no LIF and JAK inhibitor added,inhibitor treatment group: only 5 μM JAK inhibitor added,LIF+inhibitor treatment group: both 50 ng/m L LIF and 5 μM JAK inhibitor added).The oocyte proliferation of COCs in each treatment group was observed,and the differences in apoptosis,mitochondrial and ROS levels,actin integrity,sperm-egg binding ability and m RNA expression of Stat3,a key factor of JAK-STAT3 signaling pathway and apoptosis-related genes(Bax,Bcl-2,Bax/Bcl-2)were detected in each group of oocytes,and the first polar body expulsion rate was counted.The results were as follows: compared with the inhibitor-treated group,mitochondrial and actin levels,first pole body efflux rate,sperm binding ability and Stat3 m RNA expression levels were significantly upregulated(P < 0.05/ P < 0.01)and ROS and apoptosis levels were significantly downregulated(P < 0.01)in the LIF+ inhibitor-treated oocytes.These results indicated that LIF promoted oocyte meiotic maturation by upregulating Stat3,a JAK-STAT3 signaling pathway,decreasing ROS levels and upregulating mitochondrial levels in oocytes,decreasing oocyte apoptosis levels,and increasing oocyte actin levels,ability to bind sperm,first polar body expulsion rate and oocyte spreading in oocytes.4.50 ng/m L of LIF and/or 5 μM JAK inhibitor was added to yak embryo development medium,and three experimental groups were set up(control group: no LIF and JAK inhibitor added,inhibitor treatment group: only JAK inhibitor added,LIF+inhibitor treatment group: both 50 ng/m L of LIF and 5 μM JAK inhibitor added)to observe the embryos of each treatment group.The expression of JAK-STAT3pathway-related genes(Stat3,Klf4,C-MYC)and pluripotency-related genes(Oct4,Nanog,Sox2,Cdx2)and OCT4 and CDX2 proteins in blastocysts were detected.The results were as follows: the oogenesis and blastocyst rates of embryos in the LIF+inhibitor-treated group were significantly higher compared with the inhibitor-treated group(P < 0.05).Meanwhile,JAK-STAT3 pathway-related genes(Stat3,Klf4,C-MYC)and pluripotency-related genes(Oct4,Nanog,Sox2,Cdx2)were significantly higher in blastocysts of the LIF+ inhibitor-treated group compared with those of the inhibitortreated group.In addition,the distribution of OCT4 protein in the inner cell mass was more concentrated in the blastocysts of the LIF + inhibitor-treated group compared to the inhibitor group.These results indicated that LIF can increase the oogenesis rate,blastocyst rate and the expression of Oct4,Nanog,Sox2 and Cdx2 in blastocysts of yak embryos by regulating the expression of Stat3,Klf4 and C-MYC in the JAK-STAT3 signaling pathway.In summary,50 ng/m L LIF increased mitochondrial and actin levels,decreased ROS levels and reduced early apoptosis levels in yak oocytes,thereby improving the maturation and developmental capacity of yak oocytes and the developmental quality of subsequent parthenogenetic activated blastocysts.LIF upregulated STAT3 expression through the JAK-STAT3 signaling pathway,downregulated ROS levels and upregulated mitochondrial and actin levels in yak oocytes,thereby reducing the level of apoptosis mediated by the mitochondrial pathway and promoting the ability of oocytes to mature and develop in vitro.LIF upregulated Stat3,Klf4 and C-MYC through the JAK-STAT3 signaling pathway to promote the expression of pluripotencyrelated factors Oct4,Nanog,Sox2 and Cdx2 in yak blastocysts,maintaining pluripotency and self-renewal of blastocysts,and increase the cleavage rate and blastocyst rates of yak preimplantation embryos,improving the development of preimplantation embryos and the quality of blastocysts.