| Intracellular calcium homeostasis has a critical role in maintaining cell function.Calcium homeostasis is easily affected by many factors,which can induce the dysfunction of calcium homeostasis system.The main characterization is that abnormal increase of intracellular calcium concentration far beyond the usual range,the so-called calcium overload,which could interfere calcium signal regulation function,and subsequently result in cell function disorder,even induce cell damage or apoptosis.Cryopreservation results in calcium homeostasis dyregulation of oocytes,but the mechanism and its relationship with freezing injury remain unclear.Therefore,the study blocks endoplasmic reticulum calcium channel before vitrification of mature bovine oocytes,in order to identify the mainly involved calcium channel and mechanism of calcium homeostasis dyregulation.In order to ensure the cryopreservation efficiency,obtaining sufficient high quality oocytes is indispensable.Therefore,the study probes the effects of LIF on in vitro maturation and development of bovine oocytes.Matured bovine oocytes pretreated with Ca2+-ATPase(SERCA)blocker TG before vitrification,the survival rate after thawed significantly decreased.Pretreated with 1 0 μmol/L 2-APB before vitrification,the survival rate significantly improved.After thawed,CG anomaly distribution proportion significantly decreased in the experimental group(42.40%)than the control group(51.81%,p<0.05).whaf’s more,2-APB significantly improved the intracellular GSH level,and significantly decreased the content of intracellular ROS.Besides,2-APB pretreatment significantly improved the cleavage rate(87.83%vs.61.45%)and blastocyst rate(47.31%vs.25.95%)after PA compared with the vitrification group.In addition,pretreating bovine oocytes with 2-APB,the ER restruction obviously recovered(ER cluster distribution),the expression of ERS protein GRP78 and CHOP,as well as the cell apoptosis protein cytC,Caspase-9 and Caspase 3 significantly reduced after thawed.Compared with control group,adding 25 ng/mL LIF in the maturation COCs and DOs significantly improved the rates of nuclear maturation(72.53%vs.81.23%,and 60.59%vs.70.04%).Additionally,LIF also increased the migration rates of CG,intracellular GSH levels(cytoplasmic maturation parameters)and the expression of CD9 protein.After in vitro fertilization,LIF treatment significantly improved the cleavage rate(76.03%vs.66.52%),blastocyst rate(35.35%vs.25.82%)and blastocyst cells(67.52 vs.80.75)(p<0.05).To further dissect the molecular mechanisms of oocyte maturation,we examined the involvement of MAPK3/1 and STAT3 dependent pathways.The results showed that LIF significantly improved phosphorylation level of MAPK3/1 and STAT3.Inhibition of MAPK3/1 activation with MEK inhibitor U0126 only partially blocked LIF-induced nuclear maturation,although it attenuated oocyte cytoplasmic maturation.Inhibition of JAK/STAT3 activation with JAK inhibitor completely abolished the nuclear and cytoplasmic maturation in bovine oocyte.Adding LIF in the IVM、IVC and IVM/IVC group to dissect whether LIF has a cumulative effect on oocytes maturation and early embryonic development.After parthenogenetic activation(PA),the cleavage rate and blastocyst rate were significantly higher in IVM group than the control group.The IVM/IVC group has the highest blastocyst rate among all groups,and the expressions of pluripotent genes POU5F1 and NANOG are significantly higher than the control group.In conclusion,IP3R channel blocker 2-APB maintained the balance of calcium homeostasis,significantly increased the survival rate and developmental competence of bovine oocytes,and reduced the expression of ERs proteins and cell apoptosis related proteins,thus oocytes avoided suffering excessive endoplasmic reticulum stress.LIF promoted the bovine oocyte nuclear maturation and cytoplasmic maturation by activating MAPK and JAK/STAT3 signaling pathway. |
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