Effect Of NSC668394 On Egg Maturation,Early Embryo And Gonadal Development In Mice

Ezrin,a member of ERM protein family,connecting F-actin cytoskeleton to the membrane,plays a role in many cellular process,such as cell remodeling,cell adhesion,cell motility and cell signal transduction.Recent researches demonstrate that Ezrin has close relation to animal reproduction and regulates oocyte maturation,early embryo development,spermatogenesis and capacitation,endometrial development and embryo implantation.In our recent study,p-Ezrin Thr567 was found expressed in mouse oocyte,early embryo and gonad,however,its role in these tissue still remains unclear.Recently,NSC668394,a small molecule,was reported couldspecifically inhibit phosphorylation of Ezrin Thr567.So,we deceide to research the role of Ezrin Thr567 phosphorylation in mice oocytes maturation,fertilization,gonado and early embryo development with NSC668394.This work will help us to understand the regulation of mouse oocyte maturation,fertilization and early embryonic development.Experiment I.Effection of NSC668394 on mice oocytes in vitro maturation,in vitro fertilization and early embryo development.GV-stage oocytes,in vivo MII-stage oocytes and pronuclear stage embryos were randomly divided into five groups and were respectively cultured in medium supplemented with 0,2.5,5.0,10.0μM of NSC668394 or 0.1%DMSO.Results showed that adding NSC668394 to the medium not only decreased oocyte maturation rate,but also decreased the fertilization rate of oocytes matured in vivo or in vitro and the potential of further development.The mature rate and fertilization rate of the group Ⅰ(medium containing 2.5μM NSC668394)was lower remarkably than all other groups(P<0.05);most embryos of group IV(medium containing 10.0μM NSC668394)wereblocked at the 2-cell stage;morula and blastocyst rates of group Ⅱ and group III(medium containing 5.0μM NSC668394)were significantly lower than that of control group(P<0.05).Experiment II.Recure of Oocytes maturation failure,in vitro fertilization failure and embryo development retardation caused by NSC668394.oocytes at GV-stage,MII-stage orpronuclear stage were randomly assigned to four groups,oocytes of group I,II and Illwere cultured inmedium supplemented with 0(),2.5μM(group II)NSC668394 or 0.1%DMSO(group Ⅲ)respectively,oocytesof group IV were microinjected with Ezrin T567D mRNA,then cultured in medium containing 2.5μM NSC668394.Results showed that oocyte maturation rate,fertilization rate morulaand blastocyst rates of group IV were significantly higher than group II(P<0.05),but remarkably lower than group I and III(P<0.05);fertilization rate and morula-blastocyst rate of MII-stage oocyte derived from in vivo,of group IV were significantly higher than group II(P<0.05),but lower than group I and III(P<0.05);morula-blastocyst rates of pronuclear stage embryo of group IV were significantly higherthan group II(P<0.05),but lower than group I and III(P<0.05).Experiment III.Effectof intraperitoneal injection of NSC668394 on oocytes in vitro maturation,in vitro fertilization and early embryo development in mouse.Mice were continuously intraperitoneally injected NSC668394(test-group)or 0.1%DMSO(control-group)from 3w old to sexual maturity.Results indicated that NSC668394 did not affect the oocytes maturation rate,but reduced the rate of in vitro fertilization and morula-blastocyst rates remarkably;west bloting showed the expression level of p-Ezrin(Thr567)in test group was lower than control group.Experiment IV.Effect of intraperitoneally injecting NSC668394 on growth,gonadal development and expression of Ezrin and p-Ezrin(Thr567)..Mice at gestation of 10.5 d or at the age of 3w were intraperitoneally injected NSC668394(test group)or 0.1%DMSO(control group)respectively,Results revealed the weight-gain,diameter and thickness of seminiferous tubule of test group maternal mice’ offsprings were lower significantly than those of control group(P<0,05),.In contrast,seminiferous tubule diameter and thickness of weaning mice of test group was significantly higher than control group.There were no significant difference in ovaries between test and control groups.In addition,distribution of Ezrin was changed by intraperitoneal injected NSC668394,however,expression and distribution of p-Ezrin(Thr567)was not changed.Conclusions:Oocyte maturation,fertilization and early embryo rates were significantly decreased by NSC668394 and microinjecting Ezrin T567D mRNA into cell could rescure mice oocyte maturation failure,in vitro fertilization failure and early embryonic block caused by NSC668394.Pregnant and weaning mice were treated with NSC668394 through intraperitoneal injection affected growth and gonad development of new born and sex mature mice and also could significantly reduce the rate of oocytes in vitro fertilization and early embryo development rate,but did not affect the in vitro oocyte maturation.