Study On The Roles Of Nek5 And Pthlh During Mouse Oocyte Maturation And Preimplantation Embryonic Development

Mammalian oocyte maturation and preimplantation embryonic development are very important for the transmission of genetic material and reproduction of offspring.Previous studies have shown that NEK5 and PTHLH play an important role in mitosis.We studied the role and mechanism of NEK5 and PTHLH during mouse oocyte maturation and preimplantation embryonic development in this study.The main works and results of the study are as follows:(1)The role of NEK5 during mouse oocyte maturation and preimplantation embryonic developmentWe investigated the expression,localization and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development.The protein expression of NEK5 was examined by Western blotting during mouse oocytes maturation.The results showed that NEK5 was expressed from germinal vesicle(GV)to metaphase II(MII)stages with the highest level of expression at GV stage oocytes.In order to show the subcellular localization of NEK5,we microinjected Myc-Nek5 m RNA at a low concentration(400 ng/?l)into GV stage oocytes and immunostained with anti-Myc antibody.The results showed that NEK5 localized in the cytoplasm of oocytes at GV stage,concentrated around chromosomes at germinal vesicle breakdown(GVBD)stage,and localized to the entire spindle at prometaphase I(pro-MI),MI and MII stages.To investigate the role of NEK5 during mouse oocyte maturation,Nek5 expression was depleted in GV stage oocytes by the microinjection of Nek5 si RNA.The results indicated that Nek5 depletion resulted in the decrease of GVBD rate during oocyte maturation,the increase of p-CDK1(Tyr15)protein level and the failture of mouse meiotic resumption,while Nek5 depletion did not affect the localization of CDC25 B in the germ vesicle of oocytes.Studies further indicated that the GV arrest caused by Nek5 depletion could be rescued by Wee1 B knockdown and cyclin B1 overexpression,whereas could not be rescued by Cdc25 overexpression.Moreover,we investigated the role of NEK5 in the metaphase/anaphase transition of oocyte meiosis.The results showed that the first polar body extrusion was significantly lower in the Nek5 depletion group than the control group.Nek5-depleted oocytes were arrested at MI stage of meiosis with the shorter length and smaller area of spindle.The results also showed that Nek5 depletion resulted in the continued retention of Bub3 at the kinetochores,a component of spindle assembly checkpoint(SAC).We investigated the expression and role of NEK5 during preimplantation embryonic development.The protein expression of NEK5 was examined by Western blotting during preimplantation embryonic development.Nek5 depletion was performed by the microinjection of Nek5 si RNA into 1-cell embryos.The results showed that NEK5 was expressed from 1-cell to blastocyst with the highest level of expression at blastocyst stage during mouse preimplantation embryonic development.Nek5-depleted embryos arrested at 2-cell stage.Nek5 depletion severely impaired mouse preimplantation embryonic development.This study demonstrated for the first time that NEK5 plays important roles during mouse oocytes meiosis and preimplantation embryonic development.(2)The role of PTHLH during mouse oocyte maturation and preimplantation embryonic developmentOur previous study reported that Pthlh depletion did not affect the mouse oocyte maturation and PTHLH plays an important role in the development of preimplantation embryos.However,the regulatory mechanism of PTHLH on blastocyst formation,transcription factor/pluripotent genes expression and histone acetylation modification is not clear.In this study,we explored the mechanism by which PTHLH regulates mouse preimplantation embryonic development.Firstly,Pthlh expression was depleted by the microinjection of Pthlh si RNA into 1-cell embryos.We found that Pthlh depletion resulted in a significant decrease of blastocyst formation,which was consistent with our previous study.The results indicated that the developmental rates of embryos in the Pthlhdepleted group were lower than the control group at 2-cell,4-cell,8-cell and morula stages.We also found that Pthlh depletion decreased the cell number of embryos at morula stage.We examined the expression level of cyclin D1 and Akt1/2 m RNA by quantitative real-time PCR during preimplantation embryonic development.We also examined the protein levels of cyclin D1,AKT and p-AKT(Thr308)by Western blotting and immunofluorescence analysis.These results indicated that the protein expression of cyclin D1 and p-AKT(Thr308)was significantly decreased in the Pthlh si RNA-injected group compared with the control group.RT-PCR results indicated that there were no significant differences in the m RNA levels of cyclin D1,Akt1 and Akt2 between the Pthlh si RNA-injected group and the control group.We examined the expression level and localization of HDAC4 in the control group and Pthlh si RNA-injected group by RT-PCR,Western blotting and immunofluorescence.Western blotting and RT-PCR results indicated that Pthlh depletion did not affect the m RNA and protein levels of HDAC4 at 2-cell,4-cell,8-cell and morula stages.Immunofluorescence results showed that the nuclear accumulation of HDAC4 was significantly increased in the Pthlh si RNA-injected group compared to the control group.We found that there were no significant differences in the expression of ?-catenin,RUNX2 and CDK4 between the control group and the Pthlh si RNA-injected group,but Pthlh depletion significantly decreased the m RNA level of E2 f.We investigated the role of Pthlh overexpression during mouse oocyte maturation and preimplantation embryonic development.Pthlh overexpression was conducted by the microinjection of Myc-Pthlh m RNA into GV stage oocytes and 1-cell embryos.The results presented that Pthlh overexpression did not affect the mouse oocyte meiosis and preimplantation embryonic development.In this study,we demonstrated for the first time that PTHLH regulates the embryonic cleavage division,blastocyst formation,pluripotency genes expression and histones acetylation by AKT/cyclin D1 pathway and HDAC4 nuclear tranlocation during mouse preimplantation embryonic development.We also demonstrated that Pthlh overexpression has no effect on mouse oocyte maturation and preimplantation embryonic development.In summary,we found for the first time that NEK5 and PTHLH considered as potential targets of cancer treatment play an important role in mouse oocyte maturation and preimplantation embryonic development.This study will help people to understand the molecular mechanism of mammalian oocyte maturation and early embryonic development,provide reference and guidance for the research of large animals,and provides a theoretical basis for human health.