The Expression And Role Of MTOR And Its Substrate S6K1 In The Mouse Zygotes

IntroductionMammalian target of rapamycin (mTOR) is a structural and functional conservative protein, and is a member of phosphatidylinositol kinase related kinase family. mTOR, a serine/threonine protein kinase, is the critical regulator of cell growth. mTOR is regulated by growth factor, insulin, nutrient, ATP etc, and regulates protein translation through its downstream effector, S6K1 and 4EBP1. mTOR promotes cell growth and inhibits apoptosis, and is involved in cancer, for in cells transfected by proto - oncogene mTOR is constructive activated. mTOR also controls cell cycle progression, promote cell in G1 phase progress to S phase. Furthermore, mTOR has important role in the early development of mouse embryos, for mTOR null mouse embryos die shortly after implantation. But the exact role of mTOR in embryo development and when the role begin to exert are all elusive. And mTOR's downstream effector S6K1 is also important for the development of embryos, such as Drosophila and mouse. But the effect of S6K1 on the development of mouse zygote is unknown. In the present experiment we examine the expression, distribution and role of mTOR and S6K1 in the first mitosis of the mouse zygotes. The research will promote a better understanding of the physiological role of mTOR and the developmental mechanism of mammalian zygote.MaterialKUNMING strain mice, were provided by the Department of Experimental Animal, China Medical University.QuickPrep micro mRNA Purification Kit was purchased from Amersham Bi-osciences.RT-PCR Kit was purchased from TaKaRa Biotechnology (Dalian) Co. Ltd.Antibodies;anti - FRAP rabbit polyclonal antibody and anti - p70S6K polyclonal antibody were purchased from Santa Cruz.HRP - conjugated anti - rabbit IgG secondary antibody was purchased from Santa Cruz.ECL Kit was purchased from Piece Biotechnology Co.MgC12, EDTA, EGTA, are analytical reagent made in China, DTT was purchased from Promega Co. (*y -32P) ATPwas purchased from Beijing Furui biomedical Co. histone HI (type III - S), polyvinyl alcohol, Na3VO4, NaF, P - glycerophosphate, P - nitrophenylphosphate, 3 - [ N - morpholino ] -propanesulfonic acid ( MOPS ) , genistein, ML - 9, leupeptin, aprotonin, pep-statin , chymostatin, trypsin - chymotrypsin inhibitor were purchase ï¿¡rom Sigma Co.Methods1. Superovulation, natural mating, collection of fertilized eggs;2. RT - PCR, anaylysis the expression of mTOR and S6K1 in mRNA level in the mouse eggs, collect 200 eggs of each stages in a 2ml microcentrfuge, performed as< the Kit protocol describe, breifly: Extraction of sample, Isolation of mRNA, wash the oligo(dT) -cellulose that already binds mRNA respectively with high salt buffer and low salt buffer, elution of mRNA. RT - PCR was performed with TaKaRa RNA PCR (AMV) Ver3.0 Kit as the protocol described: Reverse transcription reaction volume is 10|x, reaction condition was: 50t, 30min;99