title: Oocyte spindle proteomics analysis leading to rescue of chromosome congression defects in cloned embryos.
abstract: Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown, we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos.
authors.affiliation: The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine.
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Han
authors.affiliation: The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine.
authors.forename: Cheng-Guang
authors.initials: CG
authors.lastname: Liang
authors.affiliation: The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine.
authors.forename: Yong
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Department of Biochemistry, Temple University School of Medicine.
authors.forename: Xunbao
authors.initials: X
authors.lastname: Duan
authors.affiliation: The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine.
authors.forename: Zhisheng
authors.initials: Z
authors.lastname: Zhong
authors.affiliation: The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine.
authors.forename: Santhi
authors.initials: S
authors.lastname: Potireddy
authors.affiliation: Department of Biochemistry, Temple University School of Medicine.
authors.forename: Camilo
authors.initials: C
authors.lastname: Moncada
authors.affiliation: Department of Biochemistry, Temple University School of Medicine.
authors.forename: Salim
authors.initials: S
authors.lastname: Merali
authors.affiliation: The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine.|Department of Biochemistry, Temple University School of Medicine.
authors.forename: Keith E
authors.initials: KE
authors.lastname: Latham
mesh_terms: D000818:Animals; D064107:Aurora Kinase B; D064127:Aurora Kinases; D047389:Casein Kinase I / Q000172:deficiency; D020090:Chromosome Segregation / Q000187:drug effects* / Q000235:genetics; D033922:Clathrin Heavy Chains / Q000032:analysis / Q000187:drug effects; D019976:Cloning, Organism; D000072159:Dynactin Complex; D004622:Embryo, Mammalian; D051379:Mice; D008815:Mice, Inbred Strains; D008869:Microtubule-Associated Proteins / Q000172:deficiency; D009865:Oocytes / Q000737:chemistry / Q000648:ultrastructure*; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000008:administration & dosage / Q000627:therapeutic use; D008941:Spindle Apparatus / Q000737:chemistry*
chemical_list: D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D011506:Proteins; D012333:RNA, Messenger; D033922:Clathrin Heavy Chains; C576924:Aurkb protein, mouse; D064107:Aurora Kinase B; D064127:Aurora Kinases; D047389:Casein Kinase I; D017346:Protein Serine-Threonine Kinases