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.

Do nuclear envelope and intranuclear proteins reorganize during mitosis to form an elastic, hydrogel-like spindle matrix?

“…A large number of studies spanning from early microscopy studies in the 1960s (Forer and Goldman 1969) to recent proteomic analysis (Sauer et al 2005;Han et al 2010) confirm that the spindle is a very complex structure containing significantly more than microtubules. A "spindle remnant" can be isolated in the absence of microtubules (Rebhun and Palazzo 1988;Leslie et al 1987;Wein et al 1998) and in Drosophila, nuclear proteins (e.g., Megator, Skeletor, Chromator, and EAST) have been shown to adopt a spindle-like structure that can persist independently of the microtubule spindle (Walker et al 2000;Rath et al 2004;Qi et al 2004Qi et al , 2005.…”

Association of Maternal mRNA and Phosphorylated EIF4EBP1 Variants With the Spindle in Mouse Oocytes: Localized Translational Control Supporting Female Meiosis in Mammals

“…We note that the list of SCC-enriched mRNAs includes those encoding proteins found previously enriched on the SCC, such as calmodulin, as well as proteins involved in endocytosis, also previously found to be related to spindle formation and function (Miyara et al 2006;Han et al 2010). Several of the genes associated with the plasma membrane are involved in interaction of the cell surface with the cytoskeleton or spindle.…”

Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition

“…Proteomic approaches have been used to monitor protein synthesis in oocytes (Vitale et al 2007;Zhang et al 2009;Han et al 2010;Wang et al 2010), but the resolution achieved thus far provides insufficient insight into the regulation of maturation, as low-abundance proteins are difficult to quantitate by this approach. Although nucleic acid detection is quite sensitive, the strategy most frequently used involves the analysis of the transcriptome of oocytes and embryos (Latham et al 2000;Oh et al 2000;Evsikov et al 2006;Su et al 2007).…”