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.

Fig 1

Expression of proteins that show differences between ooSCCs and pmSCCs. (A) Confocal immunofluorescent analysis. Green, spindle protein (DCTN4, AURKB, CLTC, CSNK1A1). red, DNA. Clone 1 h, 2 h, 3 h represents time after SCNT. The scale bar = 10 μm. (B) Western blot analysis of CLTC in oocytes, fertilized embryos and clone constructs.

Fig 2

Confocal microscopic imaging of tetraploid embryos stained with antibody for DCTN4, AURKB, CLTC and Casein kinase 1 alpha. Green, spindle proteins, red, DNA. Clone 1 h, 2 h, 3 h represents recovery time after cumulus nuclei injection. The spindles corresponding to the ooSCC and pmSCC are indicated. The scale bar = 10 μm

Fig 3

Effect of Cltc siRNA on oocyte maturation and chromosome congression. (A), Confocal microscopic imaging of knockdown effect of Cltc siRNA. Green: CLTC (row 1 and 3) and beta tubulin (row 5), Red: DNA. Lagging chromosomes are indicated by arrows. The scale bar = 5 μm. (B), Western blot analysis of knockdown effect of Cltc siRNA. Control group was injected with non-specific siRNA. GAPDH was used as a loading control. Each lane received 100 oocytes. (C), Effect of CLTC knockdown on oocyte maturation (a), chromosome misalignment (b) and MII plate thickness (c). T-test was used to test significance of difference for oocyte maturation and MII plate thickness (P < 0.05). Chi square test was used to test significance of difference for chromosome misalignment (P < 0.01).

Fig 4

Effect of CLTC mRNA on embryo development. (A) Western blot analysis of CLTC in cloned or fertilized late 1-cell embryos uninjected or injected with different amounts of CLTC mRNA at 1 h after SCC removal. 0.1mM EDTA was injected as vehicle injection control. GAPDH was used as loading control. Each lane received 20 embryos of indicated type. (B): Rescue effect of CLTC mRNA on the percentage of late 1-cell stage embryos with lagging chromosome(s). Number of embryos with lagging chromosome and total embryos are indicated in each bar. Chi square was used to test the significance of difference. Bars with different letter are significantly different (P < 0.01). (C) Confocal microscopic imaging of rescue effect of CLTC mRNA in 1-cell stage embryos. 1.0 fmol CLTC mRNA was injected into ooplasm at 1 hour after SCC removal. Embryos were fixed at 1-cell to 2-cell stage. Left to right: early prophase, late prophase, metaphase, anaphase, telophase and early 2-cell stage. First row: fertilized embryos; Second row, cloned embryos injected with 0.1 mM EDTA vehicle control; Third row, cloned embryos injected with CLTC mRNA. Green, CLTC; Red, DNA. Lagging chromosomes were indicated by arrows. The scale bar = 10 μm.