DDX3X regulates cell survival and cell cycle during mouse early embryonic development

Abstract

DDX3X is a highly conserved DEAD-box RNA helicase that participates in RNA transcription, RNA splicing, and mRNA transport, translation, and nucleo-cytoplasmic transport. It is highly expressed in metaphase II (MII) oocytes and is the predominant DDX3 variant in the ovary and embryo. However, whether it is important in mouse early embryo development remains unknown. In this study, we investigated the function of DDX3X in early embryogenesis by cytoplasmic microinjection with its siRNA in zygotes or single blastomeres of 2-cell embryos. Our results showed that knockdown of Ddx3x in zygote cytoplasm led to dramatically diminished blastocyst formation, reduced cell numbers, and an increase in the number of apoptotic cells in blastocysts. Meanwhile, there was an accumulation of p53 in RNAi blastocysts. In addition, the ratio of cell cycle arrest during 2-cell to 4-cell transition increased following microinjection of Ddx3x siRNA into single blastomeres of 2-cell embryos compared with control. These results suggest that Ddx3x is an essential gene associated with cell survival and cell cycle control in mouse early embryos, and thus plays key roles in normal embryo development.

Keywords: DDX3X; apoptosis; cell cycle; early embryo; p53.

Fig. 1. The expression of DDX3X in oocytes and different stage early embryos.

A: Western blotting assays showed that DDX3X was expressed in the ovary and oocyte; there is a single band around 72 kDa as predicted both in the ovarian protein sample (left panel) and oocyte sample (right panel). B: Immunofluorescence staining of DDX3X in germinal vesicle (GV) oocyte, metaphase II (MII) oocyte and different stages of early embryo from 1-cell zygote to blastocyst. Each image was photographed at the same parameter settings. Scale bar  = 10 μm.

Fig. 2. Verification of the knockdown efficiency of Ddx3x siRNA.

A: RT-PCR of Ddx3x; the three pairs of siRNA were verified in MII oocytes by microinjection and after 18 hours the RNA of oocytes was extracted and RT-PCR was performed. Non-silencing siRNA was injected as negative control. The knockdown effects were more obvious in #02 siRNA and #09 siRNA compared with #04 siRNA when compared with negative control (CTL). The levels of endogenous β-actin mRNA were used as an internal control. B: Relative expression values for each sample in Fig. 2A were normalized to the level of mouse β-actin expression relative to that in negative control. C: The knockdown efficiency of #02 siRNA and #09 siRNA continued until the blastocyst stage after siRNA was injected at the zygote stage.

Fig. 3. Ddx3x siRNA microinjection in zygote cytoplasm reduced blastocyst formation.

A: Representative images of embryo development in #02 siRNA, #09 siRNA, negative control, and normal non-treatment control. There was impaired blastocyst formation in the #02 siRNA and #09 siRNA groups. B: Repeated experiments were performed and the number of each stage embryo was counted and calculated into four groups (n = 128, 134, 144, and 139 in #02 siRNA, #09 siRNA, negative control, and non-treatment control, respectively). The blastocyst ratios per total 2-cell embryo of the #02 siRNA and #09 siRNA groups were decreased to 41.4% and 44.6%, respectively. *P<0.001. Scale bar  = 100 μm.

Fig. 4. Ddx3x knockdown led to a reduction in the number of cells, an increase in apoptosis signals, and p53 accumulation in blastocysts.

A: Immunofluorescence staining and TUNEL assay in the four groups of blastocysts. Red, Hoechst; green, TUNEL signal. B: Cell numbers were counted according to the nuclei stained by Hoechst; blastocysts in the RNAi group contained significantly fewer total cells compared with controls. C: The apoptosis signals were much greater in the RNAi groups compared with controls (n = 29, 26, 29, and 29 in #02 siRNA, #09 siRNA, negative control, and non-treatment control, respectively), ***P<0.001. D: Western blot analysis showed an accumulation of p53 in RNAi blastocysts. The same number of blastocysts (n = 50) were loaded per panel, and the levels of endogenous β-actin were used as an internal control. Scale bar  = 20 μm.

Fig. 5. Single blastomere microinjection of 2-cell embryo with Ddx3x siRNA induced cell cycle arrest.

A: Representative images of embryo development in #09 siRNA and negative control after single blastomere injection with Ddx3x siRNA, indicated by mixed FITC. Green, FITC; white, DIC. The arrow indicats the cell cycle arrest embryo with FITC. Scale bar  = 100 μm. B: The number of cell cycle arrest embryos was counted and calculated in the #09 siRNA and negative control groups (n = 163 and 137 in #09 siRNA and negative control, respectively). C(a): Representative sample for subsequent arrested development of 2-cell to 4-cell embryos, the monoplast with FITC was the blastomere injected by Ddx3x siRNA. C(b): TUNEL staining of the same embryo which showed that the RNAi blastomere was apoptotic when the normal blastomere developed to the morula stage. Scale bar  = 20 μm. PI, propidium iodide.