Overexpression of CDC14B causes mitotic arrest and inhibits zygotic genome activation in mouse preimplantation embryos

Abstract

Following fertilization the transition from a highly differentiated oocyte to a totipotent 2-cell embryo requires two unique mitotic cell cycles. The first cell cycle is characterized by a prolonged G(1) phase, DNA replication (S phase) that occurs separately in the female and male pronuclei, and a short G(2) phase that occur in the absence of cell growth. During the second cell cycle, G(1) is short whereas G(2) is prolonged and occurs concurrently with zygotic genome activation, which is essential for progression past the 2-cell stage. CDC14B, a dual specificity phosphatase that counteracts cyclin dependent kinase 1 (CDK1/CDC2A) action, regulates mitosis in somatic cells and prevents premature meiotic resumption in mouse oocytes. It is not known if CDC14B plays a role during the unique mitotic cell cycles of preimplantation development. We report that CDC14B is present in mouse embryos and localizes to mitotic centrosomes and spindles. Overexpressing CDC14B in 1-cell embryos results in 40% and 60% of the embryos arresting at the 1- and 2-cell stages, respectively. Embryos arrested at the 1-cell stage contained reduced CDC2A activity, whereas embryos arrested at the 2-cell stage were in G(2) and failed to activate the zygotic genome. In contrast, overexpressing CDC14B in meiotically-incompetent oocytes, which are arrested in a G(2)-like state and are transcriptionally active, does not repress global transcription. These data suggest that CDC14B is a negative regulator of the 1-to-2-cell transition and of zygotic genome activation in mouse embryogenesis.

Figure 1

expression of CDC14B during mouse preimplantation development. (A) Relative amounts of Cdc14b mRNA during preimplantation development were determined by qRt-pCR. In each experiment, 50 oocytes or embryos were used to isolate mRNA. this experiment was repeated three times and the data are expressed as mean ± standard deviation relative to the value obtained for GV oocytes. (B) Western blot detection of CDC14B in different stages of preimplantation embryogenesis. the membrane was stripped and re-probed with an anti-β-tubulin antibody for loading standard. Note that much less total protein was loaded in the blastocyst (BL) lane as indicated by the loading control. this experiment was conducted three times with a total of 30 embryos per stage. GV, germinal vesicle (prophase-arrested oocyte); MII, metaphase II; 1C, 1-cell embryo; 2C, 2-cell embryo; 8C, 8-cell embryo; BL, blastocyst.

Figure 2

temporal-spatial localization of CDC14B during mouse preimplantation embryonic development. embryos at the indicated stages were fixed in 3.7% paraformaldehyde prior to immunocytochemical detection. (A) Localization of CDC14B during preimplantation embryogenesis. In the merged images CDC14B is red and DNA is green. the arrows point to localization at midbodies and the asterisk indicates localization at a mitotic spindle. (B) Localization of CDC14B during the cell cycle of the first two embryonic divisions. One or two-cell embryos were collected at specific times after hCG injection as described in the methods section. In merged images CDC14B is red, γ-tubulin is green and DNA is blue. In the 2-cell embryo in the last row, the top blastomere is in anaphase and the lower blastomere is in prometaphase (pro-M). the arrows indicate co-localization at microtubule organizing centers (MtoCs).

Figure 3

overexpression of CDC14B causes mitotic arrest at the one or two-cell embryonic stage. 1-cell embryos were microinjected with the Cdc14b or Gfp cRNA and incubated in KSOM. (A) Injected embryos were incubated for the indicated amount of time and were classified according to their developmental stage. These experiments were repeated 5 times, and the error bars indicate S.E.M. (B) Bright field images of microinjected embryos 96 h after eCG injection. (C) CDC14B overexpression was confirmed by immunocytochemistry (top) and immunoblotting (bottom). To estimate the level of overexpression for the immunocytochemistry, the average intensities of CDC14B were compared between injected and non-injected 2-cell embryos using Image J software. Image J was also used to normalize the β-tubulin signals in the immunoblot to estimate the amount of CDC14B overexpression compared to non-injected embryos at the stage.

Figure 4

overexpression of CDC14B caused mitotic arrest in one blastomere of the 2-cell embryo. one blastomere of a 2-cell embryo was microinjected with a mixture of Cdc14b and mCherry cRNAs and incubated for a time in which non-injected 2-cell embryos had reached either the 8-cell or blastocyst stages. As a control, we injected one blastomere of 2-cell embryos with mCherry cRNA alone (left). these live-cell images were obtained using a spinning disc confocal microscope that is equipped with an environmental chamber to support life. this experiment was repeated three times with a total of 20 embryos.

Figure 5

embryos overexpressing CDC14B that are arrested at the 1-cell stage have low CDC2A activity. (A) one-cell embryos were collected at the indicated cell cycle stage and CDC2A and MApK activities for histone H1 or MBp, respectively, were measured by in vitro kinase assays. The graph represents quantification of the band intensity as measured in Image J for CDC2A of three independent experiments. (B) Immunoblotting analysis of levels of CCNB1 protein between control embryos in G₂ phase to 1-cell arrested embryos that were overexpressing CDC14B. this experiment was conducted two times. (C) embryos overexpressing CDC14B that did not contain visible pronuclear membranes were fixed in 3.7% paraformaldehyde and stained with an antibody against α-tubulin to visualize the spindles and mounted in propidium iodide to visualize the DNA. the images were obtained on a laser-scanning confocal microscope.

Figure 6

embryos overexpressing CDC14B that arrest at the 2-cell embryonic stage complete the second S phase. embryos overexpressing CDC14B or GFp were incubated with BrdU for 1 h as described in materials and methods. Distribution of incorporated BrdU in the pronuclei was observed by laser-scanning confocal microscopy following the immunostaining with anti-BrdU antibody.

Figure 7

two-cell embryos overexpressing CDC14B fail to activate the zygotic genome. BrUtp incorporation of 2-cell embryos (A) or meioticallyincompetent oocytes (C) microinjected with Cdc14b or Gfp cRNAs. (A) the left panels show examples of images obtained by laser-scanning confocal microscopy and the graph on the right is the quantification of the nuclear anti-BrUTP signals from 3 experiments as determined by Image J. (B) The decrease in the overall transcription in 2-cell embryos was confirmed at the gene expression level with the analysis of one zygotically-expressed transcript, C-myc, by qRt-pCR analysis. (C) Laser-scanning confocal microscope images of anti-BrUtp staining in meiotically-incompetent oocytes either injected Cdc14b cRNA or not injected as a control. The amount of BrUTP incorporation was quantified using Image J as in (A).