Cleavage of Early Mouse Embryo with Damaged DNA

Abstract

The preimplantation period of embryogenesis is crucial during mammalian ontogenesis. During this period, the mitotic cycles are initiated, the embryonic genome is activated, and the primary differentiation of embryonic cells occurs. All cellular abnormalities occurring in this period are the primary cause of fetal developmental disorders. DNA damage is a serious cause of developmental failure. In the context of DNA damage response on the cellular level, we analyzed the course of embryogenesis and phenotypic changes during the cleavage of a preimplantation embryo. Our results document that DNA damage induced before the resumption of DNA synthesis in a zygote can significantly affect the preimplantation development of the embryo. This developmental ability is related to the level of the DNA damage. We showed that one-cell embryos can correct the first cleavage cycle despite low DNA damage and incomplete replication. It seems that the phenomenon creates a predisposition to a segregation disorder of condensed chromatin that results in the formation of micronuclei in the developmental stages following the first cleavage. We conclude that zygote tolerates a certain degree of DNA damage and considers its priority to complete the first cleavage stage and continue embryogenesis as far as possible.

Keywords: DNA damage; micronucleus; mouse embryogenesis; neocarzinostatin; γH2A.X.

Figure 1

Immunofluorescence labeling the of5-ethynyl-2’-deoxyuridine (EdU) incorporated into DNA separately during the G1-phase, S-phase, or G2-phase in one-cell mouse embryos. The Z-projection of the confocal sections across the whole pronuclei is shown. Scale bar = 10 µm.

Figure 2

(A) Double immunostaining of γH2AX^Ser139/MDC1 (arrowheads) in pronuclei during the G1- and G2-phases of one-cell stage embryos. The DAPI staining (blue) shows pronuclei. The Z-projections of confocal section across whole nuclei are shown. Scale bar = 10 µm. (B) Graph represents the fluorescence image profile and co-localization analyses for γH2AXSer139 /MDC1 in the immunopositive foci. The x-axis represents the length of analyzed line on the picture “Control G2” (red bar).

Figure 2

(A) Double immunostaining of γH2AX^Ser139/MDC1 (arrowheads) in pronuclei during the G1- and G2-phases of one-cell stage embryos. The DAPI staining (blue) shows pronuclei. The Z-projections of confocal section across whole nuclei are shown. Scale bar = 10 µm. (B) Graph represents the fluorescence image profile and co-localization analyses for γH2AXSer139 /MDC1 in the immunopositive foci. The x-axis represents the length of analyzed line on the picture “Control G2” (red bar).

Figure 3

Quantification of γH2AX^Ser139/ MDC1 immunopositive foci in the male or female pronuclei of control and NCS-treated one-cell embryos analyzed in the G1-phase (A) and G2-phase (B). The values show the average number of foci in the one-cell embryos of each experimental group.

Figure 4

Cleavage ability of control and NCS-treated embryos in in vitro conditions. The data show a percentage of cleavage stages in the individual experimental groups at the periods after hCG administration. Total numbers of embryos examined in each group: control = 112; 20 ng NCS = 135; 50 ng NCS= 140; 100 ng NCS= 145. The experiment was repeated three times.

Figure 5

Propidium iodide staining of pronuclei in control (in the G1- vs.the G2-phase) and NCS-treated (in the G2-phase) one-cell embryos. The Z-projection of the confocal sections across the whole pronuclei is shown. Scale bar =10 µm.

Figure 6

Quantification of the total volume of DNA in control and NCS-treated one-cell embryos. The graph shows the relativized values of the measured fluorescent signal densities.

Figure 7

Still images form a time-lapse recording of one-cell mouse embryo expressing H2B-GFP during the first mitosis. The misalignment of chromatin is visible before the cleavage process (arrowheads) as is a micronuclear body in one blastomere of a two-cell embryo (arrow). Additionally, see Movie S1 in the Supplementary Data. Scale bar = 2 µm.

Figure 8

Percentage of the control and NCS-treated one-cell embryos they contain chromosome segregation errors.

Figure 9

Still images from a time-lapse recording of the first mitosis in the control and NCS-treated one-cell embryo expressing securin-EGFP (green) and H2B-mCHERRY (red) mRNA. The Z-projections show the maximum intensity of the confocal sections. Scale bar = 10 µm. Additionally, see Movie S2 in the Supplementary Data.

Figure 10

Time course of the changes in the level of securin during the first cleavage in control and NCS-treated embryos.The graph shows relativized values of the measured fluorescent signal densities.

Figure 11

Double immunostaining of two-cell embryo after NCS treatment in the G1-phase of the one-cell stage. DAPI staining (blue) shows the nuclei and the micronucleus (arrowhead)positive forγH2A.X^Ser139 (red fluorescence—arrowhead). The green immunofluorescence documents the incorporation of EdU into DNA of the nuclei only, but not in the micronucleus (green fluorescence—arrowhead). Polar body in the control embryo stained with DAPI (arrow). Scale bar = 10 µm.

Figure 12

Percentage of the control and NCS-treated two- and four-cell embryos that contain micronuclei in at least one blastomere.

Figure 13

DNA degradation visualized by TUNEL staining (green—arrowhead) in mouse blastocysts. DAPI staining (blue) shows nuclei of blastomeres. That Z-stack projections of the serial confocal sections across whole blastocysts are shown. Scale bar = 10 µm.

Figure 14

Quantification of TUNEL signal intensity in control, actinomycin D-treated blastocysts, and blastocysts after previous treatment with NCS during one-cell stage. The values show the average relative density of the fluorescent signal in the blastocyst of each experimental group.

Figure 15

Average number of blastomeres per blastocyst in control, actinomycin D-treated blastocysts, and blastocysts after previous treatment with NCS during one-cell stage.

Figure 16

Experimental design. All one-cell embryos were collected at time 18 h post hCG administration and each experiment (as reported in Material and Methods) was performed separately in triplicate.