Development of mouse preimplantation embryos in space

Abstract

The development of life beyond planet Earth is a long-standing quest of the human race, but whether normal mammalian embryonic development can occur in space is still unclear. Here, we show unequivocally that preimplantation mouse embryos can develop in space, but the rate of blastocyst formation and blastocyst quality are compromised. Additionally, the cells in the embryo contain severe DNA damage, while the genome of the blastocysts developed in space is globally hypomethylated with a unique set of differentially methylated regions. The developmental defects, DNA damage and epigenetic abnormalities can be largely mimicked by the treatment with ground-based low-dose radiation. However, the exposure to simulated microgravity alone does not cause major disruptions of embryonic development, indicating that radiation is the main cause for the developmental defects. This work advances the understanding of embryonic development in space and reveals long-term extreme low-dose radiation as a hazardous factor for mammalian reproduction.

Keywords: DNA damage; DNA methylation; microgravity; preimplantation development; radiation; space-flight.

Figure 1.

In vitro development of mouse pre-implantation embryos in space. (A) The embryonic culture incubator used in space experiments. The incubator consists of four imaging culture chambers (indicated by the yellow dotted line and the numbers 1, 2, 3 and 4), two groups of culture fixation units (yellow dotted lines), a microscope (red arrow), and two module reservoirs of fixative solutions (red arrow). This experimental apparatus provides temperature stability inside the incubator, the ability to replace culture medium and automatic image acquisition. (B) The imaging culture chamber was filled with gas-saturated medium, and it consisted of a sample culture cavity with a red O-ring and a cover with a window. (C) The fixation culture unit is a cylindrical perfusion chamber with a top part of the chamber, main body and bottom part of the chamber. (D) Timeline of the SJ-10 satellite space mission showing the time points for embryos loading, payload transferring, embryos fixation, sample recovery and arrival to the laboratory. (E) Representative time-lapse images of embryonic development in imaging culture chambers during spaceflight, with highlighted images showing key stages of pre-implantation development. Scale bars, 100 μm.

Figure 2.

Embryonic development and blastocyst quality are compromised in space culture condition. (A) Analysis of embryonic development in space in fixation culture units after satellite return to Earth. Data represent means of all embryos acquired from three independent units of fixative (1: indicates unit 1; 2: indicates unit 2 and 3: indicates unit 3). CC: indicates conventional culture, SC: indicates sealed culture and SS indicates space sealed culture. (B) The total cell number in blastocysts under CC, SC and SS conditions calculated from 3D reconstruction analysis. Two-tailed Student's t-tests were used for statistical analysis. The SS group had significantly fewer cells than the CC (P = 0.047) and SC groups (P = 0.021). Each dot represents one embryo, and black bars indicate the mean cell number for each group. (C) Representative 3D images of Cdx2 and Oct4 immunofluorescence in blastocysts (>64-cell stage) developed under CC, SC and SS conditions. Oct4 stains the ICM (green), while Cdx2 stains the TE (red). Nuclei were stained with Hoechst33342 (blue). Arrowheads denote colocalization of Oct4 and Cdx2 (yellow). Scale bar, 20 μm. The percentage of Oct4-positive cells (D), Cdx2-positive cells (E) and double-positive cells (F) in CC, SC and SS embryos at the 16–32 cell, 32–64 cell and >64 cell blastocyst stages, respectively. The results are shown as the means ± SEM. P values are determined by Student's t-test (two-tailed); (SS vs. CC and SS vs.SC). n.s., not significant (P > 0.05).

Figure 3.

DNA damage and DNA methylome alterations in mouse embryos in space. (A–C) Representative images of CC, SC and SS blastocysts stained with γH2AX, 53bp1 and XRCC1 antibodies, respectively. Distinct staining patterns of cells were observed in blastocysts developed in space (white arrowheads). Scale bars, 50 μm. (D–F) Quantification of γH2AX (D), 53bp1 (E) and XRCC1 (F) immunofluorescence intensity normalized to DNA staining by Hoechst and analysed with ImageJ software. One imaging section was selected for each embryo for quantification, and the pooled data from embryos were plotted in the graphs. n = number of embryos. The results are shown as the means ± SEM. P values are from two-tailed unpaired Student's t-test. (G) The levels of genome-wide DNA methylation in CC, SC and SS blastocysts are shown. Three blastocysts were used for each group. The result are shown as the means ± SEM. Asterisks indicate the significance level of P < 0.05 (t-test). (H) Distribution of DNA methylation in the CpG context among various genomic element regions, including promoters, 5'UTR, exons, introns and 3'UTRs in CC, SC and SS blastocysts. The numbers denote the ID of specific blastocysts. (I) The number of differentially methylated regions (DMRs) in each pair-wise comparison of groups are shown. Blue and red bars indicate the proportion of hypermethylated and hypomethylated DMRs.

Figure 4.

Effects of ground-based radiation on embryonic development. (A) To investigate the responses of mouse embryos to the accumulated low- doses of radiation, we irradiated 2-cell embryos with Cs-137 gamma at the doses of 0.1 mGy, 0.5 mGy and 2 mGy in ground-based experiments. (B) The percentage of 2-cell embryos that successfully developed to the blastocyst stage during in vitro cultivation with radiation exposure. Data are presented as the means ± SEM from four independent experiments. ^*P < 0.05; ^**P < 0.01; n.s., not significant (P > 0.05). (C) Representative fluorescence images of γH2AX in blastocysts that developed from 2-cell embryos with or without exposure to different doses of radiation for 64 h. Scale bars, 50 μm. (D) The level of genome-wide DNA methylation in blastocysts exposed to 0, 0.1, 0.5 and 2 mGy radiation. The results are shows as the means ± SEM. ^*P < 0.05; n.s., not significant (P < 0.05) (t-test). (E) The number of differentially methylated regions (DMRs) in each pair-wise comparison of groups are shown. Blue and red bars indicate the proportion of hypermethylated and hypomethylated DMRs.

Figure 5.

Effects of simulated microgravity on preimplantation embryonic development. (A) An image of the experimental apparatus, in which the embryos were inoculated into 10 mL of the culture vessel of RCCS for the simulated microgravity culture and static culture in CO₂ incubators. (B) The rates of development of embryos to the blastocyst stages under normal gravity (NG) and simulated microgravity (SMG) conditions. Data are presented as the means ± SEM from four independent experiments (embryos analysed: n = 899 for NG; n = 886 for SMG). (C) Representative fluorescence images of γH2AX in blastocysts that were developed from 2-cell embryos exposed to NG and SMG conditions for 64 h. Scale bars, 50 μm. (D) The levels of genome-wide DNA methylation in NG and SMG blastocysts are shown. Data are presented as the means ± SEM from four blastocysts. (E) The number of differentially methylated regions (DMRs) between SMG and NG group are shown. Blue and red bars indicate the proportion of hypermethylated and hypomethylated DMRs. Throughout, a Student's t-test (two-tailed) was used for statistical analysis; n.s., not significant.