Fig. 1.

Preservation of spermatozoa on the ISS and assessment of DNA integrity after return to Earth. (A and B) Ampules of freeze-dried spermatozoa were wrapped with polyimide film, and then four ampules from each donor mouse were wrapped together. Twelve groups of ampules derived from 12 male mice were selected for this study. The small white square (right side of B) represents the PADLES monitor, used to detect the irradiation dose. (C) All ampules were inserted into a small case, and a PADLES radiation monitor was placed on top of the case. (D) The etch pits corresponding to the tracks of atomic nuclei produced during space flight. (E and F) Observation of ground control (E) and space-preserved spermatozoa (F) by light microscopy. (G–I) Comet DNA breakage assays of ground control (G) and space-preserved spermatozoa (H). The lengths of DNA in the comet tails were standardized against the mean lengths of ground control sperm results for each mouse strain (I). The orange bars represent the mean lengths of ground control sperm samples after standardization, and blue bars indicate the space sperm samples. The asterisk denotes significant differences between samples (*P < 0.001). (J and K) Immunostaining of zygotes derived from ground control sperm samples (J) or space sperm samples (K) by the anti–gamma-H2AX antibody. Both male and female pronuclei were detected by nuclear staining with DAPI (Upper Left, blue). Female pronuclei were detected by H3K9me2 immunostaining (Upper Right, green). The foci of gamma-H2AX signals show DNA double-strand breaks (Lower Left, red), and merged images (Lower Right). (L) The brightness of each male pronucleus was plotted. Black circles indicate zygotes derived from ground control sperm samples; white circles, zygotes derived from space sperm samples. The brightness of the male pronucleus in K was 1.3. In I and L, mouse strains: B6, C57BL/6N; BD, B6D2F1; BC, B6C3F1; Tg, 129B6F1 expressing GFP. Asterisks indicate significant differences (I, *P < 0.001; L, *P < 0.05).