Fig. 1. Schematic diagram of the preparation of FD sperm and the type of experiments conducted.

(A) Preparation of FD sperm. The 12 best males were selected from 66 males by the quality lot check of ampules, and the 24 best ampoules were selected from each male, which were then divided into six boxes. Three boxes were launched and preserved on the ISS, and the other three boxes were preserved on Earth. The first box was returned from the ISS 9 months later, the second box was returned 2 years and 9 months later, and the final box was returned 5 years and 10 months later. The ground-control boxes were moved from Japan Aerospace Exploration Agency (JAXA; Tsukuba) to our laboratory at the same time. (B) Tolerance of FD sperm against x-ray irradiation on ground. Fresh and FD sperm were irradiated up to 30 Gy, and then comet, fertilization potential, γ-H2AX, full-term development, and gene expression analyses were performed. (C) Examination of sperm damage and the quality of embryos after preservation on the ISS. To detect the DNA damage in the FD sperm, comet, γ-H2AX, and abnormal chromosome segregation (ACS) assays were performed at each stage. Cytoplasmic damage to FD sperm was detected by the potential of fertilization after the intracytoplasmic sperm injection (ICSI). To detect the quality of embryos, developmental potential in vitro, cell number of blastocysts, apoptosis rate, full-term development, and NGS analyses were performed. Photo credit: Sayaka Wakayama, University of Yamanashi.