Animals

Five-week-old C57BL/6J male mice (Stock #000664) were purchased from The Jackson Laboratory (USA) for the space experiments and from Charles River Laboratories (Japan) for ground experiments. These mice were used for the experiments as described previously20. In brief, 12 male mice caged in the TCU were launched aboard the SpaceX Falcon 9 rocket (SpX9) on 18 June 2016. The Dragon space vehicle from SpX9 reached the ISS on June 20, and then mice were transferred to the HCU by the crew. We divided the mice into 2 groups of six mice each [AG (≈1G, at a centrifugation speed of 77 rpm) and MG], and they were caged in the ISS for about 35 days. This time span was decided by the flight schedule of this mission [mission #: Mouse habitat Unit-1 (MHU-1)]. All the mice returned to the Pacific Ocean offshore from California on 26 August 2016, and then they were sacrificed at the laboratory 2 days after splash down. The radiation load during the space experiment was measured with a ‘Bio Passive Dosimeter for Life Science Experiments in Space’ (Bio PADLES) (0.23 ± 0.02 mGy/d for the absorbed dose rate, and 0.43 ± 0.03 mSv/d for the dose equivalent rate). For replicating the housing conditions of the space flight experiment, we caged 6 males for 35 days at the JAXA Tsukuba Space Center in Japan.

Eight-week-old C57BL/6J female mice were purchased from SLC (Japan) for in vitro fertilization. ICR pseudopregnant female mice were purchased from SLC for embryo transfer. All animal experiments were conducted in accordance with the guidelines of “Animal experiment rules” established by the Research Institute for Microbial Diseases, Osaka University, and were approved by the Animal Care and Use Committee of the Research Institute for Microbial Diseases, Osaka University (#Biken-AP-H30-01).

Body weight and sample collection

Body weight and sample collection were performed as described previously20. Testes, epididymides, coagulating glands (also known as the anterior prostate), prostates (mixture of dorsal, lateral, and ventral regions), and seminal vesicles were used for this study. The production of frozen spermatozoa was performed as described previously38. Specifically, R18S3 medium (ARK Resource) and PB1 (ARK Resource) were used to freeze spermatozoa.

Histology

Testes, epididymides, seminal vesicles, prostates, and coagulating glands were fixed in 4% paraformaldehyde (Wako) for 1 day. After fixation, these samples were placed in PBS for 5 days, and then subjected to plastic embedding using Technovit® 8100 (Mitsui chemicals). For histological analysis of testes, 5 µm sections were stained with 1% periodic acid solution (Wako) for 10 minutes, followed by treatment with Schiff’s reagent (Wako) for 20 minutes, and then Mayer’s hematoxylin solution (Wako) for 5 minutes. For histological analysis of other tissues, 5 µm sections were stained in Mayer’s hematoxylin solution for 3 minutes, followed by treatment with eosin solution [53% (v/v) ethanol, 0.3% (v/v) eosin, and 0.5% (v/v) acetic acid] for 3 minutes. After dehydration with ethanol, these slides were mounted with Entellan® new (Merck Millipore) and observed under a BZ-X710 microscope (Keyence).

Gene expression analysis

Total RNA from testes, coagulating glands, prostates, and seminal vesicles were obtained using TRIZOL® reagent (Thermo Fisher Scientific). RNA quality was examined using an RNA 6000 Pico kit (Agilent). Total RNA from 3 males in each experimental group was mixed. Total RNAs (50 ng) were used for RNA-seq library preparation using the NEBNext® rRNA Depletion Kit (New England Biolabs) and NEBNext® Ultra Directional RNA Library Prep Kit (New England Biolabs), and then 2 × 36 base paired-end sequencing was performed with NextSeq500 (Illumina) by Tsukuba i-Laboratory LLP (Tsukuba). FASTQ files were analyzed using CLC Genomics Workbench (Version 7.5.1; Qiagen). Sequences were mapped to the mouse genome (mm10) and quantified for annotated genes. Transcription expression values were estimated as “reads per kilobase per million reads”.

Sperm motility and morphology

The frozen-thawed spermatozoa were incubated in TYH medium39. After 10 minutes and 2 hours of incubation, the sperm motility was recorded using an Olympus CX41 microscope equipped with a MiniTherm stage warmer (Sony) and a CM-040 GE CCD camera (JAI A/S) for 0.75 seconds at 37 °C. Spermatozoa were analyzed using the default program (Mouse CytoD 4x dark field settings) of the CEROS II sperm analysis system (software version 1.5.2; Hamilton Thorne Biosciences). More than 200 spermatozoa were analyzed in each sample. The morphology of the remaining spermatozoa was observed under a BX50F phase contrast microscopy (Olympus).

Sperm comet assay

The sperm comet assay was done using the CometAssay® Kit (Trevigen). Specifically, the frozen-thawed spermatozoa were incubated in TYH medium for 30 minutes at 37 °C in an incubator with 5% CO 2 . The spermatozoa were diluted with 50 µl PBS (final conc.: 1 × 105/ml), mixed with molten low-melting-point-agarose (Trevigen), and then put on the comet slide. Sample slides were immersed in lysis solution (Trevigen) with 40 mM dithiothreitol at 4 °C for 20 minutes, and then Actinase (final conc.: 10 µg/ml) was added. After incubation for 2 hours at room temperature, the slides were immersed in alkaline solution for an hour at 4 °C. After electrophoresis for 40 minutes at 1 Volt/cm, the slides were stained by SYBR Gold (Thermo Fisher Scientific). The comet tail length of about 30 spermatozoa per slide was measured with ImageJ (NIH).

In vitro fertilization and embryogenesis

Pregnant mare serum gonadotropin (PMSG) (5 units, ASKA Pharmaceutical) was injected into the abdominal cavity of C57BL/6J female mice, followed by human chorionic gonadotropin (hCG) (5 units, ASKA Pharmaceutical) 48 hours after PMSG. After 14 hours of hCG injection, the oocytes were collected from the ampulla of each oviduct, and then incubated in CARD MEDIUM (KYUDO). Frozen-thawed spermatozoa were incubated in FERTIUP medium (KYUDO) for 30 minutes. The sperm suspension was added to CARD MEDIUM. After 3 hours of insemination, the eggs were moved to KSOM medium, and then incubated for 4 days at 37 °C in an incubator with 5% CO 2 . Each day after IVF, the embryos were observed under a phase contrast microscopy (Olympus).

Embryo transfer

Two-cell stage embryos were transferred to the ampulla of pseudo-pregnant ICR mice. After 19 days of embryo transfer, offspring were obtained by caesarean section and natural childbirth.

Immunostaining of blastocysts

After 4 days of IVF, the quality of blastocysts was examined as described previously40. Specifically, rabbit anti-mouse OCT3/4 polyclonal antibody (a marker for ICM cells, 1:200, MBL Code No. PM048) and mouse anti-CDX2 monoclonal antibody (a maker for TE cells, 1:150, MBL Code No. B-MU392AUC) were used as the primary antibodies. The secondary antibodies were Alexa Fluor 488-labeled goat anti-rabbit IgG (Thermo Fisher Scientific) or Alexa Fluor 546-labeled goat anti-mouse IgG (Thermo Fisher Scientific). The 70 images of embryos were captured at 1.2 µm intervals along the z-axis using a BZ-X710 microscope (Keyence). The cell number of ICM and TE in Z-stack images was counted.

Body weight and fecundity of F1 generation

The body weight of the F1 generation was recorded at postnatal days 0, 3, 7, 14, 21, 28, 42, and 56. F1 mice were housed together with their foster mothers up to 4 weeks after birth. At 10 weeks of age, individual males were caged with 2 females for 2 months.

Statistical analysis

All values are shown as the mean ± SD of at least three independent experiments. Statistical analyses were performed using Dunnett T3 method (for Figs 1a, 2b,d) and Tukey method (for Figs 1a, 2f, 3c,d,g), after examining the normal distribution and variance. Significance was defined as p-value < 0.05 using the following notations: *p < 0.05, **p < 0.01, and ***p < 0.001.