Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months

Animals.

C56BL/6N, BDF1 (C57BL/6 × DBA/2), BCF1 (C57BL/6 × C3H/He), and 129B6F1 male mice carrying the green fluorescent protein (GFP) gene (GFP-tg-129/Sv × GFP-tg-C57BL/6), aged 3 mo, were used to collect spermatozoa. The C57BL/6N, BDF1, and BCF1 mice were purchased from Shizuoka Laboratory Animal Center, and the 129B6F1 mice were bred in our mouse facility. The C57BL/6N and BDF1 mice, age 8–10 wk, were used to produce oocytes. The surrogate pseudopregnant females used as embryo transfer recipients were ICR strain mice mated with vasectomized males of the same strain. On the day of the experiments, or after completion of all experiments, the mice were euthanized by CO₂ inhalation or by cervical dislocation. All animal experiments were performed in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Committee of Laboratory Animal Experimentation of the RIKEN Center for Developmental Biology and by the Institutional Committee of Laboratory Animal Experimentation of the University of Yamanashi.

Preparation of Freeze-Dried Spermatozoa.

Both epididymides were collected from male mice, and the ducts were cut with sharp scissors. A few drops of the dense sperm mass were placed into a centrifuge tube with 2 mL of CZB medium (43), followed by incubation for 30 min at 37 °C in 5% CO₂. After sperm concentration measurements, 50-μL aliquots of the sperm suspension were divided into 30 glass ampules. These ampules were frozen using liquid nitrogen and then freeze-dried (EYELA FDU-2200; Tokyo Rikakikai and FreeZone; Labconco). The cock of the freeze-drying machine was opened for at least 3 h until the samples were completely dry. After drying, the ampules were sealed by melting the necks using a gas burner under vacuum conditions, then kept in a –30 °C freezer until use.

Sperm samples were prepared from 70 male mice of four different strains. The quality of each lot was checked by examining its fertility (see below) using one or two ampules from each mouse. From these results, we selected samples from 12 mice: three from the C57BL/6 strain, four from the BDF1 strain, three from the BCF1 strain, and two from the 129B6F1 strain expressing GFP. These ampules and their data are referred to as the “original sperm” samples. Each ampule was wrapped in polyimide film and then the ampules were further wrapped in groups of four (Fig. 1 A and B). All ampules from 12 male mice (total of 48 ampules) were placed into small cases for space preservation (Fig. 1C) or as a ground control.

Launch to the ISS and Return to Earth.

The sample cases, including the Bio PADLES radiation monitors (Fig. 1C), were launched aboard the H-II Transfer Vehicle KOUNOTORI 4 on August 4, 2013, at room temperature. After arrival at the ISS, astronauts stored them in a –95 °C freezer on August 10, 2013. Nine months later (on May 19, 2014), the sample case was retrieved from the freezer and then returned to Earth on the SpaceX-3 vehicle at ambient temperature. Therefore, these space sperm samples were exposed to cosmic radiation for 288 d. On May 21, 2014, the sample case was delivered to NASA and placed into a –80 °C freezer. This was then transported to our laboratory. These samples are designated the space sperm samples.

At the same times and for the same durations as the launching and storing of the sample case, a control sample case was exposed to room temperature, then frozen at –95 °C and stored in a freezer at the Tsukuba Space Center in Japan. These are designated the ground control sperm samples.

Space Radiation Dosimetry.

Bio PADLES (TLD/CR39) monitoring devices (Fukuvi Chemical Industry) were used to measure radiation dosages. These include CR-39 PNTDs. The devices were placed inside the cases (Fig. 1C) of space and ground control sperm samples. After the sample cases were recovered, the analysis was initiated on June 2, 2014. Pictures of radiation-etched pits reflecting the tracks of atomic nuclei accumulated during space flight (Fig. 1D) were used to calculate the total radiation dose (44).

Analysis and Scoring of Comet Slides.

The single-cell gel electrophoresis technique (i.e., the comet assay) measures DNA damage, including double- and single-strand breaks (45). Here comet assays to detect sperm DNA damage were performed according to the manufacturer’s protocol (Trevigen). In brief, sperm specimens were collected from ampules immediately after opening and then rehydrated in water. Space and ground control sperm samples derived from the same male mouse were mounted on six slides, and 100–300 sperm heads were analyzed following electrophoresis. In some cases, spermatozoa had aggregated and were difficult to measure. To standardize the results among the different mouse strains, the length of each DNA comet “tail” was divided by the mean length of the ground control results in each strain.

Immunostaining of Zygotes.

In histone H2AX, an H2A variants, the serine at position 139 is rapidly phosphorylated within minutes after DNA damage. The phosphorylated form of H2AX, designated gamma-H2AX, forms foci at the sites of DNA damage, which then serve as platforms to recruit various repair and cell cycle checkpoint proteins (46). Therefore, gamma-H2AX foci formation was used as a marker of DNA double-strand breaks in male and female pronuclei, and histone H3K9me2 signals were used to distinguish female pronuclei. The primary antibodies used were an anti–phospho-H2AX (Ser139) rabbit polyclonal antibody (1:500; Millipore Merck) and an anti-histone H3 (dimethyl K9) mouse monoclonal antibody (1:500; Abcam). The secondary antibodies used were Alexa Fluor 488-labeled goat anti-mouse IgG (1:500; Molecular Probes) and Alexa Fluor 568-labeled goat anti-rabbit IgG (1:500 dilution; Molecular Probes). DNA was stained with DAPI (2 μg/mL; Molecular Probes). The brightness of whole male pronuclei was measured using ImageJ and then subtracted from the brightness of the zygote cytoplasm.

ICSI and Embryo Transfer.

To generate offspring from preserved spermatozoa, oocytes were collected from C57BL/6N or BDF1 female mice. Most experiments used BDF1 oocytes irrespective of the male mouse strain. Only when next-generation sequencing was performed for genomic analysis were C57BL/6 oocytes were used for C57BL/6 male mouse ICSI to generate pure inbred offspring. When ampules were opened, 50 μL of water was immediately added to rehydrate the sperm samples, and ICSI was initiated using four or five micromanipulators as described previously (47).

At 0.5 d postcoitum (dpc), embryos were transferred into the oviducts of pseudopregnant ICR strain female mice that had been mated with a vasectomized male the night before transfer. At 18.5–19.5 dpc, the offspring were delivered by caesarean section. Some embryos were cultured in 5% CO₂ in air for 5 d in CZB medium at 37 °C to examine their in vitro developmental potential and the quality of blastocysts.

Immunostaining of Blastocysts.

To evaluate the quality of blastocysts derived from space sperm samples, immunofluorescence staining of blastocysts was performed as described previously (48). The primary antibodies used were an anti-CDX2 mouse monoclonal antibody (1:200; BioGenex) to detect TE cells and an anti-Oct3/4 rabbit polyclonal antibody (1:500; MBL) to detect ICM cells. The secondary antibodies used were Alexa Fluor 488-labeled goat anti-mouse IgG (1:500; Molecular Probes) and Alexa Fluor 564-labeled goat anti-rabbit IgG (1:500; Molecular Probes). DNA was stained with DAPI (2 μg/mL; Molecular Probes).

RNA Isolation and RNA Sequencing.

In this study, both space and ground control sperm samples were obtained from the same male C57BL/6N mouse strain, and fresh oocytes were collected from C57BL/6N female mice. Therefore, the offspring were of a pure C57BL/6N strain. At 18.5 dpc, fetuses were collected by cesarean section. Several organs from seven pups (four females, neonate IDs S2, S3, S4, S7, and three males, neonate IDs S1, S3, S6) from space sperm samples and six pups (one female, neonate ID G1; and five males, neonate IDs G2–G5) from ground control sperm samples were collected, immediately frozen in liquid nitrogen, and kept at –30 °C until use. Brain tissues were used for further analysis. Total RNA was purified using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The library for RNA sequencing (RNA-seq) studies was prepared using the Kapa Stranded RNA/mRNA-Seq Kit (Illumina). The RNA-seq library was sequenced for 36-base single-end RNAs using an Illumina GAIIx machine with a TruSeq SBS kit v5–GA kit (Illumina). The resulting sequence data were mapped against the mouse reference genome sequence (GRCm38/mm10) using bowtie2 (49), followed by calculation of the reads per kilobase of exon per million mapped reads (RPKM) value for each gene using the Bioconductor package DEGseq (50). Clustering analysis and principal component analysis were performed with Cluster 3.0 (51).

Statistical Analysis.

The comet DNA breakage assay results were evaluated using the Wilcoxon–Mann–Whitney nonparametric test, the gamma-H2AX assay results were evaluated using the Student t test, and the birth rate was evaluated using the χ² test. RNA-seq analyses were evaluated using the Wilcoxon–Mann–Whitney nonparametric test adjusted for a false discovery rate, and differentially expressed genes between space and control pups were statistically analyzed using the DESeq2 package (52) with the raw sequence reads count. P < 0.05 (birth rate and gamma-H2AX staining) or P < 0.01 (others) was considered statistically significant.