Male mice, caged in the International Space Station for 35 days, sire healthy offspring

The effect on the reproductive system and fertility of living in a space environment remains unclear. Here, we caged 12 male mice under artificial gravity (≈1 gravity) (AG) or microgravity (MG) in the International Space Station (ISS) for 35 days, and characterized the male reproductive organs (testes, epididymides, and accessory glands) after their return to earth. Mice caged on earth during the 35 days served as a “ground” control (GC). Only a decrease in accessory gland weight was detected in AG and MG males; however, none of the reproductive organs showed any overt microscopic defects or changes in gene expression as determined by RNA-seq. The cauda epididymal spermatozoa from AG and MG mice could fertilize oocytes in vitro at comparable levels as GC males. When the fertilized eggs were transferred into pseudo-pregnant females, there was no significant difference in pups delivered (pups/transferred eggs) among GC, AG, and MG spermatozoa. In addition, the growth rates and fecundity of the obtained pups were comparable among all groups. We conclude that short-term stays in outer space do not cause overt defects in the physiological function of male reproductive organs, sperm function, and offspring viability.

mouse spermatozoa stored on the international space station (ISS) for 9 months are damaged by radiation 17 . Abnormal spermatogenesis and a decrease in sperm counts were observed in rats caged in outer space for 13 days 18,19 . However, the effects of space environment on the male reproductive organs at the molecular level remain unknown due to a limited sample size. Further, the effect of space environment on sperm fertilizing ability, male accessory glands, and progeny is also unclear.
To address those concerns, our project team has developed the Transportation Cage Unit (TCU) and Habitat Cage Unit (HCU) 20,21 . The HCU is capable of being installed in the Centrifuge-equipped Biological Experiment Facility (CBEF) on the ISS. This platform allowed us to cage mice under artificial gravity (AG) by centrifugation. In this mission, 12 male mice were caged individually under AG (≈1 gravity) and MG (≈μg) at The Japanese Experimental Module on the ISS for 35 days, and then transported back safely from space. The living mice in outer space are subjected to some effects of space travel (such as radiation, stress from landing and launching), but we can specifically evaluate the effect of changes in gravity by comparing between the AG and MG mice. Here, we discovered that the physiological function of male reproductive organs at the molecular level and sperm fertilizing ability of mice that return from the ISS were normal, and that the next generation (F1 generation) did not show any overt defects in growth rates and fecundity.
These results indicate that accessary gland weight is susceptible to a space environment, but histological features and gene expression of male reproductive organs in mice housed in space are normal.

Sperm fertilizing ability of space mice in vitro.
To reveal the influence of space environment on male fecundity, we characterized cauda epididymal spermatozoa from mice housed in space. Cauda epididymal sperm count from AG and GC males was comparable to GC males (Fig. 1b). We could not see any defects in sperm morphology among GC, AG, and MG males (Fig. 2a), but some parameters of sperm motility from AG and MG mice decreased 2 hours after incubation (Fig. 2b). The lengths of DNA in the comet tails of AG and MG spermatozoa were comparable to the GC spermatozoa (GC: 41.7 ± 9.1 µm, AG: 41.0 ± 7.1 µm, MG: 43.4 ± 9.6 µm) (Fig. 2c,d).
These results indicate that space environment including radiation has minimal damage on spermatozoa in vivo. The AG and MG spermatozoa could efficiently fertilize oocytes at a comparable level as GC spermatozoa (GC: 86.7 ± 14.3%, AG: 88.5 ± 10.3%, MG: 88.6 ± 9.6%) (Fig. 2e,f). Thus, we revealed that spermatozoa from males caged in outer space have normal fertilizing ability.
Embryogenesis of fertilized eggs, and fecundity of the next generation. To further analyze the developmental ability of eggs fertilized with spermatozoa from mice living in space, we observed embryogenesis for 4 days after in vitro fertilization. The developmental speed of fertilized eggs with AG and MG spermatozoa was comparable to that with GC spermatozoa, and these embryos efficiently developed to blastocysts (GC: 58.3%, AG: 80.9%, MG: 73.5%) (Fig. 3a). Wakayama et al. showed that ICSI embryos with freeze-dried mouse spermatozoa preserved in the ISS have decreased cell numbers in the trophectoderm (TE) 17 (Fig. 3b,c). When we transferred these embryos into a pseudo-pregnant female, we obtained pups (F1 generation) from fertilized eggs with AG and MG spermatozoa at a comparable level to GC (GC: 41.0 ± 6.1%, AG: 44.3 ± 7.6%, MG: 36.9 ± 3.6%) (Fig. 3d). These offspring looked healthy (Fig. 3e), and their growth curves were normal (Fig. 3f). These results indicate that quality of fertilized eggs with spermatozoa from space males is normal. We did not see any significant differences among offspring obtained from F1 × F1 intercrosses (GC: 5.56 ± 3.04, AG: 7.15 ± 2.58, MG: 6.81 ± 2.27) (Fig. 3g). This result demonstrates that the effects of space environment on the F0 generation are not carried over to the fecundity of future generations.

Discussion
There are some reports on the effects of space environment (e.g., microgravity, stress due to launching and landing, and radiation) on living mice. Specifically, the female and/or male mice that were caged for up to 90 days in the missions of The National Aeronautics and Space Administration (NASA) (https://www.nasa.gov/ames/ rodent-research) 22 . The Russian Bion-M 1 biosatellite accommodated male mice in groups for 30 days 23,24 . The www.nature.com/scientificreports www.nature.com/scientificreports/ Italian Mice Drawer System housed 6 male mice individually for 91 days on the ISS [25][26][27] . However, in the latter two experiments, more than 50% of male mice died due to various reasons, such as health status and payload-related reasons [23][24][25][26][27] . Thus, the effect of space environment on a living body remains unclear due to a limited sample size. To overcome this issue, our project team newly developed two mouse habitat cages (TCU and HCU) 20,21 , that made it possible to house the mice under artificial gravity by centrifugation. In this mission, our project team caged individual male mice under AG (≈1G) and MG (≈μg) for 35 days on the ISS, and then all the mice were www.nature.com/scientificreports www.nature.com/scientificreports/ returned to Earth, indicating that we can purely compare the effect of gravity on the living body between AG and MG mice. Here, we analyzed the effect of space environment on the male reproductive system and sperm fertilizing ability at the molecular level.
The weights of the male accessory gland (seminal vesicles, prostates, and coagulating glands) decreased in MG males (Fig. 1a). It should be noted that the accessory gland weight slightly decreased in AG males (Fig. 1a). Meanwhile, the weights of the testes and epididymides did not decrease in either group. It is well-known that the majority of accessory gland weight is composed of secretions 28,29 . A previous report showed a decrease in www.nature.com/scientificreports www.nature.com/scientificreports/ plasma volume and total blood volume during spaceflight 5 . Thus, our results suggest that the weight of those organs which store secretions is also susceptible to space environment (gravity and other factors). Previous data showed that the accessory gland weight in castrated male mice is lower than controls due to a decrease in serum testosterone concentrations 30 . The decrease of testosterone levels has been observed during flight and postflight in rats and humans 31,32 . It would be interesting to examine the effect of space environment on male reproductive hormone levels, and whether changes of testosterone levels may lead to the decrease in accessory gland weight. Previous work examined the amount of cytoskeletal proteins, sperm-specific proteins, and epigenetic event-related proteins (DNA-methylases, DNA demethylases, DNA acetylases, and histone deacetylases) in testes and vas deferens of mice exposed to space flight conditions for 21-24 days 33 . In fact, they showed that the expression levels of several genes in testes [Beta-tubulin, DNA methyltransferase (cytosine-5) 1, histone aminotransferase 1, histone deacetylase 1, lysine (K)-specific demethylase 5B, and tet methylcytosine dioxygenase 2] changed with qPCR, but that the quantitative change in these proteins was not detected. Meanwhile, we could not detect the change of the expression level of these genes with RNA-seq. The differences in some conditions (such as living environment, period for space flight) may lead to the discrepancy between the previous paper and our study.
The previous report showed that freeze-dried spermatozoa stored in the ISS for 9 months have DNA damage, and that the cell number in TE in embryos generated by these spermatozoa decreased 17 . However, we could not observe these defects in spermatozoa and embryos with AG and MG males caged in ISS for 35 days (Figs 2c,d, 3b,c). The discrepancy between the previous report and this study may be caused by the length of exposure to the radiation in the ISS or the repair of DNA damage in the individuals, leading to the more severe phenotypes in freeze-dried spermatozoa. We revealed that the growth rates and fecundity of the F1 generation from AG and MG males were normal (Fig. 3f,g). As the previous studies indicated that MG induced epigenetic changes in human lymphocytes 34 and human twins (https://www.nasa.gov/feature/nasa-twins-study-investigators-to-release-integrated-paper-in-2018) 35 , in the future we need to analyze the effects of space environment on the epigenetics of male reproductive organs in mice caged in the ISS and the next generation.
Here, we revealed that the function of male reproductive organs at the molecular level and sperm fertilizing ability were normal in F0 mice caged in the ISS with AG and MG for 35 days. Further, the F1 generation from AG and MG males did not show any overt defects in growth rates and fecundity. In mice, spermatogenesis in testis and sperm maturation in the epididymis take 35 days and 10 days, respectively 36,37 . The 35-days stay in the ISS covered spermatogenesis and sperm maturation in outer space, respectively. Further analyses are required to examine the long-term effects of space environment on the male reproductive system. However, our results indicate that a short stay in space does not cause a decrease in male fertilizing ability and an undesirable effect in the next generation.

Methods
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 previously 20  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 previously 20 . 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 previously 38 . 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". www.nature.com/scientificreports www.nature.com/scientificreports/ Sperm motility and morphology. The frozen-thawed spermatozoa were incubated in TYH medium 39 .
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 × 10 5 /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 previously 40 . 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.