Impact of Female Space Travel on Progeny Health

Impact of Female Space Travel on Progeny Health | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Impact of Female Space Travel on Progeny Health Lane Christenson, Xiaoman Hong, John Prom, Michael Ponte, Fereshteh Dalouchi, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6458038/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Spaceflight presents unique microgravity, hypergravity, radiation, and isolation hazards for astronauts. Notably, spaceflight impacts on many life sustaining physiologic systems (e.g., cardiac, musculoskeletal) are known, while impacts on the reproductive systems and progeny of space travelers are largely unassessed. This study analyzes behavioral, metabolic, and functional outcomes in F1 and F2 offspring of female mice following a 42-day spaceflight on the International Space Station. Progeny from the spaceflight dams that were bred following return to earth exhibited growth, functional and behavioral differences compared to control offspring. Introduction of a secondary stressor (feeding a Western diet) revealed a transgenerational inheritance pattern in F2 pups of spaceflight granddams. This study demonstrates spaceflight can impact subsequent generations, pointing to the importance of further study to protect future astronauts’ children. Biological sciences/Physiology Biological sciences/Physiology/Reproductive biology Biological sciences/Developmental biology/Epigenetic memory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Fundamental questions regarding the long-term effects of spaceflight have garnered considerable interest as more and more individuals venture into space. The unique environmental conditions of space travel such as microgravity, increased radiation exposure, and the acute hypergravity during transitions (launch and reentry) encountered by astronauts present unique challenges to mammalian physiology 1 . While it is clear that humans can acclimate to space for extended periods (up to one year continuously or longer cumulatively) and successfully re-acclimate to Earth, the specific consequences on reproductive health remain largely unknown, and in particular the impact of space travel on the female reproductive systemand whether physiological changes sustained in space might have longer term consequences on offspring born to these space travelers 2–4 . Spaceflight studies with non-mammalian species have indicated that fertilization and development can occur in space to produce viable offspring 5–7 . In mammals, our understanding in this area are anecdotal in the case of individual human experiences or come from very short-term (< 12 d) spaceflight exposures of already pregnant rodents followed by ground birth of phenotypically normal offspring 8–12 . Consistent with late gestational exposures, when 2-cell mouse embryos were cultured in microgravity, they were able to develop to the blastocyst stage 8,9 , although in one study ( 9 ) a decrease in embryo quality was observed. In the only mammalian study to date to examine spaceflight effects on F1 offspring, frozen sperm from male mice exposed to microgravity on the International Space Station for 35 days were used to fertilize oocytes and the resultant embryos were transferred to recipient females 13 . These F1 offspring exhibited no changes in growth rates or viability when compared to F1 offspring sired by ground controls, nor were there any differences in litter sizes of F1 x F1 intercrosses 13 . Transcriptomic analysis of F1 offspring originating from 2 microgravity exposed (C57BL6/J) males and 2 ground control (C57BL6/NCrl) males, identified 24 differentially expressed genes in liver tissue of F1 males, while F1 females were not evaluated 14 . To date, no phenotypic differences have been reported in these F1 offspring. This work leaves many questions unanswered, particularly with respect to the possible effects on the females, and this is of particular importance as females bring greater genetic, cellular and environmental contributions (i.e., mitochondria, nutritional) to the offspring. Epigenetic changes occur in response to spaceflight, with changes seen in Arabidopsis 15,16 , C. elegans 17 , tissues/cells from male mice 14 and in the NASA human Twin Study, where DNA methylation changes were present in immune and oxidative stress–related pathways in white blood cells 18 . Given that environmental insults (mal-/over-nutrition, chemical exposures, lifestyle) can reprogram the epigenome of the germline (eggs and sperm), which then transmits to future generations 19–22 , we set out to test the hypothesis that spaceflight might also impact future generations. Recently, our group completed a comprehensive analysis of the impacts of microgravity and spaceflight on the female reproductive system, as part of NASA Rodent Research-20 mission. In this study a group of 20 female mice after having spent 42 days in space (on the International Space Station; roughly equivalent to 8 months of a woman’s reproductive lifespan), were returned to Earth and then mated to ground-based males. The resultant offspring (F1) and subsequent offspring of these F1 mice (i.e., F2 generation) were extensively phenotyped with respect to growth, behavioral changes, tissue function and to the impact of a secondary metabolic stress on the adaptability of these mice. Results Offspring originating from spaceflight (FLT) dams that experienced 42 days in microgravity on the International Space Station (ISS) were compared to two groups of ground control offspring (Fig. 1 ). Habitat ground control dams (HGC) were housed in identical habitats as the FLT females, but in a ground-based ISS Environmental Simulator that mimics ISS FLT conditions (light schedule, temperature, humidity, CO 2 levels, with the obvious exception of microgravity). Additionally, vivarium ground control dams (VGC) were housed in standard mouse cages with key modifications (see Methods), these were also placed in the ISS Environmental Simulator to mimic ISS FLT conditions. Five days following return to Earth, FLT dams (C57BL6/J) were mated to ground-based DBA2/J males. The resultant offspring (F1) from these matings and intercrosses of F1-FLT females to F1-FLT males were used to generate F2-FLT mice, this same paradigm was repeated for the HGC and VGC dams and their F1 offspring (Fig. 1 ). Maternal spaceflight exposure leads to increased offspring body weights. Body weights of F0 offspring were not different at 5 days of age across the three environmental conditions, however, by 10 days of age, F1-FLT pups were heavier than F1-HGC and F1-VGC pups (Fig. 2 ). At 15 and 21 days of age female F1-FLT pups remained heavier than control pups, and this remained into adulthood (93–106 d; Fig. 2 b). In contrast, F1-FLT male weights were not different from controls (F1-HGC/F1-VGC) at 21 days of age or thereafter (Fig. 2 a, b). Body composition of the F0 offspring also revealed sex-specific impacts associated with maternal spaceflight exposure, with F1-FLT females having greater fat mass than F1-HGC females (P < 0.05) and F1-VGC females (p = 0.08) (Fig. 2 c). Comparison of % fat of the F1-FLT females had a greater level of fat compared to F1-HGC females, but were not different than F1-VGC females (Fig. 2 c). F1-FLT females tended (p = 0.12) and had greater (p < 0.05) lean mass than F1-HGC and F1-VGC females, respectively. When adjusted for total body weight, F1-FLT females exhibited a lower percentage of lean mass compared to F1-HGC (p < 0.05) and F1-VGC (p = 0.06) females (Fig. 2 d). In contrast, body composition did not vary for the F1-FLT, F1-HGC, and F1-VGC males (Extended Data Fig. 1 ). The data indicate that maternal spaceflight exposure predominantly affected female offspring, resulting in increased body weight, fat mass, and altered lean mass distribution, while male body weight and composition remained unaffected after day 15 of life. Impaired muscle force production in offspring following maternal spaceflight exposure Soleus muscles isolated from 21-day-old male and female pups of FLT, HGC and VGC dams were tested for their ability to generate force, because postural muscles such as the soleus are well-known to be most susceptible to muscle atrophy and weakness after spaceflight. However, whether these effects may be passed down to spaceflight offspring has never been examined. Soleus muscle mass (absolute and relative to body mass) was greater in F1-FLT male pups versus F1-HGC and F1-VGC male pups, whereas female pups showed no differences (Fig. 3 a-b). Force-frequency analysis demonstrated a significant reduction in specific force production (i.e., made relative to muscle cross-sectional area) at submaximal and maximal frequencies in the F1-FLT pups compared to F1-HGC and F1-VGC pups, being significantly different for female and combined male and female datasets (Fig. 3 c). Force production by male F1-FLT pups compared to F1-HGC and F1-VGC pups did not reach statistical significance. Behavioral, locomotor and aerobic fitness impacts of maternal spaceflight exposure on offspring Behavior and locomotor activity were examined in FLT, HGC and VGC offspring (n = 17–20/sex/treatment group, average of 70-days-old). Locomotor and behavioral activity in offspring born to F0 spaceflight dams demonstrated sex-specific differences. F1-FLT females demonstrated a 15% decrease in average locomotion relative to their F1-HGC counterparts (Fig. 4 a). In contrast, F1-FLT, F1-HGC and F1-VGC males showed no significant difference in locomotion (Fig. 4 a), indicating sex-dependent differences in response to their mother's spaceflight exposure. The marble burying test, a measurement for anxiety and compulsive behaviors, revealed that F1-FLT females buried approximately 37% fewer marbles compared to F1-HGC and F1-VGC females (Fig. 4 b). Conversely, F1-FLT males displayed no notable variations in marble burial behavior, hence reinforcing a sex-dependent impact (Fig. 4 b). Measure of anxiety and risk-taking behavior via elevated plus maze indicated that F1-FLT males allocated approximately 90% more time to the open arms than F1-HGC and F1-VGC males (Fig. 4 c), implying diminished anxiety levels or heightened risk-taking behavior. No notable differences were detected in F1-FLT females, suggesting a possible sex-specific reaction to stresses caused by spaceflight (Fig. 4 c). Aerobic fitness of F1 females and males (90–100 days of age) was tested on a rodent treadmill. Though there were sex-related variations in total distance run or time to exhaustion (Extended Data Fig. 2 ), there were no differences related to the maternal environmental (FLT/HGC/VGC) exposures. Likewise, there were no differences in isolated soleus muscle endurance between the F1-FLT, F1-HGC, and F1-VGC male, female and combined male/female datasets (Extended Data Fig. 3 ). Transgenerational impacts of maternal spaceflight exposure and ability of F1 offspring (F2 pups) to respond to a western diet metabolic stressor. In contrast to F0 offspring (F1 pups), adult F1 offspring or F2 pups (n = 25–43/sex/group, averaging 95-days-old) did not exhibit any striking differences in behavioral or locomotion based on their maternal granddam’s environmental exposure (FLT, HGC and VGC; Extended Data Fig. 4 ). Because F1-FLT, F1-HGC and F1-VGC mice are genetically C57BL/6 X DBA2 hybrids, the intercross progeny (i.e.,F2-FLT, F2-HGC and F2-VGC) generated will display more individual genetic variation than their parents. This genetic variation could contribute to an observed greater variance in phenotypes (Extended Data Fig. 4 ). To test whether a secondary stressor might elicit differences, F2 mice were fed a Western diet (high fat, high sucrose, low/simple fiber; HFD) for 8 weeks. Briefly, 8–10-week-old F2 mice were maintained in a thermoneutral temperature (~ 28ºC) and fed control (LFD) or HFD. All F2 mice fed a HFD (n = 72) exhibited increased weight gain over those fed a LFD (n = 72; Fig. 5 a). Female F2 mice exhibited no differences in 8-week weight gain within diet based on their maternal granddam’s spaceflight exposure. In contrast, HFD-fed F2-FLT males had increased weight gain throughout the eight-week feeding trial when compared to HFD-fed F2-HGC and F2-VGC males (Fig. 5 a, p < 0.05). As expected, HFD feeding resulted in greater fat mass gain in males and females compared to LFD (p < 0.05, Fig. 5 c). Additionally, change in fat-free mass (FFM) was increased in HFD males compared to LFD (p < 0.05), however, F2-FLT males had a greater change in FFM than F2-HGC and F2-VGC controls (p < 0.05, Fig. 5 d). Conversely, while both HFD-fed F2-HGC females (p = 0.06) and F2-VGC females (p < 0.05) gained FFM after 8 weeks compared to LFD, the HFD-fed F2-FLT females did not (Fig. 5 d). As such, HFD-fed F2-HGC females (p < 0.05) and F2-VGC females (p = 0.07) gained more FFM than F2-FLT females. Interestingly, while HFD increased energy intake and percent metabolic efficiency in all groups compared to LFD (Extended Data Fig. 5 ), no group differences within diet were observed. Examination of basal blood glucose in female mice showed increased blood glucose in HFD-fed F2-HGC females (p < 0.05) and F2-VGC females (p = 0.16) compared to their LFD controls (Fig. 5 b), these differences were not observed in the F2-FLT females. Therefore, F2-HGC (p < 0.05) and F2-VGC (p = 0.12) female mice fed a HFD had higher blood glucose than F2-FLT females. Blood glucose levels in the male mice showed no difference across the maternal granddam groups or across the dietary intervention. These data suggest transgenerational sex-specific differences in diet-induced weight gain and glucose homeostasis following female (granddam) spaceflight. Importantly, these effects are not related to the typical diet-induced increase in fat mass, but specifically to differences in diet-induced gain of FFM. Discussion Maternal spaceflight exposure results in observable functional and phenotypic changes in mammalian (F1) offspring conceived and born to spaceflight females and ground-based males on earth following 42 days of spaceflight. In particular, F1-FLT females were heavier at weaning and throughout adulthood compared to F1-HGC and F1-VGC female mice. F1-FLT females also showed significant reductions in isolated soleus muscle force production, which is consistent with overall declines observed in locomotion and marble burying, although neither treadmill endurance nor soleus fatigue were affected. In males, these effects were less apparent. While F1-FLT males were larger at 15 days of age, this difference did not extend to weaning and into adulthood. Furthermore, F1-FLT males did not show differences in locomotion or marble burying, and although soleus muscle force production seemed to be impaired, this did not reach statistical significance. However, it is important to note that the soleus muscles from F1-FLT males were significantly larger compared to those from F1-HGC and F1-VGC male mice. This may indicate a pseudohypertrophic phenotype at weaning, which will require further and more detailed investigation. Overall, these data illustrate functional and phenotypic changes in F1-FLT mice that are observant soon after birth and extends into adulthood in a sex-dependent manner. Moreover, when offspring produced by intercrossing F1-FLT mice were metabolically stressed this F2 generation also exhibited marked functional and phenotypic differences that were not observed in the control groups. This later transmission to F2 offspring is consistent with a preconception transgenerational inheritance pattern 23 . Interestingly and consistent with other experimental environmental/nutritional treatments and observations in humans where grandmothers were subjected to a nutritional stress (famine, over feeding), effects could be seen in their grandchildren 24-26 . These transgenerational changes seen in F2 mice are not often observed unless the animal is put into a stressful situation 27 . In this study, F2 mice were not provide a chronic secondary challenge or stress prior to their behavioral/locomotion analyses, which might explain the lack of effect in those F2 analyses versus that observed in their parents (F1 mice). Considering the greater individual genetic variation and potential greater epigenetic compositions of the F2 generation versus that of their parents which are genetically C57BL/6 X DBA2 hybrids, the observation of a spaceflight effect transmitted from the maternal granddam is quite remarkable. The mechanism by which this transgenerational effect is mediated being is under investigation as are more detailed molecular and biochemical studies which might provide deeper insights into how spaceflight is altering the maternal system. Indeed, whether this effect is induced by microgravity specifically or other spaceflight stressors (i.e., return) is not known, nor is it understood if this change in the F1-FLT mice was at the gamete (oocyte) level or changes in uterine support by F0 mothers during their pregnancies that gave rise to the F1 mice. What can be assured is that the oocytes that gave rise to the F1 mice, developed from the primary follicles that transitioned from primordial follicles in microgravity in their F0 mothers, as it is known that leaving the quiescent primordial follicle pool to ovulation takes 46.8 days to occur in mice 28 , and our experimental FLT dams were in space for 42 days followed by a 5 day acclimation to Earth gravity prior to mating. Ultimately, while the mouse system provides insights into mammalian reproductive phenotypes and responses to environmental conditions, it cannot predict whether similar impacts of spaceflight might occur in humans. Mitigating this effect of spaceflight in mice might be as simple as waiting additional time prior to conception, or by modifying the diets of the space females or their progeny. Because of the length of time between human generations it is important that these observations be understood better, in order to provide astronauts better health reccomendation on whether preservation of gametes prior to long-term exploration missions should be considered 29,30 . Lastly, this study primarily investigates the impact of microgravity and spaceflight (launch and return), while not addressing the role of space irradiation associated with long-term exploration missions outside of the Van Allen belts protection. As male and female gametes are both highly susceptible to irradiation, combined irradiation and microgravity studies using mice can provide meaningful insight for our next generation of human space travelers. Declarations Acknowledgements We thank Dr. Joseph Tash for the initial inspiration, guidance and submission of this project in 2013. We thank the Roskamp Institute Animal Care Team (Alexandra Goldring, Scott Ferguson, Molly Petit, Henry Sarau, Lisa Wallace) and Veterinarian’s (Joanna Maza, Howard Small); the Leidos Team (Nicholas Cole, Hailey Wiley, William McFadden, Steve Ortiz, Matthew Trisnadi, Nelson Cole, Estevan Fitzpatrick, Miranda McMickens, Ethan Baseden, Ben Wichmann and others); NASA Veterinarian’s (Chad Foster and Russell Higbee); the SpaceX CRS-29 payload integration, launch, and return teams; the Expedition 70 Astronauts for the excellent care and coordination of the work completed in Florida and on the ISS. We thank Ronca, Alwood and Christenson lab members Steffy Tabares-Ruiz, Jade Nguyen and Ashley Teatefor their assistance. We thank the University of Kansas Medical Center Animal Care Team (Megan Brown, Jill Courtney, Allison Rodecap, Zachary Bailey, Mary Kroh, Seth Barlett, Travis Hagedorn, Lea Vacek, Allison Neely); the Smith Intellectual and Developmental Disabilities Research Center Behavioral Core (Alexandra Frazier, Michelle Winter and Kenneth McCarson (NIH U54 HD 090216) and the KC MORE Metabolic Core (Colin McCoin; NIH COBRE P20GM144269) who provided critical support for behavioral and metabolic analyses; and Christenson lab members Shlok Chauhan and Savannah Siceloft for data assistance. We thank NASA Management, Ames and Space Biology (Yasaman Shirazi-Fard, Jonathan Galazka, Amy Gresser, Jenna Comella, Mary Lou James, Lynn Clary, John Howard, Mellodee White, Yi-Chun Chen and Christina Lim and so many others) for providing a supportive environment. We thank the NASA Space Radiation Analysis Group (SRAG) at JSC for radiation dosimetry estimates. We thank K. Roby and V. Chennathukuzhi for helpful discussions. Funding Principal Funding acquisition: NASA Space Biology grant #NNX15AB48G / #80NSSC24M0072 (PI: L.K.C., Co-I A.E.R., J.S.A., and S.P.); Secondary Funding: NASA Ames Research Innovation Award (A.E.R, J.S.A, and S.P); NIH COBRE P20GM144269 and University of Kansas Diabetes Institute Pilot Grant (E.M.M.); NSERC Alliance International Catalyst Grant ALLRP 591061 and Canadian Space Agency Grant 21HLSRM01 (VAF); NSERC CGS-D awards (B.L.H. and J.L.B.). Author contributions Conceptualization: L.K.C., J.S.A., A.E.R., S.P.; Funding acquisition: L.K.C., J.S.A., A.E.R.; Investigation: X.H., J.P., M.P., F.D., J.L.B., B.L.H., P.N., A.S., S.Y.C. S.P.; Methodology: L.K.C., J.S.A., A.E.R., S.P., E.M.M., V.F.; Project Administration: L.K.C., J.S.A., S.P., E.M.M., V.F. S.Y.C.; Data curation: F.D., S.P., E.M.M., V.F.; Supervision: L.K.C., J.S.A., S.P., E.M.M., V.F.; Visualization: F.D., S.P., E.M.M., V.F.; Writing - original draft: F.D., L.K.C., J.S.A., S.P., E.M.M., V.F. Competing Interests The authors declare that they have no competing interests. Data and Material Availability Metadata for animals is available upon request. All other data needed to evaluate the conclusions are available in the main text or Extended Data. Additional information Correspondence and requests for materials should be addressed to Lane K. Christenson, [email protected] Reprints and permissions information is available at www.nature.com/reprints. References National Academies of Sciences, E. & Medicine. Thriving in Space: Ensuring the Future of Biological and Physical Sciences Research: A Decadal Survey for 2023-2032 . (2023). Drago-Ferrante, R. et al. Extraterrestrial gynecology: could spaceflight increase the risk of developing cancer in female astronauts? 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Extraterrestrial gynecology: could spaceflight increase the risk of developing cancer in female astronauts? An updated review. International journal of molecular sciences 23 , 7465 (2022). https://doi.org/10.3390/ijms23137465 Chaplia, O., Mathyk, B. A., Nichols-Burns, S., Basar, M. & Halicigil, C. Beyond Earth’s bounds: navigating the frontiers of Assisted Reproductive Technologies (ART) in space. Reproductive Biology and Endocrinology 22 , 123 (2024). https://doi.org/10.1186/s12958-024-01290-y Method and Materials All experimental plans were approved by the Institutional Animal Care and Use Committees (IACUC) of NASA, the Roskamp Institute, and University of Kansas Medical Center (KUMC) and experiments carried out within applicable ethical guidelines for animal welfare. Preflight Acclimation of Animals Six-week old female C57BL/6J mice were purchased from the Jackson Labs (Bar Harbor, ME). The animals were implanted with BMDS IMI-500 transponders (Avidity Science, Waterford, WI) prior to shipping. One week after arriving at the Roskamp Institute, the animals were acclimated to the NASA Type 12 Nutrient upgraded rodent foodbars (NuRFB), water bottles outfitted with lixits and raised wire floors for about 4 weeks before being transferred to the Rodent Transporters for launch. Flight and Ground Control Animals As illustrated in Fig. 1, in total, 20 female were launched (FLT) on SpaceX Commercial Resupply Service (CRS)-29 at 11 weeks of age and resided on the International Space Station (ISS) for a duration of 42 days as part of Rodent Research 20 (RR-20) mission with live animal return, using the NASA Rodent Research platform described in Choi, et al 2020 31 . During the mission, radiation dosimetry performed at locations on the ISS nearby the Rodent Habitat estimated a total absorbed dose of approximately 10.1 mGy for the 42-day mission. Within 12 h of the Dragon capsule splashdown, female mice were transported to the NASA facility at the Roskamp Institute in Sarasota, FL. There 16 randomly selected females were transferred from the NASA Rodent Transporter which maintained the mice on GMT consistent with their tenure on the ISS, to standard vivarium cages that were maintained at EST. To enhance acclimation a small piece of the spaceflight nutrient food bar was placed in the cage in addition to regular breeding chow (Envigo 2919). On the 5 th day following return to Earth, these females were transferred to cages containing singly housed, fertility-proven DBA2/J male mice (Jackson Labs, Bar Harbor, ME). From these 16 breeding FLT F0 females, 10 F0 female mice ultimately gave birth to the F1-FLT offspring. Habitat ground control F0 (HGC; n=16) and vivarium ground control (VGC; n=16) remained on Earth, residing in either the NASA’s Rodent Habitat which was placed in a vertical orientation such that the doors were easily assessable or in standard filter top vivarium cages, respectively. The VGC mouse cages had elevated wire flooring (~10 mm above standard bedding) and were supplied with identical food bars and water lixits as the FLT and HGC animals. The HGC and VGC mice were maintained at a temperature of 28-29°C, within the ISS Environmental Simulator (ISSES) chamber that mimics the conditions (light schedule, temperature, humidity, CO2 levels, with the obvious exception of microgravity) consistent with the FLT mice are experiencing on the ISS. The VGC mice on RR20 vary from prior missions’ VGC cohorts, in that we placed the VGC mice into the ISSES chamber to ensure we mimicked the elevated temperatures seen in the FLT and HGC groups, as environmental temperature is known to have widespread physiological effects. F1-Offspring Husbandry Breeding cages were examined twice daily (without touching) at ~7 am and 3:30 pm to determine day of birth. At 5 days of age pup weights were taken using a balance that was maintained under a heat lamp and under barrier conditions in order to prevented cold shock. The entire cohort of pups was weighed in under 30 sec to minimize offspring and parental stress. No loss in pups was noted from day 5 through weaning for any litters. At 15 days of age, offspring sex was recorded while weighing. At weaning, 22 F1-FLT male offspring and 22 F1-FLT female mice were selected for transfer to the University of Kansas Medical Center (KUMC). To ensure a robust cohort of F1 mice, every effort was given to select 2 males and 2 females from each of the original F0- spaceflown dams. Similarly, the F0-HGC (n=10) and -VGC (n=10) dams, which were on a 5 and 8 day delay from the F0-FLT animals, respectively, in order to match the ISS environmental conditions, were mated and their F1 offspring were selected for shipment to KUMC. At weaning, randomly selected F1 mice were lightly sedated, and an RFID tag was injected under the skin near the shoulder blade and these F1 mice were then group housed in cohorts of 5 animals based on sex in preparation for shipping to KUMC. Cohorts of 5 male or 5 female F1 mice contained mixed F1-FLT, F1-HGC and F1-VGC mice. In total, 132 pups were shipped to KUMC via ground courier. At KUMC the animals were quarantined for two weeks and then transferred into the KUMC barrier facility, in preparation for behavioral analyses. Animals had alibitum access to food PicoLab Rodent Diet 20 (5053) and water. For the F1 behavioral study, n= 17, 19 and 22 for males and n=20, 19 and 24 for females for the F1-FLT, F1-HGC and F1-VGC experimental groups, respectively. All remaining F1 mice that were not transferred to KUMC, were euthanized at 21 days of age and blood samples collected and select tissues were collected (i.e., F1 soleus muscle). Following completion of the F1 mice behavioral analyses half of the males and females in each group were intercross mated within their same experimental group (i.e., FLT, HGC, VGC). Animals had ad libitum access to PicoLab Mouse Diet 20 (5058, breeding chow) and water. While the other half (~66 in total) of the F1-FLT, F1-HGC and F1-VGC mice were transferred to another facility at KUMC, adjacent to the metabolic testing facility. There animals completed qMRI and VOmax treadmill running between 90 and 100 days of age. Body composition was determined by qMRI (EchoMRI, Houston, TX). Following treadmill running this second cohort of F1-FLT, F1-HGC and F1-VGC were intercross mated as shown in Fig. 1. The resultant F2 pups from these matings were weighed at weaning, and then ~400 pups from the first litters of these intercross matings were RFID and either designated for behavioral analyses (n=180) or metabolic testing (n=144). Soleus force frequency and fatigue curves Left soleus muscles were carefully dissected and stimulated at 22-24°C using an Aurora Scientific contractile apparatus with a biphasic stimulator (model 701C). Muscles were subjected to a force frequency curve of 1, 20, 40, 80, and 160 Hz (350 ms) with a sampling rate of 1 kHz where peak isometric force was obtained. After a 2 min rest period, soleus muscles were then subjected to a fatigue protocol consisting of 70 Hz volleys (450 ms) every 2s for 160s. For force-frequency curves, specific isometric force was determined by normalizing peak force to cross-sectional area (CSA). CSA was calculated using the following formula: where m is muscle mass (mg); l is muscle length (mm), d is mammalian skeletal muscle density (1.06 mg/mm 3 ), Lf/Lo is the fiber length-to-muscle ratio (0.71 for soleus). For fatigue curves, data were presented as percent of initial force. Locomotor Activity Locomotor activity was measured using a BASi Force-Plate Actimeter (Bioanalytical Systems, Inc., Mount Vernon, IN) as described previously in 32,33 . The Actimeter is enclosed with a sound attenuating chamber to prevent outside influences on the animal’s behavior. The arena consists of a horizontal plate (42 cm × 42 cm) attached at the corners to force transducers that record the center of mass (X, Y position) of the animal subject. Mice were individually placed into the testing arena and allowed to move freely for 10 minutes. The average movement was quantified. All recorded data was processed using FPAAnalysis software version 9.10.04. Marble Bury Marble burying test is used to test for anxiety and compulsive behaviors. Mice that display behaviors associated with high anxiety or compulsive behaviors tend to dig in the bedding more, resulting in a greater percentage of marbles buried. The arena consists of a micro-isolator cage (30.8 cm x 19 cm) filled 2/3 of the way with clean cobb bedding. Prior to the test, animals were acclimated to the test room for a minimum of 30 minutes. Animals were then acclimated to the test arena for 5 minutes, then removed briefly and 20 marbles were placed in the arena in a regular pattern (5 rows of 4 across). Each animal was then placed in the arena for a total of 20 minutes, and the number of marbles buried to at least ¾ of their diameter beneath the bedding were counted. Elevated Zero Maze The elevated zero maze is used to evaluate anxiety-related behaviors in rodents. The maze consists of an open junction connected to four equal arms. Two opposing arms are open and two are enclosed by walls. Prior to the test, animals were acclimated to the test room for a minimum of 30 minutes. Each animal was placed on the maze in the junction facing one of the open arms. The mouse was allowed to freely explore for 5 minutes. The test was recorded and analyzed using Noldus EthoVision XT tracking software (version 16.0.1536). The relative time spent (%) in open arms versus closed arms was calculated. Western Diet Feeding Male and female 6-week-old F2 mice were individually housed at 28°C utilizing a reverse light cycle (dark 10:00-22:00) prior to the start of the experiment. Between 8-10 weeks of age, mice were assigned to either a low-fat diet (LFD, 10% kcal fat, 3.5% kcal sucrose, fiber (5%/w cellulose, 1%/w inulin, 1%/w pectin), Research diets, D24061901) or a western diet (HFD, 45% kcal fat, 7% kcal sucrose, fiber (5%/w cellulose), Research diets, D12451) (n=12/sex/diet /treatment group), with ad libitum access to food and water. Body weight (BW) and food weight were recorded at the start of every week prior to the dark cycle (8:00-10:00). Body composition (fat mass and fat-free mass) was determined by qMRI (EchoMRI, Houston, TX) every two weeks. Fat -free mass (FFM) was calculated as the difference between body weight and fat mass. HFD was replaced twice a week (every 3-4 days). Energy intake was calculated using the energy density of the LFD (3.85 kcal/g) or HFD (4.73 kcal/gram) times the food intake over the course of the 8 weeks. Percent metabolic efficiency was calculated as: (change in fat mass (kcal) + change in lean mass (kcal)/EI, with the change fat and lean kcal calculated as 9.32 kcal/g & 1.19 kcal/g, respectively 34 . Blood glucose levels were measured following a 4-hour food withdrawal (Contour Next EZ, Ascensia Diabetes Care). Statistics For F1 and F2 behavior all datasets were tested for normal distribution. For normally distributed data, we performed an ANOVA followed by Dunnett's posthoc. For non-normal data, we performed a Kruskal-Wallis followed by Dunn's posthoc. For the F2 western diet study, data are presented as means and standard error. The two-standard deviation test was utilized to test for outliers within group. Data were analyzed by two-way ANOVA. Where interactions and/or main effects were observed, post-hoc analysis was performed using Fisher’s LSD. Main effects are only designated when all pairwise comparisons were significant. 31 Choi, S. Y. et al. Validation of a new rodent experimental system to investigate consequences of long duration space habitation. Scientific reports 10 , 2336 (2020). https://doi.org/10.1038/s41598-020-58898-4 32 Fowler, S. et al. A force-plate actometer for quantitating rodent behaviors: illustrative data on locomotion, rotation, spatial patterning, stereotypies, and tremor. Journal of neuroscience methods 107 , 107-124 (2001). https://doi.org/10.1016/s0165-0270(01)00359-4 33 McCarson, K. E., Winter, M. K., Abrahamson, D. R., Berman, N. E. & Smith, P. G. Assessing complex movement behaviors in rodent models of neurological disorders. Neurobiology of learning and memory 165 , 106817 (2019). https://doi.org/10.1016/j.nlm.2018.02.025 34 Luijten, I. H. et al. Glucocorticoid-induced obesity develops independently of UCP1. Cell Reports 27 , 1686-1698. e1685 (2019). https://doi.org/10.1016/j.celrep.2019.04.041 Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6458038","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":463110861,"identity":"4c1dbf8b-ad05-4826-8a90-00e169ad1f6f","order_by":0,"name":"Lane 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"April","middleName":"","lastName":"Ronca","suffix":""},{"id":463110872,"identity":"cfe2268f-077d-4d80-b9d3-d7b2be396e00","order_by":11,"name":"Joshua Alwood","email":"","orcid":"https://orcid.org/0000-0002-6702-6673","institution":"Space Biosciences Division, NASA Ames Research Center","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Alwood","suffix":""},{"id":463110873,"identity":"b0c00a63-61e9-4747-a38d-fba745878d8e","order_by":12,"name":"Val Fajardo","email":"","orcid":"https://orcid.org/0000-0003-4500-3347","institution":"Brock University","correspondingAuthor":false,"prefix":"","firstName":"Val","middleName":"","lastName":"Fajardo","suffix":""},{"id":463110874,"identity":"4aa0366b-fd86-49e5-be10-4c3629954567","order_by":13,"name":"E Morris","email":"","orcid":"","institution":"University of Kansas Medical Center","correspondingAuthor":false,"prefix":"","firstName":"E","middleName":"","lastName":"Morris","suffix":""},{"id":463110875,"identity":"297178a6-9082-4c95-96b5-00689d19b82e","order_by":14,"name":"Stephanie Puukila","email":"","orcid":"","institution":"Blue Marble Space Institute for Science","correspondingAuthor":false,"prefix":"","firstName":"Stephanie","middleName":"","lastName":"Puukila","suffix":""}],"badges":[],"createdAt":"2025-04-15 21:55:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6458038/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6458038/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83610112,"identity":"17af8d06-cce4-457f-a36c-f378553ca23b","added_by":"auto","created_at":"2025-05-29 12:06:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnimal mating and experimental design for generation of F1 and F2 offspring. \u003c/strong\u003e11-week-old C57BL/6J females (F0) were assigned to be launched aboard SpX29 and reside on the ISS as flight (FLT) mice, or in the ISS Environmental Simulator for the habitat ground control (HGC) or vivarium ground control (VGC) groups. Proven fertile DBA/2J males were mated to FLT, HGC and VGC females to generate F1 offspring, 120 of these F1 offspring underwent detailed behavioral assessments followed by intercross breeding to generate F2 offspring.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458038/v1/e40ff4c340b5c0dce6766cf2.jpg"},{"id":83610117,"identity":"cc91befd-84d1-497a-bfaf-78525de1ba84","added_by":"auto","created_at":"2025-05-29 12:06:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFLT offspring exhibit sex-dependent difference in body weight and composition compared to controls.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Combined offspring (female and male) weights at 5 and 10 days of age and sex-specific offspring weights from FLT (n=10) HGC (n=10) and VGC (n=9) dams at 15 and 21 days of age. Pup weight data includes only 1\u003csup\u003est\u003c/sup\u003e litter F1-FLT males (n=26) and females (n=40), F1-HGC males (n=45) and females (n=41), F1-VGC males (n=27) and females (n=46).\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e) Body weights for adult (93-106 d) female and male F1-FLT (n=10, 11 respectively), F1-HGC (n=10, 11 respectively) and F1-VGC (n=10, 9 respectively). (\u003cstrong\u003ec\u003c/strong\u003e) Adult body composition (fat mass and % fat mass) and (\u003cstrong\u003ed\u003c/strong\u003e) lean mass and percentage lean mass are shown for females, male data are shown in (Extended Data Fig. 1). \u003csup\u003e* \u003c/sup\u003eMeans ± SEM are different (p\u0026lt; 0.05, respectively) with individual offspring represented by open circles.\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458038/v1/78ec710fc4ac810e72c6c569.jpg"},{"id":83611199,"identity":"c8b2d120-b45b-4579-ad9d-fd8b88c4f2c2","added_by":"auto","created_at":"2025-05-29 12:22:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSoleus muscle mass and isometric force production in 21-day-old pups. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Absolute soleus muscle weights in male and female F1-FLT (n = 4, 12 respectively), F1-HGC (n = 12, 7 respectively) and F1-VGC (n = 4, 5 respectively) pups. (\u003cstrong\u003eb\u003c/strong\u003e) Soleus: body mass ratio in male and female F1-FLT, F1-HGC and F1-VGC pups. (\u003cstrong\u003ec\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eIsometric soleus force-frequency curves in male, female and combined male/female datasets from male and female F1-FLT (n = 4, 12 respectively), F1-HGC (n = 11, 7 respectively) and F1-VGC (n = 3, 5 respectively) pups. For all bar graphs,\u003csup\u003e * \u003c/sup\u003emeans ± SEM are different (p\u0026lt; 0.05) with individual offspring represented by open circles. For force-frequency curves, \u003csup\u003e* \u003c/sup\u003eFLT vs VGC comparisons and \u003csup\u003e†\u003c/sup\u003e FLT vs HGC are different (p\u0026lt; 0.05, respectively), using a Bonferonni post-hoc test with data displayed as means ± SEM.\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458038/v1/d7bfb600b40a4874f8238037.jpg"},{"id":83610114,"identity":"5d55f671-d8b2-412b-8718-42d7e812830f","added_by":"auto","created_at":"2025-05-29 12:06:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":69844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBehavioral and locomotor consequences of maternal spaceflight exposure on offspring.\u003cbr\u003e\n\u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Average locomotor activity for the three maternal environmental exposures were compared within sex for adult (64-77 day old) F1-FLT, F1-HGC and F1-VGC mice (n=17-20/sex/group). Similarly, (\u003cstrong\u003eb\u003c/strong\u003e) marble burying and (\u003cstrong\u003ec\u003c/strong\u003e) time in open arms during elevated plus maze were compared within sex across the treatment groups. \u003csup\u003e* \u003c/sup\u003eMeans ± SEM are different (p\u0026lt; 0.05) with individual offspring represented by open circles.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458038/v1/df0d965cbaf4fe12abf15d0f.jpg"},{"id":83610121,"identity":"b672a740-a6a8-44ca-a1fd-6776952c38ad","added_by":"auto","created_at":"2025-05-29 12:06:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":134226,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysiological and Metabolic Responses in F2 mice as Influenced by Maternal Granddam Spaceflight Exposure and Diet. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eWeight gain patterns in female and male F2 mice fed a HFD or LFD, and cumulative weight change at 8-weeks for F2-FLT, F2-HGC and F2-VGC mice. (\u003cstrong\u003eb\u003c/strong\u003e) Blood glucose levels completed by tail vein collection at 8-weeks of dietary intervention. (\u003cstrong\u003ec\u003c/strong\u003e) Change in fat mass, (\u003cstrong\u003ed\u003c/strong\u003e) fat-free mass were determined bi-weekly using qMRI. Additionally, energy intake and metabolic efficiency (%) were determined (see Extended Data Fig. 5\u003cstrong\u003e)\u003c/strong\u003e. Data are presented as means ± SEM (n=10-12). † p\u0026lt;0.05 main effect of diet group, \u003csup\u003e*\u003c/sup\u003e p\u0026lt;0.05 effect of group within diet.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6458038/v1/4e8847ef52d6304cdd793268.jpg"},{"id":83612532,"identity":"ba89d02b-ae59-4acf-a9a9-64a871520135","added_by":"auto","created_at":"2025-05-29 12:38:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1489146,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6458038/v1/207ae717-d9e9-4a82-b269-836c1e6b0d6b.pdf"},{"id":83610119,"identity":"73743804-9e8a-46ff-ab07-609f0f87108d","added_by":"auto","created_at":"2025-05-29 12:06:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":646527,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6458038/v1/7e3d945ecd97185cbd5c730a.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Impact of Female Space Travel on Progeny Health","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFundamental questions regarding the long-term effects of spaceflight have garnered considerable interest as more and more individuals venture into space. The unique environmental conditions of space travel such as microgravity, increased radiation exposure, and the acute hypergravity during transitions (launch and reentry) encountered by astronauts present unique challenges to mammalian physiology \u003csup\u003e1\u003c/sup\u003e. While it is clear that humans can acclimate to space for extended periods (up to one year continuously or longer cumulatively) and successfully re-acclimate to Earth, the specific consequences on reproductive health remain largely unknown, and in particular the impact of space travel on the female reproductive systemand whether physiological changes sustained in space might have longer term consequences on offspring born to these space travelers \u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. Spaceflight studies with non-mammalian species have indicated that fertilization and development can occur in space to produce viable offspring \u003csup\u003e5\u0026ndash;7\u003c/sup\u003e. In mammals, our understanding in this area are anecdotal in the case of individual human experiences or come from very short-term (\u0026lt;\u0026thinsp;12 d) spaceflight exposures of already pregnant rodents followed by ground birth of phenotypically normal offspring \u003csup\u003e8\u0026ndash;12\u003c/sup\u003e. Consistent with late gestational exposures, when 2-cell mouse embryos were cultured in microgravity, they were able to develop to the blastocyst stage \u003csup\u003e8,9\u003c/sup\u003e, although in one study (\u003cem\u003e9\u003c/em\u003e) a decrease in embryo quality was observed. In the only mammalian study to date to examine spaceflight effects on F1 offspring, frozen sperm from male mice exposed to microgravity on the International Space Station for 35 days were used to fertilize oocytes and the resultant embryos were transferred to recipient females \u003csup\u003e13\u003c/sup\u003e. These F1 offspring exhibited no changes in growth rates or viability when compared to F1 offspring sired by ground controls, nor were there any differences in litter sizes of F1 x F1 intercrosses \u003csup\u003e13\u003c/sup\u003e. Transcriptomic analysis of F1 offspring originating from 2 microgravity exposed (C57BL6/J) males and 2 ground control (C57BL6/NCrl) males, identified 24 differentially expressed genes in liver tissue of F1 males, while F1 females were not evaluated \u003csup\u003e14\u003c/sup\u003e. To date, no phenotypic differences have been reported in these F1 offspring. This work leaves many questions unanswered, particularly with respect to the possible effects on the females, and this is of particular importance as females bring greater genetic, cellular and environmental contributions (i.e., mitochondria, nutritional) to the offspring.\u003c/p\u003e \u003cp\u003eEpigenetic changes occur in response to spaceflight, with changes seen in Arabidopsis \u003csup\u003e15,16\u003c/sup\u003e, C. elegans \u003csup\u003e17\u003c/sup\u003e, tissues/cells from male mice \u003csup\u003e14\u003c/sup\u003e and in the NASA human Twin Study, where DNA methylation changes were present in immune and oxidative stress\u0026ndash;related pathways in white blood cells \u003csup\u003e18\u003c/sup\u003e. Given that environmental insults (mal-/over-nutrition, chemical exposures, lifestyle) can reprogram the epigenome of the germline (eggs and sperm), which then transmits to future generations \u003csup\u003e19\u0026ndash;22\u003c/sup\u003e, we set out to test the hypothesis that spaceflight might also impact future generations. Recently, our group completed a comprehensive analysis of the impacts of microgravity and spaceflight on the female reproductive system, as part of NASA Rodent Research-20 mission. In this study a group of 20 female mice after having spent 42 days in space (on the International Space Station; roughly equivalent to 8 months of a woman\u0026rsquo;s reproductive lifespan), were returned to Earth and then mated to ground-based males. The resultant offspring (F1) and subsequent offspring of these F1 mice (i.e., F2 generation) were extensively phenotyped with respect to growth, behavioral changes, tissue function and to the impact of a secondary metabolic stress on the adaptability of these mice.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eOffspring originating from spaceflight (FLT) dams that experienced 42 days in microgravity on the International Space Station (ISS) were compared to two groups of ground control offspring (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Habitat ground control dams (HGC) were housed in identical habitats as the FLT females, but in a ground-based ISS Environmental Simulator that mimics ISS FLT conditions (light schedule, temperature, humidity, CO\u003csub\u003e2\u003c/sub\u003e levels, with the obvious exception of microgravity). Additionally, vivarium ground control dams (VGC) were housed in standard mouse cages with key modifications (see Methods), these were also placed in the ISS Environmental Simulator to mimic ISS FLT conditions. Five days following return to Earth, FLT dams (C57BL6/J) were mated to ground-based DBA2/J males. The resultant offspring (F1) from these matings and intercrosses of F1-FLT females to F1-FLT males were used to generate F2-FLT mice, this same paradigm was repeated for the HGC and VGC dams and their F1 offspring (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMaternal spaceflight exposure leads to increased offspring body weights.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBody weights of F0 offspring were not different at 5 days of age across the three environmental conditions, however, by 10 days of age, F1-FLT pups were heavier than F1-HGC and F1-VGC pups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At 15 and 21 days of age female F1-FLT pups remained heavier than control pups, and this remained into adulthood (93\u0026ndash;106 d; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In contrast, F1-FLT male weights were not different from controls (F1-HGC/F1-VGC) at 21 days of age or thereafter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Body composition of the F0 offspring also revealed sex-specific impacts associated with maternal spaceflight exposure, with F1-FLT females having greater fat mass than F1-HGC females (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and F1-VGC females (p\u0026thinsp;=\u0026thinsp;0.08) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Comparison of % fat of the F1-FLT females had a greater level of fat compared to F1-HGC females, but were not different than F1-VGC females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). F1-FLT females tended (p\u0026thinsp;=\u0026thinsp;0.12) and had greater (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lean mass than F1-HGC and F1-VGC females, respectively. When adjusted for total body weight, F1-FLT females exhibited a lower percentage of lean mass compared to F1-HGC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and F1-VGC (p\u0026thinsp;=\u0026thinsp;0.06) females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In contrast, body composition did not vary for the F1-FLT, F1-HGC, and F1-VGC males (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The data indicate that maternal spaceflight exposure predominantly affected female offspring, resulting in increased body weight, fat mass, and altered lean mass distribution, while male body weight and composition remained unaffected after day 15 of life.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eImpaired muscle force production in offspring following maternal spaceflight exposure\u003c/h2\u003e \u003cp\u003eSoleus muscles isolated from 21-day-old male and female pups of FLT, HGC and VGC dams were tested for their ability to generate force, because postural muscles such as the soleus are well-known to be most susceptible to muscle atrophy and weakness after spaceflight. However, whether these effects may be passed down to spaceflight offspring has never been examined. Soleus muscle mass (absolute and relative to body mass) was greater in F1-FLT male pups versus F1-HGC and F1-VGC male pups, whereas female pups showed no differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). Force-frequency analysis demonstrated a significant reduction in specific force production (i.e., made relative to muscle cross-sectional area) at submaximal and maximal frequencies in the F1-FLT pups compared to F1-HGC and F1-VGC pups, being significantly different for female and combined male and female datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Force production by male F1-FLT pups compared to F1-HGC and F1-VGC pups did not reach statistical significance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBehavioral, locomotor and aerobic fitness impacts of maternal spaceflight exposure on offspring\u003c/h3\u003e\n\u003cp\u003eBehavior and locomotor activity were examined in FLT, HGC and VGC offspring (n\u0026thinsp;=\u0026thinsp;17\u0026ndash;20/sex/treatment group, average of 70-days-old). Locomotor and behavioral activity in offspring born to F0 spaceflight dams demonstrated sex-specific differences. F1-FLT females demonstrated a 15% decrease in average locomotion relative to their F1-HGC counterparts (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, F1-FLT, F1-HGC and F1-VGC males showed no significant difference in locomotion (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea), indicating sex-dependent differences in response to their mother\u0026apos;s spaceflight exposure. The marble burying test, a measurement for anxiety and compulsive behaviors, revealed that F1-FLT females buried approximately 37% fewer marbles compared to F1-HGC and F1-VGC females (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). Conversely, F1-FLT males displayed no notable variations in marble burial behavior, hence reinforcing a sex-dependent impact (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). Measure of anxiety and risk-taking behavior via elevated plus maze indicated that F1-FLT males allocated approximately 90% more time to the open arms than F1-HGC and F1-VGC males (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec), implying diminished anxiety levels or heightened risk-taking behavior. No notable differences were detected in F1-FLT females, suggesting a possible sex-specific reaction to stresses caused by spaceflight (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). Aerobic fitness of F1 females and males (90\u0026ndash;100 days of age) was tested on a rodent treadmill. Though there were sex-related variations in total distance run or time to exhaustion (Extended Data Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), there were no differences related to the maternal environmental (FLT/HGC/VGC) exposures. Likewise, there were no differences in isolated soleus muscle endurance between the F1-FLT, F1-HGC, and F1-VGC male, female and combined male/female datasets (Extended Data Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransgenerational impacts of maternal spaceflight exposure and ability of F1 offspring (F2 pups) to respond to a western diet metabolic stressor.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn contrast to F0 offspring (F1 pups), adult F1 offspring or F2 pups (n\u0026thinsp;=\u0026thinsp;25\u0026ndash;43/sex/group, averaging 95-days-old) did not exhibit any striking differences in behavioral or locomotion based on their maternal granddam\u0026rsquo;s environmental exposure (FLT, HGC and VGC; Extended Data Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Because F1-FLT, F1-HGC and F1-VGC mice are genetically C57BL/6 X DBA2 hybrids, the intercross progeny (i.e.,F2-FLT, F2-HGC and F2-VGC) generated will display more individual genetic variation than their parents. This genetic variation could contribute to an observed greater variance in phenotypes (Extended Data Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). To test whether a secondary stressor might elicit differences, F2 mice were fed a Western diet (high fat, high sucrose, low/simple fiber; HFD) for 8 weeks. Briefly, 8\u0026ndash;10-week-old F2 mice were maintained in a thermoneutral temperature (~\u0026thinsp;28\u0026ordm;C) and fed control (LFD) or HFD. All F2 mice fed a HFD (n\u0026thinsp;=\u0026thinsp;72) exhibited increased weight gain over those fed a LFD (n\u0026thinsp;=\u0026thinsp;72; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Female F2 mice exhibited no differences in 8-week weight gain within diet based on their maternal granddam\u0026rsquo;s spaceflight exposure. In contrast, HFD-fed F2-FLT males had increased weight gain throughout the eight-week feeding trial when compared to HFD-fed F2-HGC and F2-VGC males (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As expected, HFD feeding resulted in greater fat mass gain in males and females compared to LFD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). Additionally, change in fat-free mass (FFM) was increased in HFD males compared to LFD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), however, F2-FLT males had a greater change in FFM than F2-HGC and F2-VGC controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). Conversely, while both HFD-fed F2-HGC females (p\u0026thinsp;=\u0026thinsp;0.06) and F2-VGC females (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) gained FFM after 8 weeks compared to LFD, the HFD-fed F2-FLT females did not (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). As such, HFD-fed F2-HGC females (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and F2-VGC females (p\u0026thinsp;=\u0026thinsp;0.07) gained more FFM than F2-FLT females. Interestingly, while HFD increased energy intake and percent metabolic efficiency in all groups compared to LFD (Extended Data Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), no group differences within diet were observed. Examination of basal blood glucose in female mice showed increased blood glucose in HFD-fed F2-HGC females (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and F2-VGC females (p\u0026thinsp;=\u0026thinsp;0.16) compared to their LFD controls (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb), these differences were not observed in the F2-FLT females. Therefore, F2-HGC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and F2-VGC (p\u0026thinsp;=\u0026thinsp;0.12) female mice fed a HFD had higher blood glucose than F2-FLT females. Blood glucose levels in the male mice showed no difference across the maternal granddam groups or across the dietary intervention. These data suggest transgenerational sex-specific differences in diet-induced weight gain and glucose homeostasis following female (granddam) spaceflight. Importantly, these effects are not related to the typical diet-induced increase in fat mass, but specifically to differences in diet-induced gain of FFM.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMaternal spaceflight exposure results in observable functional and phenotypic changes in mammalian (F1) offspring conceived and born to spaceflight females and ground-based males on earth following 42 days of spaceflight. In particular, F1-FLT females were heavier at weaning and throughout adulthood compared to F1-HGC and F1-VGC female mice. F1-FLT females also showed significant reductions in isolated soleus muscle force production, which is consistent with overall declines observed in locomotion and marble burying, although neither treadmill endurance nor soleus fatigue were affected. In males, these effects were less apparent. While F1-FLT males were larger at 15 days of age, this difference did not extend to weaning and into adulthood. Furthermore, F1-FLT males did not show differences in locomotion or marble burying, and although soleus muscle force production seemed to be impaired, this did not reach statistical significance. However, it is important to note that the soleus muscles from F1-FLT males were significantly larger compared to those from F1-HGC and F1-VGC male mice. This may indicate a pseudohypertrophic phenotype at weaning, which will require further and more detailed investigation. Overall, these data illustrate functional and phenotypic changes in F1-FLT mice that are observant soon after birth and extends into adulthood in a sex-dependent manner.\u003c/p\u003e\n\u003cp\u003eMoreover, when offspring produced by intercrossing F1-FLT mice were metabolically stressed this F2 generation also exhibited marked functional and phenotypic differences that were not observed in the control groups. This later transmission to F2 offspring is consistent with a preconception transgenerational inheritance pattern \u003csup\u003e23\u003c/sup\u003e. Interestingly and consistent with other experimental environmental/nutritional treatments and observations in humans where grandmothers were subjected to a nutritional stress (famine, over feeding), effects could be seen in their grandchildren \u003csup\u003e24-26\u003c/sup\u003e. These transgenerational changes seen in F2 mice are not often observed unless the animal is put into a stressful situation \u003csup\u003e27\u003c/sup\u003e. In this study, F2 mice were not provide a chronic secondary challenge or stress prior to their behavioral/locomotion analyses, which might explain the lack of effect in those F2 analyses versus that observed in their parents (F1 mice). Considering the greater individual genetic variation and potential greater epigenetic compositions of the F2 generation versus that of their parents which are genetically C57BL/6 X DBA2 hybrids,\u0026nbsp; the observation of a spaceflight effect transmitted from the maternal granddam is quite remarkable. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mechanism by which this transgenerational effect is mediated being is under investigation as are more detailed molecular and biochemical studies which might provide deeper insights into how spaceflight is altering the maternal system. Indeed, whether this effect is induced by microgravity specifically or other spaceflight stressors (i.e., return) is not known, nor is it understood if this change in the F1-FLT mice was at the gamete (oocyte) level or changes in uterine support by F0 mothers during their pregnancies that gave rise to the F1 mice. What can be assured is that the oocytes that gave rise to the F1 mice, developed from the primary follicles that transitioned from primordial follicles in microgravity in their F0 mothers, as it is known that leaving the quiescent primordial follicle pool to ovulation takes 46.8 days to occur in mice \u003csup\u003e28\u003c/sup\u003e, and our experimental FLT dams were in space for 42 days followed by a 5 day acclimation to Earth gravity prior to mating.\u003c/p\u003e\n\u003cp\u003eUltimately, while the mouse system provides insights into mammalian reproductive phenotypes and responses to environmental conditions, it cannot predict whether similar impacts of spaceflight might occur in humans. Mitigating this effect of spaceflight in mice might be as simple as waiting additional time prior to conception, or by modifying the diets of the space females or their progeny. Because of the length of time between human generations it is important that these observations be understood better, in order to provide astronauts better health reccomendation on whether preservation of gametes prior to long-term exploration missions should be considered \u003csup\u003e29,30\u003c/sup\u003e. Lastly, this study primarily investigates the impact of microgravity and spaceflight (launch and return), while not addressing the role of space irradiation associated with long-term exploration missions outside of the Van Allen belts protection. As male and female gametes are both highly susceptible to irradiation, combined irradiation and microgravity studies using mice can provide meaningful insight for our next generation of human space travelers. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe thank Dr. Joseph Tash for the initial inspiration, guidance and submission of this project in 2013. We thank the Roskamp Institute Animal Care Team (Alexandra Goldring, Scott Ferguson, Molly Petit, Henry Sarau, Lisa Wallace) and Veterinarian’s (Joanna Maza, Howard Small); the Leidos Team (Nicholas Cole, Hailey Wiley, William McFadden, Steve Ortiz, Matthew Trisnadi, Nelson Cole, Estevan Fitzpatrick, Miranda McMickens, Ethan Baseden, Ben Wichmann and others); NASA Veterinarian’s (Chad Foster and Russell Higbee); the SpaceX CRS-29 payload integration, launch, and return teams; the Expedition 70 Astronauts for the excellent care and coordination of the work completed in Florida and on the ISS. We thank Ronca, Alwood and Christenson lab members Steffy Tabares-Ruiz, Jade Nguyen and Ashley Teatefor their assistance. We thank the University of Kansas Medical Center Animal Care Team (Megan Brown, Jill Courtney, Allison Rodecap, Zachary Bailey, Mary Kroh, Seth Barlett, Travis Hagedorn, Lea Vacek, Allison Neely); the Smith Intellectual and Developmental Disabilities Research Center\u0026nbsp;Behavioral Core (Alexandra Frazier, Michelle Winter and Kenneth McCarson (NIH U54 HD 090216) and the KC MORE Metabolic Core (Colin McCoin; NIH COBRE P20GM144269)\u0026nbsp;who provided critical support for behavioral and metabolic analyses; and Christenson lab members Shlok Chauhan and Savannah Siceloft for data assistance. We thank NASA Management, Ames and Space Biology (Yasaman Shirazi-Fard, Jonathan Galazka, Amy Gresser, Jenna Comella, Mary Lou James, Lynn Clary, John Howard, Mellodee White, Yi-Chun Chen and Christina Lim and so many others) for providing a supportive environment. We thank the NASA Space Radiation Analysis Group (SRAG) at JSC for radiation dosimetry estimates. We thank K. Roby and V. Chennathukuzhi for helpful discussions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e Principal Funding acquisition:\u0026nbsp;NASA Space Biology grant #NNX15AB48G / #80NSSC24M0072 (PI: L.K.C., Co-I A.E.R., J.S.A., and S.P.); Secondary Funding: NASA Ames Research Innovation Award (A.E.R, J.S.A, and S.P); NIH COBRE P20GM144269 and\u0026nbsp;University of Kansas Diabetes Institute Pilot Grant (E.M.M.);\u0026nbsp;NSERC Alliance International Catalyst Grant ALLRP 591061 and Canadian Space Agency Grant 21HLSRM01 (VAF); NSERC CGS-D awards (B.L.H. and J.L.B.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e Conceptualization: L.K.C., J.S.A., A.E.R., S.P.; Funding acquisition: L.K.C., J.S.A., A.E.R.; Investigation: X.H., J.P., M.P., F.D., J.L.B., B.L.H., P.N., A.S., S.Y.C. S.P.; Methodology: L.K.C., J.S.A., A.E.R., S.P., E.M.M., V.F.; Project Administration: L.K.C., J.S.A., S.P., E.M.M., V.F. S.Y.C.; Data curation: F.D., S.P., E.M.M., V.F.; Supervision: L.K.C., J.S.A., S.P., E.M.M., V.F.; Visualization: F.D., S.P., E.M.M., V.F.; Writing - original draft: F.D., L.K.C., J.S.A., S.P., E.M.M., V.F.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and Material Availability\u003c/strong\u003e Metadata for animals is available upon request. All other data needed to evaluate the conclusions are available in the main text or Extended Data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u0026nbsp;\u003c/strong\u003eshould be addressed to Lane K. Christenson, [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permissions information\u0026nbsp;\u003c/strong\u003eis available at www.nature.com/reprints.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNational Academies of Sciences, E. \u0026amp; Medicine. \u003cem\u003eThriving in Space: Ensuring the Future of Biological and Physical Sciences Research: A Decadal Survey for 2023-2032\u003c/em\u003e. (2023).\u003c/li\u003e\n\u003cli\u003eDrago-Ferrante, R.\u003cem\u003e et al.\u003c/em\u003e Extraterrestrial gynecology: could spaceflight increase the risk of developing cancer in female astronauts? 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One week after arriving at the Roskamp Institute, the animals were acclimated to the NASA Type 12 Nutrient upgraded rodent foodbars (NuRFB), water bottles outfitted with lixits and raised wire floors for about 4 weeks before being transferred to the Rodent Transporters for launch.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlight and Ground Control Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Fig. 1, in total, 20 female were launched (FLT) on SpaceX Commercial Resupply Service (CRS)-29 at 11 weeks of age and resided on the International Space Station (ISS) for a duration of 42 days as part of Rodent Research 20 (RR-20) mission with live animal return, using the NASA Rodent Research platform described in Choi, et al 2020 \u003csup\u003e31\u003c/sup\u003e. During the mission, radiation dosimetry performed at locations on the ISS nearby the Rodent Habitat estimated a total absorbed dose of approximately 10.1 mGy for the 42-day mission. Within 12 h of the Dragon capsule splashdown, female mice were transported to the NASA facility at the Roskamp Institute in Sarasota, FL. There 16 randomly selected females were transferred from the NASA Rodent Transporter which maintained the mice on GMT consistent with their tenure on the ISS, to standard vivarium cages that were maintained at EST. To enhance acclimation a small piece of the spaceflight nutrient food bar was placed in the cage in addition to regular breeding chow (Envigo 2919). On the 5\u003csup\u003eth\u003c/sup\u003e day following return to Earth, these females were transferred to cages containing singly housed, fertility-proven DBA2/J male mice (Jackson Labs, Bar Harbor, ME). From these 16 breeding FLT F0 females, 10 F0 female mice ultimately gave birth to the F1-FLT offspring.\u003c/p\u003e\n\u003cp\u003eHabitat ground control F0 (HGC; n=16) and vivarium ground control (VGC; n=16) remained on Earth, residing in either the NASA’s Rodent Habitat which was placed in a vertical orientation such that the doors were easily assessable or in standard filter top vivarium cages, respectively. The VGC mouse cages had elevated wire flooring (~10 mm above standard bedding) and were supplied with identical food bars and water lixits as the FLT and HGC animals. The HGC and VGC mice were maintained at a temperature of 28-29°C, within the ISS Environmental Simulator (ISSES) chamber that mimics the conditions (light schedule, temperature, humidity, CO2 levels, with the obvious exception of microgravity) consistent with the FLT mice are experiencing on the ISS. The VGC mice on RR20 vary from prior missions’ VGC cohorts, in that we placed the VGC mice into the ISSES chamber to ensure we mimicked the elevated temperatures seen in the FLT and HGC groups, as environmental temperature is known to have widespread physiological effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF1-Offspring Husbandry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBreeding cages were examined twice daily (without touching) at ~7 am and 3:30 pm to determine day of birth. At 5 days of age pup weights were taken using a balance that was maintained under a heat lamp and under barrier conditions in order to prevented cold shock. The entire cohort of pups was weighed in under 30 sec to minimize offspring and parental stress. No loss in pups was noted from day 5 through weaning for any litters. At 15 days of age, offspring sex was recorded while weighing. At weaning, 22 F1-FLT male offspring and 22 F1-FLT female mice were selected for transfer to the University of Kansas Medical Center (KUMC). To ensure a robust cohort of F1 mice, every effort was given to select 2 males and 2 females from each of the original F0- spaceflown dams. Similarly, the F0-HGC (n=10) and -VGC (n=10) dams, which were on a 5 and 8 day delay from the F0-FLT animals, respectively, in order to match the ISS environmental conditions, were mated and their F1 offspring were selected for shipment to KUMC. At weaning, randomly selected F1 mice were lightly sedated, and an RFID tag was injected under the skin near the shoulder blade and these F1 mice were then group housed in cohorts of 5 animals based on sex in preparation for shipping to KUMC. Cohorts of 5 male or 5 female F1 mice contained mixed F1-FLT, F1-HGC and F1-VGC mice. In total, 132 pups were shipped to KUMC via ground courier. At KUMC the animals were quarantined for two weeks and then transferred into the KUMC barrier facility, in preparation for behavioral analyses. Animals had alibitum access to food PicoLab Rodent Diet 20 (5053) and water. \u0026nbsp;For the F1 behavioral study, n= 17, 19 and 22 for males and n=20, 19 and 24 for females for the F1-FLT, F1-HGC and F1-VGC experimental groups, respectively. All remaining F1 mice that were not transferred to KUMC, were euthanized at 21 days of age and blood samples collected and select tissues were collected (i.e., F1 soleus muscle).\u003c/p\u003e\n\u003cp\u003eFollowing completion of the F1 mice behavioral analyses half of the males and females in each group were intercross mated within their same experimental group (i.e., FLT, HGC, VGC). Animals had \u003cem\u003ead libitum\u003c/em\u003e access to PicoLab Mouse Diet 20 (5058, breeding chow) and water. While the other half (~66 in total) of the F1-FLT, F1-HGC and F1-VGC mice were transferred to another facility at KUMC, adjacent to the metabolic testing facility. There animals completed qMRI and VOmax treadmill running between 90 and 100 days of age. Body composition was determined by qMRI (EchoMRI, Houston, TX). Following treadmill running this second cohort of F1-FLT, F1-HGC and F1-VGC were intercross mated as shown in Fig. 1. The resultant F2 pups from these matings were weighed at weaning, and then ~400 pups from the first litters of these intercross matings were RFID and either designated for behavioral analyses (n=180) or metabolic testing (n=144). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoleus force frequency and fatigue curves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeft soleus muscles were carefully dissected and stimulated at 22-24°C using an Aurora Scientific contractile apparatus with a biphasic stimulator (model 701C). Muscles were subjected to a force frequency curve of 1, 20, 40, 80, and 160 Hz (350 ms) with a sampling rate of 1 kHz where peak isometric force was obtained. After a 2 min rest period, soleus muscles were then subjected to a fatigue protocol consisting of 70 Hz volleys (450 ms) every 2s for 160s. For force-frequency curves, specific isometric force was determined by normalizing peak force to cross-sectional area (CSA). CSA was calculated using the following formula:\u0026nbsp;\u0026nbsp;\u0026nbsp;where m is muscle mass (mg); l is muscle length (mm), d is mammalian skeletal muscle density (1.06 mg/mm\u003csup\u003e3\u003c/sup\u003e), Lf/Lo is the fiber length-to-muscle ratio (0.71 for soleus). For fatigue curves, data were presented as percent of initial force.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLocomotor Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLocomotor activity was measured using a BASi Force-Plate Actimeter (Bioanalytical Systems, Inc., Mount Vernon, IN) as described previously in \u003csup\u003e32,33\u003c/sup\u003e. The Actimeter is enclosed with a sound attenuating chamber to prevent outside influences on the animal’s behavior. The arena consists of a horizontal plate (42 cm × 42 cm) attached at the corners to force transducers that record the center of mass (X, Y position) of the animal subject. Mice were individually placed into the testing arena and allowed to move freely for 10 minutes. The average movement was quantified. All recorded data was processed using FPAAnalysis software version 9.10.04.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMarble Bury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMarble burying test is used to test for anxiety and compulsive behaviors. Mice that display behaviors associated with high anxiety or compulsive behaviors tend to dig in the bedding more, resulting in a greater percentage of marbles buried. The arena consists of a micro-isolator cage (30.8 cm x 19 cm) filled 2/3 of the way with clean cobb bedding. Prior to the test, animals were acclimated to the test room for a minimum of 30 minutes. Animals were then acclimated to the test arena for 5 minutes, then removed briefly and 20 marbles were placed in the arena in a regular pattern (5 rows of 4 across). Each animal was then placed in the arena for a total of 20 minutes, and the number of marbles buried to at least ¾ of their diameter beneath the bedding were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElevated Zero Maze\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe elevated zero maze is used to evaluate anxiety-related behaviors in rodents. The maze consists of an open junction connected to four equal arms. Two opposing arms are open and two are enclosed by walls. Prior to the test, animals were acclimated to the test room for a minimum of 30 minutes. Each animal was placed on the maze in the junction facing one of the open arms. The mouse was allowed to freely explore for 5 minutes. The test was recorded and analyzed using Noldus EthoVision XT tracking software (version 16.0.1536). The relative time spent (%) in open arms versus closed arms was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Diet Feeding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale and female 6-week-old F2 mice were individually housed at 28°C utilizing a reverse light cycle (dark 10:00-22:00) prior to the start of the experiment. Between 8-10 weeks of age, mice were assigned to either a low-fat diet (LFD, 10% kcal fat, 3.5% kcal sucrose, fiber (5%/w cellulose, 1%/w inulin, 1%/w pectin), Research diets, D24061901) or a western diet (HFD, 45% kcal fat, 7% kcal sucrose, fiber (5%/w cellulose), Research diets, D12451) (n=12/sex/diet\u003cbr\u003e/treatment group), with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. Body weight (BW) and food weight were recorded at the start of every week prior to the dark cycle (8:00-10:00). Body composition (fat mass and fat-free mass) was determined by qMRI (EchoMRI, Houston, TX) every two weeks. Fat -free mass (FFM) was calculated as the difference between body weight and fat mass. \u0026nbsp;HFD was replaced twice a week (every 3-4 days). Energy intake was calculated using the energy density of the LFD (3.85 kcal/g) or HFD (4.73 kcal/gram) times the food intake over the course of the 8 weeks. Percent metabolic efficiency was calculated as: (change in fat mass (kcal) + change in lean mass (kcal)/EI, with the change fat and lean kcal calculated as 9.32 kcal/g \u0026amp; 1.19 kcal/g, respectively \u003csup\u003e34\u003c/sup\u003e. Blood glucose levels were measured following a 4-hour food withdrawal (Contour Next EZ, Ascensia Diabetes Care).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor F1 and F2 behavior all datasets were tested for normal distribution. For normally distributed data, we performed an ANOVA followed by Dunnett's posthoc. For non-normal data, we performed a Kruskal-Wallis followed by Dunn's posthoc. For the F2 western diet study, data are presented as means and standard error. The two-standard deviation test was utilized to test for outliers within group. Data were analyzed by two-way ANOVA. Where interactions and/or main effects were observed, post-hoc analysis was performed using Fisher’s LSD. Main effects are only designated when all pairwise comparisons were significant.\u003c/p\u003e\n\u003cp\u003e31\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Choi, S. 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H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Glucocorticoid-induced obesity develops independently of UCP1. \u003cem\u003eCell Reports\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 1686-1698. e1685 (2019). https://doi.org/10.1016/j.celrep.2019.04.041\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6458038/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6458038/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Spaceflight presents unique microgravity, hypergravity, radiation, and isolation hazards for astronauts. Notably, spaceflight impacts on many life sustaining physiologic systems (e.g., cardiac, musculoskeletal) are known, while impacts on the reproductive systems and progeny of space travelers are largely unassessed. This study analyzes behavioral, metabolic, and functional outcomes in F1 and F2 offspring of female mice following a 42-day spaceflight on the International Space Station. Progeny from the spaceflight dams that were bred following return to earth exhibited growth, functional and behavioral differences compared to control offspring. Introduction of a secondary stressor (feeding a Western diet) revealed a transgenerational inheritance pattern in F2 pups of spaceflight granddams. This study demonstrates spaceflight can impact subsequent generations, pointing to the importance of further study to protect future astronauts’ children.","manuscriptTitle":"Impact of Female Space Travel on Progeny Health","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-29 12:06:36","doi":"10.21203/rs.3.rs-6458038/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d68e6e7b-f5f2-4c18-a30f-8b5ebba18293","owner":[],"postedDate":"May 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49174865,"name":"Biological sciences/Physiology"},{"id":49174866,"name":"Biological sciences/Physiology/Reproductive biology"},{"id":49174867,"name":"Biological sciences/Developmental biology/Epigenetic memory"}],"tags":[],"updatedAt":"2025-05-29T12:06:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-29 12:06:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6458038","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6458038","identity":"rs-6458038","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}