Spaceflight presents unique gravitational, radiation, and isolation hazards for human exploration of the Moon, Mars, and beyond, yet its impact on the female reproductive system and successive generations has been largely unassessed. In the NASA Rodent Research 20 mission, we examined the impact of a 42-d spaceflight on the female reproductive axis including ovulatory capacity, implantation rate, and fecundity as well as behavioral, metabolic, and functional outcomes in F1 and F2 offspring. Females bred 5 d after return to Earth became pregnant but only exhibited a slight decline in fecundity compared to ground controls. In contrast, F1 offspring from spaceflight dams exhibited marked growth, functional, and behavioral differences compared to F1 offspring from control dams. Moreover, F1 female offspring from spaceflight dams exhibited decreased ovarian reserves as evidenced by reduced anti-Mullerian hormone levels early in life (21 d of age) and premature ovarian failure or an early loss in fertility, as indicated by reduced numbers of litters and total number of pups born to females over a 9-mo period. Strikingly, transgenerational metabolic and reproductive disturbances were also observed in F2 pups of spaceflight granddams, including persistent reductions in ovarian reserve, suggesting germline-level effects. Together these findings reveal significant short- and long-term impacts of spaceflight on the female reproductive system and on their offspring across generations, demonstrating biological transmission of reproductive vulnerability following maternal spaceflight exposure, and raising concerns for space travelers and colonization missions.

Exposure to the space environment leads to cardiovascular deconditioning in the absence of mitigation strategies. During deep space missions, crew will be exposed to prolonged periods of microgravity and space radiation. In this study, we sought to define the molecular signatures of the cardiac response to deep space mission-relevant doses of galactic cosmic radiation (GCR) simulation to gain insight into potential latent effects of space radiation exposure and sex-dependent responses. We hypothesized that exposure to simulated space radiation increases cardiovascular disease risk in a sexually dimorphic manner by altering key pathways involved in CVD progression and aging. Six-month-old female and male mice were exposed to a single dose of 5, 15 or 50 cGy of 5-ion simplified GCR simulation (simGCRsim) or sham-treated (0 cGy). Animals were euthanized at four months post-irradiation and cardiac tissues collected. RNA sequencing revealed sex differences in the cardiac transcriptome, with a limited subset of genes exhibiting radiation dose-dependent expression changes. Several differentially expressed genes in simGCRsim-exposed groups have established roles in the development of cardiovascular disease. Analysis of immune-modulating cytokine protein levels showed sexually dimorphic expression changes but no differences between sham and 50 cGy groups. However, correlation analyses of cytokine protein levels revealed altered cytokine signaling networks in 50 cGy-exposed animals compared with sham controls. In males, mitochondrial DNA copy number and telomere length were reduced in the 5 and 15 cGy groups relative to sham controls, whereas values in the 50 cGy groups were comparable to sham controls. In contrast, females exhibited no differences in both measures across sham and simGCRsim groups. Collectively, these findings suggest the potential for delayed cardiovascular health risks following space radiation exposure and underscore the importance of accounting for sex, radiation dose, and tissue-specific responses in cardiovascular risk assessment for deep space missions.

NASA has a robust history of biomedical investigations using rodent models in spaceflight. The results from these experiments have provided significant insights into the effects of spaceflight on mammalian physiological systems for fundamental, biomedical and commercial research interests. To date, more than 19 long-duration (> 30-day) Rodent Research (RR) missions have been successfully conducted on board ISS using the NASA Rodent Habitat. Recent installation of digital cameras within the Rodent Habitat to support in-flight video observations can provide recordings during spaceflight. We hypothesized that application of unique fur dye patterns would enable individual identification of animals and small, implantable data loggers could be used to collect and store body temperature data. We designed and performed two ground-based validation studies. First, animals were housed in the Rodent Habitat in groups of five; fur dye markings enabled identification of individual mice. Body temperature was acquired using implanted dataloggers. Video was recorded and species typical behaviors were quantified for individual mice during both light and dark phases. Second, centrifugation was used to mimic launch. For the first time, we validated the biocompatibility of datalogger implants, fur dye pattern application and mouse visual identification methods using digital video and behavioral analysis protocols.

The effects of galactic cosmic radiation on reproductive physiology remain largely unknown. We determined the impact of near-continuous low-dose-rate Californium-252 neutron irradiation (1 mGy/day) as a space-relevant analog on litter size and number of resorptions at embryonic day (E) 12.5 (n = 19 radiated dams, n = 20 controls) and litter size, number of resorptions, fetal growth, and placental signaling and transcriptome (RNA sequencing) at E18.5 (n = 21 radiated dams, n = 20 controls) in pregnant mice. A significantly increased early resorption rate and decreased placental weight were observed in irradiated mice. There were no statistically significant differences in litter size, fetal weight, length, or malformation rate between the groups. Near-continuous radiation had no significant effects on the mechanistic target of rapamycin (mTOR), endoplasmic reticulum stress or inflammatory signaling, rate of double-stranded DNA breaks, and had minimal effects on gene expression in the placenta. These data suggest that near-continuous, low-level galactic cosmic radiation has a limited impact on pregnancy outcomes.

Men and women have different cardiovascular responses to spaceflight; however, few studies have focused on direct comparisons between sexes. We investigated the mechanisms of aortic stiffening in socially and sexually mature 20-week-old male and female Sprague Dawley (SD) rats exposed to hindlimb unloading (HLU) for 14 days. Pulse wave velocity (PWV) was greater in the aortic arch of females after HLU versus control females (n = 6-8). HLU had no effect on aortic PWV in males (n = 5-6). Aortic alpha smooth muscle actin, myosin, collagen, elastin, and collagen-to-elastin ratio were not different in rats of either sex following HLU. The levels of G protein-coupled estrogen receptor (GPER) were lower in the aorta of SD females exposed to HLU compared with female controls but were not altered in males. HLU females also had lower aortic PPAR gamma, increased oxidative stress markers, and diastolic dysfunction compared with control females. GPER agonist G1 prevented the increase in PWV and 8-hydroxy-2'-deoxyguanosine without altering PPAR gamma or p47phox in HLU females (n = 4 in each group) suggesting that lower GPER may contribute to arterial stiffening in the setting of simulated microgravity. This study highlights sex-specific vascular adaptations to the state of simulated microgravity.

Exposure to cosmic ionizing radiation is an innate risk of the spaceflight environment that can cause DNA damage and altered cellular function. In astronauts, longitudinal monitoring of physiological systems and interactions between these systems are important to consider for mitigation strategies. In addition, assessments of sex-specific biological responses in the unique environment of spaceflight are vital to support future exploration missions that include both females and males. Here we assessed sex-specific, multi-system immune and endocrine responses to simulated cosmic radiation. For this, 24-week-old, male and female C57Bl/6J mice were exposed to simplified five-ion, space-relevant galactic cosmic ray (GCRsim) radiation at 15 and 50 cGy, to simulate predicted radiation exposures that would be experienced during lunar and Martian missions, respectively. Blood and adrenal tissues were collected at 3- and 14-days post-irradiation for analysis of immune and endocrine biosignatures and pathways. Sexually dimorphic adrenal gland weights and morphology, differential total RNA expression with corresponding gene ontology, and unique immune phenotypes were altered by GCRsim. In brief, this study offers new insights into sexually dimorphic immune and endocrine kinetics following simulated cosmic radiation exposure and highlights the necessity for personalized translational approaches for astronauts during exploration missions.

The recent acceleration of commercial, private and multi-national spaceflight has created an unprecedented level of activity in low Earth orbit, concomitant with the largest-ever number of crewed missions entering space and preparations for exploration-class (lasting longer than one year) missions. Such rapid advancement into space from many new companies, countries and space-related entities has enabled a 'second space age'. This era is also poised to leverage, for the first time, modern tools and methods of molecular biology and precision medicine, thus enabling precision aerospace medicine for the crews. The applications of these biomedical technologies and algorithms are diverse, and encompass multi-omic, single-cell and spatial biology tools to investigate human and microbial responses to spaceflight. Additionally, they extend to the development of new imaging techniques, real-time cognitive assessments, physiological monitoring and personalized risk profiles tailored for astronauts. Furthermore, these technologies enable advancements in pharmacogenomics, as well as the identification of novel spaceflight biomarkers and the development of corresponding countermeasures. In this Perspective, we highlight some of the recent biomedical research from the National Aeronautics and Space Administration, Japan Aerospace Exploration Agency, European Space Agency and other space agencies, and detail the entrance of the commercial spaceflight sector (including SpaceX, Blue Origin, Axiom and Sierra Space) into aerospace medicine and space biology, the first aerospace medicine biobank, and various upcoming missions that will utilize these tools to ensure a permanent human presence beyond low Earth orbit, venturing out to other planets and moons.

Exposure to space galactic cosmic radiation is a principal consideration for deep space missions. While the effects of space irradiation on the nervous system are not fully known, studies in animal models have shown that exposure to ionizing radiation can cause neuronal damage and lead to downstream cognitive and behavioral deficits. Cognitive health implications put humans and missions at risk, and with the upcoming Artemis missions in which female crew will play a major role, advance critical analysis of the neurological and performance responses of male and female rodents to space radiation is vital. Here, we tested the hypothesis that simulated Galactic Cosmic Radiation (GCRSim) exposure disrupts species-typical behavior in mice, including burrowing, rearing, grooming, and nest-building that depend upon hippocampal and medial prefrontal cortex circuitry. Behavior comprises a remarkably well-integrated representation of the biology of the whole animal that informs overall neural and physiological status, revealing functional impairment. We conducted a systematic dose-response analysis of mature (6-month-old) male and female mice exposed to either 5, 15, or 50 cGy 5-ion GCRSim (H, Si, He, O, Fe) at the NASA Space Radiation Laboratory (NSRL). Behavioral performance was evaluated at 72 h (acute) and 91-days (delayed) postradiation exposure. Specifically, species-typical behavior patterns comprising burrowing, rearing, and grooming as well as nest building were analyzed. A Neuroscore test battery (spontaneous activity, proprioception, vibrissae touch, limb symmetry, lateral turning, forelimb outstretching, and climbing) was performed at the acute timepoint to investigate early sensorimotor deficits postirradiation exposure. Nest construction, a measure of neurological and organizational function in rodents, was evaluated using a five-stage Likert scale 'Deacon' score that ranged from 1 (a low score where the Nestlet is untouched) to 5 (a high score where the Nestlet is completely shredded and shaped into a nest). Differential acute responses were observed in females relative to males with respect to species-typical behavior following 15 cGy exposure while delayed responses were observed in female grooming following 50 cGy exposure. Significant sex differences were observed at both timepoints in nest building. No deficits in sensorimotor behavior were observed via the Neuroscore. This study revealed subtle, sexually dimorphic GCRSim exposure effects on mouse behavior. Our analysis provides a clearer understanding of GCR dose effects on species typical, sensorimotor and organizational behaviors at acute and delayed timeframes postirradiation, thereby setting the stage for the identification of underlying cellular and molecular events.

The multi-organ physiological responses to spaceflight stressors resemble aging on Earth. We simulate space environment together with environmental stress, as a model of accelerated aging. Redox dys-homeostasis was shown to contribute to aging-related pathologies such as Alzheimer’s and Parkinson’s. The MCAT transgenic mice (overexpressing human catalase in the mitochondria), have increased life span, delayed age-related pathology and enhanced hippocampal spatial learning and memory. Our study uses 1-year old C57BL/6NJ male and female mice that underwent exposure to 0.5 gray of gamma radiation together with social isolation. In order to determine ROS contribution to neuro-behavioral stress response, we used the longevity MCAT mouse model in which human catalase is overexpressed in the mitochondria. We aimed to determine whether in older mice quenching ROS, will mitigate the neuro-behavioral consequences of low dose ionizing radiation and social isolation and whether sex differences will be detected. We have performed multiple behavioral tests which focused on performance, memory, physical stance, and stress, together with plasma and hippocampal neuro-immune panels. We have detected both sex and radiation/isolation effects; the older females look physically better, are faster and perform better almost in all behavioral tasks compared to their male counterparts. On the other hand, they are more sensitive to low dose radiation and isolation in many cases. Interestingly, many of the neuro-immune changes caused by radiation and social isolation were mitigated in the MCAT mice. In a plasma multiplex cytokine panel, corticosterone (7- and 90-days post radiation), and hippocampal cytokine and microglial activation at the end of the experiment, we have detected significant changes due to radiation, isolation, sex and genotype in both short and long post radiation period. Our focus is now on applying advanced statistical modeling to correlate the behavioral tests with our recent molecular findings to look for specific biomarkers that could predict behavioral deficits caused by various environmental stresses. Quenching ROS in older mice partially mitigates neuro-immune consequences of environmental stresses. Significant sex differences were detected, pointing out the importance of examining both sexes, for better personal medicine. The study points out that anti-oxydant meassures could help older isolated population.

As human space exploration advances to establish a permanent presence beyond the Low Earth Orbit (LEO) with NASA's Artemis mission, researchers are striving to understand and address the health challenges of living and working in the spaceflight environment. Exposure to ionizing radiation, microgravity, isolation and other spaceflight hazards pose significant risks to astronauts. Determining neurobiological and neurobehavioral responses, understanding physiological responses under Central Nervous System (CNS) control, and identifying putative mechanisms to inform countermeasure development are critically important to ensuring brain and behavioral health of crew on long duration missions. Here we provide a detailed and comprehensive review of the effects of spaceflight and of ground-based spaceflight analogs, including simulated weightlessness, social isolation, and ionizing radiation on humans and animals. Further, we discuss dietary and non-dietary countermeasures including artificial gravity and antioxidants, among others. Significant future work is needed to ensure that neural, sensorimotor, cognitive and other physiological functions are maintained during extended deep space missions to avoid potentially catastrophic health and safety outcomes.

Long duration spaceflight poses potential health risks to astronauts during flight and re-adaptation after return to Earth. There is an emerging need for NASA to provide successful and reliable therapeutics for long duration missions when capability for medical intervention will be limited. Clinically relevant, human placenta-derived therapeutic stromal cells (PLX-PAD) are a promising therapeutic alternative. We found that treatment of adult female mice with PLX-PAD near the onset of simulated weightlessness by hindlimb unloading (HU, 30 d) was well-tolerated and partially mitigated decrements caused by HU. Specifically, PLX-PAD treatment rescued HU-induced thymic atrophy, and mitigated HU-induced changes in percentages of circulating neutrophils, but did not rescue changes in the percentages of lymphocytes, monocytes, natural killer (NK) cells, T-cells and splenic atrophy. Further, PLX-PAD partially mitigated HU effects on the expression of select cytokines in the hippocampus. In contrast, PLX-PAD failed to protect bone and muscle from HU-induced effects, suggesting that the mechanisms which regulate the structure of these mechanosensitive tissues in response to disuse are discrete from those that regulate the immune- and central nervous system (CNS). These findings support the therapeutic potential of placenta-derived stromal cells for select physiological deficits during simulated spaceflight. Multiple countermeasures are likely needed for comprehensive protection from the deleterious effects of prolonged spaceflight.

Isolation on Earth can alter physiology and signaling of organs systems, including the central nervous system. Although not in complete solitude, astronauts operate in an isolated environment during spaceflight. In this study, we determined the effects of isolation and simulated microgravity solely or combined, on the inflammatory cytokine milieu of the hippocampus. Adult female wild-type mice underwent simulated microgravity by hindlimb unloading for 30 days in single or social (paired) housing. In hippocampus, simulated microgravity and isolation each regulate a discrete repertoire of cytokines associated with inflammation. Their combined effects are not additive. A model for mitochondrial reactive oxygen species (ROS) quenching via targeted overexpression of the human catalase gene to the mitochondria (MCAT mice), are protected from isolation- and/or simulated microgravity-induced changes in cytokine expression. These findings suggest a key role for mitochondrial ROS signaling in neuroinflammatory responses to spaceflight and prolonged bedrest, isolation, and confinement on Earth.

Ovarian steroids dramatically impact normal homeostatic and metabolic processes of most tissues within the body, including muscle, bone, neural, immune, cardiovascular, and reproductive systems. Determining the effects of spaceflight on the ovary and estrous cycle is, therefore, critical to our understanding of all spaceflight experiments using female mice. Adult female mice (n = 10) were exposed to and sacrificed on-orbit after 37 days of spaceflight in microgravity. Contemporary control (preflight baseline, vivarium, and habitat; n = 10/group) groups were maintained at the Kennedy Space Center, prior to sacrifice and similar tissue collection at the NASA Ames Research Center. Ovarian tissues were collected and processed for RNA and steroid analyses at initial carcass thaw. Vaginal wall tissue collected from twice frozen/thawed carcasses was fixed for estrous cycle stage determinations. The proportion of animals in each phase of the estrous cycle (i.e., proestrus, estrus, metestrus, and diestrus) did not appreciably differ between baseline, vivarium, and flight mice, while habitat control mice exhibited greater numbers in diestrus. Ovarian tissue steroid concentrations indicated no differences in estradiol across groups, while progesterone levels were lower (p < 0.05) in habitat and flight compared to baseline females. Genes involved in ovarian steroidogenic function were not differentially expressed across groups. As ovarian estrogen can dramatically impact multiple non-reproductive tissues, these data support vaginal wall estrous cycle classification of all female mice flown in space. Additionally, since females exposed to long-term spaceflight were observed at different estrous cycle stages, this indicates females are likely undergoing ovarian cyclicity and may yet be fertile.

Animal models are useful for exploring the health consequences of prolonged spaceflight. Capabilities were developed to perform experiments in low earth orbit with on-board sample recovery, thereby avoiding complications caused by return to Earth. For NASA’s Rodent Research-1 mission, female mice (ten 32 wk C57BL/6NTac; ten 16 wk C57BL/6J) were launched on an unmanned vehicle, then resided on the International Space Station for 21/22d or 37d in microgravity. Mice were euthanized on-orbit, livers and spleens dissected, and remaining tissues frozen in situ for later analyses. Mice appeared healthy by daily video health checks and body, adrenal, and spleen weights of 37d-flight (FLT) mice did not differ from ground controls housed in flight hardware (GC), while thymus weights were 35% greater in FLT than GC. Mice exposed to 37d of spaceflight displayed elevated liver mass (33%) and select enzyme activities compared to GC, whereas 21/22d-FLT mice did not. FLT mice appeared more physically active than respective GC while soleus muscle showed expected atrophy. RNA and enzyme activity levels in tissues recovered on-orbit were of acceptable quality. Thus, this system establishes a new capability for conducting long-duration experiments in space, enables sample recovery on-orbit, and avoids triggering standard indices of chronic stress.

Now that orbital spaceflight is a continuous reality, deep space exploration and extra-Earth colonization have become the aspirational activities of space agencies and private entities. Understanding the impact of the extraterrestrial environment on human reproduction is a vital precursor to extra-Earth colonization, however, the effects of microgravity and cosmic radiation on reproductive physiology remain largely unknown. Relevant forms of cosmic radiation include protons, heavy ions, and the secondary neutron irradiation that occurs as protons bombard spacecrafts, habitats, or the human body, and these forms of radiation are fundamentally different than gamma or x-irradiation. We hypothesize that exposure of pregnant mice throughout gestation to chronic low-dose neutron irradiation would result in increased miscarriage and restricted fetal growth. Following copulatory plug positivity, eighty 9-13-week old female C57BL/6 mice were randomized to near-continuous neutron irradiation (21 hours/day) using Califonium-252 at a dose rate of 1 mGy/day vs. control (background: 0.005 mGy/day) for the duration of pregnancy (E0.5-E18.5) at NASA's Colorado State University Neutron Radiation Facility. The dams were then randomized to euthanasia at either E12.5 to determine the rate of early miscarriage, or E18.5 to assess the rate of late miscarriage, fetal anomalies, and growth restriction. Placentas were weighed and stored for future secondary analyses including placental immunohistochemistry, cell signaling and nutrient transport, and gene expression studies. Phenotypic characteristics are provided in Table 1. Chronic low-dose neutron irradiation caused increased early resorption rate and significantly decreased placental weight. In contrast, there were no differences in birth length, birth weight, or anomaly rate between groups. Although this data cannot be directly extrapolated to humans, for the first time, we show that near-continuous low-dose neutron irradiation pan-pregnancy at a space-relevant dose rate leads to increased risk of miscarriage in a mouse model.

Space flight missions are becoming longer and more common and evidence points to the physiological toll the missions have on the human body. Aging, sedentary lifestyle, and spaceflight have similar degenerative effects on almost every part of our body; both exposure to the space environment and aging result in cardiovascular deconditioning, bone loss, muscle atrophy, brain changes, and immune response impairment. We hypothesize that exposure to the space environment generates excessive Reactive Oxygen Species (ROS), which results in neuroinflammation and aging‐like degenerative symptoms in the brain. We used the hindlimb unloading (HU) model to mimic microgravity with either paired or single housed animals (social isolation). Responses to 30d of HU were compared in wildtype or transgenic MCAT mice, in which mitochondrial ROS is quenched by over‐expression of human catalase. Expression of 4‐Hydroxynonenal (4HNE) and Park7 (redox‐sensitive chaperone and sensor of oxidative stress) were measured by ELISA, a protein array quantified from the hippocampal cytokines and 8‐hydroxy‐2’‐deoxyguanosine in serum was measured by ELISA to assess oxidative DNA damage. Preliminary analysis of cage behavior patterns from video collected at the end of the study showed that MCAT HU mice (socially housed) were more active and conducted more exploratory activities compared to NL. Our biochemical results showed simulated microgravity and/or social isolation caused changes in levels of cytokines related to immune responses. Two‐way ANOVA revealed significant interaction effects of HU and genotype in expression levels of five cytokines (out of 35) in socially‐housed animals. Elevation of these generally pro‐inflammatory cytokines by HU in WT mice was mitigated in MCAT mice, suggesting a role for mitochondrial ROS signaling in inflammatory CNS responses to microgravity. Interestingly, some of these cytokines in the hippocampus displayed strong correlations to the 4HNE levels. We also found substantive cytokine responses to social isolation in the hippocampus; housing and genotype interaction effects were significant (by 2‐Factor ANOVA) for 15 cytokines, most of which were mitigated in MCAT mice.Taken together, our results showed that both simulated microgravity and social isolation influenced cytokine levels in the hippocampus and MCAT mice were at least partially protected from these changes. These findings implicate a potentially important role for mitochondrial ROS in CNS responses to the challenges posed by long duration spaceflight.Support or Funding InformationThis work is supported by a NASA Space Biology Grant to RKG (NNH14ZTT001N). LG was supported by a Space Biology NASA Postdoctoral Fellowship AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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The hindlimb unloading (HU) model has been used extensively to simulate the cephalad fluid shift and musculoskeletal disuse observed in spaceflight with its application expanding to study immune, cardiovascular and central nervous system responses, among others. Most HU studies are performed with singly housed animals, although social isolation also can substantially impact behavior and physiology, and therefore may confound HU experimental results. Other HU variants that allow for paired housing have been developed although no systematic assessment has been made to understand the effects of social isolation on HU outcomes. Hence, we aimed to determine the contribution of social isolation to tissue responses to HU. To accomplish this, we developed a refinement to the traditional NASA Ames single housing HU system to accommodate social housing in pairs, retaining desirable features of the original design. We conducted a 30-day HU experiment with adult, female mice that were either singly or socially housed. HU animals in both single and social housing displayed expected musculoskeletal deficits versus housing matched, normally loaded (NL) controls. However, select immune and hypothalamic-pituitary-adrenal (HPA) axis responses were differentially impacted by the HU social environment relative to matched NL controls. HU led to a reduction in % CD4(+) T cells in singly housed, but not in socially housed mice. Unexpectedly, HU increased adrenal gland mass in socially housed but not singly housed mice, while social isolation increased adrenal gland mass in NL controls. HU also led to elevated plasma corticosterone levels at day 30 in both singly and socially housed mice. Thus, musculoskeletal responses to simulated weightlessness are similar regardless of social environment with a few differences in adrenal and immune responses. Our findings show that combined stressors can mask, not only exacerbate, select responses to HU. These findings further expand the utility of the HU model for studying possible combined effects of spaceflight stressors.

Oxidative stress has been implicated in the pathophysiology of numerous terrestrial disease processes and associated with morbidity following spaceflight. Furthermore, oxidative stress has long been considered a causative agent in adverse reproductive outcomes. The purpose of this review is to summarize the pathogenesis of oxidative stress caused by cosmic radiation and microgravity, review the relationship between oxidative stress and reproductive outcomes in females, and explore what role spaceflight-induced oxidative damage may have on female reproductive and developmental outcomes.