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Effects of spaceflight aboard the International Space Station on mouse estrous cycle and ovarian gene expression

Effects of spaceflight aboard the International Space Station on mouse estrous cycle and ovarian... www.nature.com/npjmgrav ARTICLE OPEN Effects of spaceflight aboard the International Space Station on mouse estrous cycle and ovarian gene expression 1 1 2 1 3,4 3 Xiaoman Hong , Anamika Ratri , Sungshin Y. Choi , Joseph S. Tash , April E. Ronca , Joshua S. Alwood and Lane K. Christenson 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. npj Microgravity (2021) 7:11 ; https://doi.org/10.1038/s41526-021-00139-7 INTRODUCTION temporal changes in expression of steroidogenic pathway proteins and other critically important ovarian genes known to Spaceflight microgravity is known to impact numerous physiolo- play critical roles in ovulation, oocyte development and corpus gical systems. These include cardiovascular changes, decreased luteum development and function are well known and may be bone mineral density, loss of muscle tone, adipogenesis, insulin evaluated to provide information regarding ovarian function. resistance, vision and vestibular disturbances, altered fluid and Understanding these molecular changes is vital for informing electrolyte balance, and altered kidney function to name a few . female reproductive health during and after spaceflight. Many, if not all, normal homeostatic processes occurring within To date, studies of ovarian function and fertility in mice exposed mammals, both male and female, are dramatically impacted by to microgravity during NASA Space Shuttle flights were of short gonadal hormones. Indeed, the reproductive system is often duration (<12 days) and, importantly, all included the effects of considered one of the best markers of overall health and fitness of live reentry prior to collection of tissues and analysis of animal an animal . However, research is limited regarding the effects of 3,7 behavior . Similarly, pregnant female rats flown on Cosmos 1514 spaceflight on the reproductive system and its consequences on 3,4 (1982), NASA-NIH Rodent (R)1 (STS-66 in 1994), and NASA-NIH R2 normal physiology . (STS-70 in 1995) were of short duration (4.5–11 days) and had live The ovarian steroid hormones, estrogen and progesterone, play 8–10 animal return . Notably, in the pregnant rats no effects of a significant role in regulating a myriad of physiological functions, spaceflight on healthy and atretic ovarian antral follicle popula- and are subject to cyclic changes based on the hypothalamic/ tions, fetal wastage in utero, plasma concentrations of progester- pituitary regulation of ovarian folliculogenesis . Moreover, ovarian one and luteinizing hormone (LH) or pituitary content of follicle steroidogenic output can be dramatically impacted by stress as stimulating hormone (FSH) were noted . Spaceflight, however, well as other internal homeostatic processes . In cycling rodents, significantly increased plasma concentrations of FSH and the antral follicle is the primary steroidogenic tissue within the decreased pituitary content of LH analyzed postpartum (day ovary, unless mating occurs at which time the corpus luteum 22–23; ). In these prior studies, it is not possible to separate the becomes the major producer of progesterone. The mural effects of microgravity from that of reentry with the associated granulosa cells and the vascularized theca cells within the antral hypergravity, turbulence, and the likely ensuing stress responses ovarian follicles are responsible for the enzymatic conversion of linked to these events. Moreover, these studies while evaluating cholesterol to its ultimate endpoint product estradiol. In addition maintenance of pregnancy, provide no information regarding the to steroidogenesis, these follicular cells also carry out the critical fertility of these animals and their ability to exhibit normal estrous missions of nurturing the enclosed growing oocyte, as well as facilitating the process of ovulation, or expulsion of oocyte, and cycles and ovarian function. The current investigation provides subsequent formation of the ephemeral corpus luteum. Cyclic and insights into the endocrine status and ovarian cyclicity of female 1 2 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA. KBR, NASA-Ames Research Center, Moffett Field, CA, USA. 3 4 Space Biosciences Division, NASA-Ames Research Center, Moffett Field, CA, USA. Department of Obstetrics & Gynecology, Wake Forest Medical School, Winston-Salem, NC, USA. email: lchristenson@kumc.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; X. Hong et al. ab Fig. 1 Hematoxylin and eosin staining of vaginal walls used for estrous cycle staging. Vaginal wall estrous cycle staging was completed on all mice (n = 40). Representative sections from two flight and two habitat control mice depict each stage of the estrous cycle a proestrus, b estrus, c metestrus, and d diestrus. *Marks the vaginal lumen. Images were taken at ×20 using brightfield microscopy; scale bar measures 100 μm. mice following an extended period of exposure to microgravity in Table 1. Number of mice at each stage of the estrous cycle (n = 10/ the absence of reentry effects. Addressing the effects of extended group). duration spaceflight on endocrine status are significant and of high impact to long-term female astronaut health before and after Proestrus Estrus Metestrus Diestrus ND flight, interpretation of spaceflight findings derived from female mice, and for long-term plans for multigenerational mammalian Baseline 2 6 0 1 1 studies in spaceflight. Vivarium 3 5 0 0 2 Habitat 2 3 2 3 0 Flight 0 6 4 0 0 RESULTS Estrous cycle staging ND not determinable. Estrous cycle stage for each mouse was determined by three independent investigators blinded to treatment group categories. Ovarian RNA quality and tissue steroid levels Figure 1 shows representative H&E stained cross sections of Ovarian RNA quality varied appreciably depending upon whether vaginal wall tissue from the individual flight and habitat control it was flash-frozen in LN or placed in RNAlater (Fig. 2). animals following collection after the second carcass thaw during Representative electropherograms from the Agilent 2100 are March of 2016. Each stage of the estrous cycle is depicted in shown for 2 mice collected in either RNAlater or LN. Overall, RNA Fig. 1. Proestrus is characterized by the presence of a thick layer quality as determined by Agilent RNA integrity numbers (RIN of stratified squamous epithelium, with an absence of luminal values) from tissues frozen in LN were greater (p < 0.05) in every cells and any cornified epithelial cells (Fig. 1a). Estrous stage animal, and overall averaged around 5.71 ± 0.12 compared to animals exhibit a stratified squamous epithelium with a layer of 4.08 ± 0.11 for RNAlater preserved ovarian tissue (Fig. 2b). To cornified epithelial [red eosin stained cells above the nucleated evaluate ovarian hormone production without access to serum, stratified squamous epithelial cells (Fig. 1b)]. Often, this cornified whole ovarian tissue steroid levels were determined. Ovarian layer is delaminated from the underlying epithelial cells. In tissue E2 levels were not significantly different across experi- metestrus, leukocytes appear and traverse the nucleated epithe- mental groups (Fig. 3a). In contrast, whole ovarian tissue P4 levels lial layer to reach the delaminated mass of cornified stratified were significantly lower in the habitat control (p = 0.005) and squamous epithelium in the lumen (Fig. 1c). During diestrus, the flight mice (p = 0.013) in comparison to baseline control mice vaginal wall is characterized by columnar mucified cells next to a (Fig. 3b). Due to insufficient animal numbers at individual stages thin layer of nucleated epithelium (Fig. 1d). Staging of the mice within treatment groups, we were unable to evaluate stage effects indicated that the majority of the animals in the baseline control alone and stage-treatment interactions. group (6/9), the vivarium control group (5/8), and the flight group (6/10) were in estrus (Table 1). In comparison, habitat control Ovarian tissue gene expression analyses mice presented at all stages of the cycle in approximately equal amounts (n = 2 or 3/stage). Flight group mice presented at estrus Quantitative RT-PCR analysis of critical proteins involved in ovarian (n = 6) and metestrus (n = 4; Table 1). function and steroidogenesis were compared across treatment npj Microgravity (2021) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; X. Hong et al. b e c d RNA later LN RNALater LN Fig. 2 RNA quality of liquid nitrogen snap-frozen and RNAlater preserved ovarian tissues. Representative electropherograms from 2 animals (M13 vivarium control a, b and M22 flight c, d) perserved in RNAlater (a, c)orLN(b, d). e Histogram of RIN values from analysis of paired LN and RNALater samples (n = 12; 4BL, 4VC, 2HC, 2FL). *Means ± SEM RIN values were significantly (p ≤ 0.05) different based on the preservation method. where P450 aromatase (Cyp19a1) expression was elevated a b E2 P4 (p ≤ 0.05) ~4-fold compared to all other stages (Fig. 5). Lastly, as 400 10 each group of animals had at least three animals staged at estrus, we compared the gene expression across the four treatment a,b groups at this stage (Fig. 6). Levels of steroidogenic acute 200 regulatory protein (Star), P450 cholesterol side chain cleavage (Cyp11a1), 3β-hydroxysteroid dehydrogenase (Hsd3b1), Cyp19a1, 100 and liver receptor homolog-1 (Nr5a2) expression did not change b b appreciably across experimental conditions. Expression of Cyp17a1 0 under baseline and vivarium control conditions were significantly VC VC BL HC FL BL HC FL decreased (p < 0.05) compared to habitat control conditions (p < 0.05). Ovarian Cyp17a1 levels in flight animals in estrus were not Fig. 3 Whole ovarian tissue estrogen and progesterone concen- notably different from estrus stage animals in the other experi- trations. a Estradiol (E2; pg/ml/mg tissue) and b progesterone mental conditions (Fig. 6). Examination of other critical ovarian (P4; ng/ml/mg tissue) levels for all mice within a treatment group a,b (n = 8-9/treatment; 9BL, 8VC, 9HC, 9FL). Means ± SEM with genes during estrus showed no significant changes in expression different superscripts are significantly different (p ≤ 0.05). levels across control and flight conditions (Supplementary Fig. 2). groups irrespective of stage of the cycle (Fig. 4, Supplementary DISCUSSION Fig. 2). Spaceflight had no demonstrable effect on gene Rodent Research-1 (RR-1), a Validation mission launched aboard expression of any of the key enzymatic steps of steroidogenesis SpaceX’s fourth cargo resupply mission, was the first to transport or mitochondrial cholesterol uptake expression. Ovarian Cyp17a1 rodents to the International Space Station (ISS) in an unmanned mRNA levels were elevated (p ≤ 0.05) in mice housed in the commercial vehicle. Lasting 37 days in microgravity, RR-1 was, at habitat maintained on the ground (HC) when compared to the time, the longest space rodent study conducted by NASA. In- baseline and vivarium controls but were not different from flight flight studies of RR1 mouse behavior and post-flight physiology animals that were housed in the habitat on the ISS (Fig. 4). revealed the Validation mice showed no overt physiological signs Analysis of other critically important ovarian genes involved in of chronic stress or compromised health or welfare . Moreover, steroidogenic production and action, including estrogen receptors mice remained highly active and mobile throughout the experi- (Esr1 and Esr2), scavenger receptor class B type 1 (Scarb1), luteinizing hormone receptor (Lhcgr) as well as oocyte-specific ment, engaging in feeding, drinking, self-grooming, huddling, proteins, growth differentiation factor 9 (Gdf9), zona pellucida and social interactions . Our results provide detailed conse- glycoprotein 3 (Zp3) showed no differences across the treatment quences of long-term spaceflight microgravity on rodent estrous groups (Supplementary Fig. 1). cycle, ovarian steroidogenesis, and gene expression. Notably, Comparison of ovarian gene expression at different stages of the previous observations examining the potential impacts of micro- estrous cycle did not vary significantly with exception of proestrus, gravity on ovarian function and its target tissue the uterus were of Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 11 E2 Levels (pg/ml/mg) M22 M13 P4 Levels (ng/ml/mg) RNA Integrity Number (RIN value) X. Hong et al. a bc Cyp11a1 Hsd3b1 Star 3 3 1.5 2 1.0 1 0.5 0 0 0 VC BL HC FL VC HC FL BL HC FL BL VC df e 1.5 Cyp17a1 Cyp19a1 Nr5a2 5 1.5 1.0 1.0 a,b 0.5 0.5 HC FL HC FL BL HC FL BL VC BL VC VC Fig. 4 Whole ovarian tissue steroidogenic gene expression was not altered by spaceflight. Histograms depict relative fold change (ΔΔCt method) for a Star, b Cyp11a1, c Hsd3b1, d Cyp17a1, e Cyp19a1, and f Nr5a2 following normalization to 18S; n = 8–10/treatment group; 10BL, a,b 10VC, 8HC, 10FL). Means ± SEM with different superscripts are significantly different (p ≤ 0.05). a short-term nature and had the confounding effects of reentry serum at the time of sacrifice or longitudinally over time is 3,9,13 prior to tissue collection . It is well known that estrous cycle severely restricted logistically. We, therefore, analyzed whole staging in female rodents can be influenced by a variety of factors ovarian tissue levels of estrogen and progesterone. We detected such as age, light, temperature, humidity, ambient noise, nutrition, no striking difference in tissue estrogen concentrations across and social relationships . Indeed, mice that are group housed the four treatment groups, and it should be noted that serum exhibit greater estrous cycle irregularities, than those individually estrogen levels in rodents also do not exhibit wide variations 15,16 housed . Nonetheless, many labs using contemporary strains of across the cycle, often ranging from ND to 8–50 pg/ml in 24–27 mice are able to stage mice in group housing conditions, albeit it total . In contrast, we did observe that flight and habitat 17–19 may not be as ideal as single housed mice . Again, group control animals had lower overall P4 levels in ovarian tissue. housing was necessitated by animal husbandry concerns and One might predict this as these two groups had greater limitations of the NASA’s flight hardware. Our results point to an numbers of mice in metestrus and diestrus, in total 4 and 5 for ability of female mice to exhibit estrous cycles after an extended flight and habitat groups, respectively. During both of these duration in space, as evidenced by our observation that flight stages of estrous cycle the ovary has reduced steroidogenic 26–28 animals exhibited two different stages of the cycle (estrus and activity . This was in contrast to the vivarium and baseline metestrus). Typically, under conditions of exogenous treatments/ control groups, which both had 8 animals each in proestrus and stress, mice may exhibit constant vaginal estrus or an extended or estrus in total, stages associated with increased ovarian 14,20 constant diestrus , but not metestrus. In contrast to mice, follicular steroidogenesis. We recognize that it would have rats exhibit a greater synchrony when group housed, making it been ideal to examine the interaction of estrous cycle stage on difficult to extrapolate finding across these two species . ovarian steroidogenesis as well as ovarian gene expression, but While measuring true estrous cyclicity in space is logistically due to the insufficient numbers of animals at individual stages, daunting due to the intensive daily monitoring required, we we were unable to do so. It is worth noting the experimental posit that the analysis of the vaginal wall provides a robust design for RR1 as a hardware demonstration flight determined methodology that can indicate the estrous stage, and thus the how tissues were preserved as well as group sizes and thus were endocrine status of the animal at the moment of sacrifice. Indeed beyond our control. Whole ovarian gene expression analyses the morphology of these twice frozen and thawed vaginal wall were also dictated by the RR1 design. This type of analysis is tissues rivaled that of freshly isolated and fixed tissues from highly confounded as whole ovarian tissue contains an contemporary animals collected in our laboratory (data not abundance of stromal tissue and smaller follicles, which shown) and in those laboratories who pioneered the use of predominate over the few selected follicles that become highly 20,22,23 vaginal wall histology as a mechanism to track cyclicity . steroidogenic. Thus, when whole ovarian RNA is used, it was not Ovarian tissue steroid levels are also not commonly mea- surprising to see no major differences in ovarian gene sured, as most investigators use serum levels of estrogen and expression across the treatment groups or even during different progesterone as a measure of ovarian steroidogenic activity. stages of the cycle. Future studies need to take into considera- Again, in animals exposed to microgravity, the ability to collect tion this limitation, and collect and fix tissues for analysis, so npj Microgravity (2021) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Fold Change Fold Change Fold Change Fold Change Fold Change Fold Change X. Hong et al. a bc 2.5 Star Cyp11a1 Hsd3b1 1.5 2.0 1.0 1.5 1.0 0.5 0.5 0 0 Proestrus Metestrus Diestrus Estrus Proestrus Estrus Metestrus Diestrus Proestrus Estrus Metestrus Diestrus d f Cyp17a1 Cyp19a1 Nr5a2 0.8 1.5 0.6 a 1.0 0.4 4 0.5 0.2 0 0 Proestrus Estrus Metestrus Diestrus Metestrus Diestrus Proestrus Estrus Metestrus Diestrus Proestrus Estrus Fig. 5 Whole ovarian tissue steroidogenic gene expression by stage of estrous cycle. Histograms depict relative fold change (ΔΔCt method) for a Star, b Cyp11a1, c Hsd3b1, d Cyp17a1, e Cyp19a1, and f Nr5a2 following normalization to 18S; n = 8–10/treatment group; 10BL, a,b 10VC, 8HC, 10FL). Means ± SEM with different superscripts are significantly different (p ≤ 0.05). they can be properly analyzed using in situ hybridization/ which are composed of highly dynamic tissues (follicles, corpora RNAscope and immunohistochemistry. Cyp17a1,the enzyme lutea, endometrium), we recommend that they be preserved located in the theca cell compartment of the ovarian follicle and primarily in fixatives or in a manner where tissue/cell specific responsible for androgen production was elevated in the aspects can be determined afterward. Lastly, the ability of these habitat control animals. This difference was however, mani- females to exhibit estrous cycle activity provides a tractable fested primarily in two of the three HC animals in estrus. mammalian model system to test whether fertility is actually Safeguarding RNA tissue quality is acutely dependent on tissue impacted by microgravity. Reproductive cycles characterized by storage methods, which ideally would minimize RNase activity alterations in hormone levels can exert significant effects on throughout the process. Tissue collection and preservation during experimental outcomes, including emotion and the response to spaceflight presents a unique challenge in the analysis of these environmental stressors . Determining the effects of spaceflight valuable specimens. Our current analyses of the right and left on estrous cyclicity is thus critical to making accurate interpreta- ovaries processed in RNALater or snap-frozen in liquid nitrogen tions of experimental endpoints for all spaceflight experiments (LN) indicated that LN preserved tissues always had greater RIN using female mice. values when compared to those of RNALater. Previous studies utilizing RR1 RNAlater preserved spleen tissues showed higher RIN values than those observed here . This may be due to a time of METHODS collection aspect with respect to spleen vs ovary following thawing Animals of the carcass, or just to tissue differences in RNA susceptibility to Forty 12-week old C57BI/6J female mice (Jackson Lab, Bar Harbor, ME) the freeze/thaw. We note that freshly isolated ovarian tissues that were selected based on similar body weights for 4 experimental groups were snap-frozen in LN and then simultaneously processed with (16-week old at launch, n = 10/group): baseline control, vivarium control, the RR1 samples yielded RIN values >9. habitat control, and flight. Flight mice were launched on SpaceX-4 (Rodent Research 1) on September 21, 2014, as part of NASA’s Validation study. In conclusion, our analyses of ovarian tissues and vaginal wall Details of RR1 mission timeline (i.e., mouse pre-adaptation to cages, food) tissues of mice exposed to extended periods of microgravity and 11,12,31 as well as mouse spaceflight hardware details are available . Briefly, sacrificed in space indicate that these female mice were exhibiting vivarium control mice were maintained in standard mouse cages (5 mice/ estrous cyclic activity. These studies point positively to the ability cage) for the duration of spaceflight at Kennedy Space Center. Habitat of these animals to either continue or regain estrous cycle activity control mice were housed in a Rodent Research Transporter and Habitat during extended microgravity exposure. We posit that vaginal wall Hardware on Earth for durations consistent with flight times at the same estrous cycle activity be collected and provided to investigators to density of mice as those in flight (n = 10/habitat). The habitat control was include as covariate on all female mice used in future space placed in the ISS Environmental Simulator at KSC on a 4-day delay to missions, much the same as weight and age. The current studies mimic flight temperature, CO , and humidity conditions on the ISS for the also provide critical logistical information regarding tissue duration of spaceflight. Baseline control mice were euthanized one day collection, particularly for organs such as the ovary and uterus, after launch and then partially dissected prior to freezing of the animal Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 11 Fold Change Fold Change Fold Change Fold Change Fold Change Fold Change X. Hong et al. a b Cyp11a1 Hsd3b1 4 Star 4 1.5 3 3 1.0 2 2 0.5 1 1 0 0 HC FL BL VC BL VC HC FL VC HC FL BL d f Cyp17a1 Cyp19a1 2.5 Nr5a2 4 1.5 2.0 1.0 1.5 1.0 a,b 0.5 0.5 0 0 HC FL VC HC FL BL VC BL VC HC FL BL Fig. 6 Whole ovarian tissue steroidogenic gene expression at estrus across all four treatment groups. Histograms depict relative fold change (ΔΔCt method) for a Star, b Cyp11a1, c Hsd3b1, d Cyp17a1, e Cyp19a1, and f Nr5a2 following normalization to 18S; n = 3–6/treatment a,b group; 6BL, 5VC, 3HC, 6FL). Means ± SEM with different superscripts are significantly different (p ≤ 0.05). carcasses mimicking the procedures to be conducted on flight and for all RNA isolation and quantitative RT-PCR analysis other animals at the conclusion of experiment. Flight mice were exposed Ovaries tissues were transferred directly from RNAlater to tubes containing to microgravity for a total of 37 days (33 days on ISS and 4 days in the 1 ml of Trizol (Sigma) or 1 ml of Trizol was added directly to ovarian tissue Dragon Capsule). Mice were euthanized by injection of Euthasol followed snap-frozen in liquid nitrogen. Tissues were briefly dissociated on ice by cervical dislocation and immediately fast frozen intact (n = 8) or (3 bursts, 62 W) in a PowerGen 700 homogenizer (Fisher Scientific). RNA partially dissected (n = 2) prior to carcasses being frozen. Mice carcasses was isolated from Trizol as per manufacturer protocol. To facilitate were frozen using pre-chilled Ice Bricks prior to transfer to the Minus Eighty precipitation 1 μL of glycogen (20ug/ul; Invitrogen) was added to aqueous Degree Laboratory Freezer (MELFI) aboard the ISS. All ground mice were fraction and the resulting pellet was then resuspended in 15 μL of RNase processed similarly. Flight mice carcasses returned to Earth February 2015 free water (ThermoFisher 18064022). An aliquot (1 μL) of this resuspended aboard the SpaceX CRS-5 and were then maintained along with the control RNA was used to determine RNA concentration and RNA integrity number mice frozen at the Biospecimen Sharing Program (BSP) at the Ames (RIN) values using the Agilent RNA 6000 Pico kit and Agilent 2100 Research Center until dissection . All animal procedures performed were Bioanalyzer (Agilent Technologies). Total RNA (400 ng) from each sample approved by the Institutional Animal Care and Use Committees (IACUC) for was reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen/ThermoFisher) with random hexamer primers (IDT). flight at the NASA Ames Research Center (ARC) and the Kennedy Space Quantitative PCR (qPCR) was performed using a 1:5 dilution of cDNA on Center (KSC) and the methods were carried out in accordance with an Applied Biosystems HT7900 sequence detector. Genes interrogated relevant guidelines and regulations . included 3β-hydroxysteroid dehydrogenase (Hsd3b1), P450 cholesterol side chain cleavage (Cyp11a1), 17α-hydroxylase (Cyp17a1), P450 aromatase Tissue collection (Cyp19a1), steroidogenic acute regulatory protein (Star), liver receptor Female reproductive tissues (ovary and uterus) were collected from all homolog-1 (Nr5a2), estrogen receptor 1 (Esr1), estrogen receptor 2 (Esr2), carcasses at about 35–45 min after removal from the −80 °C freezer. One scavenger receptor class B member 1 (Scarb1), luteinizing hormone ovary was placed into RNAlater (Thermo Fisher Scientific, Waltham, MA) receptor (Lhcgr), zona pellucida glycoprotein 3 (Zp3), and growth and the other was flash-frozen in liquid nitrogen (LN). Similarly, the right differentiation factor 9 (Gdf9). Primers were designed using Primer Express and left uterine horns were placed in RNAlater or snap-frozen in LN; uterine 3.0 (Applied Biosystems) and purchased from IDT (see Supplementary tissues are part of an independent study by another investigator and will Table 1). qPCR was performed for Hsd3b1, Cyp11a1, Cyp17a1, Cyp19a1, not be further described here. Tissues preserved in RNAlater were kept at Nr5a2, Esr1, Esr2, Scarb1, Zp3, Lhcgr, and Gdf9 using PowerSYBR Green PCR 4 °C for 2 days then frozen and stored at −80 °C, tissues snap-frozen in Master Mix (Applied Biosystems). qPCR on Star, and 18 S rRNA levels were liquid nitrogen were also stored at −80 °C. Following primary tissue determined using the TaqMan Universal PCR Master Mix (Applied dissections in April of 2015, the remaining carcass was refrozen and stored Biosystems) protocol and Applied Biosystems (Eukaryotic 18S rRNA at −80 °C. In March of 2016, carcasses were thawed again for a secondary Endogenous Control [VIC® ⁄ MGB Probe, Primer Limited]) probes and tissue dissection. At this time the combined cervix and vaginal wall were primers. Samples were run in triplicate, and the ΔΔCt method was used to dissected free of the pelvic cavity, and these tissues were fixed in 4% calculate the relative expression between the samples after normalization paraformaldehyde in phosphate-buffered saline. Following overnight with 18S levels. All SYBR Green reactions were evaluated for the presence fixation these tissues were transferred to 70% EtOH, in preparation for of a single dissociation curve, to confirm the amplification of a single shipping to the University of Kansas Medical Center. transcript and lack of primer dimers. npj Microgravity (2021) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Fold Change Fold Change Fold Change Fold Change Fold Change Fold Change X. Hong et al. Histological analysis 9. Macho, L. et al. Effect of space flight on the development of endocrine functions in rats. Endocr. Regul. 27,17–22 (1993). Upon receipt of tissues from the BSP, the vaginal walls samples were 10. Ronca, A. E. & Alberts, J. R. Physiology of a microgravity environment selected transferred to tissue embedding cassettes, and processed through a contribution: effects of spaceflight during pregnancy on labor and birth at 1 G. J. standard series of dehydration steps prior to being embedded in paraffin. Appl. Physiol. 89, 849–854 (2000). Tissue blocks were then sectioned on a Leica microtome at a thickness of 11. Choi, S. Y. et al. Validation of a new rodent experimental system to investigate 6 μm and mounted on microscope slides (Superfrost Plus, Fisher Scientific). consequences of long duration space habitation. Sci. Rep. 10, 2336 (2020). Tissue sections were then deparaffinized, rehydrated, and stained with 12. Ronca, A. E. et al. Behavior of mice aboard the International Space Station. Sci. hematoxylin and eosin prior to histological examination. Slides were Rep. 9, 4717 (2019). randomized and sample (animal and treatment) labels were blinded 13. Ronca, A. et al. Effects of sex and gender on adaptations to space: reproductive prior to examination by three independent reviewers for estrous cycle health. J. 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(UVA School of Medicine) on dry ice and the rest were stored as 50 μL Behav. 59, 114–122 (2011). aliquots at −80 °C. Estradiol (E2) and progesterone (P4) were determined 20. Goldman, J. M., Murr, A. S. & Cooper, R. L. The rodent estrous cycle: character- by commercial ELISAs (E2: Calbiotech, El Cajon, CA. Cat # ES180S-100; P4: ization of vaginal cytology and its utility in toxicological studies. Birth Defects Res. IBL, Minneapolis, MN, Cat # IB79105). E2 assay characteristics were as Part B: Dev. Reprod. Toxicol. 80,84–97 (2007). follows: sensitivity = 3 pg/ml; intra-assay coefficient of variation (CV)= 21. McClintock, M. K. Social control of the ovarian cycle and the function of estrous 7.5%; inter-assay CV = 10.1%. For P4, sensitivity= 0.15 ng/ml; intra-assay synchrony. Am. Zool. 21, 243–256 (1981). CV= 6.5%; inter-assay CV= 10.3%. 22. Gal, A., Lin, P.-C., Barger, A. M., MacNeill, A. L. & Ko, C. Vaginal fold histology reduces the variability introduced by vaginal exfoliative cytology in the classifi- cation of mouse estrous cycle stages. Toxicol. Pathol. 42, 1212–1220 (2014). Statistical analysis 23. Merkwitz, C. et al. A simple method for inducing estrous cycle stage-specific One-way ANOVA was used to determine differences amongst treatment morphological changes in the vaginal epithelium of immature female mice. Lab. groups (flight status) or estrous cycle stage. Tukey’s mean separation tests Anim. 50, 344–353 (2016). were used to determine differences between mean once a significant 24. Wood, G. A., Fata, J. E., Watson, K. L. M. & Khokha, R. Circulating hormones and F-test was observed. All statistical analyses and graphs for the RIA were estrous stage predict cellular and stromal remodeling in murine uterus. Repro- performed using GraphPad Prism v6 (GraphPad, San Diego, CA). duction 133, 1035–1044 (2007). 25. Nelson, J. F., Felicio, L. S., Osterburg, H. H. & Finch, C. E. Differential contributions of ovarian and extraovarian factors to age-related reductions in plasma estradiol Reporting summary and progesterone during the estrous cycle of C57BL/6J mice. Endocrinology 130, Further information on experimental design is available in the Nature 805–810 (1992). Research Reporting Summary linked to this paper. 26. Nilsson, M. E. et al. Measurement of a comprehensive sex steroid profile in rodent serum by high-sensitive gas chromatography-tandem mass spectrometry. Endocrinology 156, 2492–2502 (2015). DATA AVAILABILITY 27. Zenclussen, M. L., Casalis, P. A., Jensen, F., Woidacki, K. & Zenclussen, A. C. Hor- Individual mouse estrous cycle stage results can be found in Supplementary Table 2. monal fluctuations during the estrous cycle modulate heme oxygenase-1 expression in the uterus. Front. Endocrinol. 5, 32 (2014). 28. DeLeon, D. D., Zelinski-Wooten, M. B. & Barkley, M. S. Hormonal basis of variation in Received: 2 July 2020; Accepted: 17 February 2021; oestrous cyclicity in selected strains of mice. J. Reprod. Fertil. 89,117–126 (1990). 29. Choi, S., Ray, H. E., Lai, S. H., Alwood, J. S. & Globus, R. K. Preservation of multiple mammalian tissues to maximize science return from ground based and space- flight experiments. PLoS ONE 11, e0167391 (2016). 30. ter Horst, J. P., de Kloet, E. R., Schächinger, H. & Oitzl, M. S. Relevance of stress and REFERENCES female sex hormones for emotion and cognition. Cell. Mol. Neurobiol. 32, 725–735 1. Demontis, G. C. et al. Human pathophysiological adaptations to the space (2012). environment. Front. Physiol. 8, 547 (2017). 31. Beheshti, A. et al. Exploring the effects of spaceflight on mouse physiology using 2. Rajkovic, A. & Pangas, S. Ovary as a biomarker of health and longevity: insights the open access NASA GeneLab platform. J. Vis. Exp. 143, 58447 (2019). from genetics. Semin. Reprod. Med. 35, 231–240 (2017). 3. Steller, J. G., Alberts, J. R. & Ronca, A. E. Oxidative stress as cause, consequence, or biomarker of altered female reproduction and development in the space envir- ACKNOWLEDGEMENTS onment. Int. J. Mol. Sci. 19, 3729 (2018). 4. Mishra, B. & Luderer, U. Reproductive hazards of space travel in women and men. Rodent Research 1 was a joint operation between NASA and the ISS National Lab. Nat. Rev. Endocrinol. 15, 713–730 (2019). This work was supported by the NASA Space Biology Program, Spaceflight Research 5. Messinis, I. E. Ovarian feedback, mechanism of action and possible clinical Opportunities in Space Biology—NNX15AB48G to L.K. Christenson and University of implications. Hum. Reprod. Update 12, 557–571 (2006). Kansas Medical School research funds to L.K. Christenson. 6. Whirledge, S. & Cidlowski, J. A. Glucocorticoids, stress, and fertility. Minerva Endocrinol. 35, 109–125 (2010). 7. Ronca, A. E., Alwood,J.S., Globus,R.K.& Souza, K. A. Mammalianrepro- AUTHOR CONTRIBUTIONS duction and development on the International Space Station (ISS): pro- X.H., A.E.R., J.S.A., J.S.T., and L.K.C. designed the research; X.H., A.R., S.Y.C., and L.K.C., ceedings of the rodent Mark III Habitat Workshop. Gravitational Space Res. 1, processed tissues and performed data collection; A.R. and L.K.C. analyzed data and 107–123 (2013). 8. Burden, H. W. et al. Effects of space flight on ovarian–hypophyseal function in prepared figures; A.R., J.S.A., A.E.R., J.S.T., and L.K.C. wrote the paper. All authors postpartum rats. Reproduction 109, 193–197 (1997). reviewed the manuscript. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 11 X. Hong et al. 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Effects of spaceflight aboard the International Space Station on mouse estrous cycle and ovarian gene expression

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www.nature.com/npjmgrav ARTICLE OPEN Effects of spaceflight aboard the International Space Station on mouse estrous cycle and ovarian gene expression 1 1 2 1 3,4 3 Xiaoman Hong , Anamika Ratri , Sungshin Y. Choi , Joseph S. Tash , April E. Ronca , Joshua S. Alwood and Lane K. Christenson 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. npj Microgravity (2021) 7:11 ; https://doi.org/10.1038/s41526-021-00139-7 INTRODUCTION temporal changes in expression of steroidogenic pathway proteins and other critically important ovarian genes known to Spaceflight microgravity is known to impact numerous physiolo- play critical roles in ovulation, oocyte development and corpus gical systems. These include cardiovascular changes, decreased luteum development and function are well known and may be bone mineral density, loss of muscle tone, adipogenesis, insulin evaluated to provide information regarding ovarian function. resistance, vision and vestibular disturbances, altered fluid and Understanding these molecular changes is vital for informing electrolyte balance, and altered kidney function to name a few . female reproductive health during and after spaceflight. Many, if not all, normal homeostatic processes occurring within To date, studies of ovarian function and fertility in mice exposed mammals, both male and female, are dramatically impacted by to microgravity during NASA Space Shuttle flights were of short gonadal hormones. Indeed, the reproductive system is often duration (<12 days) and, importantly, all included the effects of considered one of the best markers of overall health and fitness of live reentry prior to collection of tissues and analysis of animal an animal . However, research is limited regarding the effects of 3,7 behavior . Similarly, pregnant female rats flown on Cosmos 1514 spaceflight on the reproductive system and its consequences on 3,4 (1982), NASA-NIH Rodent (R)1 (STS-66 in 1994), and NASA-NIH R2 normal physiology . (STS-70 in 1995) were of short duration (4.5–11 days) and had live The ovarian steroid hormones, estrogen and progesterone, play 8–10 animal return . Notably, in the pregnant rats no effects of a significant role in regulating a myriad of physiological functions, spaceflight on healthy and atretic ovarian antral follicle popula- and are subject to cyclic changes based on the hypothalamic/ tions, fetal wastage in utero, plasma concentrations of progester- pituitary regulation of ovarian folliculogenesis . Moreover, ovarian one and luteinizing hormone (LH) or pituitary content of follicle steroidogenic output can be dramatically impacted by stress as stimulating hormone (FSH) were noted . Spaceflight, however, well as other internal homeostatic processes . In cycling rodents, significantly increased plasma concentrations of FSH and the antral follicle is the primary steroidogenic tissue within the decreased pituitary content of LH analyzed postpartum (day ovary, unless mating occurs at which time the corpus luteum 22–23; ). In these prior studies, it is not possible to separate the becomes the major producer of progesterone. The mural effects of microgravity from that of reentry with the associated granulosa cells and the vascularized theca cells within the antral hypergravity, turbulence, and the likely ensuing stress responses ovarian follicles are responsible for the enzymatic conversion of linked to these events. Moreover, these studies while evaluating cholesterol to its ultimate endpoint product estradiol. In addition maintenance of pregnancy, provide no information regarding the to steroidogenesis, these follicular cells also carry out the critical fertility of these animals and their ability to exhibit normal estrous missions of nurturing the enclosed growing oocyte, as well as facilitating the process of ovulation, or expulsion of oocyte, and cycles and ovarian function. The current investigation provides subsequent formation of the ephemeral corpus luteum. Cyclic and insights into the endocrine status and ovarian cyclicity of female 1 2 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA. KBR, NASA-Ames Research Center, Moffett Field, CA, USA. 3 4 Space Biosciences Division, NASA-Ames Research Center, Moffett Field, CA, USA. Department of Obstetrics & Gynecology, Wake Forest Medical School, Winston-Salem, NC, USA. email: lchristenson@kumc.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; X. Hong et al. ab Fig. 1 Hematoxylin and eosin staining of vaginal walls used for estrous cycle staging. Vaginal wall estrous cycle staging was completed on all mice (n = 40). Representative sections from two flight and two habitat control mice depict each stage of the estrous cycle a proestrus, b estrus, c metestrus, and d diestrus. *Marks the vaginal lumen. Images were taken at ×20 using brightfield microscopy; scale bar measures 100 μm. mice following an extended period of exposure to microgravity in Table 1. Number of mice at each stage of the estrous cycle (n = 10/ the absence of reentry effects. Addressing the effects of extended group). duration spaceflight on endocrine status are significant and of high impact to long-term female astronaut health before and after Proestrus Estrus Metestrus Diestrus ND flight, interpretation of spaceflight findings derived from female mice, and for long-term plans for multigenerational mammalian Baseline 2 6 0 1 1 studies in spaceflight. Vivarium 3 5 0 0 2 Habitat 2 3 2 3 0 Flight 0 6 4 0 0 RESULTS Estrous cycle staging ND not determinable. Estrous cycle stage for each mouse was determined by three independent investigators blinded to treatment group categories. Ovarian RNA quality and tissue steroid levels Figure 1 shows representative H&E stained cross sections of Ovarian RNA quality varied appreciably depending upon whether vaginal wall tissue from the individual flight and habitat control it was flash-frozen in LN or placed in RNAlater (Fig. 2). animals following collection after the second carcass thaw during Representative electropherograms from the Agilent 2100 are March of 2016. Each stage of the estrous cycle is depicted in shown for 2 mice collected in either RNAlater or LN. Overall, RNA Fig. 1. Proestrus is characterized by the presence of a thick layer quality as determined by Agilent RNA integrity numbers (RIN of stratified squamous epithelium, with an absence of luminal values) from tissues frozen in LN were greater (p < 0.05) in every cells and any cornified epithelial cells (Fig. 1a). Estrous stage animal, and overall averaged around 5.71 ± 0.12 compared to animals exhibit a stratified squamous epithelium with a layer of 4.08 ± 0.11 for RNAlater preserved ovarian tissue (Fig. 2b). To cornified epithelial [red eosin stained cells above the nucleated evaluate ovarian hormone production without access to serum, stratified squamous epithelial cells (Fig. 1b)]. Often, this cornified whole ovarian tissue steroid levels were determined. Ovarian layer is delaminated from the underlying epithelial cells. In tissue E2 levels were not significantly different across experi- metestrus, leukocytes appear and traverse the nucleated epithe- mental groups (Fig. 3a). In contrast, whole ovarian tissue P4 levels lial layer to reach the delaminated mass of cornified stratified were significantly lower in the habitat control (p = 0.005) and squamous epithelium in the lumen (Fig. 1c). During diestrus, the flight mice (p = 0.013) in comparison to baseline control mice vaginal wall is characterized by columnar mucified cells next to a (Fig. 3b). Due to insufficient animal numbers at individual stages thin layer of nucleated epithelium (Fig. 1d). Staging of the mice within treatment groups, we were unable to evaluate stage effects indicated that the majority of the animals in the baseline control alone and stage-treatment interactions. group (6/9), the vivarium control group (5/8), and the flight group (6/10) were in estrus (Table 1). In comparison, habitat control Ovarian tissue gene expression analyses mice presented at all stages of the cycle in approximately equal amounts (n = 2 or 3/stage). Flight group mice presented at estrus Quantitative RT-PCR analysis of critical proteins involved in ovarian (n = 6) and metestrus (n = 4; Table 1). function and steroidogenesis were compared across treatment npj Microgravity (2021) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; X. Hong et al. b e c d RNA later LN RNALater LN Fig. 2 RNA quality of liquid nitrogen snap-frozen and RNAlater preserved ovarian tissues. Representative electropherograms from 2 animals (M13 vivarium control a, b and M22 flight c, d) perserved in RNAlater (a, c)orLN(b, d). e Histogram of RIN values from analysis of paired LN and RNALater samples (n = 12; 4BL, 4VC, 2HC, 2FL). *Means ± SEM RIN values were significantly (p ≤ 0.05) different based on the preservation method. where P450 aromatase (Cyp19a1) expression was elevated a b E2 P4 (p ≤ 0.05) ~4-fold compared to all other stages (Fig. 5). Lastly, as 400 10 each group of animals had at least three animals staged at estrus, we compared the gene expression across the four treatment a,b groups at this stage (Fig. 6). Levels of steroidogenic acute 200 regulatory protein (Star), P450 cholesterol side chain cleavage (Cyp11a1), 3β-hydroxysteroid dehydrogenase (Hsd3b1), Cyp19a1, 100 and liver receptor homolog-1 (Nr5a2) expression did not change b b appreciably across experimental conditions. Expression of Cyp17a1 0 under baseline and vivarium control conditions were significantly VC VC BL HC FL BL HC FL decreased (p < 0.05) compared to habitat control conditions (p < 0.05). Ovarian Cyp17a1 levels in flight animals in estrus were not Fig. 3 Whole ovarian tissue estrogen and progesterone concen- notably different from estrus stage animals in the other experi- trations. a Estradiol (E2; pg/ml/mg tissue) and b progesterone mental conditions (Fig. 6). Examination of other critical ovarian (P4; ng/ml/mg tissue) levels for all mice within a treatment group a,b (n = 8-9/treatment; 9BL, 8VC, 9HC, 9FL). Means ± SEM with genes during estrus showed no significant changes in expression different superscripts are significantly different (p ≤ 0.05). levels across control and flight conditions (Supplementary Fig. 2). groups irrespective of stage of the cycle (Fig. 4, Supplementary DISCUSSION Fig. 2). Spaceflight had no demonstrable effect on gene Rodent Research-1 (RR-1), a Validation mission launched aboard expression of any of the key enzymatic steps of steroidogenesis SpaceX’s fourth cargo resupply mission, was the first to transport or mitochondrial cholesterol uptake expression. Ovarian Cyp17a1 rodents to the International Space Station (ISS) in an unmanned mRNA levels were elevated (p ≤ 0.05) in mice housed in the commercial vehicle. Lasting 37 days in microgravity, RR-1 was, at habitat maintained on the ground (HC) when compared to the time, the longest space rodent study conducted by NASA. In- baseline and vivarium controls but were not different from flight flight studies of RR1 mouse behavior and post-flight physiology animals that were housed in the habitat on the ISS (Fig. 4). revealed the Validation mice showed no overt physiological signs Analysis of other critically important ovarian genes involved in of chronic stress or compromised health or welfare . Moreover, steroidogenic production and action, including estrogen receptors mice remained highly active and mobile throughout the experi- (Esr1 and Esr2), scavenger receptor class B type 1 (Scarb1), luteinizing hormone receptor (Lhcgr) as well as oocyte-specific ment, engaging in feeding, drinking, self-grooming, huddling, proteins, growth differentiation factor 9 (Gdf9), zona pellucida and social interactions . Our results provide detailed conse- glycoprotein 3 (Zp3) showed no differences across the treatment quences of long-term spaceflight microgravity on rodent estrous groups (Supplementary Fig. 1). cycle, ovarian steroidogenesis, and gene expression. Notably, Comparison of ovarian gene expression at different stages of the previous observations examining the potential impacts of micro- estrous cycle did not vary significantly with exception of proestrus, gravity on ovarian function and its target tissue the uterus were of Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 11 E2 Levels (pg/ml/mg) M22 M13 P4 Levels (ng/ml/mg) RNA Integrity Number (RIN value) X. Hong et al. a bc Cyp11a1 Hsd3b1 Star 3 3 1.5 2 1.0 1 0.5 0 0 0 VC BL HC FL VC HC FL BL HC FL BL VC df e 1.5 Cyp17a1 Cyp19a1 Nr5a2 5 1.5 1.0 1.0 a,b 0.5 0.5 HC FL HC FL BL HC FL BL VC BL VC VC Fig. 4 Whole ovarian tissue steroidogenic gene expression was not altered by spaceflight. Histograms depict relative fold change (ΔΔCt method) for a Star, b Cyp11a1, c Hsd3b1, d Cyp17a1, e Cyp19a1, and f Nr5a2 following normalization to 18S; n = 8–10/treatment group; 10BL, a,b 10VC, 8HC, 10FL). Means ± SEM with different superscripts are significantly different (p ≤ 0.05). a short-term nature and had the confounding effects of reentry serum at the time of sacrifice or longitudinally over time is 3,9,13 prior to tissue collection . It is well known that estrous cycle severely restricted logistically. We, therefore, analyzed whole staging in female rodents can be influenced by a variety of factors ovarian tissue levels of estrogen and progesterone. We detected such as age, light, temperature, humidity, ambient noise, nutrition, no striking difference in tissue estrogen concentrations across and social relationships . Indeed, mice that are group housed the four treatment groups, and it should be noted that serum exhibit greater estrous cycle irregularities, than those individually estrogen levels in rodents also do not exhibit wide variations 15,16 housed . Nonetheless, many labs using contemporary strains of across the cycle, often ranging from ND to 8–50 pg/ml in 24–27 mice are able to stage mice in group housing conditions, albeit it total . In contrast, we did observe that flight and habitat 17–19 may not be as ideal as single housed mice . Again, group control animals had lower overall P4 levels in ovarian tissue. housing was necessitated by animal husbandry concerns and One might predict this as these two groups had greater limitations of the NASA’s flight hardware. Our results point to an numbers of mice in metestrus and diestrus, in total 4 and 5 for ability of female mice to exhibit estrous cycles after an extended flight and habitat groups, respectively. During both of these duration in space, as evidenced by our observation that flight stages of estrous cycle the ovary has reduced steroidogenic 26–28 animals exhibited two different stages of the cycle (estrus and activity . This was in contrast to the vivarium and baseline metestrus). Typically, under conditions of exogenous treatments/ control groups, which both had 8 animals each in proestrus and stress, mice may exhibit constant vaginal estrus or an extended or estrus in total, stages associated with increased ovarian 14,20 constant diestrus , but not metestrus. In contrast to mice, follicular steroidogenesis. We recognize that it would have rats exhibit a greater synchrony when group housed, making it been ideal to examine the interaction of estrous cycle stage on difficult to extrapolate finding across these two species . ovarian steroidogenesis as well as ovarian gene expression, but While measuring true estrous cyclicity in space is logistically due to the insufficient numbers of animals at individual stages, daunting due to the intensive daily monitoring required, we we were unable to do so. It is worth noting the experimental posit that the analysis of the vaginal wall provides a robust design for RR1 as a hardware demonstration flight determined methodology that can indicate the estrous stage, and thus the how tissues were preserved as well as group sizes and thus were endocrine status of the animal at the moment of sacrifice. Indeed beyond our control. Whole ovarian gene expression analyses the morphology of these twice frozen and thawed vaginal wall were also dictated by the RR1 design. This type of analysis is tissues rivaled that of freshly isolated and fixed tissues from highly confounded as whole ovarian tissue contains an contemporary animals collected in our laboratory (data not abundance of stromal tissue and smaller follicles, which shown) and in those laboratories who pioneered the use of predominate over the few selected follicles that become highly 20,22,23 vaginal wall histology as a mechanism to track cyclicity . steroidogenic. Thus, when whole ovarian RNA is used, it was not Ovarian tissue steroid levels are also not commonly mea- surprising to see no major differences in ovarian gene sured, as most investigators use serum levels of estrogen and expression across the treatment groups or even during different progesterone as a measure of ovarian steroidogenic activity. stages of the cycle. Future studies need to take into considera- Again, in animals exposed to microgravity, the ability to collect tion this limitation, and collect and fix tissues for analysis, so npj Microgravity (2021) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Fold Change Fold Change Fold Change Fold Change Fold Change Fold Change X. Hong et al. a bc 2.5 Star Cyp11a1 Hsd3b1 1.5 2.0 1.0 1.5 1.0 0.5 0.5 0 0 Proestrus Metestrus Diestrus Estrus Proestrus Estrus Metestrus Diestrus Proestrus Estrus Metestrus Diestrus d f Cyp17a1 Cyp19a1 Nr5a2 0.8 1.5 0.6 a 1.0 0.4 4 0.5 0.2 0 0 Proestrus Estrus Metestrus Diestrus Metestrus Diestrus Proestrus Estrus Metestrus Diestrus Proestrus Estrus Fig. 5 Whole ovarian tissue steroidogenic gene expression by stage of estrous cycle. Histograms depict relative fold change (ΔΔCt method) for a Star, b Cyp11a1, c Hsd3b1, d Cyp17a1, e Cyp19a1, and f Nr5a2 following normalization to 18S; n = 8–10/treatment group; 10BL, a,b 10VC, 8HC, 10FL). Means ± SEM with different superscripts are significantly different (p ≤ 0.05). they can be properly analyzed using in situ hybridization/ which are composed of highly dynamic tissues (follicles, corpora RNAscope and immunohistochemistry. Cyp17a1,the enzyme lutea, endometrium), we recommend that they be preserved located in the theca cell compartment of the ovarian follicle and primarily in fixatives or in a manner where tissue/cell specific responsible for androgen production was elevated in the aspects can be determined afterward. Lastly, the ability of these habitat control animals. This difference was however, mani- females to exhibit estrous cycle activity provides a tractable fested primarily in two of the three HC animals in estrus. mammalian model system to test whether fertility is actually Safeguarding RNA tissue quality is acutely dependent on tissue impacted by microgravity. Reproductive cycles characterized by storage methods, which ideally would minimize RNase activity alterations in hormone levels can exert significant effects on throughout the process. Tissue collection and preservation during experimental outcomes, including emotion and the response to spaceflight presents a unique challenge in the analysis of these environmental stressors . Determining the effects of spaceflight valuable specimens. Our current analyses of the right and left on estrous cyclicity is thus critical to making accurate interpreta- ovaries processed in RNALater or snap-frozen in liquid nitrogen tions of experimental endpoints for all spaceflight experiments (LN) indicated that LN preserved tissues always had greater RIN using female mice. values when compared to those of RNALater. Previous studies utilizing RR1 RNAlater preserved spleen tissues showed higher RIN values than those observed here . This may be due to a time of METHODS collection aspect with respect to spleen vs ovary following thawing Animals of the carcass, or just to tissue differences in RNA susceptibility to Forty 12-week old C57BI/6J female mice (Jackson Lab, Bar Harbor, ME) the freeze/thaw. We note that freshly isolated ovarian tissues that were selected based on similar body weights for 4 experimental groups were snap-frozen in LN and then simultaneously processed with (16-week old at launch, n = 10/group): baseline control, vivarium control, the RR1 samples yielded RIN values >9. habitat control, and flight. Flight mice were launched on SpaceX-4 (Rodent Research 1) on September 21, 2014, as part of NASA’s Validation study. In conclusion, our analyses of ovarian tissues and vaginal wall Details of RR1 mission timeline (i.e., mouse pre-adaptation to cages, food) tissues of mice exposed to extended periods of microgravity and 11,12,31 as well as mouse spaceflight hardware details are available . Briefly, sacrificed in space indicate that these female mice were exhibiting vivarium control mice were maintained in standard mouse cages (5 mice/ estrous cyclic activity. These studies point positively to the ability cage) for the duration of spaceflight at Kennedy Space Center. Habitat of these animals to either continue or regain estrous cycle activity control mice were housed in a Rodent Research Transporter and Habitat during extended microgravity exposure. We posit that vaginal wall Hardware on Earth for durations consistent with flight times at the same estrous cycle activity be collected and provided to investigators to density of mice as those in flight (n = 10/habitat). The habitat control was include as covariate on all female mice used in future space placed in the ISS Environmental Simulator at KSC on a 4-day delay to missions, much the same as weight and age. The current studies mimic flight temperature, CO , and humidity conditions on the ISS for the also provide critical logistical information regarding tissue duration of spaceflight. Baseline control mice were euthanized one day collection, particularly for organs such as the ovary and uterus, after launch and then partially dissected prior to freezing of the animal Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 11 Fold Change Fold Change Fold Change Fold Change Fold Change Fold Change X. Hong et al. a b Cyp11a1 Hsd3b1 4 Star 4 1.5 3 3 1.0 2 2 0.5 1 1 0 0 HC FL BL VC BL VC HC FL VC HC FL BL d f Cyp17a1 Cyp19a1 2.5 Nr5a2 4 1.5 2.0 1.0 1.5 1.0 a,b 0.5 0.5 0 0 HC FL VC HC FL BL VC BL VC HC FL BL Fig. 6 Whole ovarian tissue steroidogenic gene expression at estrus across all four treatment groups. Histograms depict relative fold change (ΔΔCt method) for a Star, b Cyp11a1, c Hsd3b1, d Cyp17a1, e Cyp19a1, and f Nr5a2 following normalization to 18S; n = 3–6/treatment a,b group; 6BL, 5VC, 3HC, 6FL). Means ± SEM with different superscripts are significantly different (p ≤ 0.05). carcasses mimicking the procedures to be conducted on flight and for all RNA isolation and quantitative RT-PCR analysis other animals at the conclusion of experiment. Flight mice were exposed Ovaries tissues were transferred directly from RNAlater to tubes containing to microgravity for a total of 37 days (33 days on ISS and 4 days in the 1 ml of Trizol (Sigma) or 1 ml of Trizol was added directly to ovarian tissue Dragon Capsule). Mice were euthanized by injection of Euthasol followed snap-frozen in liquid nitrogen. Tissues were briefly dissociated on ice by cervical dislocation and immediately fast frozen intact (n = 8) or (3 bursts, 62 W) in a PowerGen 700 homogenizer (Fisher Scientific). RNA partially dissected (n = 2) prior to carcasses being frozen. Mice carcasses was isolated from Trizol as per manufacturer protocol. To facilitate were frozen using pre-chilled Ice Bricks prior to transfer to the Minus Eighty precipitation 1 μL of glycogen (20ug/ul; Invitrogen) was added to aqueous Degree Laboratory Freezer (MELFI) aboard the ISS. All ground mice were fraction and the resulting pellet was then resuspended in 15 μL of RNase processed similarly. Flight mice carcasses returned to Earth February 2015 free water (ThermoFisher 18064022). An aliquot (1 μL) of this resuspended aboard the SpaceX CRS-5 and were then maintained along with the control RNA was used to determine RNA concentration and RNA integrity number mice frozen at the Biospecimen Sharing Program (BSP) at the Ames (RIN) values using the Agilent RNA 6000 Pico kit and Agilent 2100 Research Center until dissection . All animal procedures performed were Bioanalyzer (Agilent Technologies). Total RNA (400 ng) from each sample approved by the Institutional Animal Care and Use Committees (IACUC) for was reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen/ThermoFisher) with random hexamer primers (IDT). flight at the NASA Ames Research Center (ARC) and the Kennedy Space Quantitative PCR (qPCR) was performed using a 1:5 dilution of cDNA on Center (KSC) and the methods were carried out in accordance with an Applied Biosystems HT7900 sequence detector. Genes interrogated relevant guidelines and regulations . included 3β-hydroxysteroid dehydrogenase (Hsd3b1), P450 cholesterol side chain cleavage (Cyp11a1), 17α-hydroxylase (Cyp17a1), P450 aromatase Tissue collection (Cyp19a1), steroidogenic acute regulatory protein (Star), liver receptor Female reproductive tissues (ovary and uterus) were collected from all homolog-1 (Nr5a2), estrogen receptor 1 (Esr1), estrogen receptor 2 (Esr2), carcasses at about 35–45 min after removal from the −80 °C freezer. One scavenger receptor class B member 1 (Scarb1), luteinizing hormone ovary was placed into RNAlater (Thermo Fisher Scientific, Waltham, MA) receptor (Lhcgr), zona pellucida glycoprotein 3 (Zp3), and growth and the other was flash-frozen in liquid nitrogen (LN). Similarly, the right differentiation factor 9 (Gdf9). Primers were designed using Primer Express and left uterine horns were placed in RNAlater or snap-frozen in LN; uterine 3.0 (Applied Biosystems) and purchased from IDT (see Supplementary tissues are part of an independent study by another investigator and will Table 1). qPCR was performed for Hsd3b1, Cyp11a1, Cyp17a1, Cyp19a1, not be further described here. Tissues preserved in RNAlater were kept at Nr5a2, Esr1, Esr2, Scarb1, Zp3, Lhcgr, and Gdf9 using PowerSYBR Green PCR 4 °C for 2 days then frozen and stored at −80 °C, tissues snap-frozen in Master Mix (Applied Biosystems). qPCR on Star, and 18 S rRNA levels were liquid nitrogen were also stored at −80 °C. Following primary tissue determined using the TaqMan Universal PCR Master Mix (Applied dissections in April of 2015, the remaining carcass was refrozen and stored Biosystems) protocol and Applied Biosystems (Eukaryotic 18S rRNA at −80 °C. In March of 2016, carcasses were thawed again for a secondary Endogenous Control [VIC® ⁄ MGB Probe, Primer Limited]) probes and tissue dissection. At this time the combined cervix and vaginal wall were primers. Samples were run in triplicate, and the ΔΔCt method was used to dissected free of the pelvic cavity, and these tissues were fixed in 4% calculate the relative expression between the samples after normalization paraformaldehyde in phosphate-buffered saline. Following overnight with 18S levels. All SYBR Green reactions were evaluated for the presence fixation these tissues were transferred to 70% EtOH, in preparation for of a single dissociation curve, to confirm the amplification of a single shipping to the University of Kansas Medical Center. transcript and lack of primer dimers. npj Microgravity (2021) 11 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Fold Change Fold Change Fold Change Fold Change Fold Change Fold Change X. Hong et al. Histological analysis 9. Macho, L. et al. Effect of space flight on the development of endocrine functions in rats. Endocr. Regul. 27,17–22 (1993). Upon receipt of tissues from the BSP, the vaginal walls samples were 10. Ronca, A. E. & Alberts, J. R. Physiology of a microgravity environment selected transferred to tissue embedding cassettes, and processed through a contribution: effects of spaceflight during pregnancy on labor and birth at 1 G. J. standard series of dehydration steps prior to being embedded in paraffin. Appl. Physiol. 89, 849–854 (2000). Tissue blocks were then sectioned on a Leica microtome at a thickness of 11. Choi, S. Y. et al. Validation of a new rodent experimental system to investigate 6 μm and mounted on microscope slides (Superfrost Plus, Fisher Scientific). consequences of long duration space habitation. Sci. Rep. 10, 2336 (2020). Tissue sections were then deparaffinized, rehydrated, and stained with 12. Ronca, A. E. et al. Behavior of mice aboard the International Space Station. Sci. hematoxylin and eosin prior to histological examination. Slides were Rep. 9, 4717 (2019). randomized and sample (animal and treatment) labels were blinded 13. Ronca, A. et al. Effects of sex and gender on adaptations to space: reproductive prior to examination by three independent reviewers for estrous cycle health. J. Women’s Health 23, 967–974 (2014). staging (proestrus, estrus, metestrus, diestrus or not determinable). Non- 14. Li, S. & Davis, B. Evaluating rodent vaginal and uterine histology in toxicity stu- determinable samples were the result of tissues not containing sufficient dies. Birth Defects Res. Part B: Dev. Reprod. Toxicol. 80, 246–252 (2007). vaginal wall tissue to yield reliable results. Following independent reviews, 15. Whitten, W. K. Occurrence of anoestrus in mice caged in groups. J. Endocrinol. 18, those samples (~10%) without a consensus stage call were reevaluated by 102–107 (1959). the reviewers and a consensus stage agreed upon. 16. Champlin, A. K. Suppression of oestrus in grouped mice: the effects of various densities and the possible nature of the stimulus. J. Reprod. Fertil. 27, 233–241 Radioimmunoassay (1971). The steroid content (estrogen/progesterone) content of the ovary, as 17. Wilson, K. et al. 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(UVA School of Medicine) on dry ice and the rest were stored as 50 μL Behav. 59, 114–122 (2011). aliquots at −80 °C. Estradiol (E2) and progesterone (P4) were determined 20. Goldman, J. M., Murr, A. S. & Cooper, R. L. The rodent estrous cycle: character- by commercial ELISAs (E2: Calbiotech, El Cajon, CA. Cat # ES180S-100; P4: ization of vaginal cytology and its utility in toxicological studies. Birth Defects Res. IBL, Minneapolis, MN, Cat # IB79105). E2 assay characteristics were as Part B: Dev. Reprod. Toxicol. 80,84–97 (2007). follows: sensitivity = 3 pg/ml; intra-assay coefficient of variation (CV)= 21. McClintock, M. K. Social control of the ovarian cycle and the function of estrous 7.5%; inter-assay CV = 10.1%. For P4, sensitivity= 0.15 ng/ml; intra-assay synchrony. Am. Zool. 21, 243–256 (1981). CV= 6.5%; inter-assay CV= 10.3%. 22. Gal, A., Lin, P.-C., Barger, A. M., MacNeill, A. L. & Ko, C. Vaginal fold histology reduces the variability introduced by vaginal exfoliative cytology in the classifi- cation of mouse estrous cycle stages. Toxicol. Pathol. 42, 1212–1220 (2014). Statistical analysis 23. Merkwitz, C. et al. A simple method for inducing estrous cycle stage-specific One-way ANOVA was used to determine differences amongst treatment morphological changes in the vaginal epithelium of immature female mice. Lab. groups (flight status) or estrous cycle stage. Tukey’s mean separation tests Anim. 50, 344–353 (2016). were used to determine differences between mean once a significant 24. Wood, G. A., Fata, J. E., Watson, K. L. M. & Khokha, R. Circulating hormones and F-test was observed. All statistical analyses and graphs for the RIA were estrous stage predict cellular and stromal remodeling in murine uterus. Repro- performed using GraphPad Prism v6 (GraphPad, San Diego, CA). duction 133, 1035–1044 (2007). 25. Nelson, J. F., Felicio, L. S., Osterburg, H. H. & Finch, C. E. 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Mammalianrepro- AUTHOR CONTRIBUTIONS duction and development on the International Space Station (ISS): pro- X.H., A.E.R., J.S.A., J.S.T., and L.K.C. designed the research; X.H., A.R., S.Y.C., and L.K.C., ceedings of the rodent Mark III Habitat Workshop. Gravitational Space Res. 1, processed tissues and performed data collection; A.R. and L.K.C. analyzed data and 107–123 (2013). 8. Burden, H. W. et al. Effects of space flight on ovarian–hypophyseal function in prepared figures; A.R., J.S.A., A.E.R., J.S.T., and L.K.C. wrote the paper. All authors postpartum rats. Reproduction 109, 193–197 (1997). reviewed the manuscript. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2021) 11 X. Hong et al. 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