Sex and estrous cycle influence fasting-induced torpor via hypothalamic estrogen signalling

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Marshall, Anthony E. Pickering, Michael T. Ambler This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7356338/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Torpor is a state of transient hypometabolism and hypothermia that is engaged by many species in adverse conditions such as food scarcity. Neurons in the hypothalamic preoptic area (POA) are capable of driving entry into torpor when activated. Estrogens, principally estradiol, are capable of modulating thermogenesis and energy balance by central actions, and POA neurons express the canonical estrogen receptor ERα. We find that torpor depth and duration vary across the estrus cycle in mice, whereby the torpor response is greatest during the diestrus phase in which circulating estradiol is at its peak. Torpor responses are longer and deeper in female mice compared to males, and this is potentiated by exogenous estradiol, which lengthens torpor bouts in female mice, but not males. Knockdown of ERα within the POA blunts torpor in female mice, suggesting that estradiol acting via ERα modulates the activity of hypothalamic neurons that generate torpor. We speculate that this cyclical oscillation in torpor propensity which is lowest in the estrous phase may be an adaptive change that preserves reproduction in periods of moderate environmental stress. Biological sciences/Neuroscience Biological sciences/Physiology Biological sciences/Zoology Figures Figure 1 Figure 2 Figure 3 Introduction Maintenance of core body temperature is an energetically demanding physiological process for warm blooded animals (endotherms) that accounts for a large proportion of daily energy expenditure, particularly in small animals with a larger surface area relative to their volume. For example, mice use around half of their total energy expenditure when maintaining their core body temperature at a typical room temperature of 20–22˚C 1 . Where adverse environmental conditions lead to energy deficit, the metabolic demand of maintaining temperature homeostasis can present a major challenge to survival. A strategy employed frequently across animal species, including mammals, is a controlled and reversable suppression of metabolic demand known as torpor 2 , 3 . During torpor, metabolism may decrease to as little as 2% of basal rate, and internal body temperature is defended at a much lower set point, in some species dropping to near ambient temperatures, even in freezing conditions 4 , 5 . Torpor is an extreme physiological adaptation, and a growing body of evidence has converged on the preoptic area of the hypothalamus (POA) as the driving locus for this phenomenon 6 – 11 . Alongside identification of the mechanisms, there is a growing interest in artificially activating them to induce torpor-like states for potential applications in humans 12 – 14 . Several groups, including our own, have demonstrated that activation of excitatory neurons in the POA drives torpor-like states in rats, which are non-hibernators 10 , 11 , 15 , and so far one group has achieved a modest synthetic torpor-like state in rhesus macaques, demonstrating that synthetic torpor is achievable in primates 16 . An interesting observation is that many torpor studies primarily or exclusively use female mice. It has been suggested that female mice more readily enter torpor and are thus preferred as experimental subjects for their consistency 17 . Despite this observation, the effects of sex or indeed circulating sex hormones on torpor response has not been closely examined. Estrogens, principally estradiol, modulate thermoregulation by tuning BAT thermogenesis and vasomotor activity 7 , 18 , 19 . Estrogens cross the blood brain barrier and can exert their thermoregulatory effects via central mechanisms 20 , 21 . Indeed, estrogen receptors (ERs) are expressed throughout the central thermoregulatory pathways and are particularly enriched within the preoptic area (POA) of the hypothalamus 22 , 23 . Downstream of the POA, high ERα expression is also found within other hypothalamic regions with roles in thermoregulation and energy balance such as the ventromedial and arcuate nuclei 18 , 24 – 26 , and is moderately expressed in the dorsomedial hypothalamic nucleus 22 . The expression of ER in both energy balance and thermoregulatory circuits, as well as their abundance in the POA, has made these neurons an interesting population for further study. One such recent study demonstrated that ERα + POA neurons are active during fasting-induced torpor and are necessary and sufficient for torpor induction 7 . What remains unclear, however, is whether estradiol signalling via ERα in these neurons influences torpor induction and maintenance to explain the observations that female mice enter torpor more readily, or whether the estrogen sensitivity in these neurons is unrelated to their role in torpor. This study aimed to explore the sex differences in mouse torpor, that have thus far been mainly described anecdotally, and to determine whether estradiol modulates entry into or maintenance of torpor. We hypothesised that estradiol levels and ERα-signalling in the POA potentiates torpor in female mice. To test this, we fasted female mice to induce torpor at each of the three phases of the estrus cycle where estradiol levels are naturally oscillating (and compared to males under equivalent conditions); additionally, we examined the effect of exogenous estradiol administration on torpor, and conversely the effect of POA ERα knockdown on torpor. Methods Ethical statement Experiments were conducted according to the guidelines, and with approval, of the University of Bristol Welfare and Ethical review board and in accordance with the Animals (Scientific Procedures) Act 1986, under a UK Home Office Project Licence. This study is reported in accordance with current ARRIVE guidelines for the reporting of animal experiments. Mice For all experiments, C57/BL6j mice were used. Adult (60-100 day old) mice were group housed unless otherwise stated, with ad libitum access to water and standard rodent chow. Mice were housed in a 12h:12h light:dark cycle (lights on at 10:00am, ZT+0 h) at a standard temperature of 21 °C ± 1 °C. At the point of entry to experiment, the mean body mass was 21.7 ± 0.5 g for female mice, and 25.9 ± 0.7 g for male mice. Estrous cycle staging The mouse estrous cycle was determined by vaginal cytology as described previously 27 . Briefly, daily vaginal lavage was performed with ~20 µL sterile saline for a period of up to 14 days, flushing the canal several times to obtain a sufficient sample, then transferring 2-3 drops onto a glass slide. Samples were allowed to air dry and then stained with 0.1% crystal violet for 5 minutes before washing with ddH 2 O and cover slipping. Specimens were assessed by brightfield microscopy with the relative presence of leukocytes, cornified or nucleated epithelial cells being used to determine the stage of estrous on the day of sample collection. Examples of cytology present during diestrus, estrus and proestrus are shown in Supplemental Figure 1. Torpor induction and thermography For recording of torpor induction, mice were transferred into custom built Perspex chambers, each containing inserts that divided the space into quadrants and allowed simultaneous recording of four mice per chamber (as per 9 ). On the day of recording, mice were placed into the chamber at ZT+7 h, initiating the 24 h fast. Where mice were recorded multiple times over the estrus cycle, the order was pseudorandomised by initiating the first torpor bout at different phases of the estrus cycle between mice. When multiple recordings occurred, a minimum of four days recovery was allowed between fasts. Surface body temperature was recorded using a FLIR C2 thermal camera (Teledyne FLIR LLC) positioned above the recording chamber. Temperature data was obtained from captured footage using ResearchIR software (FLIR), using the region of interest (ROI) and maximum temperature functions. Further analysis of surface temperature data was carried out using MATLAB R2021a software (The MathWorks Inc., https://www.mathworks.com), using processing-pipeline described by our group previously, defining torpor as periods of >30 min where surface temperature was <29 °C. Estradiol treatments A stock solution of 17-β-estradiol (cat# 8875, Sigma-Aldrich) was prepared by first dissolving 1 mg/mL in absolute ethanol, which was then added to 50 mL of chen oil; the ethanol was evaporated within a vacuum centrifuge for ~60 min giving a final stock solution of 20 µg/mL. Male and female mice were treated with 2 µg / 20 g body mass s.c. bolus (or chen oil vehicle), administered at 10:00am (ZT+0 h) to ‘mimic’ the diestrus peak of estradiol described in C57/BL6 mice recently 28 . Female mice were treated on days they were in estrus, determined by vaginal cytology as described above. Viral constructs Lentiviral vectors were obtained from Santa-Cruz Biotechnology (Lenti-shERα; cat# sc-29306-V) prepared at a titre of 1.0×10 6 IFUs. This vector contained four Esr1- specific constructs encoding 19-25 nt short hairpin RNAs designed to knock down the expression of ERα in mouse tissues 29 . The AAV5-GFP (pAAV.CMV.PI.EGFP.WPRE.bGH) was obtained from Addgene (RRID:Addgene_105530, a gift from James M. Wilson), at a titre of 7×10¹² vg/mL. AAV5-GFP delivers the EGFP gene under the control of the constitutively expressed CMV promotor and was used to assess the placement of stereotaxic injections. Stereotaxic viral injections Mice were anaesthetized with ketamine (Ketavet; 70 mg/kg, i.p.) and medetomidine (Domitor; 0.5 mg/kg, i.p.) and maintained with additional injections if needed. Following preparation of the surgical site, mice were placed in a stereotaxic frame (Model 963 Small Animal Stereotaxic Instrument, David Kopf Instruments) and their body temperature maintained using a heat pad (Harvard Apparatus). Glass pipettes were filled with mineral oil and affixed onto a robotic microinjector (Nano-W wireless capillary microinjector, Neurostar), then backfilled with either AAV5-GFP diluted in saline (1:4, control mice, n=8) or a mixture of AAV5-GFP and Lenti-shERα (1:4, ERα knockdown mice, n=7). Burr holes were made at AP +0.4 mm, ML ±0.5 mm relative to Bregma. Bilateral injections were then made at DV -5.1 mm and -5.0 mm relative to the surface of the brain, targeting the medial POA. Virus was delivered at 62.5 nL/minute to a final volume of 125 nL per injection site and therefore 250 nL per hemisphere. Within each hemisphere, 2 minutes were allowed before delivery of the second injection; following the second injection, 5 minutes were allowed before retraction of the pipette. Atipamezole (1 mg/kg, i.p) was used to reverse anaesthesia and carprofen (5 mg/kg, s.c.) was given for pain management up to 3 days post-surgery. Mice were individually housed for five days following surgery and were allowed to recover for a least two weeks following surgery before commencing experiments, torpor was assessed between two- and four-weeks post-surgery. Tissue collection and immunohistochemistry Mice were killed with a terminal dose of pentobarbital (Euthetal; 175 mg/kg, i.p.), followed by transcardial perfusion of heparinised saline (~15 mL, 50 units/mL heparin in 0.9% NaCl) and 15-20 mL neutral buffered formalin (VWR Chemicals). Brains were removed and stored in 10% formalin overnight, before being transferred to a 30% sucrose solution with 0.02% sodium azide and stored at 4°C. Coronal brain slices (30 µm thickness) containing the POA were collected using a freezing microtome and mounted onto Superfrost Plus slides (Epredia). Slices were washed twice for 2 mins in PBS-T (PBS + 0.1% Triton), then blocked with 0.25% bovine serum albumin and 10% normal donkey serum in PBS-T for 1 hour at RT. Slices were then incubated with a primary rabbit anti-ERα antibody (1:1000; Merck Millipore, cat# 06-935, RRID: AB_310305) overnight at 4°C. Slices were then washed three times for 5 mins in PBS-T, then incubated with secondary donkey anti-rabbit 568 for two hours at RT. Slices were washed, counterstained with DAPI (1:10,000) for 5 min, then cover slipped using Fluoromount-G hard set mounting medium. Microscopy and image analysis Images of sections throughout the POA were collected using an Olympus VS200 slide scanner microscope. Image files were registered against the Allen Mouse Brain Common Coordinate Framework 30 (https://atlas.brain-map.org/) using the Aligning Big Brains & Atlases (ABBA; https://biop.github.io/ijp-imagetoatlas/) plugin for Fiji 31 . Registered images were further analysed in QuPath 32 (https://qupath.github.io/) using the cell detection function to quantify ERα+ cells. Cell count .csv files were exported and collated in RStudio (R version 4.2.1) and pre-processed to determine the total ERα+ cells in POA nuclei per mouse before statistical analysis. Statistics Statistical analysis and data visualisation was performed in RStudio and GraphPad Prism 10 (GraphPad Software). Power calculations for assessing group sizes for the ERα knockdown study were performed in G*Power 33 . Prior to any statistical comparisons, normality was assessed by Shapiro-Wilk test. The effect of estrous cycle stage on female mouse torpor was compared using one-way repeated measures ANOVA with Tukey’s multiple comparisons tests. Within-animal comparisons of vehicle and estradiol treatment effects on torpor was conducted either using paired two-tailed t tests or Wilcoxon matched-pairs signed rank tests. Comparisons between torpor in male and female mice and virally treated control vs knockdown mice were carried out by unpaired two-tailed t tests or Mann-Whitney U tests. Results are presented as either mean ± SD or median and quartiles (as appropriate). P values ≤ 0.05 were considered statistically significant. Results Depth, duration and latency of torpor varies through the mouse estrus cycle To assess the effect of the estrous cycle on torpor, induced by a 24-hour period of fasting, we recorded surface temperature in female mice (n = 8) across the diestrus, proestrus and estrus phases (Figure 1A, B). Mice in diestrus exhibited deeper torpor bouts sustained for longer, compared to the same mice in proestrus and estrus (Figure 1A, B). We found the duration of time spent in torpor was significantly influenced by estrous cycle stage (Figure 1C, RM one-way ANOVA, F (2, 23) = 12.75, p = 0.0021) where mice spend longer in torpor during diestrus (389.3 ± 143.3 min) than in proestrus (282.1 ± 113.4 min, p < 0.01) or estrus (172.8 ± 103.2 min, p < 0.05). Similarly, estrous cycle stage also significantly influenced the magnitude of temperature fall from baseline during bouts of torpor (Figure 1D, RM one-way ANOVA, F (2, 23) = 2.362, p = 0.043) where there was a greater reduction temperature in diestrus (-6.59 ± 1.08 °C) vs. estrus (-5.6 ± 1.57 °C, p < 0.05, Tukey’s post hoc). The integral of surface temperature over time was calculated, generating a ‘torpor score’ for each recording session per animal. This torpor score was significantly affected by estrous cycle stage in female mice (Figure 1D, RM one-way ANOVA, F (2, 23) = 10.54 , p = 0.0016) where it was greatest during diestrus (27,737 ± 13,834 °C.s) compared with proestrus (15,665 ± 10,014 °C.s, p < 0.05) and estrus (10,042 ± 6,166 °C.s, p < 0.01, Tukey’s post hoc). The stage of the cycle also impacted the time to the onset of the first torpor bout following the removal of food (Figure 1E, RM one-way ANOVA) where onset of torpor was significantly earlier in diestrus (12.32 ± 1.62 h after food removal) compared with estrus (14.67 ± 1.51 h) (p < 0.01, Tukey’s post hoc). Female mice display longer and deeper bouts of torpor than male mice We assessed sex differences in torpor by comparing female mice in diestrus to male mice over the course of a 24 hour fast. Interestingly, just five out of eight male mice entered torpor compared to all of the female mice indicating that male mice may have a lower probability of entering torpor when challenged with a single fasting period (Figure 2A). Where torpor did occur, female mice sustained torpor for a longer duration than males (Figure 2B, 389.3 ± 143.3 vs. 186.1 ± 98.2 min; t = 2.77, p = 0.018, Unpaired t-test), and reached cooler temperatures from baseline (Figure 2C, -6.59 ± 1.08 °C vs. -4.8 ± 0.78 °C; t = 3.10, p = 0.01, Unpaired t-test). This was also reflected in markedly greater torpor scores in female mice than male mice (Figure 2D) (27,737 ± 13,834 °C.s vs 7,785 ± 4,667 °C.s, U = 1.00, p = 0.0031, Mann-Whitney U test). At the time of experiment, the age matched male mice had greater body mass than females (23.2 ± 1.28 g vs. 20.5 ± 0.77 g, t = 2.61, p < 0.05, Unpaired t-test). Exogenous estradiol treatment lengthens torpor bouts in female, but not male mice Given that female mice displayed a greater propensity to enter and stay in torpor than male mice, and that this varied across the estrous cycle, we tested the effect of exogenous estradiol on fasting-induced torpor in male mice and in female mice in estrus phase (where estradiol levels are lowest 28 ). Estradiol administration the morning prior to fasting extended the time that female mice spent in torpor (compared to a chen oil vehicle Figure 2E; p = 0.018, F (3, 14) = 3.21, Two-way ANOVA). Torpor scores for estradiol-treated female mice were similarly greater (Figure 2G; p = 0.0042, F (3, 14) = 4.45, Two-way ANOVA). Estradiol treatment appeared to also increase the depth of torpor in some female mice, but this was not uniform across mice, and there was no significant group level effect compared to vehicle (Figure 2F). In contrast, estradiol treatment of male mice had no effect on depth or duration of torpor (Figure 2E-G). Knockdown of ERα in the POA blunts torpor response following a fast Given that activation of estradiol-sensitive neurons in the POA induces a torpor-like state 7 , we tested the hypothesis that estradiol-signalling in the POA causes female mice to display a robust torpor response. Lentivirus containing short-hairpin RNA to knock down ERα (sh-ERα) was delivered into the POA of female mice and compared with control female mice where only GFP was delivered by AAV. The number of ERα+ cells in the POA of sh-ERα mice was reduced compared to GFP control mice (Figure 3A, B; 237.1 ± 42.0 cells vs 287.6 ± 34.2 cells, p = 0.023, t = 2.57, Unpaired t-test), and similarly the mean fluorescence intensity of positive cells was reduced in sh-ERα mice compared to controls (84.14 ± 5.14 AU vs. 69.13 ± 14.38 AU; p = 0.016, t = 2.77). Surface temperature thermography profiles during fasting in ERα knockdown mice were distinct from control mice; torpor bouts were shorter (Figure 3D). Overall, knockdown mice had shorter torpor bouts (Figure 3E; p = 0.035, t = 2.35, Unpaired t-test), as well as shallower bouts (Figure 3F; -7.16 ± 0.85 °C vs. -8.19 ± 0.72 °C; p = 0.024, t = 2.55, unpaired t-test). Torpor scores were also considerably lower than control mice (Figure 3G; 9,771 ± 8,660 vs 26,738 ± 16,454 °C.s, p = 0.029, t = 2.44, unpaired t-test). In contrast, ERα knockdown did not change the time to onset of torpor following fasting (Figure 3H). Discussion The aim of this study was to examine whether the cyclical variation in circulating estradiol in female mice modulates entry into, and maintenance of, torpor. The data presented here support prior incidental descriptions that female mice enter torpor more readily than male mice 17 . We observed that female mice exhibit deeper and longer bouts of torpor during the phase of their estrous cycles where estradiol is highest, and they exhibited longer and deeper bouts than male mice. We confirmed that estradiol signalling mediates this association: exogenous estradiol lengthened torpor bouts in female mice. Finally, we show that estradiol exerts this effect via the POA: knocking down ERα in the POA reduced torpor length and depth in female mice. Together, these results build on the growing body of work implicating the POA as the key site for torpor induction, and indicate that in female mice, activity of POA torpor-inducing neurons is enhanced by the action of estradiol on ERα. Neurons in the POA that induce torpor have a distinct transcriptomic signature, including expression of Qrfp, Ptger3, Lepr, Opn5 , and Tacr3 , which has led to them being termed the QPLOT neurons 34 . QPLOT neurons co-express a host of receptors including ERα, positioning them to integrate hormonal, metabolic, thermal, and immune inputs that may influence whether an animal enters torpor 34 . ERα may influence activity and excitability of neurons through both genomic and second messenger mediated pathways. To act via the latter, ERα may be trafficked to the plasma membrane via post-translational palmitoylation and subsequent association with the scaffold protein calveolin-1 35,36 , where it forms functional dimers that associate with G-protein complexes 37 . Membrane associated ERs facilitate estradiol’s rapid modulation of cellular processes by activating secondary pathways including metabotropic glutamate receptors 38 . Functionally, this has mostly been studied in the context of cognition, memory and motivation, however, there is evidence that estradiol acting via metabotropic glutamate receptors in the medial POA decreases food intake 39 . ERs also function as intracellular receptors capable of modulating neural activity and excitability by stimulating calcium release from the endoplasmic reticulum, or by modulating ion channel activity 40 . Whether this leads to up or down-regulation of excitability is context dependent. For example, again with respect to energy balance in the hypothalamus, estradiol disinhibits pro-opiomelanocortin neurons by attenuating GABA B -mediated activation of inwardly-rectifying potassium channels, while the opposite is true of agouti-related peptide neurons where GABAergic inhibition is enhanced 41 . Hence, estradiol is capable of acting in the hypothalamus to modulate neural activity and change behaviour via multiple cellular pathways. Our results support the hypothesis that, in the case of POA torpor-inducing neurons, activation of ERα is excitatory in nature. Studies of neurons in the POA consistently report that their activation drives torpor output 6-8 , and POA neural activity is elevated during torpor when examined by cFos expression or in vivo recording of calcium events by fibre photometry 6,8,9 . It follows then that estradiol increasing excitability of QPLOT neurons in the POA would promote torpor. Our results indicate that estradiol plays a role in maintenance of torpor, whereby torpor duration was longer in in estrous phases where estradiol would have been highest, or when estradiol signalling was intact. We also present evidence that estradiol reduces the latency between fasting onset and entry into torpor and drives larger drops in temperature during torpor compared to euthermic baselines. Torpor is an active process associated with a higher frequency of calcium events in the POA 7,8 , and nadir temperature may be reduced during optogenetic stimulation of POA neurons by increasing either the power or frequency of light stimulus 10 , hence lower nadir temperatures are indicative of higher neural activity. Our knockdown findings indicate that ERα signalling in the POA is necessary for estradiol to enhance activity of torpor neurons directly. Elucidation of this mechanism is a goal for future investigations. One question raised by our findings across the sexes is why does estradiol enhance torpor in female mice, but not males? We demonstrate that female mice enter a longer and deeper state of torpor than male mice in a similar age range, which could be explained by females simply having higher levels of circulating estradiol than male mice 42,43 . However, we also demonstrate that estradiol treatment enhances torpor in female mice but not males. The POA displays sexual dimorphism: several nuclei differ in size, neurotransmitter expression, and cell number 44 . Furthermore, ERα expression is greater in the POA of female mice compared to males 7 . Female mice may have adapted to become more energetically thrifty, making ‘savings’ in diestrus when they are not fertile. This might allow for greater energy expenditure with lower probability of torpor entry and shorter and shallower torpor bouts typically seen during proestrus and estrus, which is when mating occurs. Hence, female mice preferentially allocate energy expenditure to periods when mating occurs and prioritise energy saving when mating does not occur. An interesting consideration is whether stimuli such as the presence of male scent-marked bedding across the estrous cycle has any effect on torpor, which may allow us to tease apart the hierarchy of priorities between mating and energy savings. Alternatively (or additionally), the low metabolic activity and body temperature of torpor might be hazardous during early pregnancy, in which case torpor might be inhibited during periods of the cycle in which fertilisation and implantation occur. In this interpretation, torpor is not so much promoted during diestrus as inhibited at other times. Energy expenditure and food intake are additional factors that fluctuate over the course of the estrous cycle. Energy expenditure is lower in diestrus compared to estrus in both rats and mice and diestrus is associated with lower heat production over 24 hours in mice 45,46 . Food intake, similarly, may vary over the course of the estrous cycle by 20% and is highest during estrus 47 . |It is possible that mice experience a period of relative energy deficit during diestrus compared to other phases of the cycle. This might make torpor more advantageous at this timepoint. Hence, the POA torpor-inducing neurons might have evolved estrogen-sensitivity to align torpor with periods of the estrus cycle in which relative energy deficit occurs. Limitations An obvious consideration regarding the difference in torpor between males and females is that of body mass. Recently published work from our group has determined that body mass affects the probability of torpor onset 48 . Body mass influences the ‘lower critical limit’ of ambient thermoneutral temperature, below which mammals must increase their metabolic rate to maintain core body temperature. In mice, as body mass decreases, the temperature of the lower critical limit will increase thus narrowing the thermoneutral range 49 . Mice in the present study were housed at 21°C, and female mice had an ~11% lower body mass than male mice. While this may partially account for some of the differences in torpor response between the age-matched males and females, we still demonstrate a striking sex difference in that female mice respond to estradiol treatment while males do not. Another consideration is that our focus on estradiol does not consider other sex hormones that might have additional roles in regulating torpor. Progesterone levels are lower throughout most of the mouse estrous cycle aside from a brief peak in late proestrus 28 , likely generated by the corpus luteum following ovulation 50 . While progesterone released during the luteal phase, and taken exogenously from oral contraceptives, drives an increase in body temperature in humans (~0.5˚C) 51 , its role in mice and other animal models is not as clearly defined. Some evidence for thermoregulatory effects in rodents comes from one study in rabbits which reported that progesterone drove an increase in body temperature, with a concurrent decrease in firing rate from warm-sensitive POA neurons 52 . Given that progesterone is released in a relatively confined window of early estrus in mice before being rapidly metabolised 50 , it seems unlikely that it would regulate torpor to the same degree as estradiol. However, it does stand as a reasonable consideration for studies examining the role of sex hormones in torpor going forward. Conclusion These findings highlight estradiol as a modulator of torpor through its actions on the POA, deepening and lengthening torpor. Further work elucidating how estradiol modulates neurons in this area will be important in advancing our understanding of how these neurons integrate physiological information to modulate torpor. This work highlights the role of estrogen in modulating the fasting response in female mice via neurons in the POA. It demonstrates that torpor propensity changes throughout the estrus cycle in females, suggesting an interaction between the demands of energy balance and reproduction in mice. Declarations Acknowledgements This work was supported by the Medical Research Council grant number MR/W029138/1. Funding This work was funded by the Medical Research Council grant number MR/W029138/1. Author contributions CJM: Conceptualization, methodology, investigation, formal analysis, writing - original draft, writing - review & editing, visualization. AEP: Conceptualization, methodology, writing - review & editing, supervision, funding acquisition. MTA: Conceptualization, methodology, writing - review & editing, supervision, funding acquisition. Additional information The authors declare no competing interests. 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Mol Cell Biol 23 , 1633-1646 (2003). https://doi.org/10.1128/mcb.23.5.1633-1646.2003 Acconcia, F. et al. Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol. Mol Biol Cell 16 , 231-237 (2005). https://doi.org/10.1091/mbc.e04-07-0547 Levin, E. R. Plasma membrane estrogen receptors. Trends Endocrinol Metab 20 , 477-482 (2009). https://doi.org/10.1016/j.tem.2009.06.009 Gross, K. S. & Mermelstein, P. G. in Vitamins and Hormones Vol. 114 (ed Gerald Litwack) 211-232 (Academic Press, 2020). Santollo, J. & Daniels, D. Anorexigenic effects of estradiol in the medial preoptic area occur through membrane-associated estrogen receptors and metabotropic glutamate receptors. Horm Behav 107 , 20-25 (2019). https://doi.org/10.1016/j.yhbeh.2018.11.001 Kelly, M. J. & Rønnekleiv, O. K. Minireview: neural signaling of estradiol in the hypothalamus. Mol Endocrinol 29 , 645-657 (2015). https://doi.org/10.1210/me.2014-1397 Smith, A. W., Bosch, M. A., Wagner, E. J., Rønnekleiv, O. K. & Kelly, M. J. The membrane estrogen receptor ligand STX rapidly enhances GABAergic signaling in NPY/AgRP neurons: role in mediating the anorexigenic effects of 17β-estradiol. Am J Physiol Endocrinol Metab 305 , E632-640 (2013). https://doi.org/10.1152/ajpendo.00281.2013 Saito, T. et al. Estrogen contributes to gender differences in mouse ventricular repolarization. Circ Res 105 , 343-352 (2009). https://doi.org/10.1161/circresaha.108.190041 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). https://doi.org/10.1210/en.2014-1890 Semaan, S. J. & Kauffman, A. S. Sexual differentiation and development of forebrain reproductive circuits. Curr Opin Neurobiol 20 , 424-431 (2010). https://doi.org/10.1016/j.conb.2010.04.004 Parker, G. C., McKee, M. E., Bishop, C. & Coscina, D. V. Whole-body metabolism varies across the estrous cycle in Sprague–Dawley rats. Physiology & Behavior 74 , 399-403 (2001). https://doi.org/https://doi.org/10.1016/S0031-9384(01)00599-6 Reho, J. J. et al. Modulatory effects of estrous cycle on ingestive behaviors and energy balance in young adult C57BL/6J mice maintained on a phytoestrogen-free diet. Am J Physiol Regul Integr Comp Physiol 326 , R242-r253 (2024). https://doi.org/10.1152/ajpregu.00273.2023 Asarian, L. & Geary, N. Modulation of appetite by gonadal steroid hormones. Philos Trans R Soc Lond B Biol Sci 361 , 1251-1263 (2006). https://doi.org/10.1098/rstb.2006.1860 Wheatley, W. S. R. et al. The cold truth: torpor as a confound in studies of caloric restriction. Journal of Comparative Physiology B (2025). https://doi.org/10.1007/s00360-025-01616-1 Gordon, C. J. Thermal physiology of laboratory mice: Defining thermoneutrality. Journal of Thermal Biology 37 , 654-685 (2012). https://doi.org/https://doi.org/10.1016/j.jtherbio.2012.08.004 Bachelot, A. & Binart, N. in Current Topics in Developmental Biology Vol. 68 49-84 (Academic Press, 2005). Stachenfeld, N. S., Silva, C. & Keefe, D. L. Estrogen modifies the temperature effects of progesterone. Journal of Applied Physiology 88 , 1643-1649 (2000). https://doi.org/10.1152/jappl.2000.88.5.1643 Nakayama, T., Suzuki, M. & Ishizuka, N. Action of progesterone on preoptic thermosensitive neurones. Nature 258 , 80-80 (1975). https://doi.org/10.1038/258080a0 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigures.docx Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 19 Sep, 2025 Reviews received at journal 18 Sep, 2025 Reviews received at journal 30 Aug, 2025 Reviewers agreed at journal 21 Aug, 2025 Reviewers agreed at journal 20 Aug, 2025 Reviewers invited by journal 20 Aug, 2025 Editor assigned by journal 20 Aug, 2025 Editor invited by journal 18 Aug, 2025 Submission checks completed at journal 14 Aug, 2025 First submitted to journal 14 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7356338","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":500676707,"identity":"b2bf9242-3acf-4dae-9ba2-e1f84ad8e863","order_by":0,"name":"Christopher J. Marshall","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIie2RPWvDMBBAzxia5UCrhkL+whlD0iFp/oqMwVMwAS8eUzJkKllV2h9RKHR20JClH6tBHZypq7t5rNRm6KC4GTPoTSfB454QgMdzjnCA6ndCgGYxERDaw+LnKvxfEZRZJVgC9SvwR1HCTP0Ku7/dbzt4Ho43r9tW0Hs+Xg8+m5ZgyJYYk2vJx44Ugo4e6jzlgnRxqTC6kQSRrDAWDoV4BgpAB5IjWSWRIQYrJAgeAePqiGLC9Eyyl7gT9GaUwd4qsz6lMmGJhPnIbKmMApFVEqu4wnhtwpB0Kvl8dCUoLXiI0Z0knkp1Ubiez2QWfnWlntqwui2vc852TduWk+lmvXriDuWQ51h/7CM9Ho/HcwLfmWpcK6sP0CwAAAAASUVORK5CYII=","orcid":"","institution":"University of Bristol","correspondingAuthor":true,"prefix":"","firstName":"Christopher","middleName":"J.","lastName":"Marshall","suffix":""},{"id":500676708,"identity":"8ccfa5e0-83be-4e3b-ad32-d5091f317dae","order_by":1,"name":"Anthony E. Pickering","email":"","orcid":"","institution":"University of Bristol","correspondingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"E.","lastName":"Pickering","suffix":""},{"id":500676709,"identity":"891d7322-59e4-406e-b598-af79bf10f721","order_by":2,"name":"Michael T. Ambler","email":"","orcid":"","institution":"University of Bristol","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"T.","lastName":"Ambler","suffix":""}],"badges":[],"createdAt":"2025-08-12 13:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7356338/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7356338/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-41051-y","type":"published","date":"2026-02-27T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89277630,"identity":"4023d1f7-0c76-41ce-9ea6-f2a8ffaa46b6","added_by":"auto","created_at":"2025-08-18 09:48:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1690017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTorpor depth and duration varies over the course of the female mouse estrous cycle. \u003c/strong\u003eA)\u003cstrong\u003e \u003c/strong\u003eRepresentative temperature plots from surface temperature thermography recordings in one fasted mouse during diestrus, proestrus and estrus, bouts highlighted in red defined as surface temperature \u0026lt; 29 °C for at \u0026gt; 30 min. B) Heatmaps representing a gestalt overview of temperature over 24 hours following fasting when mice were in diestrus, proestrus and estrus. C) Total duration in hours spent in torpor during a 24-hour fast over the estrous cycle. D) The nadir temperature change from baseline in each phase of the estrous cycle. E) Torpor score (integrating temperature over time) over the course of the estrous cycle. F) The time to onset of the first bout of torpor following a 24-hour fast in each phase of the estrous cycle. Histograms show means. *p ≤ 0.05, **p ≤ 0.01.\u003c/p\u003e","description":"","filename":"Fig1stagesfinal.png","url":"https://assets-eu.researchsquare.com/files/rs-7356338/v1/9edd18046f5a101d8256056a.png"},{"id":89277629,"identity":"6b2fce16-4d5d-4c09-84ac-eed263d875a3","added_by":"auto","created_at":"2025-08-18 09:48:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1101007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFemale mice display deeper and longer torpor bouts that are potentiated by exogenous estradiol administration. \u003c/strong\u003eA)\u003cstrong\u003e \u003c/strong\u003eRepresentative temperature plots from surface temperature thermography recordings from female, responder male and non-responder male mice, bouts highlighted in red defined as surface temperature \u0026lt; 29 °C for at \u0026gt; 30 min. B) Total duration of time spent in torpor in male vs. female mice. C) Nadir temperature change from baseline in male vs. female mice during torpor. D) Torpor score in male vs female mice over the course of a 24-hour fast. E-G) the effect of exogenous estradiol on duration, change in temperature from baseline and torpor score in male vs. female mice. Box and whisker plot displays population median and quartiles, while histograms show mean ± SD. *p ≤ 0.05, **p ≤ 0.01.\u003c/p\u003e","description":"","filename":"Fig2mvffinal.png","url":"https://assets-eu.researchsquare.com/files/rs-7356338/v1/7397392a5670c8c88129fee3.png"},{"id":89277632,"identity":"7698dbc3-9e6c-4749-bb71-a0c0f7992dab","added_by":"auto","created_at":"2025-08-18 09:48:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":9876235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of ERα in the POA blunts torpor response following a 24 hour fast. \u003c/strong\u003eA) Representative 20x photomicrographs taken in the POA of control (left) and sh-ERα injected mice (right) showing ERα (red), with a DAPI counterstain (blue). Dashed lines delineate the medial preoptic area. Scale bar = 100 µm. B) ERα-positive cell counts within the POA of control vs. knockdown mice. C) The mean fluorescence intensity per cell area within the POA of control vs. knockdown mice. D) Representative temperature plots from surface temperature thermography recordings in female control and knockdown mice. E) Total duration of time spent in torpor in control vs. knockdown mice. F) Nadir temperature change from baseline in control vs. knockdown mice during torpor. G) Torpor score following 24-hour fast in control vs. knockdown mice. H) The time to onset of torpor following a 24-hour fast. \u0026nbsp;Histograms show mean ± SD. *p ≤ 0.05, **p ≤ 0.01.\u003c/p\u003e","description":"","filename":"Fig3kdfinal.png","url":"https://assets-eu.researchsquare.com/files/rs-7356338/v1/36e2fc183f1efa8f6012b0dc.png"},{"id":103765420,"identity":"118c45d7-ac0c-4ad8-8ac7-0fe99a62a78e","added_by":"auto","created_at":"2026-03-02 16:00:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16884228,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7356338/v1/ed8dd475-e50b-462d-b2de-97d2df74a43e.pdf"},{"id":89277640,"identity":"482988cb-9a47-4853-baa5-4969aeec67a8","added_by":"auto","created_at":"2025-08-18 09:48:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":39288157,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7356338/v1/fcbac7018d1eb5a3800b4760.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sex and estrous cycle influence fasting-induced torpor via hypothalamic estrogen signalling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMaintenance of core body temperature is an energetically demanding physiological process for warm blooded animals (endotherms) that accounts for a large proportion of daily energy expenditure, particularly in small animals with a larger surface area relative to their volume. For example, mice use around half of their total energy expenditure when maintaining their core body temperature at a typical room temperature of 20\u0026ndash;22˚C \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Where adverse environmental conditions lead to energy deficit, the metabolic demand of maintaining temperature homeostasis can present a major challenge to survival. A strategy employed frequently across animal species, including mammals, is a controlled and reversable suppression of metabolic demand known as torpor \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. During torpor, metabolism may decrease to as little as 2% of basal rate, and internal body temperature is defended at a much lower set point, in some species dropping to near ambient temperatures, even in freezing conditions \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTorpor is an extreme physiological adaptation, and a growing body of evidence has converged on the preoptic area of the hypothalamus (POA) as the driving locus for this phenomenon \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Alongside identification of the mechanisms, there is a growing interest in artificially activating them to induce torpor-like states for potential applications in humans \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Several groups, including our own, have demonstrated that activation of excitatory neurons in the POA drives torpor-like states in rats, which are non-hibernators \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and so far one group has achieved a modest synthetic torpor-like state in rhesus macaques, demonstrating that synthetic torpor is achievable in primates \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. An interesting observation is that many torpor studies primarily or exclusively use female mice. It has been suggested that female mice more readily enter torpor and are thus preferred as experimental subjects for their consistency \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Despite this observation, the effects of sex or indeed circulating sex hormones on torpor response has not been closely examined.\u003c/p\u003e\u003cp\u003eEstrogens, principally estradiol, modulate thermoregulation by tuning BAT thermogenesis and vasomotor activity \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Estrogens cross the blood brain barrier and can exert their thermoregulatory effects via central mechanisms \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Indeed, estrogen receptors (ERs) are expressed throughout the central thermoregulatory pathways and are particularly enriched within the preoptic area (POA) of the hypothalamus \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Downstream of the POA, high ERα expression is also found within other hypothalamic regions with roles in thermoregulation and energy balance such as the ventromedial and arcuate nuclei \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and is moderately expressed in the dorsomedial hypothalamic nucleus \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe expression of ER in both energy balance and thermoregulatory circuits, as well as their abundance in the POA, has made these neurons an interesting population for further study. One such recent study demonstrated that ERα\u0026thinsp;+\u0026thinsp;POA neurons are active during fasting-induced torpor and are necessary and sufficient for torpor induction \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. What remains unclear, however, is whether estradiol signalling via ERα in these neurons influences torpor induction and maintenance to explain the observations that female mice enter torpor more readily, or whether the estrogen sensitivity in these neurons is unrelated to their role in torpor.\u003c/p\u003e\u003cp\u003eThis study aimed to explore the sex differences in mouse torpor, that have thus far been mainly described anecdotally, and to determine whether estradiol modulates entry into or maintenance of torpor. We hypothesised that estradiol levels and ERα-signalling in the POA potentiates torpor in female mice. To test this, we fasted female mice to induce torpor at each of the three phases of the estrus cycle where estradiol levels are naturally oscillating (and compared to males under equivalent conditions); additionally, we examined the effect of exogenous estradiol administration on torpor, and conversely the effect of POA ERα knockdown on torpor.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eEthical statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were conducted according to the guidelines, and with approval, of the University of Bristol Welfare and Ethical review board and in accordance with the Animals (Scientific Procedures) Act 1986, under a UK Home Office Project Licence. This study is reported in accordance with current ARRIVE guidelines for the reporting of animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor all experiments, C57/BL6j mice were used. Adult (60-100 day old) mice were group housed unless otherwise stated, with \u003cem\u003ead libitum\u003c/em\u003e access to water and standard rodent chow. Mice were housed in a 12h:12h light:dark cycle (lights on at 10:00am, ZT+0 h) at a standard temperature of 21\u0026nbsp;\u0026deg;C \u0026plusmn; 1\u0026nbsp;\u0026deg;C. \u0026nbsp;At the point of entry to experiment, the mean body mass was 21.7 \u0026plusmn; 0.5 g for female mice, and 25.9 \u0026plusmn; 0.7 g for male mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEstrous cycle staging\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse estrous cycle was determined by vaginal cytology as described previously \u003csup\u003e27\u003c/sup\u003e. Briefly, daily vaginal lavage was performed with ~20 \u0026micro;L sterile saline for a period of up to 14 days, flushing the canal several times to obtain a sufficient sample, then transferring 2-3 drops onto a glass slide. Samples were allowed to air dry and then stained with 0.1% crystal violet for 5 minutes before washing with ddH\u003csub\u003e2\u003c/sub\u003eO and cover slipping. Specimens were assessed by brightfield microscopy with the relative presence of leukocytes, cornified or nucleated epithelial cells being used to determine the stage of estrous on the day of sample collection. Examples of cytology present during diestrus, estrus and proestrus are shown in Supplemental Figure 1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTorpor induction and thermography\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor recording of torpor induction, mice were transferred into custom built Perspex chambers, each containing inserts that divided the space into quadrants and allowed simultaneous recording of four mice per chamber (as per \u003csup\u003e9\u003c/sup\u003e). On the day of recording, mice were placed into the chamber at ZT+7 h, initiating the 24 h fast. Where mice were recorded multiple times over the estrus cycle, the order was pseudorandomised by initiating the first torpor bout at different phases of the estrus cycle between mice. When multiple recordings occurred, a minimum of four days recovery was allowed between fasts. Surface body temperature was recorded using a FLIR C2 thermal camera (Teledyne FLIR LLC) positioned above the recording chamber. Temperature data was obtained from captured footage using ResearchIR software (FLIR), using the region of interest (ROI) and maximum temperature functions. Further analysis of surface temperature data was carried out using MATLAB R2021a software (The MathWorks Inc., https://www.mathworks.com), using processing-pipeline described by our group previously, defining torpor as periods of \u0026gt;30 min where surface temperature was \u0026lt;29 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEstradiol treatments\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA stock solution of 17-\u0026beta;-estradiol (cat# 8875, Sigma-Aldrich) was prepared by first dissolving 1 mg/mL in absolute ethanol, which was then added to 50 mL of chen oil; the ethanol was evaporated within a vacuum centrifuge for ~60 min giving a final stock solution of 20 \u0026micro;g/mL. Male and female mice were treated with 2 \u0026micro;g / 20 g body mass s.c. bolus (or chen oil vehicle), administered at 10:00am (ZT+0 h) to \u0026lsquo;mimic\u0026rsquo; the diestrus peak of estradiol described in C57/BL6 mice recently \u003csup\u003e28\u003c/sup\u003e. Female mice were treated on days they were in estrus, determined by vaginal cytology as described above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eViral constructs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLentiviral vectors were obtained from Santa-Cruz Biotechnology (Lenti-shER\u0026alpha;; cat# sc-29306-V) prepared at a titre of 1.0\u0026times;10\u003csup\u003e6\u003c/sup\u003e IFUs. This vector contained four \u003cem\u003eEsr1-\u003c/em\u003especific constructs encoding 19-25 nt short hairpin RNAs designed to knock down the expression of ER\u0026alpha; in mouse tissues \u003csup\u003e29\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe AAV5-GFP (pAAV.CMV.PI.EGFP.WPRE.bGH) was obtained from Addgene (RRID:Addgene_105530, a gift from James M. Wilson), at a titre of 7\u0026times;10\u0026sup1;\u0026sup2; vg/mL. AAV5-GFP delivers the EGFP gene under the control of the constitutively expressed CMV promotor and was used to assess the placement of stereotaxic injections.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStereotaxic viral injections\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMice were anaesthetized with ketamine (Ketavet; 70 mg/kg, i.p.) and medetomidine (Domitor; 0.5 mg/kg, i.p.) and maintained with additional injections if needed. Following preparation of the surgical site, mice were placed in a stereotaxic frame (Model 963 Small Animal Stereotaxic Instrument, David Kopf Instruments) and their body temperature maintained using a heat pad (Harvard Apparatus). Glass pipettes were filled with mineral oil and affixed onto a robotic microinjector (Nano-W wireless capillary microinjector, Neurostar), then backfilled with either AAV5-GFP diluted in saline (1:4, control mice, n=8) or a mixture of AAV5-GFP and Lenti-shER\u0026alpha; (1:4, ER\u0026alpha; knockdown mice, n=7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBurr holes were made at AP +0.4 mm, ML \u0026plusmn;0.5 mm relative to Bregma. Bilateral injections were then made at DV -5.1 mm and -5.0 mm relative to the surface of the brain, targeting the medial POA. Virus was delivered at 62.5 nL/minute to a final volume of 125 nL per injection site and therefore 250 nL per hemisphere. Within each hemisphere, 2 minutes were allowed before delivery of the second injection; following the second injection, 5 minutes were allowed before retraction of the pipette. Atipamezole (1 mg/kg, i.p) was used to reverse anaesthesia and carprofen (5 mg/kg, s.c.) was given for pain management up to 3 days post-surgery. Mice were individually housed for five days following surgery and were allowed to recover for a least two weeks following surgery before commencing experiments, torpor was assessed between two- and four-weeks post-surgery.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTissue collection and immunohistochemistry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMice were killed with a terminal dose of pentobarbital (Euthetal; 175 mg/kg, i.p.), followed by transcardial perfusion of heparinised saline (~15 mL, 50 units/mL heparin in 0.9% NaCl) and 15-20 mL neutral buffered formalin (VWR Chemicals). Brains were removed and stored in 10% formalin overnight, before being transferred to a 30% sucrose solution with 0.02% sodium azide and stored at 4\u0026deg;C. Coronal brain slices (30 \u0026micro;m thickness) containing the POA were collected using a freezing microtome and mounted onto Superfrost Plus slides (Epredia). Slices were washed twice for 2 mins in PBS-T (PBS + 0.1% Triton), then blocked with 0.25% bovine serum albumin and 10% normal donkey serum in PBS-T for 1 hour at RT. Slices were then incubated with a primary rabbit anti-ER\u0026alpha; antibody (1:1000; Merck Millipore, cat# 06-935, RRID: AB_310305) overnight at 4\u0026deg;C. Slices were then washed three times for 5 mins in PBS-T, then incubated with secondary donkey anti-rabbit 568 for two hours at RT. Slices were washed, counterstained with DAPI (1:10,000) for 5 min, then cover slipped using Fluoromount-G hard set mounting medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicroscopy and image analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eImages of sections throughout the POA were collected using an Olympus VS200 slide scanner microscope. Image files were registered against the Allen Mouse Brain Common Coordinate Framework \u003csup\u003e30\u003c/sup\u003e (https://atlas.brain-map.org/) using the Aligning Big Brains \u0026amp; Atlases (ABBA; https://biop.github.io/ijp-imagetoatlas/) plugin for Fiji \u003csup\u003e31\u003c/sup\u003e. Registered images were further analysed in QuPath \u003csup\u003e32\u003c/sup\u003e (https://qupath.github.io/) using the cell detection function to quantify ER\u0026alpha;+ cells. Cell count .csv files were exported and collated in RStudio (R version 4.2.1) and pre-processed to determine the total ER\u0026alpha;+ cells in POA nuclei per mouse before statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis and data visualisation was performed in RStudio and GraphPad Prism 10 (GraphPad Software). Power calculations for assessing group sizes for the ER\u0026alpha; knockdown study were performed in G*Power \u003csup\u003e33\u003c/sup\u003e. Prior to any statistical comparisons, normality was assessed by Shapiro-Wilk test. The effect of estrous cycle stage on female mouse torpor was compared using one-way repeated measures ANOVA with Tukey\u0026rsquo;s multiple comparisons tests. Within-animal comparisons of vehicle and estradiol treatment effects on torpor was conducted either using paired two-tailed t tests or Wilcoxon matched-pairs signed rank tests. Comparisons between torpor in male and female mice and virally treated control vs knockdown mice were carried out by unpaired two-tailed t tests or Mann-Whitney \u003cem\u003eU\u003c/em\u003e tests. Results are presented as either mean \u0026plusmn; SD or median and quartiles (as appropriate). \u003cem\u003eP\u003c/em\u003e values \u0026le; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eDepth, duration and latency of torpor varies through the mouse estrus cycle\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the effect of the estrous cycle on torpor, induced by a 24-hour period of fasting, we recorded surface temperature in female mice (n = 8) across the diestrus, proestrus and estrus phases (Figure 1A, B). Mice in diestrus exhibited deeper torpor bouts sustained for longer, compared to the same mice in proestrus and estrus (Figure 1A, B). We found the duration of time spent in torpor was significantly influenced by estrous cycle stage (Figure 1C, RM one-way ANOVA, F\u003csub\u003e(2, 23) \u0026nbsp;\u003c/sub\u003e= 12.75, p = 0.0021) where mice spend longer in torpor during diestrus (389.3 \u0026plusmn; 143.3 min) than in proestrus (282.1 \u0026plusmn; 113.4 min, p \u0026lt; 0.01) or estrus (172.8 \u0026plusmn; 103.2 min, p \u0026lt; 0.05). Similarly, estrous cycle stage also significantly influenced the magnitude of temperature fall from baseline during bouts of torpor (Figure 1D, RM one-way ANOVA, F\u003csub\u003e(2, 23) \u0026nbsp;\u003c/sub\u003e= 2.362, p = 0.043) where there was a greater reduction temperature in diestrus (-6.59 \u0026plusmn; 1.08 \u0026deg;C) vs. estrus (-5.6 \u0026plusmn; 1.57 \u0026deg;C, p \u0026lt; 0.05, Tukey\u0026rsquo;s post hoc). The integral of surface temperature over time was calculated, generating a \u0026lsquo;torpor score\u0026rsquo; for each recording session per animal. This torpor score was significantly affected by estrous cycle stage in female mice (Figure 1D, RM one-way ANOVA, F\u003csub\u003e(2, 23) \u0026nbsp;\u003c/sub\u003e= 10.54\u003csub\u003e,\u003c/sub\u003e p = 0.0016) where it was greatest during diestrus (27,737 \u0026plusmn; 13,834 \u0026deg;C.s) compared with proestrus (15,665 \u0026plusmn; 10,014 \u0026deg;C.s, p \u0026lt; 0.05) and estrus (10,042 \u0026plusmn; 6,166 \u0026deg;C.s, p \u0026lt; 0.01, Tukey\u0026rsquo;s post hoc). The stage of the cycle also impacted the time to the onset of the first torpor bout following the removal of food (Figure 1E, RM one-way ANOVA) where onset of torpor was significantly earlier in diestrus (12.32 \u0026plusmn; 1.62 h after food removal) compared with estrus (14.67 \u0026plusmn; 1.51 h) (p \u0026lt; 0.01, Tukey\u0026rsquo;s post hoc).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFemale mice display longer and deeper bouts of torpor than male mice\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe assessed sex differences in torpor by comparing female mice in diestrus to male mice over the course of a 24 hour fast. Interestingly, just five out of eight male mice entered torpor compared to all of the female mice indicating that male mice may have a lower probability of entering torpor when challenged with a single fasting period (Figure 2A). Where torpor did occur, female mice sustained torpor for a longer duration than males (Figure 2B, 389.3 \u0026plusmn; 143.3 vs. 186.1 \u0026plusmn; 98.2 min; t = 2.77, p = 0.018, Unpaired t-test), and reached cooler temperatures from baseline (Figure 2C, -6.59 \u0026plusmn; 1.08 \u0026deg;C vs. -4.8 \u0026plusmn; 0.78 \u0026deg;C; t = 3.10, p = 0.01, Unpaired t-test). This was also reflected in markedly greater torpor scores in female mice than male mice (Figure 2D) (27,737 \u0026plusmn; 13,834 \u0026deg;C.s vs 7,785 \u0026plusmn; 4,667 \u0026deg;C.s, U = 1.00, p = 0.0031, Mann-Whitney U test). At the time of experiment, the age matched male mice had greater body mass than females (23.2 \u0026plusmn; 1.28 g vs. 20.5 \u0026plusmn; 0.77 g, t = 2.61, p \u0026lt; 0.05, Unpaired t-test).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExogenous estradiol treatment lengthens torpor bouts in female, but not male mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGiven that female mice displayed a greater propensity to enter and stay in torpor than male mice, and that this varied across the estrous cycle, we tested the effect of exogenous estradiol on fasting-induced torpor in male mice and in female mice in estrus phase (where estradiol levels are lowest \u003csup\u003e28\u003c/sup\u003e). Estradiol administration the morning prior to fasting extended the time that female mice spent in torpor (compared to a chen oil vehicle Figure 2E; p = 0.018, F\u003csub\u003e(3, 14)\u0026nbsp;\u003c/sub\u003e=\u003csub\u003e\u0026nbsp;\u003c/sub\u003e3.21, Two-way ANOVA). \u0026nbsp; Torpor scores for estradiol-treated female mice were similarly greater (Figure 2G; p = 0.0042, F\u003csub\u003e(3, 14)\u0026nbsp;\u003c/sub\u003e=\u003csub\u003e\u0026nbsp;\u003c/sub\u003e4.45, Two-way ANOVA). Estradiol treatment appeared to also increase the depth of torpor in some female mice, but this was not uniform across mice, and there was no significant group level effect\u003cem\u003e\u0026nbsp;\u003c/em\u003ecompared to vehicle (Figure 2F).\u003cem\u003e\u0026nbsp;\u0026nbsp;\u003c/em\u003eIn contrast, estradiol treatment of male mice had no effect on depth or duration of torpor (Figure 2E-G).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKnockdown of ER\u0026alpha; in the POA blunts torpor response following a fast\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGiven that activation of estradiol-sensitive neurons in the POA induces a torpor-like state \u003csup\u003e7\u003c/sup\u003e, we tested the hypothesis that estradiol-signalling in the POA causes female mice to display a robust torpor response. Lentivirus containing short-hairpin RNA to knock down ER\u0026alpha; (sh-ER\u0026alpha;) was delivered into the POA of female mice and compared with control female mice where only GFP was delivered by AAV. The number of ER\u0026alpha;+ cells in the POA of sh-ER\u0026alpha; mice was reduced compared to GFP control mice (Figure 3A, B; 237.1 \u0026plusmn; 42.0 cells vs 287.6 \u0026plusmn; 34.2 cells, p = 0.023, t = 2.57, Unpaired t-test), and similarly the mean fluorescence intensity of positive cells was reduced in sh-ER\u0026alpha; mice compared to controls (84.14 \u0026plusmn; 5.14 AU vs. 69.13 \u0026plusmn; 14.38 AU; p = 0.016, t = 2.77). Surface temperature thermography profiles during fasting in ER\u0026alpha; knockdown mice were distinct from control mice; torpor bouts were shorter (Figure 3D). Overall, knockdown mice had shorter torpor bouts (Figure 3E; p = 0.035, t = 2.35, Unpaired t-test), as well as shallower bouts (Figure 3F; -7.16 \u0026plusmn; 0.85 \u0026deg;C vs. -8.19 \u0026plusmn; 0.72 \u0026deg;C; p = 0.024, t = 2.55, unpaired t-test). Torpor scores were also considerably lower than control mice (Figure 3G; 9,771 \u0026plusmn; 8,660 vs 26,738 \u0026plusmn; 16,454 \u0026deg;C.s, p = 0.029, t = 2.44, unpaired t-test). In contrast, ER\u0026alpha; knockdown did not change the time to onset of torpor following fasting (Figure 3H). \u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aim of this study was to examine whether the cyclical variation in circulating estradiol in female mice modulates entry into, and maintenance of, torpor. The data presented here support prior incidental descriptions that female mice enter torpor more readily than male mice \u003csup\u003e17\u003c/sup\u003e. We observed that female mice exhibit deeper and longer bouts of torpor during the phase of their estrous cycles where estradiol is highest, and they exhibited longer and deeper bouts than male mice. We confirmed that estradiol signalling mediates this association: exogenous estradiol lengthened torpor bouts in female mice. Finally, we show that estradiol exerts this effect via the POA: knocking down ER\u0026alpha; in the POA reduced torpor length and depth in female mice. Together, these results build on the growing body of work implicating the POA as the key site for torpor induction, and indicate that in female mice, activity of POA torpor-inducing neurons is enhanced by the action of estradiol on ER\u0026alpha;.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNeurons in the POA that induce torpor have a distinct transcriptomic signature, including expression of \u003cem\u003eQrfp, Ptger3, Lepr, Opn5\u003c/em\u003e, and \u003cem\u003eTacr3\u003c/em\u003e, which has led to them being termed the QPLOT neurons \u003csup\u003e34\u003c/sup\u003e. QPLOT neurons co-express a host of receptors including ER\u0026alpha;, positioning them to integrate hormonal, metabolic, thermal, and immune inputs that may influence whether an animal enters torpor \u003csup\u003e34\u003c/sup\u003e.\u0026nbsp;ER\u0026alpha; may influence activity and excitability of neurons through both genomic and second messenger mediated pathways. To act via the latter, ER\u0026alpha; may be trafficked to the plasma membrane via post-translational palmitoylation and subsequent association with the scaffold protein calveolin-1 \u003csup\u003e35,36\u003c/sup\u003e, where it forms functional dimers that associate with G-protein complexes \u003csup\u003e37\u003c/sup\u003e. Membrane associated ERs facilitate estradiol\u0026rsquo;s rapid modulation of cellular processes by activating secondary pathways including metabotropic glutamate receptors \u003csup\u003e38\u003c/sup\u003e. Functionally, this has mostly been studied in the context of cognition, memory and motivation, however, there is evidence that estradiol acting via metabotropic glutamate receptors in the medial POA decreases food intake \u003csup\u003e39\u003c/sup\u003e. ERs also function as intracellular receptors capable of modulating neural activity and excitability by stimulating calcium release from the endoplasmic reticulum, or by modulating ion channel activity \u003csup\u003e40\u003c/sup\u003e. \u0026nbsp;Whether this leads to up or down-regulation of excitability is context dependent. For example, again with respect to energy balance in the hypothalamus, estradiol disinhibits pro-opiomelanocortin neurons by attenuating GABA\u003csub\u003eB\u003c/sub\u003e-mediated activation of inwardly-rectifying potassium channels, while the opposite is true of agouti-related peptide neurons where GABAergic inhibition is enhanced \u003csup\u003e41\u003c/sup\u003e. Hence, estradiol is capable of acting in the hypothalamus to modulate neural activity and change behaviour via multiple cellular pathways.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur results support the hypothesis that, in the case of POA torpor-inducing neurons, activation of\u0026nbsp;ER\u0026alpha; is\u0026nbsp;excitatory in nature. Studies of neurons in the POA consistently report that their activation drives torpor output \u003csup\u003e6-8\u003c/sup\u003e, and POA neural activity is elevated during torpor when examined by cFos expression or \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003erecording of calcium events by fibre photometry \u003csup\u003e6,8,9\u003c/sup\u003e. It follows then that estradiol increasing excitability of QPLOT neurons in the POA would promote torpor. Our results indicate that estradiol plays a role in maintenance of torpor, whereby torpor duration was longer in in estrous phases where estradiol would have been highest, or when estradiol signalling was intact. We also present evidence that estradiol reduces the latency between fasting onset and entry into torpor and drives larger drops in temperature during torpor compared to euthermic baselines. Torpor is an active process associated with a higher frequency of calcium events in the POA \u003csup\u003e7,8\u003c/sup\u003e, and nadir temperature may be reduced during optogenetic stimulation of POA neurons by increasing either the power or frequency of light stimulus \u003csup\u003e10\u003c/sup\u003e, hence lower nadir temperatures are indicative of higher neural activity. Our knockdown findings indicate that ER\u0026alpha; signalling in the POA is necessary for estradiol to enhance activity of torpor neurons directly. Elucidation of this mechanism is a goal for future investigations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOne question raised by our findings across the sexes is why does estradiol enhance torpor in female mice, but not males? We demonstrate that female mice enter a longer and deeper state of torpor than male mice in a similar age range, which could be explained by females simply having higher levels of circulating estradiol than male mice \u003csup\u003e42,43\u003c/sup\u003e. However, we also demonstrate that estradiol treatment enhances torpor in female mice but not males. The POA displays sexual dimorphism: several nuclei differ in size, neurotransmitter expression, and cell number \u003csup\u003e44\u003c/sup\u003e. Furthermore, ER\u0026alpha; expression is greater in the POA of female mice compared to males \u003csup\u003e7\u003c/sup\u003e. Female mice may have adapted to become more energetically thrifty, making \u0026lsquo;savings\u0026rsquo; in diestrus when they are not fertile. This might allow for greater energy expenditure with lower probability of torpor entry and shorter and shallower torpor bouts typically seen during proestrus and estrus, which is when mating occurs. Hence, female mice preferentially allocate energy expenditure to periods when mating occurs and prioritise energy saving when mating does not occur. An interesting consideration is whether stimuli such as the presence of male scent-marked bedding across the estrous cycle has any effect on torpor, which may allow us to tease apart the hierarchy of priorities between mating and energy savings. Alternatively (or additionally), the low metabolic activity and body temperature of torpor might be hazardous during early pregnancy, in which case torpor might be inhibited during periods of the cycle in which fertilisation and implantation occur. In this interpretation, torpor is not so much promoted during diestrus as inhibited at other times.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEnergy expenditure and food intake are additional factors that fluctuate over the course of the estrous cycle. Energy expenditure is lower in diestrus compared to estrus in both rats and mice and diestrus is associated with lower heat production over 24 hours in mice \u003csup\u003e45,46\u003c/sup\u003e. Food intake, similarly, may vary over the course of the estrous cycle by 20% and is highest during estrus\u0026nbsp;\u003csup\u003e47\u003c/sup\u003e. |It is possible that mice experience a period of relative energy deficit during diestrus compared to other phases of the cycle. This might make torpor more advantageous at this timepoint. Hence, the POA torpor-inducing neurons might have evolved estrogen-sensitivity to align torpor with periods of the estrus cycle in which relative energy deficit occurs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLimitations\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAn obvious consideration regarding the difference in torpor between males and females is that of body mass. Recently published work from our group has determined that body mass affects the probability of torpor onset \u003csup\u003e48\u003c/sup\u003e. Body mass influences the \u0026lsquo;lower critical limit\u0026rsquo; of ambient thermoneutral temperature, below which mammals must increase their metabolic rate to maintain core body temperature. In mice, as body mass decreases, the temperature of the lower critical limit will increase thus narrowing the thermoneutral range \u003csup\u003e49\u003c/sup\u003e. Mice in the present study were housed at 21\u0026deg;C, and female mice had an ~11% lower body mass than male mice. While this may partially account for some of the differences in torpor response between the age-matched males and females, we still demonstrate a striking sex difference in that female mice respond to estradiol treatment while males do not.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother consideration is that our focus on estradiol does not consider other sex hormones that might have additional roles in regulating torpor. Progesterone levels are lower throughout most of the mouse estrous cycle aside from a brief peak in late proestrus \u003csup\u003e28\u003c/sup\u003e, likely generated by the corpus luteum following ovulation \u003csup\u003e50\u003c/sup\u003e. While progesterone released during the luteal phase, and taken exogenously from oral contraceptives, drives an increase in body temperature in humans (~0.5˚C) \u003csup\u003e51\u003c/sup\u003e, its role in mice and other animal models is not as clearly defined. Some evidence for thermoregulatory effects in rodents comes from one study in rabbits which reported that progesterone drove an increase in body temperature, with a concurrent decrease in firing rate from warm-sensitive POA neurons \u003csup\u003e52\u003c/sup\u003e. Given that progesterone is released in a relatively confined window of early estrus in mice before being rapidly metabolised \u003csup\u003e50\u003c/sup\u003e, it seems unlikely that it would regulate torpor to the same degree as estradiol. \u0026nbsp;However, it does stand as a reasonable consideration for studies examining the role of sex hormones in torpor going forward.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThese findings highlight estradiol as a modulator of torpor through its actions on the POA, deepening and lengthening torpor. Further work elucidating how estradiol modulates neurons in this area will be important in advancing our understanding of how these neurons integrate physiological information to modulate torpor. This work highlights the role of estrogen in modulating the fasting response in female mice via neurons in the POA. It demonstrates that torpor propensity changes throughout the estrus cycle in females, suggesting an interaction between the demands of energy balance and reproduction in mice.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Medical Research Council grant number MR/W029138/1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Medical Research Council grant number MR/W029138/1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCJM: Conceptualization, methodology, investigation, formal analysis, writing - original draft, writing - review \u0026amp; editing, visualization. AEP: Conceptualization, methodology, writing - review \u0026amp; editing, supervision, funding acquisition. MTA: Conceptualization, methodology, writing - review \u0026amp; editing, supervision, funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed in the current study will be made available upon reasonable request to the corresponding author. \u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbreu-Vieira, G., Xiao, C., Gavrilova, O. \u0026amp; Reitman, M. L. 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Action of progesterone on preoptic thermosensitive neurones. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e258\u003c/strong\u003e, 80-80 (1975). https://doi.org/10.1038/258080a0\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7356338/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7356338/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTorpor is a state of transient hypometabolism and hypothermia that is engaged by many species in adverse conditions such as food scarcity. Neurons in the hypothalamic preoptic area (POA) are capable of driving entry into torpor when activated. Estrogens, principally estradiol, are capable of modulating thermogenesis and energy balance by central actions, and POA neurons express the canonical estrogen receptor ERα. We find that torpor depth and duration vary across the estrus cycle in mice, whereby the torpor response is greatest during the diestrus phase in which circulating estradiol is at its peak. Torpor responses are longer and deeper in female mice compared to males, and this is potentiated by exogenous estradiol, which lengthens torpor bouts in female mice, but not males. Knockdown of ERα within the POA blunts torpor in female mice, suggesting that estradiol acting via ERα modulates the activity of hypothalamic neurons that generate torpor. We speculate that this cyclical oscillation in torpor propensity which is lowest in the estrous phase may be an adaptive change that preserves reproduction in periods of moderate environmental stress.\u003c/p\u003e","manuscriptTitle":"Sex and estrous cycle influence fasting-induced torpor via hypothalamic estrogen signalling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-18 09:48:40","doi":"10.21203/rs.3.rs-7356338/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-19T14:10:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T09:01:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-30T13:39:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338957652084531031366345642542237468861","date":"2025-08-21T07:22:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247990288315841348527931557244470705432","date":"2025-08-20T20:57:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-20T16:06:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-20T15:51:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-18T10:53:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-14T14:20:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-14T14:16:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c8061b5d-c794-4a22-a76b-1e957cbedaae","owner":[],"postedDate":"August 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53191944,"name":"Biological sciences/Neuroscience"},{"id":53191945,"name":"Biological sciences/Physiology"},{"id":53191946,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-03-02T16:00:13+00:00","versionOfRecord":{"articleIdentity":"rs-7356338","link":"https://doi.org/10.1038/s41598-026-41051-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-27 15:57:15","publishedOnDateReadable":"February 27th, 2026"},"versionCreatedAt":"2025-08-18 09:48:40","video":"","vorDoi":"10.1038/s41598-026-41051-y","vorDoiUrl":"https://doi.org/10.1038/s41598-026-41051-y","workflowStages":[]},"version":"v1","identity":"rs-7356338","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7356338","identity":"rs-7356338","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}