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Banks et al 1 2 3 Running title: Light and thyroid regulation of reproduction and immune function. 4 5 6 Title: Photoperiod- and triiodothyronine-dependent regulation of reproductive neuropeptides, pro- 7 inflammatory cytokines and peripheral physiology in Siberian hamsters (Phodopus sungorus). 8 9 Authors: Ruth Banks1, 2, Mirela Delibegovic2, Tyler J Stevenson1 10 11 Affiliation: 1Institute for Biological and Environmental Sciences, 2Institute for Medical Sciences, 12 University of Aberdeen, Aberdeen, United Kingdom 13 14 15 16 17 18 19 20 21 22 Contact: 23 Disclosure statement: The authors have nothing to declare. Tyler Stevenson Institute for Biological and Environmental Sciences University of Aberdeen Aberdeen, United Kingdom [email protected] 24 1 Banks et al 25 Abstract 26 Seasonal trade-offs in reproduction and immunity are ubiquitous in nature. The mechanisms that 27 govern transitions across seasonal physiological states appear to involve reciprocal switches in the 28 local synthesis of thyroid hormone. In long day (LD) summer-like conditions, increased 29 hypothalamic triiodothyronine (T3) stimulates gonadal development. Alternatively, short day (SD) 30 winter-like conditions, increase peripheral leukocytes and enhance multiple aspects of immune 31 function. These data indicate that the localized effects of T3 in the hypothalamus and leukocytes are 32 photoperiod-dependent. We tested the hypothesis that increased peripheral T3 in SD conditions 33 would increase aspects of reproductive physiology and inhibit immune function, whereas T3 34 injections in LD conditions would facilitate aspects of immune function (i.e. leukocytes). 35 Additionally, we also examined whether T3 regulates hypothalamic neuropeptide expression as well 36 as hypothalamic and splenic pro-inflammatory cytokine expression. Adult male Siberian hamsters 37 were maintained in LD (15L: 9D) or transferred to SD (9L:15D) for 8 weeks. A subset of LD and 38 SD hamsters were treated daily with 5 µg T3 for 2 weeks. LD and SD controls were injected with 39 saline. Daily T3 administration in SD hamsters (SD+T3) resulted in a rapid and substantial decrease 40 in peripheral leukocyte concentrations and stimulated gonadal development. T3 treatment in LD 41 (LD+T3) had no effect on testicular volumes, but significantly increased leukocyte concentrations. 42 Molecular analyses revealed that T3 stimulated interleukin 1β mRNA expression in the spleen and 43 inhibited RFamide Related Peptide-3 mRNA expression in the hypothalamus. Moreover, there was 44 a photoperiod-dependent decrease in splenic tumour necrosis factor-α mRNA expression. These 45 findings reveal that T3 has tissue-specific and photoperiod-dependent regulation of seasonal 46 rhythms in reproduction and immune function. 47 48 Key words: Circannual, thyroid, immune, rodent, photoperiod 49 2 Banks et al 50 Introduction 51 Seasonal rhythms in reproduction and immunity are common in temperate species (Nelson, 52 2004; Stevenson & Prendergast, 2015). Seasonal temporal strategies are important as they 53 coordinate energetic activities across the year; winter is generally associated with reduced energy 54 availability but requires the highest energy demands (Nelson et al., 1998; Sheldon & Verhulst, 55 1996). Seasonal rhythms in hormones involved in energy balance, such as leptin, have been 56 implicated as mediators of yearly trade-offs according to energetic demands (Drazen et al., 2001; 57 Demas, 2004). Thyroid hormones are also key regulators of energy balance and form an integral 58 part of the neuroendocrine regulation of seasonal rhythms (Ebling, 2014). Thyroid hormones are 59 similarly potent immunomodulators (Dorshkind and Horseman, 2000). Thus, thyroid hormones are 60 poised to mediate seasonal trade-offs in energetic demands involved in timing reproduction and 61 immune function. 62 Several neuropeptides located in the hypothalamus are involved in regulating the 63 neuroendocrine timing of seasonal reproduction, such as gonadotropin-releasing hormone (gnrh; 64 Stevenson et al., 2012), kisspeptin (kiss; Greives et al., 2007) and RFamide-related peptide 3 (rfrp3; 65 Tsutsui et al., 2013). The annual change in day length is a predictive proximate cue that drives 66 seasonal variation in the timing of reproduction (Stevenson & Ball, 2011). Increased summer day 67 length (LD) triggers the local synthesis of triiodothyronine (T3) in the mediobasal hypothalamus 68 leading to gonadotropin secretion and subsequent gonadal development (Yoshimura, 2015; Rani & 69 Kumar, 2014). Moreover, increased hypothalamic T3 is proposed to regulate the expression of 70 several neuropeptides involved in the control of seasonal reproduction; such as RFRP3 and KiSS1 71 (Henson et al., 2013). Hypothalamic RFRP3 expression in both Siberian and Syrian hamsters is 72 greater in LD compared to short day conditions (SD; Ubuka et al., 2012; Mason et al., 2010, 73 respectively). T3 implants or exogenous T3 injection in SD hamsters has been shown to induce 74 growth of the gonads (Freeman et al., 2007; Barrett et al., 2007; Klosen et al., 2013). Daily T3 75 administration also acts in the hypothalamus to trigger increased anteroventral periventricular 3 Banks et al 76 nucleus KiSS1, decreased arcuate nucleus KiSS1 and increased RFRP3 expression (Henson et al., 77 2013). It is clear that thyroid hormones are an integral component of the neuroendocrine circuitry 78 that regulates seasonal reproduction; however, the local role of triiodothyronine in the timing of 79 annual rhythms in immune function is in its infancy. 80 Unlike seasonal rhythms in reproduction, immune function does not exhibit an overall 81 potentiation or involution during any phase of the yearly cycle (Nelson, 2004; Sheldon & Verhulst, 82 1996). Instead, the immune system displays a collection of trait-specific enhancements at specific 83 times across the year. Short winter-like photoperiods (SD) lead to enhanced immunosurveillance 84 capacity (e.g. leukocyte proliferation; Bilbo et al., 2002; Stevenson et al., 2014). However, in some 85 rodent species, humoral immune function is suppressed following adaptation to SD conditions 86 (Drazen et al., 2000; Drazen et al., 2001). Thyroid hormones have been implicated in the regulation 87 of multiple aspects of immune function, including T3 enhanced T-cell dependent (Type IV) 88 delayed-type hypersensitivity reactions (DTH) skin responses (Chandel & Chatterjee, 1989), 89 mitogen-induced splenocyte proliferation and blood leukocyte concentrations (Chatterjee & 90 Chandel, 1983). In Siberian hamsters, SD leukocytes have increased intracellular T3 concentrations; 91 the local synthesis is likely involved in the timing of SD enhanced immune function, such as 92 memory T-cell dependent adaptive immune responses (DTH; Stevenson et al., 2014). Furthermore, 93 exogenous T3 in LD conditions induced an increase in peripheral leukocyte concentrations to values 94 similar to SD hamsters (Stevenson et al., 2014). These data indicate that in addition to regulating 95 the neuroendocrine control of seasonal reproduction, T3 has a powerful effect on enhancing trait- 96 specific aspects of immunity. 97 These patterns indicate a dual role of thyroid hormones for the seasonal control of 98 reproduction and immune function: LD hypothalamic T3 stimulates neuroendocrine mechanisms 99 that trigger gonadal development and SD enhances leukocyte-T3 mediated components of immune 100 function. The goal of this paper was to examine the sufficiency of T 3 to drive photoperiodic 101 dependent switches in immune and reproductive function in LD and SD conditions, respectively. To 4 Banks et al 102 examine this goal, we sought to establish whether daily peripheral administration of T3 for 2 weeks 103 in LD and SD conditions would induce inverse effects on reproduction and immunosurveillance. 104 We hypothesised that T3 would facilitate measures of immune function in LD and stimulate 105 reproductive development in SD animals. Siberian hamsters (Phodopus sungorus) are a common 106 laboratory animal used to investigate seasonal biology. A simple switch in day length from LD (15 107 hrs light) to SD (9 hrs light) triggers a range of genetic and physiological changes leading to 108 gonadal involution and immune plasticity (Heldmaier et al., 1981; Bartness & Wade, 1985; Bilbo et 109 al., 2002; Prendergast et al., 2013; Stevenson et al., 2014). In order to assess the sufficiency of 110 triiodothyronine on regulating neuroendocrine control of seasonal reproduction and immune 111 function we examined photoperiodic and daily injections of T3 on hypothalamic expression of two 112 neuropeptides, gnrh and rfrp3, and downstream effects on body mass and testicular volume. We 113 also surveyed the number of circulating leukocytes and proinflammantory cytokine expression in 114 the hypothalamus and spleen, well-established measures of immune function (Dhabhar et al., 1995, 115 Yellon et al., 1999, Bilbo et al., 2002). Finally, we investigated whether daily T3 injections, would 116 in part, regulate the expression of two pro-inflammatory cytokines: interleukin-1β (il1β) and tumor- 117 necrosis factor-α (tnfα). 118 119 Materials and methods 120 Subjects 121 Adult male hamsters (N=31; 3-6 month old) were randomly selected from a colony maintained at 122 the University of Aberdeen. Hamsters were housed in polypropylene cages illuminated for 15h/day 123 (15L: 9D). Food and water were provided ad libitum; cotton-nesting material was available in the 124 cage. All procedures were approved by the Animal Welfare and Ethics Review Board at the 125 University of Aberdeen and conducted under the Home Office licence (70/7917). 126 127 Study design 5 Banks et al 128 Hamsters were group housed in LD (n=15) or transferred to SD (9L: 15D; n=16) cabinets for 8 129 weeks (Arrowmight, Hereford UK). LD and SD hamsters were then divided into two treatment 130 groups that received daily saline controls (LD+S and SD+S) or T3 injections (LD+ T3 and SD+ T3). 131 The final treatment group sample sizes were LD+S (n=9), LD+ T3 (n=6), SD+S (n=8) and SD+ T3 132 (n=8). Body mass and estimated testes volume (ETV see below) were measured to confirm SD 133 induced weight loss and gonadal involution (PRE). Body mass was determined using aeADAM 134 scales (Adam Equipment PGL2002) and measured to ±0.1g. In order to evaluate the effect of 135 exogenous T3 on peripheral physiology as well as hypothalamic and splenic mRNA expression, 136 hamsters were given daily subcutaneous (s.c.) injections of 5 µg T3/ 100g (T2877, Sigma-Aldrich) 137 for 2 weeks. This dose and injection regimen was selected based on previous work that revealed a 138 significant increase in circulating leukocytes in LD male hamsters (Stevenson et al., 2014). 139 Peripheral T3 readily crosses the blood brain barrier, mediated in part by transporters such as 140 Monocarboxylate transporter 8 (Heuer, 2007). Hypothalamic MCT8 exhibits photoperiodic 141 variation and is proposed to facilitate the local concentration of T3 in brain regions implicated in the 142 neuroendocrine control of seasonal rhythms (Herwig et al., 2013). SD and LD control hamsters 143 received s.c. saline (0.1 ml) for the same 2 week period. Body weight measurements and ETVs 144 were collected after 2 weeks of daily injections (POST). 145 146 Estimated Testes Volume (ETV) 147 ETVs were used to determine the response to photoperiod and T3, in brief; ETV is calculated by 148 testis width squared multiplied by length. Hamsters were lightly anaesthetized with 149 isofluorane:oxygen vapours (4% induction; 1% maintenance) and testes were measured using 150 analogue callipers. This approach to assess reproductive state is widely used as it is strongly 151 correlated with actual testicular volume (Gorman and Zucker, 1995). Testicular volumes of 152 hamsters indicated reproductive competence (LD) or gonadal involution (SD). 153 6 Banks et al 154 Leukocyte analyses 155 At the termination of the study, hamsters were anesthetized with isoflurane gas (4%) and 500 µl 156 whole blood was collected via the right retroorbital sinus using Natelson tubes coated with sodium 157 heparin. A 30 µl aliquot of blood was mixed with 3% acetic acid at a 1:20 dilution. Leukocyte were 158 counted under light microscope (Zeiss Axioskop) and values were obtained according to methods 159 reported previously (Stevenson et al., 2014, Prendergast et al., 2003). 160 161 Quantification of hypothalamic and splenic RNA expression 162 Hypothalamic and spleen tissue was rapidly dissected as previously described (Stevenson & 163 Prendergast, 2013) and frozen in powder dry ice. RNA was extracted from tissues using Trizol 164 (ThermoFisher Scientific). Nucleic acid concentration and quality were determined by 165 spectrophotometer (Nanodrop, Thermo Scientific). cDNA was synthesized using Superscript III 166 (Invitrogen) and cDNA was stored at -20ºC until quantitative PCR was performed. All cDNA tissue 167 samples were run in triplicate. qPCRs for mRNA expression in hypothalamic tissue were performed 168 using BIORAD CFX96, splenic mRNA were assayed using Roche LightCycler 480. qPCR 169 parameters were as follows: i) an initial denature at 95°C for 30 secs, then 39 cycles of ii) 95°C for 170 10 sec, iii) annealing dependent on target mRNA (See Table 1) for 30 secs and then iv) an extension 171 at 72°C for 30 sec. A melting curve analysis was added to determine the quality and specificity of 172 each reaction. Quantification of mRNA expression levels was accomplished with iQ Sybr Green 173 Supermix (BIORAD, UK). We used PCR Miner (Zhao & Fernald 2005) to calculate reaction 174 efficiencies (E) and cycle thresholds (CTs). According to the MIQE guidelines, samples that had 175 efficiency values below 0.8 or above 1.2 were excluded from analyses (Bustin et al., 2009). The 176 expression levels of hypothalamic transcript were determined using glyceraldehyde 3-phosphate 177 dehydrogenase (gapdh) as the reference. For splenic transcripts, we used the average of 18s and 178 hrpt cycling time. Fold change in expression for all target transcripts was calculated using 2-(delta- 179 deltaCt). 7 Banks et al 180 181 Statistical analyses 182 Repeated Two-way ANOVA was conducted to examine the effects of T3 on body mass and ETVs. 183 Leukocyte counts, hypothalamic and splenic mRNA expression were analysed using Two-way 184 ANOVAs. Post-hoc analyses were performed using Fisher’s LSD method. Log-transformation was 185 performed on mRNA values when violations of normality were observed. Statistical analyses were 186 performed using SigmaStat 12.0 and difference considered significant when P<0.05. 187 188 Results 189 Photoperiod- and T3- regulation of peripheral physiology. 190 Repeated Two-way ANOVA revealed a significant interaction of photoperiod and T3 191 treatment on hamster body mass (F=20.72; P<0.001; Fig1A). Post-hoc analyses confirmed that 192 saline injected controls did not exhibit a significant change in body mass after 2 weeks of daily 193 injections (P>0.08 for LD+S and SD+S). Daily T3 injections were found to significantly reduce 194 body weight in LD+T3 (P<0.001) and SD+T3 (P<0.001). 195 Daily T3 injections lead to a significant increase in ETV only in SD housed hamsters 196 (F=5.34; P<0.005; Fig1B). Post-hoc analyses indicated that both LD and SD hamsters injected with 197 daily saline maintained constant ETV levels (P>0.85). Similarly, daily T3 injections did not 198 significantly augment ETV in LD hamsters (P<0.05). In SD, T3 injections resulted in a significant 199 increase in ETV (P<0.001). These data suggest that T3 has photoperiod-dependent effects on 200 testicular volumes; there was a stimulatory effect only after gonadal involution had occurred. 201 Two-way ANOVA also revealed a significant interaction on leukocyte concentrations 202 (F=54.61; P<0.001; Fig1C). Consistent with previous publications, post-hoc analyses confirmed 203 that SD hamsters had significantly greater peripheral leukocyte numbers compared to LD controls 204 (P<0.001). Daily T3 injections had significant photoperiod-dependent effects; LD hamsters had 205 significantly greater leukocyte counts (P<0.001), whereas under SD, there was a reduction 8 Banks et al 206 (P<0.001). These robust findings indicate that exogenous T3 can have opposing effects on leukocyte 207 numbers that is, in part, regulated by photoperiodic history. 208 Hypothalamic mRNA expression is regulated by photoperiodic condition. 209 Next, we examined the impact of photoperiodic condition and exogenous T3 on the 210 expression of reproductive neuropeptides gnrh and rfrp3 as well as pro-inflammatory cytokines il1β 211 and tnfα expression. We did not detect significant variation in gnrh expression across photoperiodic 212 (F=0.36; P=0.55; Fig2A), T3 treatment (F=0.22; P=0.64) or interaction (F=0.61; P=0.44). We 213 observed a significant photoperiodic effect on rfrp3 expression with reduced levels in SD hamsters 214 (F=54.87; P<0.001; Fig2B). Furthermore, daily T3 injections also reduced rfrp3 expression (F=5.20; 215 P<0.05). There was no significant interaction for rfrp3 expression (F=0.33; P=0.57). 216 Analyses of hypothalamic il1β and tnfα indicate that these cytokines are constitutively 217 expressed across photoperiodic and are not regulated by T3. There was no significant photoperiod 218 (F=2.05; P=0.16; Fig2C), T3 dependent (F=0.01; P=0.98) or interaction (F=1.64; P=0.21) for il1β 219 expression. Similarly, tnfα expression was not driven by photoperiod (F=0.08; P=0.78; Fig2D), 220 daily T3 (F=0.34; P=0.56) or driven by an interaction of the two factors (F=2.81; P=0.10). Overall, 221 these data suggest that T3 does not appear to regulate pro-inflammatory cytokine expression in the 222 hamster hypothalamus. 223 T3 regulation of proinflammatory cytokines in the hamster spleen 224 We found that il1β expression was significantly upregulated in the hamster spleen after two 225 weeks of daily T3 injection (F=28.12; P<0.001; Fig3A). However, there was no significant 226 photoperiod effect (F=2.34; P=0.14) or a significant interaction (F=0.18; P=0.67). 227 Two-way ANOVA revealed a significant main effect of daily T3 on spleen tnfα expression 228 (F=5.63; P<0.05). There was no significant main effect of photoperiod (F=0.58; P=0.45) or 229 significant interaction (F=1.85; P=0.19). Post-hoc analyses indicated that T3 induced reductions in 230 tnfα expression was primarily due to lower levels in SD+T3 hamster compared to SD+S (P=0.01). 231 There was no significant difference between LD+S and LD+T3 (P=0.55). 9 Banks et al 232 233 234 Discussion In this study, we show that exposure to SD reduced body weight, testicular volume and 235 enhanced circulating leukocyte concentrations in adult male Siberian hamsters. 236 corroborates that hamsters maintained in LD photoperiods have greater hypothalamic RFRP3 237 expression compared to SD-photo-regressed animals (Ubuka et al., 2012; Mason et al., 2010). We 238 identified that T3 injections led to a significant inhibition in peripheral leukocyte concentrations in 239 SD hamster (i.e. SD+T3), a modest increase in ETV and reduced body mass. Furthermore, daily T3 240 significantly reduced spleen tnfα expression only in SD hamsters. We reveal that T3 categorically 241 decreased hypothalamic rfrp3 expression and increased spleen il1β expression, regardless of 242 photoperiodic condition. These data indicate that exogenous T3 treatment exerts distinct 243 photoperiod- as well as cell- and tissue-specific control over measures of immune and reproduction 244 function. Our work 245 This study permitted the dissection of timescale effects of T3 induced changes in multiple 246 aspects of organismal physiology. Circulating concentrations of T4 and T3 do not exhibit 247 photoperiodic variation and instead maintain constitutive levels in the periphery (O’Jile and 248 Bartness, 1992). Cell-specific expression of thyroid hormone enzymes, referred to as deiodinases 249 (DIO) are critical for regulating intracellular levels of T4 and T3 (St Germain et al., 2009). Thus, 250 photoperiodic regulation of hypothalamic and leukocytes DIO expression provides the ability to 251 enhance the effects of T3 on localized genomic and physiological function (Prendergast et al., 2013; 252 Stevenson et al., 2014; Stevenson & Prendergast, 2015). T3 injections in SD hamsters induced a 253 51% inhibition in peripheral leukocytes, 40% increase in ETV and a negligible effect on body mass 254 (3% inhibition; within margin of error). Our findings also illustrate that T3 effects on peripheral 255 physiology and immune function are photoperiod-dependent. In LD hamsters, body mass was 256 significantly reduced, there was no change in testicular volume and leukocytes increased to levels 10 Banks et al 257 comparable to SD controls. These data suggest that the diverse tissues/cells of the peripheral 258 nervous system exhibit photoperiodic- and T3- dependent regulation. 259 Previous work has illustrated that both central and peripheral exogenous T3 stimulates 260 reproductive development in the SD phenotype (Freeman et al., 2007; Klosen et al., 2013; Barrett et 261 al., 2007). Here, we confirm that daily T3 in SD hamsters (SD+T3) significantly increased ETV. 262 However, we observed that T3 reduced hypothalamic rfrp3 expression; regardless of photoperiodic 263 condition. Henson and colleagues (2013) demonstrated that the numbers of RFRP3 cells determined 264 by immunocytochemistry are greater after daily T3 in SD hamsters. Since we observed a reduction 265 in rfrp3 expression in LD+T3; it is likely that T3 has an inhibitory effect on RFRP3 function in the 266 hamster hypothalamus. We propose that T3 inhibits rfrp3 expression and the greater RFRP3 cell 267 numbers observed by Henson and colleagues (2013) likely reflects an accumulation of protein 268 content due to reduced transport/release from nerve terminals. Our findings also provide evidence 269 that chronic exposure to photoperiod and T3 does not regulate gnrh expression. Indeed 270 hypothalamic GnRH content exhibits slight variation during rapid transitions from LD to SD 271 conditions (Bernard et al., 1999). It remains possible that acute changes in local T3 could augment 272 hypothalamic GnRH expression. These data indicate that T3 mediated effects on neuroendocrine- 273 gonadal axis is likely due to the reduction in rfrp3 expression leading to gonadal development in 274 SD hamsters. The absence of any effect on testicular volumes in LD is presumably the result of a 275 ‘ceiling-effect’ in which the testes are already at, or near, maximum volume. 276 In this paper, we confirm that LD+T3 hamsters have greater blood leukocyte concentrations 277 compared to LD+S (Stevenson et al., 2014) and reveal that T3 effects are photoperiod-dependent as 278 SD+T3 exhibit significantly lower concentrations. We suggest that the reduced leukocytes in SD-T3 279 are not the result of direct inhibition on immune function by thyroid hormones. Instead, peripheral 280 T3 may be involved in diverting energy demands in SD hamsters to testicular growth and 281 subsequent steroidogenesis (e.g. testosterone). The increase energy demands for reproduction 282 and/or the increased production of testosterone reduces leukocyte counts and ultimately, immune 11 Banks et al 283 function (Prendergast et al., 2003; Trigunaite et al., 2015). At a molecular level, we demonstrate 284 that T3 significantly increased splenic il1β expression. T3 has been shown to stimulate il1β 285 expression in human bone marrow stromal cells (Kim et al., 1999) suggesting that T3 can potentiate 286 il1β expression across diverse tissues. Interestingly, we did not detect a significant difference in 287 hypothalamic il1β expression indicating a degree of tissue-specific regulation. A similar pattern was 288 observed for tnfα expression as T3 significantly reduced spleen levels in a photoperiod-dependent 289 manner and showed complete absence of an effect on hypothalamic expression. In addition to the 290 spleen, T3 increased tnfα expression in Kupffer (Valencia et al., 2004) and liver cells (Tapia et al., 291 2003). Thus, T3 has a powerful effect on multiple aspects of immune function (i.e. leukocytes and 292 pro-inflammatory cytokine expression) that is driven by photoperiodic history and is tissue-specific. 293 In conclusion, our results indicate that localised signalling of T3 differentially regulates 294 seasonal timing in immune and reproduction function. T3 has direct effects on pro-inflammatory 295 cytokine expression in spleen tissue and hypothalamic rfrp3 expression. In both cases, T3 effects on 296 rfrp3 and il1β are regulated independent of photoperiod condition. However, T3 effects on 297 peripheral measures are photoperiod-dependent; enhanced leukocytes in LD and gonadal 298 development in SD. We propose that enhanced peripheral T3 concentration overrides the presiding 299 photoperiodic signal, directly stimulates reproductive development in SD and indirectly leads to the 300 inhibition in leukocyte cells. 301 302 Acknowledgements 303 The study hypotheses and design were conceived by TJS. RB and TJS conducted, collected and 304 analysed the data. RB, MD and TJS wrote the paper. This work was financed by the University of 305 Aberdeen College of Life Sciences and Medicine fund to TJS. We thank Elisabetta Tolla for her 306 technical assistance. 307 308 References 12 Banks et al 309 Barrett P Ebling FJ Schuhler S Wilson D Ross AW Warner A Jethwa P Boelen A Visser TJ Ozanne 310 DM Archer ZA Mercer JG Morgan PJ (2007) Hypothalamic thyroid hormone catabolism acts 311 as a gatekeeper for the seasonal control of body weight and reproduction. Endocrinol. 312 148:3608-3617. 313 314 Bartness TJ Wade GN (1985) Photoperiodic control of seasonal body weight cycles in hamsters. Neurosci. 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