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Endocrinology, doi:10.1210/en.2003-0838
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*Nucleotide*Protein
Endocrinology Vol. 145, No. 1 13-20
Copyright © 2004 by The Endocrine Society

Photoperiodic Regulation of Hypothalamic Retinoid Signaling: Association of Retinoid X Receptor {gamma} with Body Weight

Alexander W. Ross, Catriona A. Webster, Julian G. Mercer, Kim M. Moar, Francis J. Ebling, Sandrine Schuhler, Perry Barrett and Peter J. Morgan

Molecular Endocrinology Group (A.W.R., C.A.W., J.G.M., K.M.M., P.B., P.J.M.), Rowett Research Institute, Aberdeen, AB21 9SB, Scotland, United Kingdom; and School of Biomedical Sciences (F.J.E., S.S.), University of Nottingham Medical School, Nottingham, NG7 2UH, United Kingdom

Address all correspondence and requests for reprints to: Professor Peter J. Morgan, Institute Director, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, United Kingdom. E-mail: p.morgan{at}rowett.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study reports novel events related to photoperiodic programming of the neuroendocrine hypothalamus. To investigate photoperiod-responsive genes, Siberian hamsters were maintained in long or short photoperiods that generate physiological states of obesity or leanness. Microarray expression analysis first identified CRBP1 as a photoperiod-responsive gene, and then further studies using in situ hybridization and immunocytochemistry revealed that expression levels of several related retinoid-signaling genes were modulated in response to photoperiod changes. Genes of the retinoid-signaling pathway, encoding nuclear receptors (RXR/RAR) and retinoid binding proteins (CRBP1 and CRABP2) are photoperiodically regulated in the dorsal tuberomamillary nucleus (DTM): Their expression is significantly lower in short photoperiods and parallels body weight decreases. Studies in pinealectomized hamsters confirm that the pineal melatonin rhythm is necessary for these seasonal changes, and studies in testosterone-treated hamsters reveal that these changes in gene expression are not the secondary consequence of photoperiod-induced changes in steroid levels. Comparative studies using Syrian hamsters, which show divergent seasonal body weight responses to Siberian hamsters when exposed to short photoperiods, showed a distinct pattern of changes in retinoid gene expression in the DTM in response to a change in photoperiod. We infer that the DTM may be an important integrating center for photoperiodic control of seasonal physiology and suggest that the changes in retinoid X receptor {gamma} expression may be associated with seasonal changes in body weight and energy metabolism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AS ADAPTATIONS TO SEASONAL conditions, many mammals exhibit profound changes in body weight, reproductive status, and pelage, which are triggered by a change in photoperiod (1, 2, 3). Melatonin is the key hormonal intermediate in the effects of photoperiod, and receptors for melatonin have been localized within the brain and pituitary (4). Although photoperiod and melatonin have been shown to affect the peak amplitude, duration or phase of expression of clock genes such as Per1/2 and Cry1/2 at melatonin target sites, such as the pars tuberalis of the pituitary (5, 6), little is known of how these effects relate to downstream physiological functions, such as energy balance or reproduction.

In seasonal mammals, two major neuroendocrine pathways, the control of reproduction and energy balance, are thought to be regulated by photoperiod and melatonin through the hypothalamus (1, 7, 8). Evidence for this stems from lesioning studies. In the Siberian hamster, ablation of the suprachiasmatic nucleus (SCN) has been shown to block both body weight loss and gonadal regression in response to short-day (SD) photoperiod and programmed melatonin infusion (9, 10, 11). In Syrian hamsters, however, lesions to the dorsomedial nuclei of the hypothalamus have been shown to block the reproductive response (12). By contrast, the seasonal regulation of prolactin secretion and pelage has been shown to be independent of the hypothalamus involving the pituitary pars tuberalis (13, 14).

In Siberian and Syrian hamsters, a switch from a long-day (LD) to a short-day photoperiod stimulates gonadal regression in both species, yet these animals exhibit quite different responses in terms of body weight. Siberian hamsters undergo progressive weight loss over 14–18 wk in a SD photoperiod, reaching a maximum body weight loss of up to 30%, with the major component being fat (15, 16). Moreover, these animals exhibit exquisitely tight control of energy balance involving a system that appears to track and define seasonally appropriate body weight (15). In contrast, Syrian hamsters exhibit less dramatic body weight changes in response to altered photoperiod. Syrian hamsters have been reported to exhibit small increases in body weight in response to a switch from LD to SD photoperiod (17), whereas in this study body weight is shown to be relatively insensitive to photoperiod change over a 12- to 14-wk period. This suggests that common hypothalamic mechanisms may be involved in photoperiodic regulation of gonadal regression in Siberian and Syrian hamsters, yet those involved in body weight regulation appear species specific. The specific aims of this study were to: 1) identify genes differentially expressed in the hypothalami of the Siberian hamsters held on different photoperiodic backgrounds [i.e. LD (16 h light, 8 h dark) vs. SD (8 h light, 16 h dark)] and thereby may be important to mediating photoperiodic responses; 2) identify hypothalamic sites involved in mediating photoperiodic responses; and 3) identify any tentative links between photoperiodic regulation of body weight and gene expression. This exploits the differential body weight response of Siberian (15) and Syrian (17) hamsters to photoperiod and is based on the rationale that genes whose expression were sensitive to change in photoperiod in Siberian hamsters but not the Syrian hamster potentially may be linked to the photoperiodic regulation of body weight.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental procedures
All procedures were licensed under the Animals (Scientific Procedures) Act, 1986, and had local ethical approval. Male Siberian hamsters (Phodopus sungorus) were drawn from the Rowett breeding colony or the University of Nottingham breeding colony and were gestated, suckled, and reared in LD photoperiod. Siberian hamsters used were 4–6 months old and were individually housed at least 2 wk before photoperiod manipulation. Siberian hamsters used in gene expression studies were either maintained in LD or transferred to SD for 2, 6, or 12–14 wk. Where specified, hamsters were transferred to the SD photoperiod but with temperature (22 C) unaltered. Food (Labsure pelleted diet, gross energy, 15.21 MJ/kg; Special Diet Services, Witham, Essex, UK) and water were available ad libitum for all animals except where indicated. After 13 wk in either LD or SD, some animals were anesthetized with halothane then implanted with interscapular silastic capsules for 5 d. For one SD group, the approximately 4-mm capsule was packed with crystalline testosterone, whereas other SD and LD groups had capsules containing cholesterol as control. Before implantation, silastic capsules were washed and then incubated overnight in saline. Serum concentrations of testosterone were analyzed using the DSL-4000 ACTIVE Testosterone RIA kit (Diagnostic Systems Laboratories, Inc., Webster, TX). Some LD Siberian hamsters were pinealectomized or sham operated under general anesthesia [ketamine (0.4 mg/kg) and xylamine (2 mg/kg, ip)], as described elsewhere (18), before transfer to SD. To mimic SD body weight changes by food restriction in LDs, animals were treated as described elsewhere (16). Briefly, Siberian hamsters were divided into three groups, matched for body weight. One group (n = 10) remained in LD and was fed ad libitum whereas a second group (n = 10) was transferred to SD and was also fed ad libitum. The final group (n = 10) remained in LD but received a restricted ration of food from wk 2 onward, such that the group mean body weight tracked that of the SD hamsters. The restriction imposed did not exceed 33% of the LD ad libitum fed animals at any point of the study. One animal in the restricted group failed to eat its daily food ration and was withdrawn from the study. Adult Male Syrian hamsters were purchased from Harlan, Shaw’s Farm (Blackthorn, Bicester, Oxon, UK) and individually housed under LD before photoperiod manipulation, whereupon these animals were either maintained in LD or transferred to SD for 14 wk.

Riboprobe templates
Siberian hamster total RNA was extracted by a procedure described elsewhere (19), from frozen (-80 C) hypothalamus/thalamus tissue blocks cut with anatomical precision and then DNase treated using DNAfree (AMS Biotechnology, Abingdon UK). Partial complementary DNAs of the cellular retinoic acid binding protein 2 (CRABP2), retinoic acid receptor (RAR), and retinoid X receptor {gamma} (RXR{gamma}) genes were amplified by RT-PCR. First-strand cDNAs were generated using Superscript II reverse transcriptase (Invitrogen, Paisley, UK) with oligo (dT) primers (Promega, Southampton, UK). PCR was performed using Pfu-turbo (Stratagene, Amsterdam, The Netherlands) with 30–35 cycles of amplification, extension time of 1.0 min at 68 C, and annealing time of 30 sec at temperatures of: CRABP2, 57 C; RAR, 60 C; RXR{gamma}, 55 C. Products were ligated into PCR-script (Stratagene) and cloned before sequence verification. CRABP2 was amplified using primers designed against nucleotides 106–129 (forward) and 393–413 bp (reverse) of the mouse CRABP2 gene (GenBank accession no. M35523). RAR primers were designed to nucleotides 739–762 (forward) and 1149–1172 bp (reverse) of the mouse RAR gene (GenBank accession no. NM009024). RXR{gamma} primers were designed to nucleotides 198–219 (forward) and 607–628 bp (reverse) of the mouse RXR{gamma} gene (GenBank accession no. S62948). The cellular retinol binding protein 1 (CRBP1) template was mouse I.M.A.G.E. Consortium Clone ID 406897 (20).

Gene expression analysis
From Siberian hamster total RNA, mRNA was extracted using Dynabeads Oligo (dT)25 using the manufacturer’s protocols (Dynal, Wirral, UK). Differential gene expression was analyzed, using Siberian hamster mRNA from LD and SD animals, by Incyte Genomics on their Mouse GEM1 cDNA microarray, which contained more than 8500 known genes and expressed sequence tags. The array source information is available from Incyte Genomics (www.genomesystems.com, Palo Alto, CA). The distributions and levels of hypothalamic mRNAs were examined by in situ hybridization using 35S-labeled riboprobes and quantification performed following methods described in detail elsewhere (21, 22), using tissues from animals held for 14 wk in LD or SD. Coronal sections including the dorsal tuberomamillary nucleus (DTM) corresponded to between -2.30 to -2.54 mm relative to Bregma (23), whereas the ventromedial nucleus (VMN)/dorsomedial nucleus (DMN)/arcuate nucleus (ARC) section shown was approximately -1.94 mm relative to Bregma (23).

For immunohistochemistry, animals were anesthetized with Euthatal (Rhone Merieux, Harlow, UK), transcardially perfused with 0.9% saline containing heparin (1000 U/liter) and then 4% paraformaldehyde. Brains were cryopreserved in 20% sucrose and then sectioned at 40 µ on a freezing microtome. Free-floating sections were processed following standard Vectastain procedures using anti-RXR{gamma} IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at 1 in 500, overnight at +4 C. Immunoreactivity was visualized using avidin-biotin-peroxidase in conjunction with diaminobenzidine with nickel as chromogen (Vector Laboratories, Peterborough, UK). Section images were captured using a Hitachi HV-C20 camera [Hitachi Denshi (U.K.) Ltd., London, UK] fitted to an Olympus BX50F4 microscope (Olympus U.K. Ltd., Southall, Middlesex, UK), linked to a PC running Image-Pro PLUS version 4.1.0.0 analysis software (Media Cybernetics, Workingham, Berkshire, UK) to count immunopositive cells.

Statistical analysis
Data were analyzed by t test and Mann-Whitney rank sum test, one- or two-way ANOVA with Tukey test, or Dunn’s multiple comparisons where appropriate, using SigmaStat statistical software (SPSS Scientific Software, Erkrath, Germany). Results are presented as means ± SE.

Accession numbers
Siberian hamster sequence data have been submitted to the GenBank database under the accession numbers: CRABP2, AY232274; RXR{gamma}, AY232275; RAR, AY232276.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Photoperiod regulates retinoid signaling
Male Siberian hamsters held in LD maintained a relatively stable body weight over a 14-wk period, attaining a mean body weight (BW) of 39.92 g (± 1.45 SE, n = 10) and testes weight of 0.696 g (± 0.040 SE, n = 10) at wk 14. In contrast, Siberian hamsters maintained on SD exhibited a progressive decrease in body weight over the same period, reaching a mean body weight of 27.08 g (± 0.73 SE, n = 10) and testes weight of 0.038 g (± 0.002 SE, n = 10) (Fig. 1AGo). After subtraction of testes weight, this represents a body weight loss of 32%.



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  		FIG. 1. Effect of photoperiod on gene expression and BW of male Siberian hamsters. A, Photoperiod effect on BW of male Siberian hamsters. Closed circles show animals on LD, open circles represent animals on SD. Data are mean ± SE, with 10 animals for each data point. B–D, Photoperiod regulates expression of CRBP1 mRNA in Siberian hamster hypothalamus. Data are expressed as percentages of LD values (LD levels of expression were normalized as 100%; then other values and all SEMs were scaled to this value). B, In situ hybridization of the CRBP1 probe. CRBP1 mRNA expression was reduced in the DTM of SD animals (***, P < 0.001). Representative coronal sections (scale bar, 2.5 mm) demonstrating CRBP1 expression with magnified ependymal layer (arrowhead), ventral ependymal layer (double arrowhead), and DTM (arrow) (scale bar, 1.0 mm). Data are from 10 animals per photoperiod. Brains were collected from animals at zeitgeber time 03 (ZT03). C, Difference in CRBP1 mRNA expression in the Siberian hamster DTM with time in respective photoperiods. SD values are expressed as percentages of corresponding LD values. Expression was significantly decreased at all time points (*, P < 0.05; ***, P < 0.001). Each data point represents results from 3–10 animals. D, CRBP1 mRNA expression in the DTM of Siberian hamsters that were food restricted to match SD BWs. Levels were lower in SD than LD and LD food restricted (LD-R) animals (***, P < 0.001). LD-R levels did not differ significantly from those of LD animals. For each treatment, 9–10 animals were used.

 
Microarray analysis of hypothalamic mRNA obtained from Siberian hamsters following 12 wk exposure to LD or SD revealed a 2-fold difference in CRBP1 mRNA levels (data not shown). Subsequent examination of CRBP1 mRNA expression levels by in situ hybridization in tissues from the animals exposed to LD and SD for 14 wk revealed not only distinct expression within specific areas and nuclei of the hypothalamus but also differential expression related to the photoperiodic background of the animals. Strong expression was observed in the ependymal layer of the third ventricle and in nuclei corresponding to the DTM of hamsters held on LD. The strong levels of CRBP1 gene expression, measured 3 h after lights on, were significantly attenuated to almost background levels, in Siberian hamsters held on SD (Fig. 1BGo). The magnitude of the difference in CRBP1 mRNA expression increased proportionately with duration in SD (Fig. 1CGo). Additional expression of CRBP1 mRNA was also detected in the median eminence, but this expression was unresponsive to altered photoperiod (Fig. 1BGo).

To investigate whether the change in CRBP1 mRNA expression was driven by food intake rather than photoperiod, a group of LD hamsters were food restricted to mimic the SD photoperiod-induced weight loss trajectory (16). CRBP1 mRNA expression in the DTM of these animals remained at the level of the ad libitum-fed LD controls rather than declining to the levels of SD hamsters (Fig. 1DGo). This indicated that the decline in CRBP1 mRNA levels reflected the duration of exposure to SD rather being the result of reduced food intake or body weight per se.

Given the dynamic regulation of CRBP1 by photoperiod, the expression of other genes involved in retinoid signaling was examined. The mRNA for CRABP2, a protein involved in the translocation of retinoic acid to the nucleus, was also strongly expressed in the DTM of Siberian hamsters on LD but markedly attenuated in SD animals (Fig. 2AGo). In contrast to CRBP1, mRNA for CRABP2 was not detected in the ependymal layer of the third ventricle.



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  		FIG. 2. A–C, Photoperiod regulates expression of genes related to retinoid signaling in the Siberian hamster DTM. In situ hybridization of CRABP2, RAR, and RXR{gamma} probes, respectively. Representative coronal sections of the hypothalamus, including the DTM (arrow) and in ARC (arrowhead) (C) are shown. The expression levels of all three genes were reduced in the DTM of SD animals (***, P < 0.001). For each gene, data are from 10 animals per photoperiod and data are expressed as percentages of the LD values. Brains were collected from animals at ZT03. Scale bar, 1.0 mm. D, RXR{gamma}-like immunoreactivity in the DTM is reduced in SDs (**, P < 0.01). Data are expressed as percentages of the LD values and are from two coronal sections per animal with three animals per photoperiod. Representative sections showing the third ventricle (3V) with staining in the DTM (circled) are shown. Scale bar, 50 µm. E, Representative coronal section from a LD Siberian hamster showing in situ hybridization with the RXR{gamma} probe in the DMN (arrow), ARC (single arrowhead), and VMN (double arrowhead). Scale bar, 1.0 mm.

 
The mRNA for the nuclear RAR was also expressed in the DTM of Siberian hamsters in a photoperiod-dependent manner, being more strongly expressed in LD relative to SD animals (Fig. 2BGo). Because the riboprobe used was designed to hybridize to a conserved region, no distinction was made among different isoforms of RAR (24).

RAR is a type II receptor that normally requires an auxiliary factor, RXR, for high-affinity binding (24, 25). Riboprobes were used to detect the expression of the specific RXR isoforms {alpha}, ß, and {gamma} in the hypothalamus. Although only weak RXR{alpha} and ß mRNA expression were detected (data not shown), RXR{gamma} mRNA was strongly expressed within the DTM of LD Siberian hamsters (Fig. 2CGo) and was significantly lower in SD (Fig. 2CGo). Concordantly, immunohistochemistry showed that at the protein level, RXR{gamma}-like immunoreactivity levels were also significantly lower in the DTM of hamsters in SD, compared with those in LD (Fig. 2DGo). RXR{gamma} mRNA was detected in the ARC (Fig. 2Go, C and E), but no significant photoperiod effect was observed. RXR{gamma} mRNA was also detected in the DMN and VMN of the hypothalamus (Fig. 2EGo), and weak expression of RAR mRNA was detected in the ARC. CRABP2 expression was restricted to the DTM only. There were no major changes in gene expression of any of these genes in hypothalamic regions other than in the DTM in response to photoperiod.

To confirm that expression differences were truly seasonal and not diurnal in nature, as occurs in the mouse liver for CRBP1 (26), expression levels of CRBP1, RAR, and RXR{gamma} mRNAs were determined every 3 h over 24 h in both photoperiods. The levels of mRNA in the DTM were significantly higher in LD relative to SD over 24 h, with no pronounced rhythms (Fig. 3Go, A–C).



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  		FIG. 3. Levels of CRBP1, RAR, and RXR{gamma} mRNAs in the DTM over 24 h in LD and SD photoperiods. In situ hybridization of CRBP1 (A), RAR (B), and RXR{gamma} (C) probes, respectively, in LD (closed circles) and SD (open triangles) animals. By two-way ANOVA, there were significant differences between photoperiods with the SD values being lower for all three probes (P < 0.001). For each probe, there was an overall effect of photoperiod on mRNA expression levels (CRBP1, F = 384.5, P < 0.0001; RAR F = 204.5, P < 0.0001; RXR{gamma}, F = 161.7, P < 0.0001), no effects of time and no interactions between photoperiod and time. At all time points, multiple pair-wise comparisons showed that the LD and SD values were significantly different (P < 0.05). Each data point represents results from four animals, and these are expressed as a percentage of the maximum value. ZT00 data are double plotted at ZT24.

 
True photoperiod-driven responses
To examine the role of melatonin in the regulation of retinoid gene expression, Siberian hamsters were pinealectomized (PINX) to remove the primary source of circulating melatonin. PINX hamsters failed to show SD-induced decreases in CRBP1, CRABP2, RAR, and RXR{gamma} mRNA levels in the DTM relative to sham-operated control animals (Fig. 4Go). Moreover, the differences between the sham-operated controls and the PINX animals were of similar magnitude to those seen between intact LD and SD animals (Fig. 1BGo and 2Go, A–C), strongly implicating melatonin in these responses.



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  		FIG. 4. Pinealectomy of Siberian hamsters (PINX) prevents SD-induced changes in mRNA expression of genes related to retinoid signaling. A–D, In situ hybridization of CRBP1, CRABP2, RAR, and RXR{gamma} probes, respectively. The mRNA levels of all four genes were higher in PINX than sham-operated (control) animals (CRBP1 and CRABP2: ***, P < 0.001; RAR and RXR: *, P < 0.05). Data are expressed as percentages of PINX values with four to five animals per treatment. Representative coronal sections from SD PINX and SD sham-operated animals are shown with arrows indicating the DTM. Scale bar, 1.0 mm.

 
The differences in gene expression between LD and SD hamsters were not the secondary consequence of the seasonal reduction in testosterone concentrations in SD animals. This was shown by giving testosterone implants to SD Siberian hamsters to restore blood concentrations of testosterone to approximately LD levels. The plasma testosterone levels of LD and SD hamsters were 1.08 ± 0.55 (SE) and 0.09 ± 0.03 (SE) ng/ml, respectively, whereas for SD hamsters receiving testosterone implants for 5 d, plasma levels were increased to 2.11 ± 0.11 (SE) ng/ml. Five days after testosterone replacement, the levels of CRBP1, CRABP2, RAR, and RXR{gamma} gene expression in SD hamsters were not statistically different from cholesterol-implanted control SD animals, i.e. levels of expression were not restored to LD values (Fig. 5Go). Confirmation that 5-d testosterone replacement to SD animals was effective in modulating gene expression was observed when mRNA levels of an unrelated gene (MC4-R), in testosterone-treated animals were restored to levels similar to those of LD animals (data not shown).



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  		FIG. 5. Effects of testosterone replacement to SD-housed Siberian hamsters on expression of genes related to retinoid signaling in the DTM. A–D, In situ hybridization of CRBP1, CRABP2, RAR, and RXR{gamma} probes, respectively. Expression of all four genes in SD-testosterone-treated (SD-t) animals did not differ significantly from those of SD-cholesterol controls (SD-c). SD mRNA levels were significantly lower than LD levels for all four genes (***, P < 0.001). Data are expressed as percentages of the LD values with eight animals per treatment.

 
Association of RXR{gamma} with body weight
To explore how retinoid gene expression might be linked to downstream physiological function, photoperiodic regulation of retinoid signaling was examined in the Syrian hamster. Although both Siberian and Syrian hamsters exhibit pronounced reproductive changes in response to photoperiod, Syrian hamsters express considerably less dramatic BW changes when compared with Siberian hamsters (compare Fig. 1AGo with Fig. 6AGo). For male Syrian hamsters held on LD and SD for 14 wk, the body and testes weights were as follows: LD: BW = 117.4 g ± 3.9 SE; testes = 4.10 g ± 0.08 SE, n = 8; SD: BW = 107.5 g ± 4.0 SE; testes = 0.94 g ± 0.08 SE, n = 8) (Fig. 6AGo). After subtraction of testes weight, this represents a BW decrease of 6.3%. In this study the SD animals continually gained weight, compared with their starting weights, and this was statistically significant (P < 0.05) until 12 wk after which the body weights were not statistically different from the starting weights. Compared with the LD animals, however, the difference in body weights of the SD animals did not reach significance at any time point. At the end of the 14-wk study, there was not a significant difference between the LD and SD animals weights, regardless of whether the testes weights were considered.



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  		FIG. 6. A, Effect of photoperiod on BW of male Syrian hamsters. Closed circles show animals on LD; open circles represent animals on SD. Data are mean ± SE, with eight animals for each data point. B–D, Effect of photoperiod on gene expression in the DTM of Syrian hamsters. In situ hybridization of CRBP1, RAR, and RXR{gamma} probes, respectively. CRBP1 (B) and RAR (C) mRNA levels were reduced in SDs (***, P < 0.001). D, RXR{gamma} mRNA levels did not differ significantly with photoperiod. Data are expressed as percentages of LD values with eight animals per photoperiod.

 
The differential BW responses between Siberian and Syrian hamsters provided the basis for identifying genes that potentially may be linked to the regulation of BW (i.e. gene expression changes shown in Siberian hamsters but not Syrian hamsters). CRBP1 and RAR mRNA levels were both reduced in expression and with similar magnitudes in the DTM of Syrian and Siberian hamsters in response to SD. This might suggest that these are not essential for seasonal BW regulation (compare Figs. 1BGo and 2BGo with 6BGo and 6CGo), although the contribution of individual isoforms of RAR has not been addressed. However, a clear species difference in expression of RXR{gamma} was evident where, in contrast to the large change in mRNA levels in Siberian hamsters (Fig. 2CGo), for Syrian hamsters no photoperiod-dependent differences in RXR{gamma} mRNA expression in the DTM were observed (Fig. 6DGo). In addition, expression of CRABP2 mRNA was photoperiod dependent in the Siberian hamster yet was barely detectable in the DTM of Syrian hamster from either LD or SD (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study reveals the profound effects of photoperiod and almost certainly melatonin on the expression of four genes involved in retinoid signaling, CRBP1, CRABP2, RAR, and RXR{gamma}, in the hypothalamus of the Siberian hamster. Unlike the pars tuberalis and SCN that express melatonin receptors (4) and the SCN, which also receives retinorecipient inputs (27), there is no evidence that the DTM has direct photic or melatonergic inputs in the hamster (Ellis, C., J. G. Mercer, and P; J. Morgan, unpublished observations). Because the SD-induced decline in retinoid gene expression did not occur in PINX Siberian hamsters, these changes were most likely dependent on melatonin. Importantly, the mRNA expression changes in CRBP1, CRABP2, RAR, and RXR{gamma} appear to be responses that are dependent on melatonin yet independent of changes in steroid hormone levels in Siberian hamsters, although we have not excluded long-term influences of testosterone on retinoid gene expression. These results suggest, therefore, that the DTM may be an important site involved in the integration of seasonal rather than diurnal or circadian responses. In addition, an area that encompasses the premammillary and tuberomamillary nuclei has been identified as an important site for the coordination of seasonal responses through melatonin in sheep (7). Unlike the sheep, however, there is no evidence of melatonin receptor expression in the DTM of either Siberian or Syrian hamsters (Ref.4 and Ellis, C., J. G. Mercer and P. J. Morgan unpublished observations). Thus, in the hamster the DTM must be downstream of either melatonin or light-responsive cells.

Although the reductions in RAR and CRBP1 expression in the DTM in response to SD are common to Siberian and Syrian hamsters, the responses of RXR{gamma} and CRABP2 expression are divergent between the two species. We tentatively propose that these differences in RXR{gamma} and CRABP2 may be associated with the differential seasonal BW responses observed between the two hamsters. These conclusions are drawn from the fact that after 14 wk on SD, Siberian hamsters lost a substantial amount of BW (32%) and RXR{gamma} expression was reduced, whereas Syrian hamsters under the same conditions and time frame exhibited no statistically significant change in BW and no change in RXR{gamma} expression.

RXRs form not only homodimers but also they are the required heterodimerizing partners for many nuclear hormone receptors (25, 28), including RARs. Thus, they have critical and pleiotropic roles in the regulation of gene expression. If RXR{gamma} is involved in a signaling pathway related to the seasonal regulation of BW, then RAR is unlikely to be the sole dimerizing partner involved because the reduced RAR expression in the DTM would be predicted to be associated with BW loss. This is not observed in Syrian hamsters in which RAR mRNA expression decreases substantially, whereas BW remains relatively constant. Thus, any role RXR{gamma} might play is likely to involve either RXR homodimers or an unidentified heterodimerizing partner of RXR, but contributions from individual RAR isoforms have not been excluded. In addition, associated coactivators and corepressors (29) may also have important roles in the RXR{gamma}-mediated photoperiodic response. Clearly, identifying dimerizing partners of RXR{gamma} in the DTM is important before a clear functional role in relation to its seasonal physiology can be assigned.

A link between RXR{gamma} expression and energy balance is not unprecedented. Elevated metabolic rate and increased plasma thyroid hormone levels have been reported in RXR{gamma}(-/-) mice, although no net effect on BW was reported (30). However, it is thought that other RXR isoforms may compensate for the lack of RXR{gamma} in these animals (30). Consistent with the inverse relationship between RXR{gamma} and metabolic rate, obese ob/ob(-/-) mice have reduced metabolic rate (31), and we have observed increased RXR{gamma} expression in the DTM of ob/ob(-/-) relative to lean mice (Ross, A. W., P. J. Morgan, J. G. Mercer, unpublished observations). Furthermore, in RXR{gamma}(-/-), RXRß(-/-), and RXR{alpha}(+/-) mice that express only a single copy of RXR{alpha}, a 20% weight deficit was observed, strongly suggesting a role for RXR in the regulation of BW (32). However, reports of seasonal changes in thyroid hormone levels in Siberian hamsters are inconsistent (33, 34, 35) and where changes with photoperiod have been seen, these are inconsistent with a role in photoperiod-induced BW change (34, 35). Interestingly, in Siberian hamsters, increased energy expenditure has been proposed as the mechanism of SD-induced weight loss (36). Others have reported reduced food intake in SD (37, 38) and concluded that reduced food intake is the primary driver of SD-induced weight loss (39). Consistent with this are studies in rats, which show that rapid and synchronous depletion of vitamin A/retinoic acid causes a dramatic decline of food intake and BW within a few of days, suggesting a role for retinoic acid in the regulation of food intake (40).

This speculative role proposed for the DTM and the retinoids in the seasonal control of adiposity appears to be distinct from the contribution that has been proposed for the direct sympathetic outflow from melatonin receptor-expressing cells of the hypothalamus to the white adipose tissue in the Siberian hamster (41). In this species, the SCN appears to be vital for mounting SD photoperiodic responses because daily SD-like melatonin signals delivered to PINX animals bearing SCN lesions do not trigger SD-like decreases in BW and gonadal regression (42). The DTM is thus a potential downstream recipient of communications from the SCN, but the output pathways from the SCN to the DTM or other brain/peripheral targets are currently unclear. Nevertheless the identification of the DTM and the ependymal layer of the third ventricle as key sites of integration for photoperiod-controlled events is a major step forward toward our understanding of how temporal seasonal information interfaces with neuroendocrine and physiological outputs. More studies are required to determine the precise functional role of retinoid gene expression changes in the DTM as well as to define the neuroanatomical DTM interactions with other brain areas. In addition, given the close anatomical relationship of the DTM to the ARC and DMN, a role in the regulation of appetite and energy balance appears plausible.


   Acknowledgments
 
The authors are grateful to Miss C. Ellis for providing tissue samples and technical assistance.


   Footnotes
 
This work was supported by the Scottish Executive Environment and Rural Affairs Department and the Biotechnology and Biological Sciences Research Council (42/S17106).

Abbreviations: ARC, Arcuate nucleus; BW, body weight; DMN, dorsomedial nucleus; DTM, dorsal tuberomamillary nucleus; LD, long day; PINX, pinealectomized; RAR, retinoic acid receptor; RXR, retinoid X receptor; SCN, suprachiasmatic nucleus; SD, short day; VMN, ventromedial nucleus.

Received July 7, 2003.

Accepted for publication August 28, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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