Photoperiodic regulation of hypothalamic retinoid signaling: association of retinoid X receptor gamma with body weight

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.     

Photoperiodic regulation of histamine H3 receptor and VGF messenger ribonucleic acid in the arcuate nucleus of the Siberian hamster

To survive winter the Siberian hamster has evolved profound physiological and behavioral adaptations, including a moult to winter pelage, regression of the reproductive axis, onset of daily torpor and increased capacity for thermogenesis. However, one of the most striking adaptations is the catabolism of intraabdominal and sc fat reserves contributing to the loss of up to 40% of body weight. These physiological and behavioral adaptations are photoperiodically driven, yet neither the site(s) in the brain nor the molecular mechanism(s) involved in the regulation of these profound adaptations is known. Here we report a dynamic regulation of gene expression in a dorsal region of the medial posterior area of the arcuate nucleus (dmpARC) of the Siberian and Syrian hamster brain in response to altered photoperiod. We show mRNA for the histamine H3 receptor is down-regulated and VGF is up-regulated in the dmpARC in hamsters switched from long- to short-day photoperiod. These data provide further evidence to support the view that the dmpARC is a major site to relay photoperiodic changes and as a site for the long-term regulation of seasonal physiology and behavior.     

RFamide-related peptide gene is a melatonin-driven photoperiodic gene

In seasonal species, various physiological processes including reproduction are organized by photoperiod via melatonin, but the mechanisms of melatonin action are still unknown. In birds, the peptide gonadotropin-inhibiting hormone (GnIH) has been shown to have inhibitory effects on reproductive activity and displays seasonal changes of expression. Here we present evidence in mammals that the gene orthologous to GnIH, the RFamide-related peptide (RFRP) gene, expressed in the mediobasal hypothalamus, is strongly regulated by the length of the photoperiod, via melatonin. The level of RFRP mRNA and the number of RFRP-immunoreactive cell bodies were reduced in sexually quiescent Syrian and Siberian hamsters acclimated to short-day photoperiod (SD) compared with sexually active animals maintained under long-day photoperiod (LD). This was contrasted in the laboratory Wistar rat, a non-photoperiodic breeder, in which no evidence for RFRP photoperiodic modulation was seen. In Syrian hamsters, the reduction of RFRP expression in SD was independent from secondary changes in gonadal steroids. By contrast, the photoperiodic variation of RFRP expression was abolished in pinealectomized hamsters, and injections of LD hamsters with melatonin for 60 d provoked inhibition of RFRP expression down to SD levels, indicating that the regulation is dependent on melatonin. Altogether, these results demonstrate that in these hamster species, the RFRP neurons are photoperiodically modulated via a melatonin-dependent process. These observations raise questions on the role of RFRP as a general inhibitor of reproduction and evoke new perspectives for understanding how melatonin controls seasonal processes via hypothalamic targets.     

Kisspeptin: A key link to seasonal breeding

In seasonal species, photoperiod (i.e. daylength) tightly regulates reproduction to ensure that birth occurs at the most favorable time of year. In mammals, a distinct photoneuroendocrine circuit controls this process via the pineal hormone melatonin. This hormone is responsible for the seasonal regulation of reproduction, but the anatomical substrate and the cellular mechanism through which melatonin modulates sexual activity is far from understood. The Syrian hamster is widely used to explore the photoneuroendocrine system, because it is a seasonal model in which sexual activity is promoted by long summer days (LD) and inhibited by short winter days (SD). Recent evidences indicate that the products of the KiSS-1 gene, kisspeptins, and their specific receptor GPR54, represent potent stimulators of the sexual axis. We have shown that melatonin impacts on KiSS-1 expression to control reproduction in the Syrian hamster. In this species, KiSS-1 is expressed in the antero-ventral-periventricular and arcuate nuclei of the hypothalamus at significantly higher levels in hamsters kept in LD as compared to SD. In the arcuate nucleus, the downregulation of KiSS-1 expression in SD appears to be mediated by melatonin and not by secondary changes in gonadal hormones. Remarkably, a chronic administration of kisspeptin restores testicular activity in SD hamsters, despite persisting photoinhibitory conditions. Overall, these findings are consistent with a role of KiSS-1/GPR54 in the seasonal control of reproduction. We propose that the photoperiod, via melatonin, modulates KiSS-1 neurons to drive the reproductive axis.     

Identification, expression, and physiological functions of Siberian hamster gonadotropin-inhibitory hormone

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. To further understand its physiological roles in mammalian reproduction, we identified its precursor cDNA and endogenous mature peptides in the Siberian hamster brain. The Siberian hamster GnIH precursor cDNA encoded two RFamide-related peptide (RFRP) sequences. SPAPANKVPHSAANLPLRF-NH(2) (Siberian hamster RFRP-1) and TLSRVPSLPQRF-NH(2) (Siberian hamster RFRP-3) were confirmed as mature endogenous peptides by mass spectrometry from brain samples purified by immunoaffinity chromatography. GnIH mRNA expression was higher in long days (LD) compared with short days (SD). GnIH mRNA was also highly expressed in SD plus pinealectomized animals, whereas expression was suppressed by melatonin, a nocturnal pineal hormone, administration. GnIH-immunoreactive (-ir) neurons were localized to the dorsomedial region of the hypothalamus, and GnIH-ir fibers projected to hypothalamic and limbic structures. The density of GnIH-ir perikarya and fibers were higher in LD and SD plus pinealectomized hamsters than in LD plus melatonin or SD animals. The percentage of GnRH neurons receiving close appositions from GnIH-ir fiber terminals was also higher in LD than SD, and GnIH receptor was expressed in GnRH-ir neurons. Finally, central administration of hamster RFRP-1 or RFRP-3 inhibited LH release 5 and 30 min after administration in LD. In sharp contrast, both peptides stimulated LH release 30 min after administration in SD. These results suggest that GnIH peptides fine tune LH levels via its receptor expressed in GnRH-ir neurons in an opposing fashion across the seasons in Siberian hamsters.     

Leptin acts on metabolism in a photoperiod-dependent manner, but has no effect on reproductive function in the seasonally breeding Siberian hamster (Phodopus sungorus)

Leptin may play a role in appetite regulation and metabolism, but its reproductive role is less clear. In photoperiodic Siberian hamsters, seasonal changes in fatness, leptin gene expression, and metabolism occur synchronously with activation or suppression of reproduction, analogous to puberty. Here, we test the hypothesis that seasonal changes in leptin secretion mediate the photoperiodic regulation of reproduction. Mature male and ovariectomized estrogen-treated female Siberian hamsters were kept in long (LD; 16 h of light, 8 h of darkness) or short days (SD; 8 h of light, 16 h of darkness) for 8 weeks, and recombinant murine leptin (15 microg/day) was infused for 2 weeks via osmotic minipumps. SD hamsters exhibited significant weight and fat losses, reduced serum leptin and food intake, and suppressed pituitary LH concentration. Leptin did not suppress food intake over the 2-week treatment on either photoperiod, but significantly reduced fat reserves in SD hamsters. Leptin had no significant effect on pituitary LH concentrations in either sex or photoperiod or on testicular size and testosterone concentrations in males. These results suggest hamsters are more responsive to leptin on SD than on LD and that effects on food intake and fat loss can be dissociated in this species. Our data suggest that leptin does not mediate photoperiodic reproductive changes.     

VGF-derived peptide, TLQP-21, regulates food intake and body weight in Siberian hamsters

The Siberian hamster survives winter by decreasing food intake and catabolizing abdominal fat reserves, resulting in a sustained, profound loss of body weight. VGF gene expression is photoperiodically regulated in the hypothalamus with significantly higher expression in lean Siberian hamsters. The aim of this study was to investigate the role of VGF in regulating these seasonal cycles by determining the effects of a VGF-derived peptide (TLQP-21) on food intake and body weight. Acute intracerebroventricular administration of TLQP-21 decreased food intake, and chronic treatment caused a sustained reduction in food intake and body weight and decreased abdominal fat depots. Behavioral analysis revealed that TLQP-21 reduced meal size but not the frequency of feeding bouts, suggesting a primary action on satiety. Hamsters treated with TLQP-21 lost a similar amount of weight as a pair-fed group in which food intake was matched to that of the TLQP-21-treated group. Central or peripheral treatment with TLQP-21 did not produce a significant effect on resting metabolic rate. We conclude that the primary action of TLQP-21 is to decrease food intake rather than increase energy expenditure. TLQP-21 treatment caused a decrease in UCP-1 mRNA in brown adipose tissue, but hypothalamic expression of orexigenic and anorexigenic neuropeptide genes remained unchanged after TLQP-21 treatment, although compensatory increases in NPY and AgRP mRNA were observed in the pair-fed hamsters. The effects of TLQP-21 administration are similar to those in hamsters in short days, suggesting that increased VGF activity may contribute to the hypophagia that underlies the seasonal catabolic state.     

Melatonin receptors and signal transduction in photorefractory Siberian hamsters (Phodopus sungorus).

In seasonally breeding mammals, seasonal alterations in day length are perceived via the pineal hormone melatonin. When exposed to short day lengths, hamsters and other long-day breeders undergo gonadal regression. With chronic exposure (greater than 20 weeks) to short days, however, the animals become photorefractory, or insensitive to the inhibitory influence of short-day melatonin patterns, and gonadal recrudescence occurs. In this report, we examined photorefractory Siberian hamsters and long-day housed control hamsters to examine whether this apparent insensitivity to melatonin is due to alterations in melatonin receptors or signal transduction. In vitro autoradiographic assessment of melatonin receptors using 125I-labeled 2-iodomelatonin (I-MEL) revealed that melatonin receptor distribution, affinity, density, and G protein coupling are unaltered in photorefractory animals. In each animal, high-affinity (dissociation constant approximately 40 pM) 2-iodomelatonin binding sites were observed in the hypophysial pars tuberalis, in the suprachiasmatic nucleus of the hypothalamus, and in the thalamus (paraventricular nucleus, reuniens nucleus, and nucleus of the stria medullaris). The nonhydrolyzable GTP analog, GTP gamma S (100 microM) caused a 10-fold reduction in melatonin receptor affinity in the pars tuberalis in both photorefractory and control hamsters, demonstrating receptor-G protein coupling in both groups. Furthermore, melatonin (10 nM) inhibited forskolin-stimulated cAMP accumulation in median eminence/pars tuberalis explants in photorefractory animals, just as previously observed in explants from long-day hamsters. These results suggest that melatonin receptors, receptor-G protein coupling, and inhibition of adenylyl cyclase by melatonin are not altered in photorefractory hamsters.     

Hypothalamic responses to peripheral glucose infusion in food-restricted sheep are influenced by photoperiod

Nutritional feedback provided by systemic hormones, such as insulin and leptin, influences reproductive neuroendocrine output within the hypothalamus, yet the mechanisms and their interaction with photoperiodic cues remain unresolved in seasonal species. Here, peripheral glucose (G) infusion was used to increase endogenous concentrations of insulin and leptin in food-restricted sheep kept in either long-day (LD) or short-day (SD) photoperiod, and responses were examined in terms of pulsatile luteinising hormone (LH) (gonadotrophin-releasing hormone by inference) output and hypothalamic gene expression for nutritionally sensitive neuropeptides and receptors. We addressed the hypothesis that these hypothalamic responses were correlated and influenced by photoperiod. Oestradiol-implanted, castrated male sheep were kept 16 weeks in SD (8 h light/day) or LD (16 h light/day) and then transferred to the opposite photoperiods for 8 weeks, during which food was restricted to 90% requirement to maintain body weight (maintenance). For the final 6 days, food was reduced to 75% maintenance, and sheep in both photoperiods were infused intravenously with G (60 mM/h) or saline (S) (n = 8/group). G-infused sheep had higher mean plasma concentrations of G, insulin and leptin than S-infused sheep, with no effect of photoperiod. In LD, but not in SD, G infusion increased LH pulse frequency and pulse amplitude. In LD, but not in SD, gene expression in the hypothalamic arcuate nucleus was lower in G- than S-infused sheep for neuropeptide Y (NPY) and agouti-related peptide (AGRP) and was higher in G- than S-infused sheep for pro-opiomelanocortin (POMC). Gene expression for leptin and insulin receptors was not affected by photoperiod or infusion. These results are consistent with the involvement of NPY, AGRP and POMC in mediating the reproductive neuroendocrine response to increased systemic nutritional feedback, and they support the hypothesis that hypothalamic responses to nutritional feedback are influenced by photoperiod in sheep.     

Differential regulation of luteinizing hormone and follicle-stimulating hormone in male siberian hamsters by exposure to females and photoperiod

Siberian hamsters have decreased gonadotropin levels and testis size after short-day (SD) exposure. Upon transfer from short to long days, FSH and testis weight increase rapidly, whereas LH and T remain low for much longer. We investigated whether an additional environmental stimulus, specifically a female, could trigger an earlier release of LH and whether the response to the female was dependent on photoperiod. An increase in serum LH was induced in long day (LD), but not SD, males within minutes of female exposure. The ability of SD males to secrete LH upon female exposure was regained within 4 d of photostimulation. FSH was not secreted after female exposure, but varied with photoperiod. Thus, FSH and LH are differentially regulated by photoperiod and female exposure. In subsequent studies melatonin injections and a GnRH antagonist were used to show that photoperiod modulates the endocrine responsiveness of a male to a female via melatonin and that female-induced LH release is GnRH dependent. Collectively, these results suggest separation of gonadotropin signaling pathways by environmental stimuli and provide an excellent model to elucidate the effects of photoperiod on the processing of social and chemosensory inputs to the GnRH neurons of the hypothalamus.     

Photoperiodic control of adipocyte alpha 2-adrenoceptors in Syrian hamsters: role of testosterone

The impact of photoperiodic manipulations and testosterone treatments on the adipocyte alpha 2-adrenergic (alpha 2-AR), beta-adrenergic (beta-AR), and A1-adenosine (A1-R) responsiveness, was explored in male Syrian hamsters (Mesocricetus auratus). Moreover, binding studies were performed with appropriate alpha 2-AR, beta-AR, and A1-R radioligands to study receptor changes. Animals were kept for 12 weeks in long day photoperiod (LD: 16 h light (L)-8 h dark (D)), in short day photoperiod (SD: 6L-18D), or in short photoperiod with testosterone treatments (1 mg/animal/day sc) 10 days before sacrifice (SD+T). The antilipolytic effect of the full alpha 2-AR agonist UK14304 and the specific binding of the alpha 2-AR radioligands [3H] RX821002 (antagonist) and [3H]UK14304 were significantly reduced in SD hamsters compared with LD hamsters. The alpha 2-site number and alpha 2-AR responsiveness were completely restored in SD+T hamsters. Whatever the experimental conditions the adipocyte beta-AR receptivity (lipolytic response of isoproterenol and [125I]cyanopindolol binding), and the A1-R receptivity (antilipolytic response initiated by (-)phenylisopropyladenosine and [3H]dipropyl-8-cyclopentylxanthine and [3H]phenylisopropyladenosine binding) remained unchanged. Moreover, the kidney and brain alpha 2-AR densities identified with [3H]RX821002 were not significantly different in LD, SD or SD+T hamsters. These results were obtained without any modification of animal weight, white adipose tissue weight, or white fat cell size. We conclude that, in the Syrian hamster, the expression of the adipocyte alpha 2-AR is under the control of the photoperiod by a testosterone-dependent mechanism probably mediated through the hypothalamic-pituitary axis, without any alteration of the animal fat stores.     

Intraovarian expression of GnRH-1 and gonadotropin mRNA and protein levels in Siberian hamsters during the estrus cycle and photoperiod induced regression/recrudescence

The hypothalamic-pituitary-gonadal (HPG) axis is the key reproductive regulator in vertebrates. While gonadotropin releasing hormone (GnRH), follicle stimulating (FSH), and luteinizing (LH) hormones are primarily produced in the hypothalamus and pituitary, they can be synthesized in the gonads, suggesting an intraovarian GnRH-gonadotropin axis. Because these hormones are critical for follicle maturation and steroidogenesis, we hypothesized that this intraovarian axis may be important in photoperiod-induced ovarian regression/recrudescence in seasonal breeders. Thus, we investigated GnRH-1 and gonadotropin mRNA and protein expression in Siberian hamster ovaries during (1) the estrous cycle; where ovaries from cycling long day hamsters (LD;16L:8D) were collected at proestrus, estrus, diestrus I, and diestrus II and (2) during photoperiod induced regression/recrudescence; where ovaries were collected from hamsters exposed to 14 weeks of LD, short days (SD;8L:16D), or 8 weeks post-transfer to LD after 14 weeks SD (PT). GnRH-1, LHβ, FSHβ, and common α subunit mRNA expression was observed in cycling ovaries. GnRH-1 expression peaked at diestrus I compared to other stages (p < 0.05). FSHβ and LHβ mRNA levels peaked at proestrus and diestrus I (p < 0.05), with no change in the α subunit across the cycle (p > 0.05). SD exposure decreased ovarian mass and plasma estradiol concentrations (p < 0.05) and increased GnRH-1, LHβ, FSHβ, and α subunit mRNA expression as compared to LD and, except for LH, compared to PT (p < 0.05). GnRH and gonadotropin protein was also dynamically expressed across the estrous cycle and photoperiod exposure. The presence of cycling intraovarian GnRH-1 and gonadotropin mRNA suggests that these hormones may be locally involved in ovarian maintenance during SD regression and/or could potentially serve to prime ovaries for rapid recrudescence.     

Effects of manipulating hypothalamic triiodothyronine concentrations on seasonal body weight and torpor cycles in Siberian hamsters

Siberian hamsters display photoperiodically regulated annual cycles in body weight, appetite, and reproduction. Previous studies have revealed a profound up-regulation of type 3 deiodinase (DIO3) mRNA in the ventral ependyma of the hypothalamus associated with hypophagia and weight loss in short-day photoperiods. DIO3 reduces the local availability of T(3), so the aim of this study was to test the hypothesis that decreased hypothalamic T(3) availability underlies the short-day-induced catabolic state. The experimental approach was to determine whether a local increase in T(3) in the hypothalamus of hamsters exposed to short days could reverse the behavioral and physiological changes induced by this photoperiod. In study 1, microimplants releasing T(3) were placed bilaterally into the hypothalamus. This treatment rapidly induced a long-day phenotype including increased appetite and body weight within 3 wk of treatment and increased fat mass and testis size by the end of the 10-wk study period. In study 2, hypothalamic T(3) implants were placed into hamsters carrying abdominal radiotelemetry implants. Again body weight increased significantly, and the occurrence of winter torpor bouts was dramatically decreased to less than one bout per week, whereas sham-implanted hamsters entered torpor up to six times a week. Our findings demonstrate that increased central T(3) induces a long-day metabolic phenotype, but in neither study was the molt cycle affected, so we infer that we had not disrupted the initial detection of photoperiod. We conclude that hypothalamic thyroid hormone availability plays a key role in seasonal regulation of appetite, body weight, and torpor.