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.     

Stimulatory effect of RFRP-3 on the gonadotrophic axis in the male Syrian hamster: the exception proves the rule

In seasonal mammals, a distinct photoneuroendocrine circuit that involves the pineal hormone melatonin tightly synchronizes reproduction with seasons. In the Syrian hamster, a seasonal model in which sexual activity is inhibited by short days, we have previously shown that the potent GnRH stimulator, kisspeptin, is crucial to convey melatonin's message; however, the precise mechanisms through which melatonin affects kisspeptin remain unclear. Interestingly, rfrp gene expression in the neurons of the dorsomedial hypothalamic nucleus, a brain region in which melatonin receptors are present in the Syrian hamster, is strongly down-regulated by melatonin in short days. Because a large body of evidence now indicates that RFamide-related peptide (RFRP)-3, the product of the rfrp gene, is an inhibitor of gonadotropin secretion in various mammalian species, we sought to investigate its effect on the gonadotrophic axis in the Syrian hamster. We show that acute central injection of RFRP-3 induces c-Fos expression in GnRH neurons and increases LH, FSH, and testosterone secretion. Moreover, chronic central administration of RFRP-3 restores testicular activity and Kiss1 levels in the arcuate nucleus of hamsters despite persisting photoinhibitory conditions. By contrast RFRP-3 does not have a hypophysiotrophic effect. Overall, these findings demonstrate that, in the male Syrian hamster, RFRP-3 exerts a stimulatory effect on the reproductive axis, most likely via hypothalamic targets. This places RFRP-3 in a decisive position between the melatonergic message and Kiss1 seasonal regulation. Additionally, our data suggest for the first time that the function of this peptide depends on the species and the physiological status of the animal model.     

Melatonin regulates type 2 deiodinase gene expression in the Syrian hamster

In seasonal species, photoperiod organizes various physiological processes, including reproduction. Recent data indicate that the expression of type 2 iodothyronine deiodinase (Dio2) is modulated by photoperiod in the mediobasal hypothalamus of some seasonal species. Dio2 is believed to control the local synthesis of bioactive T(3) to regulate gonadal response. Here we used in situ hybridization to study Dio2 expression in the hypothalamus of a photoperiodic rodent, the Syrian hamster. Dio2 was highly expressed in reproductively active hamsters in long day, whereas it was dramatically reduced in sexually inhibited hamsters maintained in short day. This contrasted with the laboratory rat, a nonphotoperiodic species, in which no evidence for Dio2 photoperiodic modulation was seen. We also demonstrate that photoperiodic variations of Dio2 expression in hamsters are independent from secondary changes in gonadal steroids. Studies in pinealectomized hamsters showed that the photoperiodic variation of Dio2 expression is melatonin dependent, and injections of long day hamsters with melatonin for only 7 d were sufficient to inhibit Dio2 expression to that of short day levels. Finally, because in some seasonal species thyroid hormones are involved in photorefractoriness, we examined Dio2 expression in short day-refractory hamsters and found that Dio2 mRNA levels remained low despite full reproductive recrudescence. Altogether, these results demonstrate that in the Syrian hamster Dio2 is photoperiodically modulated via a melatonin-dependent process. Furthermore, refractoriness to photoperiod in hamsters appears to occur independently of Dio2. These results raise new perspectives for understanding how thyroid hormones are involved in the control of photoperiodic neuroendocrine processes.     

Naloxone-induced secretion of LH in the male Syrian hamster: modulation by photoperiod and gonadal steroids

The role of endogenous opiates in the regulation of photoperiodically induced testicular regression was studied in the male Syrian hamster. In reproductively active hamsters exposed to a long photoperiod (LD; 16 h light: 8 h darkness) or to short days (SD; 8 h light: 16 h darkness) for 20 weeks or to SD after pinealectomy, administration of naloxone, a competitive opiate receptor antagonist, at doses of 2.5-20 mg/kg, significantly increased serum LH concentrations. In marked contrast, these doses of naloxone did not produce any change in LH levels in reproductively quiescent hamsters exposed to SD for 8 weeks. The influence of gonadal steroids on the LH response to naloxone was studied in hamsters castrated or castrated and implanted s.c with a capsule containing testosterone. Naloxone did not induce LH release in castrated hamsters maintained in LD or in SD, but this response was restored in LD but not SD when serum testosterone concentrations were maintained at levels similar to those observed in intact reproductively active hamsters. These results show that inhibition of reproduction by the photoperiod prevents naloxone-induced LH release in the male hamster. This lack of response to naloxone is not due, however, to the lower testosterone titres present in these animals compared with reproductively active animals. Responsiveness to naloxone can be restored when the animal is rendered insensitive to the inhibitory photoperiod either by removal of the pineal gland or by induction of photorefractoriness by extended exposure to SD.     

Temporal changes in gene expression in the arcuate nucleus precede seasonal responses in adiposity and reproduction

In anticipation of seasonal climate changes, Siberian hamsters display a strategy for survival that entails profound physiological adaptations driven by photoperiod. These include weight loss, reproductive quiescence, and pelage growth with shortening photoperiod and vice versa with lengthening photoperiod. This study reports gene expression changes in the hypothalamus of Siberian hamsters switched from short days (SD) to long days (LD), and also in photorefractory hamsters. Siberian hamsters were maintained in either LD or SD for 14 wk, conditions that generate physiological states of obesity under LD and leanness under SD. After 14 wk, SD lighting was switched to LD and gene expression investigated after 0, 2, 4, and 6 wk by in situ hybridization. Genes encoding nuclear receptors (RXR/RAR), retinoid binding proteins (CRBP1 and CRABP2), and histamine H3 receptor were photoperiodically regulated with significantly lower expression in SD, whereas VGF mRNA expression was significantly higher in SD, in the dorsomedial posterior arcuate nucleus. After a SD-to-LD switch, gene expression changes of CRABP2, RAR, H3R, and VGF occurred relatively rapidly toward LD control levels, ahead of body weight recovery and testicular recrudescence, whereas CRBP1 responded less robustly and rxrgamma did not respond at the mRNA level. In this brain nucleus in photorefractory animals, the CRABP2, RAR, H3R, and VGF mRNA returned toward LD levels, whereas CRBP1 and rxrgamma remained at the reduced SD level. Thus, genes described here are related to photoperiodic programming of the neuroendocrine hypothalamus through expression responses within a subdivision of the arcuate nucleus.     

Exposure to short days,but not short-term melatonin,enhances humoral immunity of male Syrian hamsters (Mesocricetus auratus)

Many non-tropical rodent species rely on photoperiod as the primary cue to co-ordinate seasonally appropriate changes in physiology and behavior. Among these seasonal changes, several rodent species (e.g. deer mice, prairie voles, Siberian hamsters) adjust immune function in response to changes in ambient day lengths. The goals of the present study were to examine the effects of photoperiod on immune function of Syrian hamsters (Mesocricetus auratus), and to determine the role of melatonin in mediating photoperiodic changes in immunity. In Experiment 1, male Syrian hamsters were housed in long (LD 14:10) or short days (LD 10:14) for 10 wk. In Experiment 2, hamsters were housed in long days and half of the animals were given 10 consecutive days of i.p. melatonin injections (15 microg) in the early evening, while the remaining animals received injections of the vehicle alone. After the respective experimental manipulations, animals were injected with the antigen, keyhole limpet hemocyanin (KLH), blood samples were obtained and anti-KLH IgG antibody production was assessed. In Experiment 1, short-day hamsters underwent gonadal regression and reduced serum testosterone as well as displayed increased humoral immune function compared with long-day animals. In Experiment 2, short-term melatonin treatment did not affect gonadal mass, testosterone or humoral immune function. These results confirm previous findings of photoperiodic changes in immunity in rodents and suggest that changes in humoral immunity are not due to short-term changes in melatonin.     

Melatonin mediates photoperiod control of endocrine adaptations and humoral immunity in male Siberian hamsters

Effects of photoperiod are mediated by the pineal gland in male Siberian hamsters. The hypothesis that the pineal hormone melatonin mediates the effects of short days (SD) to blunt select humoral and endocrine functions was tested. In the first study, regressed testes were found in pineal-intact controls transferred from long days (LD) to SDs (16 hr to 8 hr light/day); the rise in antigen-induced serum immunoglobulin (Ig) M was blunted and serum cortisol concentrations elevated compared with long-day controls. These effects of short-day were blocked in pinealectomized males moved from long to SDs, but restored by melatonin treatments. In a second study, males in LD were exposed to constant light (LL) to abolish the nighttime melatonin rhythm. In hamsters in LL, melatonin induced testicular regression as in males in SDs. Large testes were present in vehicle-treated controls in LL and in males that remained in LDs. Antigen-induced increases in serum IgM in vehicle and melatonin treatment males in LL were intermediate between concentrations in long- or short-day controls and not significantly different from each other. However, serum cortisol was again elevated in hamsters in SDs or in LL when treated with melatonin compared with males in LL or LDs. These findings indicate that melatonin treatments mimicked the effects of SDs to regulate adaptive physiologic functions in hamsters lacking the nocturnal melatonin rhythm. Thus, the photoneuroendocrine mechanism regulating reproductive responses to photoperiod also mediates short-day effects on T cell-dependent B-cell antibody production and processes that regulate cortisol in circulation.     

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.     

Effects of Short Photoperiod and Melatonin Treatment on Thermogenesis in the Syrian Hamster

This study investigated whether short photoperiod or melatonin-treatment could alter the thermogenic capacity of Syrian hamsters. Exposure of hamsters to short photoperiod and to exogenous melatonin treatment induced gonadal regression and hypertrophy of brown adipose tissue (BAT). Short photoperiod and melatonin-induced BAT hypertrophy was not accompanied by any change in noradrenaline (NA) turnover in this tissue. The concentration of NA was significantly decreased in hypertrophied BAT, indicating that sympathetic innervation in BAT did not effect its hypertrophy. No improvement in nonshivering thermogenic capacity was noticed in hamsters with increased BAT mass. However, capability for shivering thermogenesis seemed to be enhanced in melatonin-treated hamsters. These observations suggest that melatonin, in addition to mediating short photoperiod-induced gonadal regression in the Syrian hamster, also brings about thermoregulatory adjustments necessary for hibernation.     

Clock genes in calendar cells as the basis of annual timekeeping in mammals--a unifying hypothesis

Melatonin-based photoperiod time-measurement and circannual rhythm generation are long-term time-keeping systems used to regulate seasonal cycles in physiology and behaviour in a wide range of mammals including man. We summarise recent evidence that temporal, melatonin-controlled expression of clock genes in specific calendar cells may provide a molecular mechanism for long-term timing. The agranular secretory cells of the pars tuberalis (PT) of the pituitary gland provide a model cell-type because they express a high density of melatonin (mt1) receptors and are implicated in photoperiod/circannual regulation of prolactin secretion and the associated seasonal biological responses. Studies of seasonal breeding hamsters and sheep indicate that circadian clock gene expression in the PT is modulated by photoperiod via the melatonin signal. In the Syrian and Siberian hamster PT, the high amplitude Per1 rhythm associated with dawn is suppressed under short photoperiods, an effect that is mimicked by melatonin treatment. More extensive studies in sheep show that many clock genes (e.g. Bmal1, Clock, Per1, Per2, Cry1 and Cry2) are expressed in the PT, and their expression oscillates through the 24-h light/darkness cycle in a temporal sequence distinct from that in the hypothalamic suprachiasmatic nucleus (central circadian pacemaker). Activation of Per1 occurs in the early light phase (dawn), while activation of Cry1 occurs in the dark phase (dusk), thus photoperiod-induced changes in the relative phase of Per and Cry gene expression acting through PER/CRY protein/protein interaction provide a potential mechanism for decoding the melatonin signal and generating a long-term photoperiodic response. The current challenge is to identify other calendar cells in the central nervous system regulating long-term cycles in reproduction, body weight and other seasonal characteristics and to establish whether clock genes provide a conserved molecular mechanism for long-term timekeeping.     

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.     

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.     

Sustained melatonin treatment blocks body mass, pelage, reproductive, and fever responses to short day lengths in female Siberian hamsters

Winter imposes physiological challenges on individuals including increased thermoregulatory demands, risk of infection, and decreased food availability. To survive these challenges, animals living outside the tropics must appropriately distribute their energetic costs across the year, including reproduction and immune function. Individuals of many species use the annual cycle of changing day lengths (photoperiod), which is encoded by the nightly duration of melatonin secretion, to adjust physiology. Siberian hamsters exposed to short days (SD) (long nights/prolonged endogenous melatonin secretion) enhance some aspects of immune function, but curtail other energetically expensive immune functions including the febrile response. The purpose of this study was twofold. First, we determined whether sustained melatonin treatment would inhibit the development of the SD phenotype in female hamsters as it does in males. Second, we examined whether the SD attenuation of fever would be blocked by continuous exposure to exogenous melatonin. Hamsters were implanted with melatonin or empty capsules, housed in either long days (LD) or SD for 8-9 weeks, and then challenged with lipopolysaccharide; body temperature and locomotor activity were recorded. Unlike hamsters with empty capsules, hamsters with melatonin implants did not respond to SD and maintained a LD phenotype including summer-like spleen, uterine and body masses, and pelage characteristics. Further, sustained melatonin treatment blocked the SD attenuation of febrile responses and prolonged the behavioral components of the sickness response. These results suggest that the daily fluctuations in endogenous melatonin may be masked by continuous exposure to exogenous melatonin, thus inhibiting functional photoperiodic responses to SD.     

Daily and photoperiodic 2- 125I-melatonin binding changes in the pars tuberalis of the Syrian hamster (Mesocricetus auratus): Effect of constant light exposure and pinealectomy

Abstract: Using quantitative autoradiography, 2¹²⁵I-melatonin binding was investigated throughout the light/dark cycle in the pars tuberalis (PT) of the pituitary of adult Syrian hamsters kept for 8 weeks either in long or short photoperiod (LP or SP, respectively). Melatonin receptor density in the PT displayed photoperiod dependent daily variations (maximal values in LP). Indeed, in LP, melatonin receptor density underwent strong daily variations with maximal values during the first half of the light period and minimal values at the end of the night. These variations depended on changes in the maximal binding (B_max) without differences in the dissociation constant (K_d). In contrast, PT melatonin receptor density was constant and at a very low level throughout the 1ight:dark cycle in SP exposed animals. Daily PT melatonin receptor density variations of LP exposed animals were abolished by pinealectomy or continuous light exposure. These results show clearly that both at the daily and at the seasonal level the regulation of PT melatonin receptors is strongly dependent on circulating melatonin concentrations in the Syrian hamster, but that other regulatory factors, yet unclarified, might also play a role.     

Effects of Photoperiod or Exogenous Melatonin Administration on the Activity of N-Acetyltransferase and Hydroxyindole-O-Methyltransferase and the Melatonin Content of the Harderian Gland of Two Strains of Female Syrian Hamsters

The activities of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) and the melatonin concentration of the Harderian glands of two strains of Syrian hamster females (outbred and inbred LSH/SsLak) exposed to two different photoperiods (14:10 h and 8:16 h) were studied. The Harderian glands of the inbred hamsters showed greater NAT activity than those of the outbred animals. On the other hand, the glands of the outbred hamsters exhibited higher HIOMT activity and melatonin content than those of the inbred LSH/SsLak. Short photoperiod exposure, which produced gonadal regression in the inbred but not in the outbred hamsters, decreased the NAT activity in the inbred animals to the levels of the outbred. HIOMT activity was not affected by the lighting conditions. After the exposure to short days, the melatonin content of the inbred hamster Harderian glands increased to that in the outbred animals. Daily melatonin injections, which caused gonadal regression in the LSH/SsLak but not in the outbred hamsters, did not stimulate the effect of the short photoperiod on the Harderian gland NAT activity and melatonin content of the inbred hamsters.     

Photoperiod History, Melatonin, and Reproductive Responses of Male Syrian Hamsters

The action of melatonin (MEL) in mediating photoperiodic history (PPH) effects among male Syrian hamsters was investigated. In Exp. 1, pineal intact males in LD 14:10 received daily injections of MEL (15 micrograms) or ethanol:saline vehicle (SAL) 1 h before lights off for 8 wk to generate two groups experiencing identical photoperiods but distinctly different MEL histories. Following the cessation of injections, males were transferred to either LD 12:12 or LD 8:16 for 8 wk to evaluate whether their reproductive response to the new photoperiod would be more influenced by prior PPH or prior MEL history; MEL history was the significant variable. LD 12:12 caused gradual recrudescence in hamsters that were gonadally regressed following MEL injections. In contrast, LD 12:12 caused gonadal regression in hamsters that had large testes following SAL injections. Exp. 2 evaluated whether PPH influences might be mediated by aftereffects on the period (tau) of the circadian pacemaker regulating many behavioral and physiological rhythms. Pineal intact hamsters were exposed to long or short T cycles consisting of an 8 h photoperiod, repeated every 24.67 h (long T) or 23.33 h (short T) to mimic the aftereffects generated by short or long photoperiods. After 5 wk in these T-cycle conditions, all males were transferred to LD 12:12 for 11 wk. The reproductive response to LD 12:12 was modestly influenced by T-cycle history, even though each T-cycle generated different patterns of entrainment to LD 12:12. These findings support the hypothesis that the response of the reproductive system of male hamsters to an intermediate-duration photoperiod depends upon the duration of nocturnal melatonin secretion associated with hamsters' previous PPH.