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.     

Temporal dynamics of type 2 deiodinase expression after melatonin injections in Syrian hamsters

In many species living in temperate zones, reproduction is controlled by the photoperiod. Recent findings have clarified that type 2 iodothyronine deiodinase (Dio2) plays a significant role in the photoperiodic response of gonads in the mediobasal hypothalamus, converting the prohormone T(4) into bioactive T(3). In mammals, Dio2 expression is suppressed by long-term melatonin injections, although the signal transduction pathways that link the melatonin signal to Dio2 expression are unknown. As a first step to approach the problem, we have here investigated the temporal dynamics of the melatonin effect on Dio2 expression using male Syrian hamsters. Dio2 mRNA levels were found to show diurnal rhythms under long-day conditions in an area adjacent to the tuberoinfundibular sulcus and in the ependymal cell layer lining the ventrobasal walls of the third ventricle. Daily sc melatonin injections given in the late afternoon under long-day condition suppressed the Dio2 mRNA levels already at the first day after the onset of the treatment in the ependymal cell layer lining the ventrobasal walls of the third ventricle, and 1 d later in an area adjacent to the tuberoinfundibular sulcus. These suppressive effects were sustained for at least 2 d after a single injection. Furthermore, we examined the temporal changes of the Dio2 expression after the onset of the treatment, showing that the suppression did not occur until midday of the next day. These data suggest that melatonin is involved in the signal transduction mechanisms controlling the photoperiodic response of gonads by acting on Dio2 expression rather rapidly through indirect pathways.     

Photoperiodic regulation of type 2 deiodinase gene in Djungarian hamster: possible homologies between avian and mammalian photoperiodic regulation of reproduction

The molecular mechanisms responsible for seasonal time measurement have yet to be fully described. Recently, we used differential analysis to identify that the type 2 iodothyronine deiodinase (Dio2) gene is responsible for the photoperiodic response of gonads in Japanese quail. It was found that expression of Dio2 in the mediobasal hypothalamus is induced by light and that T(3) content in the mediobasal hypothalamus increased under long day conditions. In addition, we showed that intracerebroventricular infusion of T(3) mimics photoperiodically induced testicular growth. Because it is well known that thyroid hormone is also essential for the maintenance of the seasonal reproductive changes in a number of mammals, we examined expression of Dio2 in Djungarian hamsters and found expression in the ependymal cell layer lining the infralateral walls of the third ventricle and the cell-clear zone overlying the tuberoinfundibular sulcus. Signal intensity was high under long days and weak under short days. Although light pulse did not affect Dio2 expression, melatonin injections decreased Dio2 expression under long days. These results indicate that Dio2 may be involved in the regulation of seasonal reproduction in mammals in the same way as observed in birds.     

Long-day suppressed expression of type 2 deiodinase gene in the mediobasal hypothalamus of the Saanen goat, a short-day breeder: implication for seasonal window of thyroid hormone action on reproductive neuroendocrine axis

In most animals that live in temperate regions, reproduction is under photoperiodic control. In long-day breeders such as Japanese quail and Djungarian hamsters, type 2 deiodinase (Dio2) plays an important role in the mediobasal hypothalamus, catalyzing the conversion of prohormone T4 to bioactive T3 to regulate the photoperiodic response of the gonads. However, the molecular basis for seasonal reproduction in short-day breeders remains unclear. Because thyroid hormones are also known to be involved in short-day breeders, we examined the effect of an artificial long-day stimulus on Dio2 expression in the male Saanen goat (Capra hircus), a short-day breeder. Dio2 expression was observed in the caudal continuation of the arcuate nucleus, known as the target site for both melatonin and T4 action. In addition, expression of Dio2 and T3 content in the mediobasal hypothalamus was suppressed by artificial long-day conditions, which is the opposite of the results of long-day breeders. Thyroid hormone action on the development of neuroendocrine anestrus is known to be limited to a specific seasonal window. This long-day suppression of Dio2 may provide a mechanism that accounts for the lack of responsiveness to thyroxine during the mid to late anestrus.     

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.     

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.     

Effects of Melatonin and Hypothyroidism on Somatomedin Levels of Female Syrian Hamsters

Female Syrian hamsters maintained on a 14 h light, 10 h dark photoperiod were injected once daily (1-2 h before lights out) with melatonin (25 micrograms), alone or in combination with thiourea, or with thiourea plus thyroxine. Serum levels of the somatomedin, Insulin-like growth factor-I (IGF-I), were significantly reduced by thiourea as well as by melatonin administration. These data suggest that in the female hamster melatonin-induced reduction of circulating IGF-I depends largely on a reduction in circulating levels of thyroid hormones. However, melatonin-induced changes in secretion of thyroid hormones, gonadal hormones, and hypothalamic hormones could contribute to decreased growth hormone (GH)-stimulated somatomedin secretion.     

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.     

Melatonin controls photoperiodic changes in tanycyte vimentin and neural cell adhesion molecule expression in the Djungarian hamster (Phodopus sungorus)

The Djungarian hamster displays photoperiodic variations in gonadal size synchronized to the seasons by the nightly secretion of the pineal hormone melatonin. In short photoperiod (SP), the gonads regress in size, and circulating sex steroids levels decline. Thus, the brain is subject to seasonal variations of both melatonin and sex steroids. Tanycytes are specialized glial cells located in the ependymal lining of the third ventricle. They send processes either to the meninges or to blood vessels of the medio-basal hypothalamus. Furthermore, they are known to locally modulate GnRH release in the median eminence and to display seasonal structural changes. Seasonal changes in tanycyte morphology might be mediated either through melatonin or sex steroids. Therefore, we analyzed the effects of photoperiod, melatonin, and sex steroids 1) on tanycyte vimentin expression by immunohistochemistry and 2) on the expression of the neural cell adhesion molecule (NCAM) and polysialic acid as markers of brain plasticity. Vimentin immunostaining was reduced in tanycyte cell bodies and processes in SP. Similarly, tanycytes and their processes contained lower amounts of NCAM in SP. These changes induced by SP exposure could not be restored to long photoperiod (LP) levels by testosterone supplementation. Likewise, castration in LP did not affect tanycyte vimentin or NCAM expression. By contrast, late afternoon melatonin injections mimicking a SP-like melatonin peak in LP hamsters reduced vimentin and NCAM expression. Thus, the seasonal changes in vimentin and NCAM expression in tanycytes are regulated by melatonin independently of seasonal sex steroid changes.     

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.     

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.     

Hypothalamic thyroid hormone catabolism acts as a gatekeeper for the seasonal control of body weight and reproduction

Seasonal adaptations in physiology exhibited by many animals involve an interface between biological timing and specific neuroendocrine systems, but the molecular basis of this interface is unknown. In this study of Siberian hamsters, we show that the availability of thyroid hormone within the hypothalamus is a key determinant of seasonal transitions. The expression of the gene encoding type III deiodinase (Dio3) and Dio3 activity in vivo (catabolism of T(4) and T(3)) is dynamically and temporally regulated by photoperiod, consistent with the loss of hypothalamic T(3) concentrations under short photoperiods. Chronic replacement of T(3) in the hypothalamus of male hamsters exposed to short photoperiods, thus bypassing synthetic or catabolic deiodinase enzymes located in cells of the ependyma of the third ventricle, prevented the onset of short-day physiology: hamsters maintained a long-day body weight phenotype and failed to undergo testicular and epididymal regression. However, pelage moult to a winter coat was not affected. Type II deiodinase gene expression was not regulated by photoperiod in these hamsters. Collectively, these data point to a pivotal role for hypothalamic DIO3 and T(3) catabolism in seasonal cycles of body weight and reproduction in mammals.     

Pinealectomy and Constant Release of Melatonin or 5-Methoxytryptamine Induce Testicular Atrophy in the European Hamster (Cricetus cricetus, L.)

The role of the pineal and of photoperiod in the regulation of gonadal activity has been studied in the European hamster, a wild hamster commonly found in the vicinity of Strasbourg, France. Besides the presence of a probable endogenous circannual rhythm in reproductive capacities, it appears that, as in all other hamster species studied to date, the sexual activity of this animal is directly regulated by changes in the photoperiodic environment.
Pinealectomy as well as a constant release of melatonin or of 5-methoxytryptamine has no effect on the gonadal atrophy induced by short photoperiod, but induces testicular regression in animals kept under long photoperiod. The pineal in this species appears thus to be involved in the maintenance of sexual activity in long photoperiod, while it is apparently not required for the gonadal inhibitory effect of short photoperiod. These results are similar to those obtained in the Turkish hamster. 5-Methoxytryptophol is not implicated in the phenomenon of synchronization of the sexual activity with the photoperiodic changes.     

Interactive Effects of Stress and Photoperiod History on Gonadal Condition in Male Syrian Hamsters

A study of the effects of melatonin injections given to male Syrian hamsters (Mesocricetus auratus) late in the light phase in a 14L:10D photoperiod included control, oil-injected hamsters that had been transferred from a 16L:8D photoperiod. Many oil-injected hamsters underwent gonadal regression under these conditions. A literature review indicated that endocrine effects of 'control' injections are fairly common but that they might depend on previous photoperiodic history. A second study found that hamsters born and raised in 16L:8D had larger testes at 150 days of age than those raised in 14L:10D. Transfer from 16L:8D to 14L:10D caused some unhandled hamsters to show gonadal regression while transfer to 14L:10D combined with daily oil injections caused most hamsters to undergo gonadal regression. Injections in animals maintained in 14L:10D throughout the study did not cause gonadal regression. These results indicate that stress effects can confound interpretation of drug treatments that require daily injections. They also demonstrate that the endocrine system of male Syrian hamsters distinguishes two photoperiods that are longer than the critical daylength (12.5 h). The effects of shortening daylengths may be potentiated by environmental stressors; together these may trigger gonadal regression at variable annual phases in anticipation of the critical daylength.     

Decoding photoperiodic time through Per1 and ICER gene amplitude

The mammalian Per1 gene is expressed in the suprachiasmatic nucleus of the hypothalamus, where it is thought to play a critical role in the generation of circadian rhythms. Per1 mRNA also is expressed in other tissues. Its expression in the pars tuberalis (PT) of the pituitary is noteworthy because, like the suprachiasmatic nucleus, it is a known site of action of melatonin. The duration of the nocturnal melatonin signal encodes photoperiodic time, and many species use this to coordinate physiological adaptations with the yearly climatic cycle. This study reveals how the duration of photoperiodic time, conveyed through melatonin, is decoded as amplitude of Per1 and ICER (inducible cAMP early repressor) gene expression in the PT. Syrian hamsters display a robust and transient peak of Per1 and ICER gene expression 3 h after lights-on (Zeitgeber time 3) in the PT, under both long (16 h light/8 h dark) and short (8 h light/16 h dark) photoperiods. However, the amplitude of these peaks is greatly attenuated under a short photoperiod. The data show how amplitude of these genes may be important to the long-term measurement of photoperiodic time intervals.     

Hormonal Modulation of Pineal-Mediated Seasonal Events: Effects of Thyroid or Gonadal Manipulations

The ability of various manipulations of the endocrine system to affect pineal-mediated events was examined in the present studies. Male Syrian hamsters were analyzed for pineal-induced gonadal regression and depressions in serum thyroxine and testosterone levels after treatments which altered thyroid or gonadal function. Hamsters were thyroidectomized, received thyroxine implants (5 mg), or were thyroidectomized plus implanted with thyroxine. The animals were exposed to short photoperiods (10L:14D) for 9 wk, and plasma hormone levels and gonadal status were determined at the end of the experimental period. Likewise, hamsters were castrated, received testosterone implants (5 mg), or were castrated plus implanted with testosterone, and subsequently were exposed to short photoperiods for 9 wk. These animals' responses to photoperiod exposure were compared to animals which received identical hormonal treatments but remained in long photoperiod (14L:10D). All of the hamsters responded to the photoperiodic treatments equally, regardless of the hormonal treatment. The results of these studies indicate that experimentally induced alterations in plasma thyroxine or testosterone levels are unable to prevent or attenuate the ability of the pineal to elicit gonadal regression in response to short photoperiod exposure.     

Chronic administration of melatonin induces changes in porphyrins and in the histology of male and female hamster Harderian gland: Interrelation with the gonadal status

In this paper, we have investigated the influence of melatonin on the histology and porphyrin content of the Syrian hamster Harderian glands. Daily afternoon injections of 25 micrograms of melatonin to female hamsters for 12 weeks resulted in the discontinuity of estrous cyclicity, a marked decrease in the Harderian gland intraluminal area occupied by porphyrins, and in a significant rise in the number of Type II cells. A similar decrease in porphyrins was observed after 8 weeks of ovariectomy. However, if the melatonin injections were given for only 8 weeks (without inducing gonadal atrophy), no changes were observed in the area occupied by intraluminal porphyrins, suggesting that the effects of melatonin in female Syrian hamsters might be associated with the subsequent gonadal atrophy. Castration of male hamsters induced a significant increase in porphyrins and a clear drop in the number of Type II cells. These changes were totally prevented when melatonin was administered daily from the day of castration. Our results suggest that melatonin, at least in male Syrian hamsters, plays a role in Harderian metabolism, acting directly on the Harderian secretory cells or indirectly through pituitary hormones.