Environmental disruption of reproductive rhythms.

Reproduction is a key biological function requiring a precise synchronization with annual and daily cues to cope with environmental fluctuations. Therefore, humans and animals have developed well-conserved photoneuroendocrine pathways to integrate and process daily and seasonal light signals within the hypothalamic-pituitary-gonadal axis. However, in the past century, industrialization and the modern 24/7 human lifestyle have imposed detrimental changes in natural habitats and rhythms of life. Indeed, exposure to an excessive amount of artificial light at inappropriate timing because of shift work and nocturnal urban lighting, as well as the ubiquitous environmental contamination by endocrine-disrupting chemicals, threaten the integrity of the daily and seasonal timing of biological functions. Here, we review recent epidemiological, field and experimental studies to discuss how light and chemical pollution of the environment can disrupt reproductive rhythms by interfering with the photoneuroendocrine timing system.

Induced-ovulation in female dromedary camel involves kisspeptin neuron activation by ß nerve growth factor†.

The dromedary camel (Camelus dromedarius) is a short-day desert breeder in which female ovulation is induced by mating. Current data indicate that male-induced ovulation is triggered by its seminal plasma nerve growth factor beta (ß-NGF), but the exact mechanisms involved in the induction of ovulation are still unknown. In this study, we report that an intramuscular injection of ß-NGF in sexually active short-day-adapted female camels induces an ovulation attested by a surge of circulating LH (2-6 h after treatment) followed by an oocyte release with its cumulus oophorus (confirmed by ultrasonography 72 h after treatment) and a large and progressive increase in circulating progesterone (significant from the 2nd to the 10th days after ß-NGF injection). In addition, this ß-NGF treatment induces a broad nuclear c-FOS activation in cells located in various hypothalamic areas, notably the preoptic area, the arcuate nucleus, the dorso- and ventromedial hypothalamus, the paraventricular nucleus, and the supraoptic nucleus. A double immunostaining with neuropeptides known to be involved in the central control of reproduction indicates that ~28% kisspeptin neurons and 43% GnRH neurons in the proptic area, and ~10% RFRP-3 neurons in the dorso- and ventromedial hypothalamus are activated following ß-NGF injection. In conclusion, our study demonstrates that systemic ß-NGF induces ovulation in the female dromedary camel and indicates that this effect involves the central activation of hypothalamic neurons, notably the kisspeptin neurons.

Thyroid hormone receptors are required for the melatonin-dependent control of Rfrp gene expression in mice.

Mammals adapt to seasons using a neuroendocrine calendar defined by the photoperiodic change in the nighttime melatonin production. Under short photoperiod, melatonin inhibits the pars tuberalis production of TSHß, which, in turn, acts on tanycytes to regulate the deiodinase 2/3 balance resulting in a finely tuned seasonal control of the intra-hypothalamic thyroid hormone T3. Despite the pivotal role of this T3 signaling for synchronizing reproduction with the seasons, T3 cellular targets remain unknown. One candidate is a population of hypothalamic neurons expressing Rfrp, the gene encoding the RFRP-3 peptide, thought to be integral for modulating rodent's seasonal reproduction. Here we show that nighttime melatonin supplementation in the drinking water of melatonin-deficient C57BL/6J mice mimics photoperiodic variations in the expression of the genes Tshb, Dio2, Dio3, and Rfrp, as observed in melatonin-proficient mammals. Notably, we report that this melatonin regulation of Rfrp expression is no longer observed in mice carrying a global mutation of the T3 receptor, TRα, but is conserved in mice with a selective neuronal mutation of TRα. In line with this observation, we find that TRα is widely expressed in the tanycytes. Altogether, our data demonstrate that the melatonin-driven T3 signal regulates RFRP-3 neurons through non-neuronal, possibly tanycytic, TRα.

A refined method to monitor arousal from hibernation in the European hamster.

BACKGROUND: Hibernation is a physiological and behavioural adaptation that permits survival during periods of reduced food availability and extreme environmental temperatures. This is achieved through cycles of metabolic depression and reduced body temperature (torpor) and rewarming (arousal). Rewarming from torpor is achieved through the activation of brown adipose tissue (BAT) associated with a rapid increase in ventilation frequency. Here, we studied the rate of rewarming in the European hamster (Cricetus cricetus) by measuring both BAT temperature, core body temperature and ventilation frequency. RESULTS: Temperature was monitored in parallel in the BAT (IPTT tags) and peritoneal cavity (iButtons) during hibernation torpor-arousal cycling. We found that increases in brown fat temperature preceded core body temperature rises by approximately 48 min, with a maximum re-warming rate of 20.9℃*h-1. Re-warming was accompanied by a significant increase in ventilation frequency. The rate of rewarming was slowed by the presence of a spontaneous thoracic mass in one of our animals. Core body temperature re-warming was reduced by 6.2℃*h-1 and BAT rewarming by 12℃*h-1. Ventilation frequency was increased by 77% during re-warming in the affected animal compared to a healthy animal. Inspection of the position and size of the mass indicated it was obstructing the lungs and heart. CONCLUSIONS: We have used a minimally invasive method to monitor BAT temperature during arousal from hibernation illustrating BAT re-warming significantly precedes core body temperature re-warming, informing future study design on arousal from hibernation. We also showed compromised re-warming from hibernation in an animal with a mass obstructing the lungs and heart, likely leading to inefficient ventilation and circulation.

Daily and Estral Regulation of RFRP-3 Neurons in the Female Mice.

Female reproductive success relies on proper integration of circadian- and ovarian- signals to the hypothalamic-pituitary-gonadal axis in order to synchronize the preovulatory LH surge at the end of the ovarian follicular stage with the onset of the main active period. In this study, we used a combination of neuroanatomical and electrophysiological approaches to assess whether the hypothalamic neurons expressing Arg-Phe amide-related peptide (RFRP-3), a gonadotropin inhibitory peptide, exhibit daily and estrous stage dependent variations in female mice. Furthermore, we investigated whether arginine vasopressin (AVP), a circadian peptide produced by the suprachiamatic nucleus regulates RFRP-3 neurons. The number of c-Fos-positive RFRP-3 immunoreactive neurons is significantly reduced at the day-to-night transition with no difference between diestrus and proestrus. Contrastingly, RFRP neuron firing rate is higher in proestrus as compared to diestrus, independently of the time of the day. AVP immunoreactive fibers contact RFRP neurons with the highest density observed during the late afternoon of diestrus and proestrus. Application of AVP increases RFRP neurons firing in the afternoon (ZT6-10) of diestrus, but not at the same time point of proestrus, indicating that AVP signaling on RFRP neurons may depend on circulating ovarian steroids. Together, these studies show that RFRP neurons integrate both daily and estrogenic signals, which downstream may help to properly time the preovulatory LH surge.

A Kiss to drive rhythms in reproduction.

Reproduction, like many other biological functions, exhibits marked daily and seasonal rhythms in order to anticipate and adapt breeding activity to environmental challenges. In recent years, studies investigating the neuroendocrine mechanisms driving rhythms in reproduction have unveiled the pivotal role of hypothalamic neurons expressing kisspeptin in integrating and forwarding daily and seasonal cues to the reproductive system. The objective of this review is to summarize the knowledge on the effect and role of this neuropeptide on the mammalian hypothalamo-pituitary-gonadal axis and describe how it is involved in the daily control of ovulation in females and long-term adaptation of reproduction in seasonal breeders.

Photoperiodic regulation in a wild-derived mouse strain.

Mus musculus molossinus (MSM) is a wild-derived mouse strain which maintains the ability to synthesize melatonin in patterns reflecting the ambient photoperiod. The objective of this study was to characterize the effects of photoperiodic variation on metabolic and reproductive traits, and the related changes in pituitary-hypothalamic gene expression in MSM mice. MSM mice were kept in long (LP) or short photoperiod (SP) for 6 weeks. Our results demonstrate that MSM mice kept in LP, as compared to mice kept in SP, display higher expression of genes encoding thyrotropin (TSH) in the pars tuberalis, thyroid hormone deiodinase 2 (dio2) in the tanycytes, RFamide-related peptide (RFRP3) in the hypothalamus and lower expression of dio3 in the tanycytes, along with larger body and reproductive organ mass. Additionally, to assess the effects of the gestational photoperiodic environment on the expression of these genes, we kept MSM mice in LP or SP from gestation and studied offspring. We show that the gestational photoperiod affects the TSH/dio pathway in newborn MSM mice in a similar way to adults. This result indicates a transgenerational effect of photoperiod from the mother to the fetus in utero. Overall, these results indicate that photoperiod can influence neuroendocrine regulation in a melatonin-proficient mouse strain, in a manner similar that documented in other seasonal rodent species. MSM mice may therefore become a useful model for research into the molecular basis of photoperiodic regulation of seasonal biology.

Photoperiodic regulation in a wild-derived mouse strain.

MSM/Ms (MSM) is a mouse strain derived from Japanese wild mice, Mus musculus molossinus, that maintains the ability to synthesize melatonin in patterns reflecting the ambient photoperiod. The objective of this study was to characterize the effects of photoperiodic variation on metabolic and reproductive traits, and the related changes in pituitary-hypothalamic gene expression in MSM mice. MSM mice were kept in long (LP) or short photoperiod (SP) for 6 weeks. Our results demonstrate that MSM mice kept in LP, as compared with mice kept in SP, display higher expression of genes encoding thyrotropin (TSH) in the pars tuberalis, thyroid hormone deiodinase 2 (dio2) in the tanycytes and RFamide-related peptide (RFRP3) in the hypothalamus, and lower expression of dio3 in the tanycytes, along with larger body and reproductive organ mass. Additionally, to assess the effects of the gestational photoperiodic environment on the expression of these genes, we kept MSM mice in LP or SP from gestation and studied their offspring. We show that the gestational photoperiod affects the TSH/dio pathway in newborn MSM mice in a similar way to adults. This result indicates a transgenerational effect of photoperiod from the mother to the fetus in utero Overall, these results indicate that photoperiod can influence neuroendocrine regulation in a melatonin-proficient mouse strain, in a manner similar to that documented in other seasonal rodent species. MSM mice may therefore become a useful model for research into the molecular basis of photoperiodic regulation of seasonal biology.

Maternal photoperiod programs hypothalamic thyroid status via the fetal pituitary gland.

In wild mammals, offspring development must anticipate forthcoming metabolic demands and opportunities. Within species, different developmental strategies may be used, dependent on when in the year conception takes place. This phenotypic flexibility is initiated before birth and is linked to the pattern of day length (photoperiod) exposure experienced by the mother during pregnancy. This programming depends on transplacental communication via the pineal hormone melatonin. Here, we show that, in the Siberian hamster (Phodopus sungorus), the programming effect of melatonin is mediated by the pars tuberalis (PT) of the fetal pituitary gland, before the fetal circadian system and autonomous melatonin production is established. Maternal melatonin acts on the fetal PT to control expression of thyroid hormone deiodinases in ependymal cells (tanycytes) of the fetal hypothalamus, and hence neuroendocrine output. This mechanism sets the trajectory of reproductive and metabolic development in pups and has a persistent effect on their subsequent sensitivity to the photoperiod. This programming effect depends on tanycyte sensitivity to thyroid stimulating hormone (TSH), which is dramatically and persistently increased by short photoperiod exposure in utero. Our results define the role of the fetal PT in developmental programming of brain function by maternal melatonin and establish TSH signal transduction as a key substrate for the encoding of internal calendar time from birth to puberty.

Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2.

For many years, it was of interest to identify the sequences encoding the two melatonin receptors (MT1 and MT2) from various species. After publishing the basic molecular characterization of the human, rat, mouse, sheep, and platypus MT1, MT2, or Mel1c receptors, we began cloning the genes from other animals, such as birds, bats, and vipers. The goal was to advance the receptor crystallization, which could greatly contribute the understanding of the sequence/stability relationship. European hamster MT1 receptor was cloned for the first time from this gender, was expressed in stable form in cells, and its binding characterized with a sample of 19 melatonin ligands. Siberian hamster (Phodopus sungorus) expresses a non-functional MT2. We observed that unlike this hamster, the European hamster (Cricetus cricetus) does not have a stop codon in the MT2 sequence. Thus, we undertook the tedious task of cloning the MT2 receptor. We partially succeeded, sequencing the complete exon 2 and a fragment of exon 1 (from putative amino acids 12 to 38 and 77 to 323), after several years of efforts. In order to show that the protein parts we cloned were capable to sustain some binding capacities, we designed a chimeric MT2 receptor using a consensus sequence to replace the unknown amino acids, based on other small rodent MT2 sequences. This chimeric construct could bind melatonin in the nanomolar range. This work is meant to be the basis for attempts from other laboratories of the community to determine the complete natural sequence of the European hamster MT2 receptor. The present work is the first to show that, among the hamsters, if the Siberian is a natural knockout for MT2, the European one is not.

TSH restores a summer phenotype in photoinhibited mammals via the RF-amides RFRP3 and kisspeptin.

In mammals, melatonin is the pivotal messenger synchronizing biological functions, notably reproductive activity, with annual daylength changes. Recently, two major findings clarified melatonin's mode of action. First, melatonin controls the production of thyroid stimulating hormone (TSH) by the pars tuberalis of the adenohypophysis. This TSH regulates local thyroid hormone availability in the mediobasal hypothalamus. Second, the RF-amides kisspeptin and RFRP-3, recently discovered regulators of the gonadotropic axis, are involved in the melatonin control of reproduction. This study aims to establish a mechanistic link between the melatonin-driven TSH and the RF-amide control of reproduction. We treated short-day-adapted male Djungarian and Syrian hamsters with a chronic central infusion of TSH. In both hamster species, the central administration of 5 mIU/d TSH for 4 to 6 wk restored the summer phenotype of both testicular activity and kisspeptin and RFRP expression. Vehicle treated hamsters remain sexually inactive. Furthermore, the TSH treatment increased the body weight of lean short-day-adapted Djungarian hamsters and reduced hypothalamic somatostatin expression to the summer phenotype. In summary, our study demonstrates the pivotal role of melatonin-driven TSH for the seasonal regulation of reproduction and body weight, and uncovers the neuropeptides relaying this signal within the hypothalamus.

Hypothalamic tanycytes as mediators of maternally programmed seasonal plasticity.

In mammals, maternal photoperiodic programming (MPP) provides a means whereby juvenile development can be matched to forthcoming seasonal environmental conditions.1,2,3,4 This phenomenon is driven by in utero effects of maternal melatonin5,6,7 on the production of thyrotropin (TSH) in the fetal pars tuberalis (PT) and consequent TSH receptor-mediated effects on tanycytes lining the 3rd ventricle of the mediobasal hypothalamus (MBH).8,9,10 Here we use LASER capture microdissection and transcriptomic profiling to show that TSH-dependent MPP controls the attributes of the ependymal region of the MBH in juvenile animals. In Siberian hamster pups gestated and raised on a long photoperiod (LP) and thereby committed to a fast trajectory for growth and reproductive maturation, the ependymal region is enriched for tanycytes bearing sensory cilia and receptors implicated in metabolic sensing. Contrastingly, in pups gestated and raised on short photoperiod (SP) and therefore following an over-wintering developmental trajectory with delayed sexual maturation, the ependymal region has fewer sensory tanycytes. Post-weaning transfer of SP-gestated pups to an intermediate photoperiod (IP), which accelerates reproductive maturation, results in a pronounced shift toward a ciliated tanycytic profile and formation of tanycytic processes. We suggest that tanycytic plasticity constitutes a mechanism to tailor metabolic development for extended survival in variable overwintering environments.

Reciprocal Interactions between Circadian Clocks, Food Intake, and Energy Metabolism.

Like other biological functions, food intake and energy metabolism display daily rhythms controlled by the circadian timing system that comprises a main circadian clock and numerous secondary clocks in the brain and peripheral tissues. Each secondary circadian clock delivers local temporal cues based on intracellular transcriptional and translational feedback loops that are tightly interconnected to intracellular nutrient-sensing pathways. Genetic impairment of molecular clocks and alteration in the rhythmic synchronizing cues, such as ambient light at night or mistimed meals, lead to circadian disruption that, in turn, negatively impacts metabolic health. Not all circadian clocks are sensitive to the same synchronizing signals. The master clock in the suprachiasmatic nuclei of the hypothalamus is mostly synchronized by ambient light and, to a lesser extent, by behavioral cues coupled to arousal and exercise. Secondary clocks are generally phase-shifted by timed metabolic cues associated with feeding, exercise, and changes in temperature. Furthermore, both the master and secondary clocks are modulated by calorie restriction and high-fat feeding. Taking into account the regularity of daily meals, the duration of eating periods, chronotype, and sex, chrononutritional strategies may be useful for improving the robustness of daily rhythmicity and maintaining or even restoring the appropriate energy balance.

GnRH and the photoperiodic control of seasonal reproduction: Delegating the task to kisspeptin and RFRP-3.

Synchronization of mammalian breeding activity to the annual change of photoperiod and environmental conditions is of the utmost importance for individual survival and species perpetuation. Subsequent to the early 1960s, when the central role of melatonin in this adaptive process was demonstrated, our comprehension of the mechanisms through which light regulates gonadal activity has increased considerably. The current model for the photoperiodic neuroendocrine system points to pivotal roles for the melatonin-sensitive pars tuberalis (PT) and its seasonally-regulated production of thyroid-stimulating hormone (TSH), as well as for TSH-sensitive hypothalamic tanycytes, radial glia-like cells located in the basal part of the third ventricle. Tanycytes respond to TSH through increased expression of thyroid hormone (TH) deiodinase 2 (Dio2), which leads to heightened production of intrahypothalamic triiodothyronine (T3) during longer days of spring and summer. There is strong evidence that this local, long-day driven, increase in T3 links melatonin input at the PT to gonadotropin-releasing hormone (GnRH) output, to align breeding with the seasons. The mechanism(s) through which T3 impinges upon GnRH remain(s) unclear. However, two distinct neuronal populations of the medio-basal hypothalamus, which express the (Arg)(Phe)-amide peptides kisspeptin and RFamide-related peptide-3, appear to be well-positioned to relay this seasonal T3 message towards GnRH neurons. Here, we summarize our current understanding of the cellular, molecular and neuroendocrine players, which keep track of photoperiod and ultimately govern GnRH output and seasonal breeding.

Age-dependent change of RFRP-3 neuron numbers and innervation in female mice.

In female mammals, reproductive senescence is a complex process involving progressive ovarian dysfunction, associated with altered central control of the hypothalamic-pituitary-gonadal axis and desynchronization of the circadian system. The objective of this study was to investigate age-dependent changes in the daily regulation of Arg-Phe amide-related peptide-3 (RFRP-3), a hypothalamic peptide involved in reproduction, in female C57BL/6 J mice of different age groups (4, 13, and 19 months old) sampled at their diestrus stage. We found an age-dependent decrease in the total number of RFRP-3 neurons and in the relative number of activated (i.e. c-Fos-positive) RFRP-3 neurons. RFRP-3 neuronal activation exhibited a daily variation in young and middle-aged mice, which was abolished in 19-month-old mice. We also found a daily variation in the number of RFRP-3 neurons receiving close vasopressin (AVP)- and vasoactive intestinal peptide (VIP)-ergic fiber appositions in mice aged 4 and 13 months, but not in 19-month-old mice. However, we found no daily or age-dependent changes in the AVP and VIP fiber density in the dorsomedial hypothalamus. Plasma LH levels were similar in mice aged 4 and 13 months, but were markedly increased in 19-month-old mice. The present findings indicate that the number of RFRP-3 positive neurons is downregulated during old age and that the daily changes in their innervation by the circadian peptides AVP and VIP are abolished. This age-associated reduced (rhythmic) activity of the inhibitory RFRP-3 system could be implicated in the elevated LH secretion observed during reproductive senescence.

A noradrenergic sensitive endogenous clock is present in the rat pineal gland.

The aim of this study was to examine the occurrence of endogenous oscillations of Per1, Per2, Bmal1 and Rev-erbα genes in rat pineal explants and to investigate their regulation by adrenergic ligands. Our results show a significant and sustained rhythm of Per2,Bmal1 and Rev-erbα gene expression for up to 48 h in cultured pineal gland with a pattern similar to that observed in vivo. By contrast, the rhythms of Per1 and Aa-nat, the rate-limiting enzyme for melatonin synthesis, were strongly attenuated after 24 h in culture. Addition of the exogenous adrenergic agonist isoproterenol on cultured pineal glands induced a short-term increase in mRNA levels of Per1 and Aa-nat, but not those of Per2,Bmal1 and Rev-erbα. This study demonstrates that the rat pineal gland hosts a circadian oscillator as evidenced by the sustained, noradrenergic-independent, endogenous oscillations of Per2, Bmal1 and Rev-erbα mRNA levels in cultured tissues. Only expression of Per1 was stimulated by adrenergic ligands suggesting that, in vivo, the adrenergic input could synchronize the pineal clock by acting selectively on Per1.

WITHDRAWN: Age-dependent modulation of RFRP-3 neurons in female mice.

This article has been withdrawn: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been withdrawn at the request of the editor and publisher. The publisher regrets that an error occurred which led to the premature publication of this paper. This error bears no reflection on the article or its authors. The publisher apologizes to the authors and the readers for this unfortunate error.

Role of central kisspeptin and RFRP-3 in energy metabolism in the male Wistar rat.

Kisspeptin (Kp) and (Arg)(Phe) related peptide 3 (RFRP-3) are two RF-amides acting in the hypothalamus to control reproduction. In the past 10 years, it has become clear that, apart from their role in reproductive physiology, both neuropeptides are also involved in the control of food intake, as well as glucose and energy metabolism. To investigate further the neural mechanisms responsible for these metabolic actions, we assessed the effect of acute i.c.v. administration of Kp or RFRP-3 in ad lib. fed male Wistar rats on feeding behaviour, glucose and energy metabolism, circulating hormones (luteinising hormone, testosterone, insulin and corticosterone) and hypothalamic neuronal activity. Kp increased plasma testosterone levels, had an anorexigenic effect and increased lipid catabolism, as attested by a decreased respiratory exchange ratio (RER). RFRP-3 also increased plasma testosterone levels but did not modify food intake or energy metabolism. Both RF-amides increased endogenous glucose production, yet with no change in plasma glucose levels, suggesting that these peptides provoke not only a release of hepatic glucose, but also a change in glucose utilisation. Finally, plasma insulin and corticosterone levels did not change after the RF-amide treatment. The Kp effects were associated with an increased c-Fos expression in the median preoptic area and a reduction in pro-opiomelanocortin immunostaining in the arcuate nucleus. No effects on neuronal activation were found for RFRP-3. Our results provide further evidence that Kp is not only a very potent hypothalamic activator of reproduction, but also part of the hypothalamic circuit controlling energy metabolism.

Identification of an N-acylated-DArg-Leu-NH2 Dipeptide as a Highly Selective Neuropeptide FF1 Receptor Antagonist That Potently Prevents Opioid-Induced Hyperalgesia.

RFamide-related peptide-3 (RFRP-3) and neuropeptide FF (NPFF) target two different receptor subtypes called neuropeptide FF1 (NPFF1R) and neuropeptide FF2 (NPFF2R) that modulate several functions. However, the study of their respective role is severely limited by the absence of selective blockers. We describe here the design of a highly selective NPFF1R antagonist called RF3286, which potently blocks RFRP-3-induced hyperalgesia in mice and luteinizing hormone release in hamsters. We then showed that the pharmacological blockade of NPFF1R in mice prevents the development of fentanyl-induced hyperalgesia while preserving its analgesic effect. Altogether, our data indicate that RF3286 represents a useful pharmacological tool to study the involvement of the NPFF1R/RFRP-3 system in different functions and different species. Thanks to this compound, we showed that this system is critically involved in the development of opioid-induced hyperalgesia, suggesting that NPFF1R antagonists might represent promising therapeutic tools to improve the use of opioids in the treatment of chronic pain.