A putative new melatonin binding site in sheep brain, MTx: preliminary observations and characteristics.

In mammals, MT1 and MT2 melatonin receptors are high affinity G protein-coupled receptors and are thought to be involved in the integration of the melatonin signaling throughout the brain and periphery. In the present study, we describe a new melatonin binding site, named MTx, with a peculiar pharmacological profile. This site had a low affinity for 2-[125I]-melatonin in saturation assays in hypothalamus and retina (pKD = 9.13 {plus minus} 0.05, Bmax = 1.12 {plus minus} 0.11 fmol/mg protein and pKD = 8.81 {plus minus} 0.50, Bmax = 7.65 {plus minus} 2.64 fmol/mg protein, respectively) and a very high affinity, in competition assays, for melatonin (pKi = 13.08 {plus minus} 0.18), and other endogenous compounds. Using autoradiography, we showed a preferential localization of the MTx in periventricular areas of the sheep brain, with a density 3 to 8 times higher than those observed for ovine MT1 In addition, using a set of well-characterized ligands, we showed that this site did not correspond to any of the following receptors: MT1, MT2, MT3 , D1, D2, noradrenergic, nor 5-HT2 Based on its affinity for melatonin, MTx did not seem to be implicated in the integration of cerebral melatonin concentration variations since they were saturating for MTx. Nevertheless, it remained of prime importance because of its periventricular distribution, in close contact with the CSF, and its peculiar pharmacological profile responding to both melatoninergic and serotoninergic compounds. Significance Statement Herein a putative new melatonin binding site is described in sheep brain parts in close contact with the 3rd ventricle. The characteristics of the pharmacological profile of this site is different from anything previously reported in the literature. The present work forms the basis of future full pharmacological characterization.

Rapid effects of melatonin on hormonal and behavioral stressful responses in ewes.

Sheep are gregarious mammals with complex social interactions. As such, they are very sensitive to social isolation and constitute a relevant animal model to study specifically the biological consequences of social stress. We examined previously the behavioral and endocrine responses in ewes isolated socially in the familiar conspecific withdrawal model (FCW) and showed that stressful responses increased and maintenance behaviors decreased, confirming that social isolation is a strong stressor in sheep. Melatonin synchronizes seasonal and circadian rhythms; and several studies reported its implication in cognitive processes as emotion. Here we investigated its role in the modulation of social stressful responses. Firstly, we studied ewes in the FCW model during the day (characterized by low melatonin levels) and the night (characterized by high melatonin levels). We found lower stressful responses (significant lower levels of cortisol plasma, number of foot pawings, of circling attempts) during the night as compared to the day. To investigate whether these effects were due to melatonin or to darkness, we submitted ewes to FCW during the night with lights on, a condition that suppresses melatonin secretion. Ewes infused with melatonin under these conditions showed decreased stressful responses (significant lower levels cortisol plasma, number of vocalizations, time spent with the head out of the cage) as compared to ewes infused with saline. These findings demonstrate that melatonin diminishes the endocrine and behavioral impact of social isolation in ewes and support the idea that melatonin has a calming effect in socially stressful situations.

The luteal outcome of anoestrus ewes induced to ovulate by the male effect is not related to the population of ovarian antral follicles before male exposure.

The ovarian status and its relationship with the response to the male effect were studied in Ile-de-France ewes entering anoestrus early (becoming anovulatory on January-February, n=13) or late (becoming anovulatory on March, n=13). The male effect was performed, in each group of ewes, at the beginning of the anoestrus season (March-April), approximately 35 days after ewes became anovulatory. Transrectal ultrasonography of ovaries was done at D-7, D-5, D-3 and D0 (ram introduction day) to examine the number and size of follicles ≥2mm, from D0 to D4 to analyze the ram-induced preovulatory follicles and at D14-D16 to identify luteal structures. Plasmatic progesterone level was assessed from D-7 to D14-16 to examine the ovulatory response to the male effect. Before ram introduction, the number of medium (3.5-4.4mm) and large (>4.4mm) follicles and the maximum follicle diameter were lower (p<0.05) in ewes entering anoestrus early than in ewes entering it late. The percentage of ewes developing a short cycle at the first ram-induced ovulation was higher in those starting anoestrus early (92% vs 31%; p<0.05); normal cycles were only observed in ewes entering anoestrus late (0% vs 54%; p<0.05). The time of the onset of anoestrus did not affect (p>0.05) the ram-induced preovulatory follicle characteristics; these parameters were similar (p>0.05) between ewes developing a short or a normal cycle. Results did not show any relationship between the ovarian status preceding male introduction and the growing dynamic of the ram-induced preovulatory follicles or the category of cycle (normal or short) displayed following ovulation. In conclusion, (1) the luteal outcome following the first ovulation induced by the male effect depends on the time of onset of seasonal anoestrus and (2) the number and size of follicles ≥2mm also depend on the time of onset of seasonal anoestrus but are not related to the luteal outcome following the first ovulation induced by the male effect.

Enhancement of the male effect on reproductive performance in female Mediterranean goats with long day and/or melatonin treatment.

The aim of this study was to determine whether the male effect on reproductive performance in female Mediterranean goats could be enhanced if used in combination with long day and/or melatonin treatments. Thirty-two does were exposed to long days (16 h of light/day) for 95 days. At the end of this period, 16 received one melatonin implant (group LD-M) and 16 did not (group LD). Simultaneously, 16 does under natural photoperiod were implanted with melatonin (group M) or given no treatment (group C, n=16). On April 14th, after 49 days of isolation from males, all does were exposed to bucks fitted with marking harnesses. Oestrous activity, as detected by visual observation, was recorded daily. Transrectal ultrasonography was used to record ovulation, and ovarian activity confirmed by determining plasma progesterone concentration. Oestrous activity, ovulation and fertility were greater in all treatment groups compared with group C (P<0.05), but no differences in ovulation rate or litter size were observed (P>0.05). Productivity (mean number of kids per female) of the LD-M and M groups was significantly greater than in group C (1.50 ± 0.01 and 1.37 ± 0.01 vs. 0.69 ± 0.01 kids, respectively) (P<0.05). Combining melatonin treatment with natural or artificial long days adds to the impact of the male effect.

The interaction between photoperiod and nutrition and its effects on seasonal rhythms of reproduction in the ewe.

In sheep, the seasonal patterns of reproductive activity are driven primarily by the annual photoperiodic cycle, but can also respond to other environmental factors, such as nutrition, yet little is known about the mechanisms underlying this interaction. This study was designed to define the interaction between photoperiodic and nutritional cues on seasonal patterns of ovarian activity, and to determine if there is a central interaction between these cues. Groups of Ile-de-France ewes were maintained in two nutritional states (restricted and well fed) under a simulated annual photoperiod of 8-16 h of light per day over two breeding seasons. At the end of the first breeding season, half of the animals of each group were ovariectomized (OVX) and fitted subcutaneously with estradiol implants. Low nutritional status shortened the season of ovarian activity, determined from the pattern of progesterone concentrations, by modifying the timing of seasonal transitions between periods of ovarian activity and anestrus. The same results were observed for the seasonal rhythm of neuroendocrine activity, assessed in the OVX ewes, from the pattern of luteinizing hormone concentrations. These results were then confirmed for neuroendocrine activity induced by a photoperiodic treatment. We conclude that nutrition centrally modulates the interpretation of photoperiod to affect seasonal reproductive transitions. The mechanisms of this interaction are discussed in the paper.

Description of the constitutive activity of cloned human melatonin receptors hMT(1) and hMT(2) and discovery of inverse agonists.

Melatonin receptors have been described to activate different G protein-dependent signaling pathways, both in laboratory, heterologous, cellular models and in physiological conditions. Furthermore, the constitutive activity of G protein-coupled receptors has been shown to be key in physiological and pathological conditions. In the case of melatonin receptors, information is rather scare and concerns only MT1 receptors. In the present report, we show that the G protein-coupled melatonin receptors do have a constitutive, nonmelatonin-induced signaling activity using two cellular models of different origins, the Chinese hamster ovary cell line and Neuro2A, a neuroblastoma cell line. Furthermore, we show that this constitutive activity involves mainly Gi proteins, which is consistent with the common knowledge on the melatonin receptors. Importantly, we also describe, for the first time, inverse agonist properties for melatonin ligands. Although it is clear than more in-depth, biochemistry-based studies will be required to better understand by which pathway(s) the constitutively active melatonin receptors transfer melatonin information into intracellular biochemical events; our data open interesting perspectives for understanding the importance of the constitutive activity of melatonin receptors in physiological conditions.

Molecular pharmacology of the mouse melatonin receptors MT1 and MT2.

The main melatonin receptors are two G-protein coupled receptors named MT(1) and MT(2). Having described the molecular pharmacology of the human versions of these receptors, we turned to two of the three species most useful in studying melatonin physiology: rat and sheep (a diurnal species used to understand the relationship between circadian rhythm and depression). We also employed previously used compounds to describe the mouse melatonin receptors; despite the early cloning of mouse receptors, few molecular pharmacology studies on these receptors exist. To our surprise, we detected no major differences between the data obtained from mice and those from other species.

Multiple exposures to familiar conspecific withdrawal is a novel robust stress paradigm in ewes.

Paradigms used so far to study the effects of social isolation in sheep confound the effects of social isolation with those of other stressors (e.g. new environment) and showed contradictory effects after multiple social isolations. We propose here to characterize and examine the repeatable effects of social isolation induced by the familiar conspecific withdrawal (FCW). This latter test consists of socially isolating the ewe by the removal of group mates from the room test for 3 h. Behavioral and endocrine responses of adult ovariectomized-estradiol implanted ewes were compared 90 min before and 90 min after FCW, which was applied three times every fourteen days. We observed that each FCW induced significant increases in plasma cortisol level, in the number of vocalizations, foot pawing, circling attempts and a significant decrease in time spent lying down. An increase in plasma cortisol levels and decrease in duration of maintenance behaviors were significantly lower after the third FCW than after the first one. These differences could be explained by higher plasma cortisol levels and lower duration of maintenance behaviors before the third FCW than before the first FCW suggesting an anticipation of the social isolation period. These data indicate that social isolation is sufficient to induce distress with stable stressful responses after multiple exposures to familiar conspecific withdrawal.

The effect of nutrition on the neural mechanisms potentially involved in melatonin-stimulated LH secretion in female Mediterranean goats.

This research examines which neural mechanisms among the endogenous opioid, dopaminergic, serotonergic and excitatory amino acid systems are involved in the stimulation of LH secretion by melatonin implantation and their modulation by nutritional level. Female goats were distributed to two experimental groups that received either 1.1 (group H; n=24) or 0.7 (group L; n=24) times their nutritional maintenance requirements. Half of each group was implanted with melatonin after a long-day period. Plasma LH concentrations were measured twice per week. The effects of i.v. injections of naloxone, pimozide, cyproheptadine and N-methyl-d,l-aspartate (NMDA) on LH secretion were assessed the day before melatonin implantation and again on days 30 and 45. The functioning of all but the dopaminergic systems was clearly modified by the level of nutrition, melatonin implantation and time elapsed since implantation. Thirty days after implantation, naloxone increased LH concentrations irrespective of the level of nutrition (P<0.05), similar to NMDA in the melatonin-implanted H goats (HM; P<0.01). On day 45, naloxone increased LH concentrations in the HM animals (P<0.05), similar to cyproheptadine in both the non-implanted H (HC) and the HM animals (P<0.01). Finally, at 45 days, NMDA increased the LH concentration in all subgroups (P<0.01). These results provide evidence that the effects of different neural systems on LH secretion are modified by nutritional level and melatonin implantation. Endogenous opioids seem to be most strongly involved in the inhibition of LH secretion on days 30 and 45 after melatonin implantation. However, the serotonergic mechanism appears to be most influenced by nutritional level.

The effect of an intracerebroventricular injection of metformin or AICAR on the plasma concentrations of melatonin in the ewe: potential involvement of AMPK?

BACKGROUND: It is now widely accepted that AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis. Recently, it has been shown to regulate circadian clocks. In seasonal breeding species such as sheep, the circadian clock controls the secretion of an endogenous rhythm of melatonin and, as a consequence, is probably involved in the generation of seasonal rhythms of reproduction. Considering this, we identified the presence of the subunits of AMPK in different hypothalamic nuclei involved in the pre- and post-pineal pathways that control seasonality of reproduction in the ewe and we investigated if the intracerebroventricular (i.c.v.) injection of two activators of AMPK, metformin and AICAR, affected the circadian rhythm of melatonin in ewes that were housed in constant darkness. In parallel the secretion of insulin was monitored as a peripheral metabolic marker. We also investigated the effects of i.c.v. AICAR on the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream target of AMPK, in brain structures along the photoneuroendocrine pathway to the pineal gland. RESULTS: All the subunits of AMPK that we studied were identified in all brain areas that were dissected but with some differences in their level of expression among structures. Metformin and AICAR both reduced (p < 0.001 and p < 0.01 respectively) the amplitude of the circadian rhythm of melatonin secretion independently of insulin secretion. The i.c.v. injection of AICAR only tended (p = 0.1) to increase the levels of phosphorylated AMPK in the paraventricular nucleus but significantly increased the levels of phosphorylated ACC in the paraventricular nucleus (p < 0.001) and in the pineal gland (p < 0.05). CONCLUSIONS: Taken together, these results suggest a potential role for AMPK on the secretion of melatonin probably acting trough the paraventricular nucleus and/or directly in the pineal gland. We conclude that AMPK may act as a metabolic cue to modulate the rhythm of melatonin secretion.

The role of nutrition in the regulation of luteinizing hormone secretion by the opioidergic, dopaminergic, and serotonergic systems in female Mediterranean goats.

This study examined which neural mechanism (opioid, dopaminergic, or serotonergic system) is involved in the regulation of luteinizing hormone (LH) secretion, with and without nutritional modulation, at different times of the photoperiodic cycle. Goats were randomly distributed into two experimental groups that received either 1.1 (high group; n = 18) or 0.7 (low group; n = 18) times the nutritional maintenance requirements. The goats were exposed to alternations of 3 mo of long days and 3 mo of short days. Plasma LH concentrations were measured twice a week. The effects of intravenous injections of naloxone (endogenous opioid receptor antagonist), pimozide (dopaminergic(2) receptor antagonist), and cyproheptadine (serotonin 5-hydroxytryptamine(2) receptor antagonist) on LH secretion were assessed during challenges in three different photoperiodic situations: the onset of LH stimulation by short days (OnsetSD), the onset of LH inhibition by long days (OnsetLD), and during the LH inhibition by long days (LateLD). The role of the different neural systems was clearly modified by the level of nutrition. In the low-nutrition group, only naloxone increased LH concentrations during onsetLD (P < 0.05). However, in the high-nutrition group, naloxone increased the concentration and pulsatility of LH (P < 0.05) in onsetSD and onsetLD. Pimozide increased LH concentration and pulsatility (P < 0.05) in onsetLD and LH concentration in lateLD (P < 0.001). Finally, cyproheptadine significantly increased LH concentration at all three times (P < 0.001). These results provide evidence that all three systems are involved in the inhibition of LH release in onsetLD, and that the opioid and serotonin mechanisms are involved during the onsetSD that were enhanced by a high plane of nutrition.

Seasonal changes in cell proliferation in the adult sheep brain and pars tuberalis.

To adapt to seasonal variations in the environment, most mammalian species exhibit seasonal cycles in their physiology and behavior. Seasonal plasticity in the structure and function of the central nervous system contributes to the adaptation of this physiology in seasonal mammals. As part of these plasticity mechanisms, seasonal variations in proliferation rate and neuron production have been extensively studied in songbirds. In this report, we investigated whether this type of brain plasticity also occurs in sheep, a seasonal species, by assessing variations in cell proliferation in the sheep diencephalon. We administered the cell birth marker 5'-bromodeoxyuridine (BrdU) to adult female sheep in July and December, during long and short photoperiod, respectively. The BrdU incorporation was analyzed and quantified in the hypothalamus, a key center for neuroendocrine regulations, as well as in other structures involved in relaying neuroendocrine and sensory information, including the median eminence, the pars tuberalis of the pituitary gland, and the thalamus. In December, 2-fold and 6-fold increases in the number of BrdU+ nuclei were observed in the hypothalamus and thalamus, respectively, when compared with July. This variation is independent of the influence of peripheral gonadal estradiol variations. An inverse seasonal regulation of cell proliferation was observed in the pars tuberalis. In contrast, no seasonal variation in cell proliferation was seen in the subventricular zone of the lateral ventricle. Many of the newborn cells in the adult ovine hypothalamus and thalamus differentiate into neurons and glial cells, as assessed by the expression of neuronal (DCX, NeuN) and glial (GFAP, S100B) fate markers. In summary, we show that the estimated cell proliferation rates in the sheep hypothalamus, thalamus, and pars tuberalis are different between seasons. These variations are independent of the seasonal fluctuations of peripheral estradiol levels, unlike the results described in the brain nuclei involved in song control of avian species.

Ovulatory activity of female goats adapted to the subtropics is responsive to photoperiod.

The objective of this study was to determine whether reproductive seasonality of local well-fed female goats from subtropical Mexico (26 degrees N) can be controlled by photoperiod. The control group (n=12) remained in an open pen under natural photoperiod variations. The two experimental groups (n=8 each) were placed in light-proof rooms and exposed for 2 yr to alternations of 3 months of long days and 3 months of short days. One group was first exposed to long days, Group 1, and the other one to short days, Group 2. Blood samples were obtained twice a week to determine ovulation status by progesterone plasma concentrations. Goats from the control group displayed a seasonality of ovulations. The mean (+/-SEM) dates of the onset and end of the ovulations were September 10+/-5 d and February 16+/-4 d, respectively. In contrast, in both experimental groups, ovulations were modified (P<0.05) by the photoperiodic treatments such that ovulations started and ended during short and long days, respectively. In Groups 1 and 2, ovulations started 67+/-2 and 66+/-2 d (P>0.05), respectively, after the transfer from long to short days. In contrast, the timing of the cessation of ovulations after the transfer from short to long days differed (P<0.05) between groups (19+/-3 and 31+/-3 d for Groups 1 and 2, respectively). Local female goats from subtropical Mexico are, therefore, sensitive to photoperiodic changes that they are exposed to at this latitude and this environmental cue may control the timing of the breeding season in natural conditions.

Endogenous circannual rhythm in luteinizing hormone secretion: insight from signal analysis coupled with mathematical modelling.

In sheep, as in many vertebrates, the seasonal pattern of reproduction is timed by the annual photoperiodic cycle, characterized by seasonal changes in the day length. The photoperiodic information is translated into a circadian profile of melatonin secretion. After multiple neuronal relays (within the hypothalamus), melatonin affects gonadotrophin-releasing hormone (GnRH) secretion, which in turn controls ovarian cyclicity. The pattern of GnRH secretion is mirrored by that of luteinizing hormone (LH) secretion, whose plasmatic level can be easily measured. We addressed the question of whether there exists an endogenous circannual rhythm in a tropical sheep (Blackbelly) population that exhibits clear seasonal ovarian activity when ewes are subject to temperate latitudes. We based our analysis on LH time series collected in the course of 3 years from ewes subject to a constant photoperiodic regime. Owing to intra- and interanimal variability and unequal sampling times, the existence of an endogenous rhythm is not straightforward. We have used time-frequency signal processing methods, and especially the smooth pseudo-Wigner-Ville distribution, to extract possible hidden rhythms from the data. To further investigate the low-frequency (LF) and high-frequency (HF) components of the signals, we have designed a simple mathematical model of the LH plasmatic level accounting for the effect of experimental sampling times. The model enables us to (i) confirm the existence of an endogenous circannual rhythm as detected by the LF signal component, (ii) investigate the action mechanism of the photoperiod on the pulsatile pattern of LH secretion (control of the interpulse interval), and (iii) conclude that the HF component is mainly due to the experimental sampling protocol.

Male sexual behavior contributes to the maintenance of high LH pulsatility in anestrous female goats.

The objective of this study was to determine the importance of male sexual behavior in stimulating LH secretion in anovulatory female goats. Two groups of females (n=10 per group) were each exposed to a buck in sexual rest and submitted to natural daylength. In one group, the buck was awake, whereas in the other group, it was sedated to prevent its sexual behavior. Two other groups of goats (n=10 per group) were exposed to sexually active bucks that had been exposed to 2.5 months of long days. In one group, the buck was awake, and in the other group, it was sedated. LH secretion was determined every 15 min from 4 h before introducing the bucks to 8 h after, then every 15 min again from 20 to 24 h after introducing the bucks. The bucks submitted to natural daylength did not stimulate LH secretion (P>0.05), whether they were sedated or not. In contrast, both the awake and the sedated light-treated bucks induced an increase (P<0.05) of LH pulsatility in the first 4 h following their introduction. However, pulsatility remained elevated until 24 h in the females exposed to the light-treated awake buck, whereas in the group with the light-treated sedated buck, pulsatility diminished (P<0.05) after the first 4 h of stimulation by the buck. In conclusion, the sexual behavior of males contributes to the maintenance of a high LH pulsatility up to 24 h after introduction into a group of anovulatory goats.

Turnover rate of cerebrospinal fluid in female sheep: changes related to different light-dark cycles.

BACKGROUND: Sheep are seasonal breeders. The key factor governing seasonal changes in the reproductive activity of the ewe is increased negative feedback of estradiol at the level of the hypothalamus under long-day conditions. It has previously been demonstrated that when gonadotropin secretions are inhibited during long days, there is a higher concentration of estradiol in the cerebrospinal fluid (CSF) than during short days. This suggests an involvement of the CSF and choroid plexus in the neuroendocrine regulatory loop, but the mechanisms underlying this phenomenon remain unknown. One possible explanation of this difference in hormonal content is an effect of concentration or dilution caused by variations in CSF secretion rate. The aim of this study was thus to investigate changes in the CSF turnover rate related to light-dark cycles. METHODS: The turnover rate of the CSF was estimated by measuring the time taken for the recovery of intraventricular pressure (IVP) after removal of a moderate volume (0.5 to 2 ml) of CSF (slope in mmHg/min). The turnover rate was estimated three times in the same group of sheep: during a natural period of decreasing day-length corresponding to the initial period when gonadotropin activity is stimulated (SG1), during a long-day inhibitory period (IG), and finally during a short-day stimulatory period (SG2). RESULTS: The time taken and the speed of recovery of initial IVP differed between groups: 8 min 30 sec, 0.63 +/- 0.07 mmHg/min(SG1), 11 min 1 sec, 0.38 +/- 0.06 mmHg/min (IG) and 9 min 0 sec, 0.72 +/- 0.15 mmHg/min (SG2). Time changes of IVP differed between groups (ANOVA, p < 0.005, SG1 different from IG, p < 0.05). The turnover rate in SG2: 183.16 +/- 23.82 mul/min was not significantly different from SG1: 169. 23 +/- 51.58 mul/min (Mann-Whitney test, p = 0.41), but was significantly different from IG: 71.33 +/- 16.59 mul/min (p = 0.016). CONCLUSION: This study shows that the turnover rate of CSF in ewes changes according to the light-dark cycle; it is increased during short day periods and reduced in long day periods. This phenomenon could account for differences in hormonal concentrations in the CSF in this seasonal species.

Evidence that the choroids plexus in female sheep express P-glycoprotein.

OBJECTIVES: Previous studies in the female sheep have shown that photoperiod modulates the passage of sex steroids between the blood and the cerebrospinal fluid (CSF) and have suggested the involvement of efflux transport. The objective of the present work was to assess the presence of P-glycoprotein (Pgp), which could be responsible for this transport at the level of the choroid plexuses (CP). METHODS: We used flow cytometry and the UIC2 monoclonal antibody against Pgp, to demonstrate the presence of Pgp in the epithelial cell fraction isolated from the sheep choroid plexuses (CPEC). Thanks to the size of the brain structures in sheep, we analyse separately the CP from 4th ventricle (4V) and the CP from the lateral ventricle (LV). RESULTS: In the whole population isolated from the CP, the 4V contained a higher percentage of living, epithelial cells than the LV. The immunoreactive cells to the UIC2 antibody i.e. bearing activated form of Pgp, represented 26.8% of the CPEC in the 4V, and 39.3% in the LV (P<0.05). CONCLUSION: Pgp is expressed in the CPEC from the sheep. Differences in the expression of Pgp between CPEC from LV and 4V might suggest in vivo functional differences between LV and 4V CP in sheep.

Identification of melatonin-regulated genes in the ovine pituitary pars tuberalis, a target site for seasonal hormone control.

The pars tuberalis (PT) of the pituitary gland expresses a high density of melatonin (MEL) receptors and is believed to regulate seasonal physiology by decoding changes in nocturnal melatonin secretion. Circadian clock genes are known to be expressed in the PT in response to the decline (Per1) and onset (Cry1) of MEL secretion, but to date little is known of other molecular changes in this key MEL target site. To identify transcriptional pathways that may be involved in the diurnal and photoperiod-transduction mechanism, we performed a whole genome transcriptome analysis using PT RNA isolated from sheep culled at three time points over the 24-h cycle under either long or short photoperiods. Our results reveal 153 transcripts where expression differs between photoperiods at the light-dark transition and 54 transcripts where expression level was more globally altered by photoperiod (all time points combined). Cry1 induction at night was associated with up-regulation of genes coding for NeuroD1 (neurogenic differentiation factor 1), Pbef / Nampt (nicotinamide phosphoribosyltransferase), Hif1alpha (hypoxia-inducible factor-1alpha), and Kcnq5 (K+ channel) and down-regulation of Rorbeta, a key clock gene regulator. Using in situ hybridization, we confirmed day-night differences in expression for Pbef / Nampt, NeuroD1, and Rorbeta in the PT. Treatment of sheep with MEL increased PT expression for Cry1, Pbef / Nampt, NeuroD1, and Hif1alpha, but not Kcnq5. Our data thus reveal a cluster of Cry1-associated genes that are acutely responsive to MEL and novel transcriptional pathways involved in MEL action in the PT.

The PDZ protein mupp1 promotes Gi coupling and signaling of the Mt1 melatonin receptor.

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT(1) melatonin receptor and directly regulates its G(i)-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT(1) carboxyl terminus. This high affinity interaction (K(d) approximately 4 nm), which is independent of MT(1) activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT(1)/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT(1), it destabilizes the interaction between MT(1) and G(i) and abolishes G(i)-mediated signaling of MT(1). Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.

GPR50 is the mammalian ortholog of Mel1c: evidence of rapid evolution in mammals.

BACKGROUND: The melatonin receptor subfamily contains three members Mel1a, Mel1b and Mel1c, found in all vertebrates except for Mel1c which is found only in fish, Xenopus species and the chicken. Another receptor, the melatonin related receptor known as GPR50, found exclusively in mammals and later identified as a member of the melatonin receptor subfamily because of its identity to the three melatonin receptors despite its absence of affinity for melatonin. The aim of this study was to describe the evolutionary relationships between GPR50 and the three other members of the melatonin receptor subfamily. RESULTS: Using an in silico approach, we demonstrated that GPR50 is the ortholog of the high affinity Mel1c receptor. It was necessary to also study the synteny of this gene to reach this conclusion because classical mathematical models that estimate orthology and build phylogenetic trees were not sufficient. The receptor has been deeply remodelled through evolution by the mutation of numerous amino acids and by the addition of a long C-terminal tail. These alterations have modified its affinity for melatonin and probably affected its interactions with the other two known melatonin receptors MT1 and MT2 that are encoded by Mel1a and Mel1b genes respectively. Evolutionary studies provided evidence that the GPR50 group evolved under different selective pressure as compared to the orthologous groups Me11 a, b, and c. CONCLUSION: This study demonstrated that there are only three members in the melatonin receptor subfamily with one of them (Me11c) undergoing rapid evolution from fishes and birds to mammals. Further studies are necessary to investigate the physiological roles of this receptor.