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.

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.

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.

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.

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.

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.

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.

Development of an in vivo adeno-associated virus-mediated siRNA approach to knockdown tyrosine hydroxylase in the lateral retrochiasmatic area of the ovine brain.

We developed a new technique of gene knockdown (KD) in a specific brain area of the ewe using an adeno-associated virus (AAV)-mediated short interfering RNA (siRNA) method to elucidate the importance of key factors of seasonal reproduction. Two 19-nucleotide sequences (TH1 or TH2) were chosen from the tyrosine hydroxylase (TH) gene. TH1, TH2 or a random sequence (TH3) was incorporated into an eGFP expressing AAV vector. Firstly, 5 microl of AAV-TH1 or AAV-TH2 solutions (8-9 x 10(11)Vg/ml) were stereotaxically injected into one A15 nucleus while the other received a control treatment. Ewes were killed after 15 or 75 days. The number of TH neurons was 49% and 36% lower on the AAV-TH1 treated side than on the control side 15 and 75 days post-injection, respectively. AAV-TH2 did not induce a significant variation in TH cell population. Finally, in order to increase the KD, two groups of ewes received 10 microl of AAV-TH1 either in a bolus injection or in two 5 microl inoculations carried out 2 weeks apart. Only ewes receiving a bolus injection showed a larger KD reaching 66% 2 months after inoculation. This method proved effective in reducing TH expression and will be further developed to understand cellular mechanisms driving seasonal functions.

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.

First evidence of melatonin receptors distribution in the suprachiasmatic nucleus of tree shrew brain.

OBJECTIVE: In this study we investigated the distribution of melatonergic receptors in the tree shrew brain. The psychosocial stress in the tree shrew is a validated model of depression with disturbance of circadian rhythms. METHODS: Given the role of melatonin in the modulation of circadian rhythms, we determined by autoradiography the distribution of binding sites of 2-[125I]-MLT, a ligand for the two melatonergic receptors MT1 and MT2, in the tree shrew brain focusing mostly on the suprachiasmatic nucleus (SCN), the biological clock involved in resynchronization of circadian rhythms. We also analyzed the distribution of 2-[125I]-MLT in other brain areas involved in regulation of mood. RESULTS: Specific binding of 2-[125I]-MLT was found in the SCN. In addition, several structures in the tree shrew brain were labeled, among them the pars tuberalis, the cerebellum, structures of the hippocampal formation and dopaminergic areas such as the caudate putamen and nucleus accumbens, providing other potential targets of psychosocial stress in the tree shrew, in addition to the SCN. CONCLUSIONS: The demonstration of melatonergic receptors in these brain areas supports their probable involvement in the behavioural, neuroendocrine and circadian rhythms disturbances observed in the psychosocial stress model of depression in the tree shrew.

Melatonin secretion in the Mashona mole-rat, Cryptomys darlingi--influence of light on rhythmicity.

The hormone melatonin is synthesised and secreted from the pineal gland in darkness and triggers the daily and seasonal timing of various physiological and behavioural processes. The Mashona mole-rat, Cryptomys darlingi, lives in subterranean burrows that are completely sealed and is therefore rarely, if ever, exposed to light under natural conditions. Hence, this species is of particular interest for studies on rhythms of melatonin secretion. We investigated how plasma melatonin concentrations of the Mashona mole-rat responded to exposure to a long-term standard photoperiod of 12 h light, 12 h dark (12:12 LD), constant light (LL) and constant dark (DD). In addition, we examined whether plasma melatonin concentration was coupled to locomotor activity. Mashona mole-rats displayed rhythms of plasma melatonin concentration that appeared entrained to the standard LD photoperiod, suggesting that the mole-rat is capable of perceiving and entraining to this photic zeitgeber. Furthermore, under chronic constant lighting conditions (DD, LL), circadian rhythms in plasma melatonin concentration were observed, suggesting the possible existence of an endogenous rhythm. Light suppressed melatonin secretion, but constant light did not abolish the rhythm of plasma melatonin concentration. Between active and non-active animals, no difference in plasma melatonin concentration was found for any of the sequential photoperiods (LD1 DD, LD2, LL), tentatively suggesting that the rhythm of melatonin secretion is uncoupled from that of locomotor activity.

Melatonin enters the cerebrospinal fluid through the pineal recess.

The pineal recess (PR), a third ventricle (IIIV) evagination penetrating into the pineal gland, could constitute a site of melatonin passage to the cerebrospinal fluid (CSF) and explain the high concentrations of melatonin in this fluid. To test this hypothesis, we characterized melatonin distribution in the IIIV of sheep by CSF collection in the ventral part of IIIV (vIIIV) and in PR. At 30 microl/min collection rate, melatonin concentrations were much higher in PR than in vIIIV (19,934 +/- 6,388 vs. 178 +/- 70 pg/ml, mean +/- SEM, respectively, P < 0.005), and they increased in vIIIV when CSF collection stopped in the PR (P < 0.05). At 6 microl/min, levels increased to 1,682 +/- 585 pg/ml in vIIIV and were not influenced by CSF collection in the PR. This concentration difference between sites and the influence of PR collection on vIIIV levels suggest that melatonin reaches the PR and then diffuses to the IIIV. To confirm the role of PR, we demonstrated that its surgical sealing off decreased IIIV melatonin levels (1,020 +/- 305 pg/ml, compared with 5,984 +/- 1,706 and 6,917 +/- 1,601 pg/ml in shams or animals with a failed sealing off, respectively, P < 0.01) without changes in blood levels. Therefore, this study identified the localization of the main site of penetration of melatonin into the CSF, the pineal recess.

Cellular lining of the sheep pineal recess studied by light-, transmission-, and scanning electron microscopy: morphologic indications for a direct secretion of melatonin from the pineal gland to the cerebrospinal fluid.

In the sheep, the pineal hormone melatonin displays nocturnal levels 20 times as high in the cerebrospinal fluid of the third ventricle as in the jugular blood. Moreover, in the pineal recess, the evagination of the third ventricle into the pineal stalk, the levels of melatonin in the cerebrospinal fluid are even higher than in the ventral part of the third ventricle. This finding suggests melatonin to be secreted directly from the pineal gland to the ventricular lumen of the pineal recess of this species. We have, therefore, studied the interface between the sheep pineal gland and the cerebrospinal fluid by light-, scanning-, and electron microscopy of the pineal recess, as well as the permeability of the interface by tracer injections into the third ventricle. First, we show that the classic ependymal lining of the third ventricle disappears in the superior part of the recess. In this area, bulging pinealocytes, displaying immunoreactivity for serotonin, directly appose the cerebrospinal fluid. This pineal-cerebrospinal fluid interface of the sheep is large compared with other species, especially rodent species. Intraventricular injections of horseradish peroxidase and fluorescein isothiocyanate showed that both these tracers could permeate from the pineal recess into the sheep pineal parenchyma. This permeation was due to the presence of gap and intermediate junctions connecting the pinealocytes apposing the ventricular lumen. Thus, our results show that endocrine cells in this specialized area of the ventricular system are in direct contact with the cerebrospinal fluid. This finding supports the physiological concept of a direct secretion of melatonin into the cerebrospinal fluid of the sheep pineal recess.

Neuroanatomical organization of gonadotropin-releasing hormone neurons during the oestrus cycle in the ewe.

BACKGROUND: During the preovulatory surge of gonadotropin-releasing hormone (GnRH), a very large amount of the peptide is released in the hypothalamo-hypophyseal portal blood for 24-36H00. To study whether this release is linked to a modification of the morphological organization of the GnRH-containing neurons, i.e. morphological plasticity, we conducted experiments in intact ewes at 4 different times of the oestrous cycle (before the expected LH surge, during the LH surge, and on day 8 and day 15 of the subsequent luteal phase). The cycle stage was verified by determination of progesterone and LH concentrations in the peripheral blood samples collected prior to euthanasia. RESULTS: The distribution of GnRH-containing neurons throughout the preoptic area around the vascular organ of the lamina terminalis was studied following visualisation using immunohistochemistry. No difference was observed in the staining intensity for GnRH between the different groups. Clusters of GnRH-containing neurons (defined as 2 or more neurons being observed in close contact) were more numerous during the late follicular phase (43 +/- 7) than during the luteal phase (25 +/- 6), and the percentage of clusters was higher during the beginning of the follicular phase than during the luteal phase. There was no difference in the number of labelled neurons in each group. CONCLUSIONS: These results indicate that the morphological organization of the GnRH-containing neurons in ewes is modified during the follicular phase. This transitory re-organization may contribute to the putative synchronization of these neurons during the surge. The molecular signal inducing this plasticity has not yet been identified, but oestradiol might play an important role, since in sheep it is the only signal which initiates the GnRH preovulatory surge.

Changes in the 5-HT2A receptor system in the pre-mammillary hypothalamus of the ewe are related to regulation of LH pulsatile secretion by an endogenous circannual rhythm.

BACKGROUND: We wanted to determine if changes in the expression of serotonin 2A receptor (5HT2A receptor) gene in the premammillary hypothalamus are associated with changes in reproductive neuroendocrine status. Thus, we compared 2 groups of ovariectomized-estradiol-treated ewes that expressed high vs low LH pulsatility in two different paradigms (2 groups per paradigm): (a) refractoriness (low LH secretion) or not (high LH secretion) to short days in pineal-intact Ile-de-France ewes (RSD) and (b) endogenous circannual rhythm (ECR) in free-running pinealectomized Suffolk ewes in the active or inactive stage of their reproductive rhythm. RESULTS: In RSD ewes, density of 5HT2A receptor mRNA (by in situ hybridization) was significantly higher in the high LH group (25.3 +/- 1.4 vs 21.4 +/- 1.5 grains/neuron, P < 0.05) and 3H-Ketanserin binding (a specific radioligand) of the median part of the premammillary hypothalamus tended to be higher in the high group (29.1 +/- 4.0 vs 24.6 +/- 4.2 fmol/mg tissu-equivalent; P < 0.10). In ECR ewes, density of 5HT2A receptor mRNA and 3H-Ketanserin binding were both significantly higher in the high LH group (20.8 +/- 1.6 vs 17.0 +/- 1.5 grains/neuron, P < 0.01, and 19.7 +/- 5.0 vs 7.4 +/- 3.4 fmol/mg tissu-equivalent; P < 0.05, respectively). CONCLUSIONS: We conclude that these higher 5HT2A receptor gene expression and binding activity of 5HT2A receptor in the premammillary hypothalamus are associated with stimulation of LH pulsatility expressed before the development of refractoriness to short days and prior to the decline of reproductive neuroendocrine activity during expression of the endogenous circannual rhythm.

Molecular pharmacology of the ovine melatonin receptor: comparison with recombinant human MT1 and MT2 receptors.

The variations of the pharmacological properties of melatonin receptors between different mammalian species in transfected cell lines have been poorly investigated. In the present study, melatonin analogues have been used to characterize the pharmacology of the recombinant ovine melatonin receptor (oMT1) expressed in CHO cell lines and the native oMT1 from the pars tuberalis (PT). Studies with selective ligands on native and transfected oMT1 showed similar properties for binding affinities [r2(PT/CHO) = 0.85]. The affinities and the functional activities of these ligands were compared with the human receptors (hMT1 or hMT2) expressed in CHO cells as well. The oMT1 and hMT1 receptors had similar pharmacological profiles (r2=0.82). Nevertheless, some of the selective compounds at the human receptor presented a reduced affinity at the ovine receptor. Furthermore, some compounds showed marked different functional activities at oMT1 vs. hMT1 receptors. Our findings demonstrated differences in the pharmacological properties of melatonin receptors in ovine and human species.

Seasonal regulation of reproductive activity in sheep: modulation of access of sex steroids to the brain.

Sheep in temperate latitudes are seasonal breeders. In female sheep, ovarian activity decreases during the anestrous period due to modification of secretion of luteinizing hormone (LH). The seasonal changes in the hormonal LH pattern mainly reflect an increase in the brain responsiveness to the negative feedback exerted by estradiol during long days (LD) on the frequency of pulsatile LH secretion, under neurohormonal GnRH control. The resulting seasonal inhibition of LH secretion mainly involves the activation of dopaminergic systems by E2, which in turn inhibits the GnRH cells from the preoptico-hypothalamic structures. The increased responsiveness of the brain during LD could lead to increased expression of central E2 receptors. In addition, our study shows that steroid access to the brain could be modulated by photoperiodism, thus increasing the availability of steroids to the nervous structures during LD.

New selective ligands of human cloned melatonin MT1 and MT2 receptors.

Melatonin has a key role in the circadian rhythm relay to periphery organs. Melatonin exerts its multiple roles mainly through two seven transmembrane domain, G-coupled receptors, namely MT1 or MT2 receptors. A pharmacological characterization of these human cloned melatonin hMT1 and hMT2 receptors stably expressed in HEK-293 or CHO cells is presented using a 2-[125I]-iodo-melatonin binding assay and a [35S]-GTPgammaS functional assay. Both reference compounds and new chemically diverse ligands were evaluated. Binding affinities at each receptor were found to be comparable on either HEK-293 or CHO cell membranes. Novel non-selective or selective hMT1 and hMT2 ligands are described. The [35S]-GTPgammaS functional assay was used to define the functional activity of these compounds which included partial, full agonist and/or antagonist activity. None of the compounds acted as an inverse agonist. We report new types of selective antagonists, such as S 25567 and S 26131 for MT1 and S 24601 for MT2. These studies brought other new molecular tools such as the selective MT1 agonist, S 24268, as well as the non-selective antagonist, S 22153. Finally, we also discovered S 25150, the most potent melatonin receptor agonist, so far reported in the literature.