Molecular cloning and pharmacological characterization of rat melatonin MT1 and MT2 receptors.

In order to interpret the effects of melatonin ligands in rats, we need to determine their activity at the receptor subtype level in the corresponding species. Thus, the rat melatonin rMT(1) receptor was cloned using DNA fragments for exon 1 and 2 amplified from rat genomic DNA followed by screening of a rat genomic library for the full length exon sequences. The rat rMT(2) receptor subtype was cloned in a similar manner with the exception of exon 1 which was identified by screening a rat genomic library with exon 1 of the human hMT(2) receptor. The coding region of these receptors translates proteins of 353 and 364 amino acids, respectively, for rMT(1) and rMT(2). A 55% homology was observed between both rat isoforms. The entire contiguous rat MT(1) and MT(2) receptor coding sequences were cloned, stably expressed in CHO cells and characterized in binding assay using 2-[(125)I]-Iodomelatonin. The dissociation constants (K(d)) for rMT(1) and rMT(2) were 42 and 130 pM, respectively. Chemically diverse compounds previously characterized at human MT(1) and MT(2) receptors were evaluated at rMT(1) and rMT(2) receptors, for their binding affinity and functionality in [(35)S]-GTPgammaS binding assay. Some, but not all, compounds shared a similar binding affinity and functionality at both rat and human corresponding subtypes. A different pharmacological profile of the MT(1) subtype has also been observed previously between human and ovine species. These in vitro results obtained with the rat melatonin receptors are thus of importance to understand the physiological roles of each subtype in animal models.

Kisspeptin and its receptor: new gatekeepers of puberty.

The recent finding that the hormone kisspeptin plays a pivotal role in the onset of puberty is one of the biggest discoveries in human reproductive biology in 30 years. Mutations in the receptor for kisspeptin cause humans and mice to fail to reach puberty and to be sterile. It is the first time since the identification of gonadotrophin-releasing hormone that a single gene is found to have such a dramatic effect on reproduction. This discovery opens new possibilities in the treatment of reproductive disorders such as delayed or advanced puberty, infertility and sex hormone-dependent cancers.

Photoperiod regulates clock gene rhythms in the ovine liver.

To investigate the photoperiodic entrainment of peripheral rhythms in ruminants, we studied the expression of clock genes in the liver in the highly seasonal Soay sheep. Animals were kept under long (LD 16:8) or short photoperiod (LD 8:16). Daily rhythms in locomotor activity were recorded, and blood concentrations of melatonin and cortisol were measured by RIA. Per2, Bmal1, and Cry1 gene expression was determined by Northern blot analyses using ovine RNA probes in liver collected every 4h for 24h. Liver Per2 and Bmal1, but not Cry1, expression was rhythmic in all treatments. Under long days, peak Per2 expression occurred at end of the night with a similar timing to Bmal1, whereas, under short days the Per2 maximum was in the early night with an inverse pattern to Bmal1. There was a photoperiodxtime interaction for only Per2 (P < 0.001). The 24-h pattern in plasma cortisol matched the observed phasing of Per2 expression, suggesting that it may act as an endocrine entraining factor. The clock gene rhythms in the peripheral tissues were different in timing compared with the ovine suprachiasmatic nucleus (SCN, central pacemaker) and pars tuberalis (melatonin target tissue), and the hepatic rhythms were of lower amplitude compared with photoperiodic rodents. Thus, there are likely to be important species differences in the way the central and peripheral clockwork encodes external photoperiod.

Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54.

We have recently described a molecular gatekeeper of the hypothalamic-pituitary-gonadal axis with the observation that G protein-coupled receptor 54 (GPR54) is required in mice and men for the pubertal onset of pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion to occur. In the present study, we investigate the possible central mode of action of GPR54 and kisspeptin ligand. First, we show that GPR54 transcripts are colocalized with gonadotropin-releasing hormone (GnRH) neurons in the mouse hypothalamus, suggesting that kisspeptin, the GPR54 ligand, may act directly on these neurons. Next, we show that GnRH neurons seem anatomically normal in gpr54-/- mice, and that they show projections to the median eminence, which demonstrates that the hypogonadism in gpr54-/- mice is not due to an abnormal migration of GnRH neurons (as occurs with KAL1 mutations), but that it is more likely due to a lack of GnRH release or absence of GnRH neuron stimulation. We also show that levels of kisspeptin injected i.p., which stimulate robust LH and FSH release in wild-type mice, have no effect in gpr54-/- mice, and therefore that kisspeptin acts directly and uniquely by means of GPR54 signaling for this function. Finally, we demonstrate by direct measurement, that the central administration of kisspeptin intracerebroventricularly in sheep produces a dramatic release of GnRH into the cerebrospinal fluid, with a parallel rise in serum LH, demonstrating that a key action of kisspeptin on the hypothalamo-pituitary-gonadal axis occurs directly at the level of GnRH release. The localization and GnRH release effects of kisspeptin thus define GPR54 as a major control point in the reproductive axis and suggest kisspeptin to be a neurohormonal effector.

The GPR54 gene as a regulator of puberty.

BACKGROUND: Puberty, a complex biologic process involving sexual development, accelerated linear growth, and adrenal maturation, is initiated when gonadotropin-releasing hormone begins to be secreted by the hypothalamus. We conducted studies in humans and mice to identify the genetic factors that determine the onset of puberty. METHODS: We used complementary genetic approaches in humans and in mice. A consanguineous family with members who lacked pubertal development (idiopathic hypogonadotropic hypogonadism) was examined for mutations in a candidate gene, GPR54, which encodes a G protein-coupled receptor. Functional differences between wild-type and mutant GPR54 were examined in vitro. In parallel, a Gpr54-deficient mouse model was created and phenotyped. Responsiveness to exogenous gonadotropin-releasing hormone was assessed in both the humans and the mice. RESULTS: Affected patients in the index pedigree were homozygous for an L148S mutation in GPR54, and an unrelated proband with idiopathic hypogonadotropic hypogonadism was determined to have two separate mutations, R331X and X399R. The in vitro transfection of COS-7 cells with mutant constructs demonstrated a significantly decreased accumulation of inositol phosphate. The patient carrying the compound heterozygous mutations (R331X and X399R) had attenuated secretion of endogenous gonadotropin-releasing hormone and a left-shifted dose-response curve for gonadotropin-releasing hormone as compared with six patients who had idiopathic hypogonadotropic hypogonadism without GPR54 mutations. The Gpr54-deficient mice had isolated hypogonadotropic hypogonadism (small testes in male mice and a delay in vaginal opening and an absence of follicular maturation in female mice), but they showed responsiveness to both exogenous gonadotropins and gonadotropin-releasing hormone and had normal levels of gonadotropin-releasing hormone in the hypothalamus. CONCLUSIONS: Mutations in GPR54, a G protein-coupled receptor gene, cause autosomal recessive idiopathic hypogonadotropic hypogonadism in humans and mice, suggesting that this receptor is essential for normal gonadotropin-releasing hormone physiology and for puberty.

Gonadotrophin-releasing hormone drives melatonin receptor down-regulation in the developing pituitary gland.

Melatonin is produced nocturnally by the pineal gland and is a neurochemical representation of time. It regulates neuroendocrine target tissues through G-protein-coupled receptors, of which MT(1) is the predominant subtype. These receptors are transiently expressed in several fetal and neonatal tissues, suggesting distinct roles for melatonin in development and that specific developmental cues define time windows for melatonin sensitivity. We have investigated MT(1) gene expression in the rat pituitary gland. MT(1) mRNA is confined to the pars tuberalis region of the adult pituitary, but in neonates extends into the ventral pars distalis and colocalizes with luteinizing hormone beta-subunit (LH beta) expression. This accounts for the well documented transient sensitivity of rat gonadotrophs to melatonin in the neonatal period. Analysis of an upstream fragment of the rat MT(1) gene revealed multiple putative response elements for the transcription factor pituitary homeobox-1 (Pitx-1), which is expressed in the anterior pituitary from Rathke's pouch formation. A Pitx-1 expression vector potently stimulated expression of both MT(1)-luciferase and LH beta-luciferase reporter constructs in COS-7 cells. Interestingly, transcription factors that synergize with Pitx-1 to trans-activate gonadotroph-associated genes did not potentiate Pitx-1-induced MT(1)-luciferase activity. Moreover, the transcription factor, early growth response factor-1, which is induced by gonadotrophin-releasing hormone (GnRH) and trans-activates LH beta expression, attenuated Pitx-1-induced MT(1)-luciferase activity. Finally, pituitary MT(1) gene expression was 4-fold higher in hypogonadal (hpg) mice, which do not synthesize GnRH, than in their wild-type littermates. These data suggest that establishment of a mature hypothalamic GnRH input drives the postnatal decline in pituitary MT(1) gene expression.

Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer.

The 24-h expression of seven clock genes (Bmal1, Clock, Per1, Per2, Cry1, Cry2, and CK1 epsilon ) was assayed by in situ hybridization in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary gland, collected every 4 h throughout 24 h, from female Soay sheep kept under long (16-h light/8-h dark) or short (8-h light/16-h dark) photoperiods. Locomotor activity was diurnal, inversely related to melatonin secretion, and prolactin levels were increased under long days. All clock genes were expressed in the ovine SCN and PT. In the SCN, there was a 24-h rhythm in Clock expression, in parallel with Bmal1, in antiphase with cycles in Per1 and Per2; there was low-amplitude oscillation of Cry1 and Cry2. The waveform of only Per1 and Per2 expression was affected by photoperiod, with extended elevated expression under long days. In the PT, the high-amplitude 24-h cycles in the expression of Bmal1, Clock, Per1, Per2, Cry1, and Cry2, but not CK1 epsilon, were influenced by photoperiod. Per1 and Per2 peaked during the day, whereas Cry1 and Cry2 peaked early in the night. Hence, photoperiod via melatonin had a marked effect on the phase relationship between Per/Cry genes in the PT. This supports the conclusion that an "external coincidence model" best explains the way photoperiod affects the waveform of clock gene expression in the SCN, the central pacemaker, whereas an "internal coincidence model" best explains the way melatonin affects the phasing of clock gene expression in the PT to mediate the photoperiodic control of a summer or winter physiology.

Pituitary adenylate cyclase-activating polypeptide acts as a paracrine regulator of melatonin-responsive cells of the ovine pars tuberalis.

The pars tuberalis (PT) region of the anterior pituitary plays a physiological role in seasonal animals. The primary signal transduction mechanism of the melatonin receptor in this tissue is an inhibition of cAMP signaling. However, nothing is known about the endocrine signals that activate cAMP synthesis in the cells of the PT, as previous studies relied on the pharmacological tool, forskolin, to stimulate cAMP synthesis. Here we show that pituitary adenylate cyclase-activating polypeptide (PACAP) activates cAMP synthesis in the cells of the PT. The pharmacology of cAMP activation by PACAP peptides suggests that cAMP activation is mediated by the type I PACAP receptor. PACAP treatment of PT cells results in cellular responses that are consistent with cAMP activation in these cells, including activation of MAPK and elevation of melatonin receptor mt1 mRNA expression. These responses can be inhibited by melatonin, demonstrating that activation of cAMP occurs within the melatonin-responsive cells. However, although PACAP activates cAMP in the cells of the PT, the effect of PACAP may not be direct, as colocalization in situ hybridization studies demonstrates that the type I PACAP receptor and the melatonin mt1 receptor do not colocalize on the cells of the PT.