Sophie Messager

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 CK1ɛ) 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 CK1ɛ, 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.

Tissue-specific abolition of Per1 expression in the pars tuberalis by pinealectomy in the Syrian hamster.

Melatonin secretion by the pineal gland transduces photoperiod into a neuroendocrine signal. In the pars tuberalis (PT), we have shown that photoperiod modifies the amplitude of the clock gene Per1. The aim of this study was to test whether the endogenous melatonin signal is required for rhythmic expression of Per1 in the PT. Male Syrian hamsters housed in long days (LD, 16:8 h light:dark) were pinealectomized and Per1 mRNA expression studied by in situ hybridization. Pinealectomy abolished the rhythm of Per1 expression in the PT, but had no effect on Per1 expression in the suprachiasmatic nucleus (SCN), or the ventromedial nucleus (VMH) of the hypothalamus. Interestingly, a single night-time injection of melatonin (25 μg), given to pinealectomized animals, failed to restore Per1 expression in the PT. These data demonstrate that Per1 expression in the PT is driven by melatonin, and that the features of the endogenous signal through which the Per1 expression is achieved cannot be reproduced by a single melatonin injection.

oPer1 is an early response gene under photoperiodic regulation in the ovine pars tuberalis.

Mammalian Per1 (or RIGUI) is a recently described putative clock gene that is expressed in the suprachiasmatic nucleus. It is also expressed in the pars tuberalis (PT) of the pituitary, where melatonin appears to drive its expression. This study examines the regulation of Per1 expression. In ovine PT cells, oPer1 is an early response gene transiently expressed after stimulation with forskolin, but melatonin has no independent effect on its expression. In sheep, PT tissue photoperiodic background influences the magnitude or timing of expression of oPer1 2 h after lights‐on. These data demonstrate that oPer1 mRNA is elevated in the PT following the decline in night‐time melatonin, and that the amplitude or timing of this elevation is dependent upon the duration of the nocturnal melatonin signal.

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 MT1 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 MT1 gene expression in the rat pituitary gland. MT1 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 β-subunit (LHβ) 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 MT1 gene revealed multiple putative response elements for the transcription factor pituitary homeobox-1 (Pitx-1), which is expressed in the anterior pituitary from Rathkes pouch formation. A Pitx-1 expression vector potently stimulated expression of both MT1-luciferase and LHβ-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 MT1-luciferase activity. Moreover, the transcription factor, early growth response factor-1, which is induced by gonadotrophin-releasing hormone (GnRH) and trans-activates LHβ expression, attenuated Pitx-1-induced MT1-luciferase activity. Finally, pituitary MT1 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 MT1 gene expression.

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.

RAPID COMMUNICATION oPer1 is an Early Response Gene Under Photoperiodic Regulation in the Ovine Pars Tuberalis

Mammalian Per1 (or RIGUI) is a recently described putative clock gene that is expressed in the suprachiasmatic nucleus. It is also expressed in the pars tuberalis (PT) of the pituitary, where melatonin appears to drive its expression. This study examines the regulation of Per1 expression. In ovine PT cells, oPer1 is an early response gene transiently expressed after stimulation with forskolin, but melatonin has no independent effect on its expression. In sheep, PT tissue photoperiodic background influences the magnitude or timing of expression of oPer1 2 h after lights‐on. These data demonstrate that oPer1 mRNA is elevated in the PT following the decline in night‐time melatonin, and that the amplitude or timing of this elevation is dependent upon the duration of the nocturnal melatonin signal.

RAPID COMMUNICATION oPer1 is an Early Response Gene Under Photoperiodic Regulation in the Ovine Pars Tuberalis: RAPID COMMUNICATIONoPer1 is an Early Response Gene Under Photoperiodic Regulation in the Ovine Pars Tuberalis

Mammalian Per1 (or RIGUI) is a recently described putative clock gene that is expressed in the suprachiasmatic nucleus. It is also expressed in the pars tuberalis (PT) of the pituitary, where melatonin appears to drive its expression. This study examines the regulation of Per1 expression. In ovine PT cells, oPer1 is an early response gene transiently expressed after stimulation with forskolin, but melatonin has no independent effect on its expression. In sheep, PT tissue photoperiodic background influences the magnitude or timing of expression of oPer1 2 h after lights‐on. These data demonstrate that oPer1 mRNA is elevated in the PT following the decline in night‐time melatonin, and that the amplitude or timing of this elevation is dependent upon the duration of the nocturnal melatonin signal.

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.