MTNR1A Melatonin Receptors in the Ovine Premammillary Hypothalamus: Day-Night Variation in the Expression of the Transcripts

BIOLOGY OF REPRODUCTION 72, 393–398 (2005) Published online before print 6 October 2004. DOI 10.1095/biolreprod.104.030064 MTNR1A Melatonin Receptors in the Ovine Premammillary Hypothalamus: Day-Night Variation in the Expression of the Transcripts1 Martine Migaud,2 Agnès Daveau, and Benoı̂t Malpaux Physiologie de la Reproduction et des Comportements, UMR 6175 INRA-CNRS-Université de Tours-Haras Nationaux, 37380 Nouzilly, France ABSTRACT Melatonin regulation of reproductive functions in sheep is mediated by action in the premammillary hypothalamus (PMH). The aim of this study was to identify the high-affinity melatoninreceptor subtypes expressed in this structure. To achieve this, we used reverse transcription-polymerase chain reaction (RTPCR) and developed in situ hybridization techniques (ISH). By using RT-PCR, we detected a band corresponding to the MTNR1A melatonin-receptor cDNA in the PMH as well as in the pars tuberalis (PT). On the opposite, MTNR1B melatoninreceptor transcripts were not detected using degenerate primers in any of the structures considered, confirming the lack of expression of this receptor subtype in sheep. The expression of MTNR1A mRNA was further confirmed in the PMH by ISH with a 35S-labeled ovine MTNR1A riboprobe. We next investigated the variation in the expression of MTNR1A mRNA between the end of the day and the end of the night (absence and presence of melatonin, respectively). MTNR1A transcript expression was greater at the end of the night than at the end of the day in the PMH. In contrast, MTNR1A mRNA expression was lower at the end of the night than at the end of the day in the PT. No significant variation in the MTNR1A mRNA expression was observed in a more dorsal hypothalamic area. Overall, these results show that MTNR1A transcripts are expressed in the ovine PMH and that their expression follows a diurnal rhythm, which is different from the pattern of expression observed in the PT. hypothalamus, melatonin, neuroendocrinology, receptors, seasonal reproduction INTRODUCTION Most species that live in temperate climates use the annual variations in day length as temporal cues to initiate changes in their reproductive condition. Photoperiodic information is conveyed from the retina through a multisynaptic pathway to the pineal gland. From there, the variations in the annual lighting conditions are converted into rhythmic synthesis of the pineal hormone, melatonin. Synthesis and secretion of this indolamine follow a circadian rhythm with high levels at night and low levels during the day in both blood and cerebrospinal fluid [1]. The variations in the duration of nocturnal melatonin secretion serve as an endocrine messenger of the season to adjust the neuroendoA preliminary report appeared in Society for Neuroscience 2001, abstract 466.12. Correspondence: Martine Migaud, PRC, Domaine de l’Orfrasière, INRA UMR 6073, 37380 Nouzilly, France. FAX: 33 247 427 743; e-mail: 1 2 Received: 9 April 2004. First decision: 9 April 2004. Accepted: 3 September 2004. Q 2005 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org 393 crine status of seasonal animals and regulate the pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus [2]. In sheep, long periods of melatonin secretion (i.e., short days) stimulate the GnRH system. The use of the specific radioligand 2-[125]I melatonin in in vitro studies using autoradiography has allowed the detection of high-affinity melatonin binding sites in several areas of the brain and pituitary [3] and has revealed putative central nervous system (CNS) targets for the control of reproduction by melatonin. Although the pars tuberalis (PT) expresses a higher density of high-affinity melatonin receptors than any other CNS or pituitary areas in most species, this structure does not seem to mediate the effects of melatonin on neurons involved in neuroendocrine control of reproduction but appears rather involved in the photoperiodic control of prolactin secretion [4]. Functional studies have demonstrated the hypothalamic localization of the melatonin target in sheep and hamsters. A hypothalamic target has been strongly suggested by lesion studies in Syrian hamsters [5, 6]. In ewes, a strong correlation has been observed between the proximity of melatonin microimplants to an area of binding, the premammillary hypothalamic (PMH) [7], and the ability of the implants to stimulate luteinizing hormone secretion [8]. This area is also potentially involved in the generation of endogenous circannual rhythms [9]. Melatonin action is mediated through specific receptors. In mammals, two high-affinity melatonin-receptor subtypes have been cloned and characterized, MTNR1A melatonin receptor [10] and MTNR1B melatonin receptor [11], also known as MT1 and MT2, respectively. MTNR1A and MTNR1B share similar binding properties and pharmacological characteristics [12]. However, whether they are both involved in the central control of seasonal reproduction and expressed in the ovine PMH remains to be determined. The lack of pharmacological analogues (agonists or antagonists) highly specific for one or the other of each receptor subtype makes the identification of the subtype involved difficult. However, the implication of MTNR1B receptors does not seem likely because seasonality of reproduction is not altered in two species of hamster presenting a natural knockout of the gene encoding this receptor [13]. No expression of MTNR1B has ever been detected in sheep so far [14]; however, it has not been assessed in the hypothalamus. The purpose of this study was, therefore, to undoubtedly identify melatonin-receptor subtype(s) expressed in the ovine PMH and in other brain areas. Because no specific antibodies for MTNR1A and MTNR1B receptors are currently available to allow immunohistochemical experiments to be carried out in this model as well as in other models of seasonal mammals, we performed reverse transcriptionpolymerase chain reaction (RT-PCR) and in situ hybridization experiments to address this question. 394 MIGAUD ET AL. MATERIALS AND METHODS Animals and Tissue Preparation Plasma Melatonin Concentration Blood samples from each animal were collected before decapitation. Plasma melatonin-concentration measurements were performed using a well-validated radioimmunoassay [17]. Results are expressed in picograms/milliliter 6 SEM. Oligonucleotide Primers and RT-PCR Specific sense and antisense 19–20mer oligonucleotide primers (Sigma Genosis, UK) were directed toward selected regions of exons 1 and 2 of the gene coding MTNR1A receptor, respectively [10], to avoid the amplification of genomic DNA. The sequences 59-CTGTCCGTGTATCGGAACAAG-39 (bases 244–264) and 59-GCCATATAGTAACTAGCCAC-39 (bases 947–928) correspond to the upper-strand and lower-strand primers, respectively. The ovine GAPD (AF030943) served as a control for the quality of cDNA, and forward and rever (...truncated)