In mammals, day-length-sensitive (photoperiodic) seasonal breeding cycles depend on the pineal hormone melatonin, which modulates secretion of reproductive hormones by the anterior pituitary gland [1]. It is thought that melatonin acts in the hypothalamus to control reproduction through the release of neurosecretory signals into the pituitary portal blood supply, where they act on pituitary endocrine cells [2]. Contrastingly, we show here that during the reproductive response of Soay sheep exposed to summer day lengths, the reverse applies: Melatonin acts directly on anterior-pituitary cells, and these then relay the photoperiodic message back into the hypothalamus to control neuroendocrine output. The switch to long days causes melatonin-responsive cells in the pars tuberalis (PT) of the anterior pituitary to increase production of thyrotrophin (TSH). This acts locally on TSH-receptor-expressing cells in the adjacent mediobasal hypothalamus, leading to increased expression of type II thyroid hormone deiodinase (DIO2). DIO2 initiates the summer response by increasing hypothalamic tri-iodothyronine (T3) levels. These data and recent findings in quail [3] indicate that the TSH-expressing cells of the PT play an ancestral role in seasonal reproductive control in vertebrates. In mammals this provides the missing link between the pineal melatonin signal and thyroid-dependent seasonal biology.

The KiSS-1 gene encodes kisspeptin, the endogenous ligand of the G-protein-coupled receptor GPR54. Recent data indicate that the KiSS-1/GPR54 system is critical for the regulation of reproduction and is required for puberty onset. In seasonal breeders, reproduction is tightly controlled by photoperiod (i.e., day length). The Syrian hamster is a seasonal model in which reproductive activity is promoted by long summer days (LD) and inhibited by short winter days (SD). Using in situ hybridization and immunohistochemistry, we show that KiSS-1 is expressed in the arcuate nucleus of LD hamsters. Importantly, the KiSS-1 mRNA level was lower in SD animals but not in SD-refractory animals, which spontaneously reactivated their sexual activity after several months in SD. These changes of expression are not secondary to the photoperiodic variations of gonadal steroids. In contrast, melatonin appears to be necessary for these seasonal changes because pineal-gland ablation prevented the SD-induced downregulation of KiSS-1 expression. Remarkably, a chronic administration of kisspeptin-10 restored the testicular activity of SD hamsters despite persisting photoinhibitory conditions. Overall, these findings are consistent with a role of KiSS-1/GPR54 in the seasonal control of reproduction. We propose that photoperiod, via melatonin, modulates KiSS-1 signaling to drive the reproductive axis.

In isolated right atria of the rabbit heart, we studied the activation pattern within the sinus node, using the microelectrode technique. After the electrophysiological experiments, the preparations were subjected to a correlative morphological investigation, using light or electron microscopy. Different criteria for defining the dominant pacemaker were compared. A group of at least 5000 cells, located within the central part of the node where the most characteristic tissue architecture was found, was considered to be responsible for generation of the impulse. At the ultrastructural level, this leading cell group appeared to be part of a larger uniform cell group. The number of gap junctions observed suggests that all nodal cells are coupled by these structures. Toward the periphery, the excitation wave was propagated preferentially in an oblique cranial direction toward the crista terminalis. Neither morphologically nor electrophysiologically specific pathways were found for the conduction, but the preferential direction could be explained by the tissue architecture.

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In mammals, changing day length modulates endocrine rhythms via nocturnal melatonin secretion. Studies of the pituitary pars tuberalis (PT) suggest that melatonin-regulated clock gene expression is critical to this process. Here, we considered whether clock gene rhythms continue in the PT in the absence of melatonin and whether the effects of melatonin on the expression of these genes are temporally gated. Soay sheep acclimated to long photoperiod (LP) were transferred to constant light for 24 h, suppressing endogenous melatonin secretion. Animals were infused with melatonin at 4-h intervals across the final 24 h, and killed 3 h after infusion. The expression of five clock genes (Per1, Per2, Cry1, Rev-erbalpha, and Bmal1) was measured by in situ hybridization. In sham-treated animals, PT expression of Per1, Per2, and Rev-erbalpha showed pronounced temporal variation despite the absence of melatonin, with peak times occurring earlier than predicted under LP. The time of peak Bmal1 expression remained LP-like, whereas Cry1 expression was continually low. Melatonin infusion induced Cry1 expression at all times and suppressed other genes, but only when they showed high expression in sham-treated animals. Hence, 3 h after melatonin treatment, clock gene profiles were driven to a similar state, irrespective of infusion time. In contrast to the PT, melatonin infusions had no clear effect on clock gene expression in the suprachiasmatic nuclei. Our results provide the first example of acute sensitivity of multiple clock genes to one endocrine stimulus and suggest that rising melatonin levels may reset circadian rhythms in the PT, independently of previous phase.

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Melatonin, secreted only during the night by the pineal gland, transduces the photoperiodic message to the organism. One important target for the hormone is the pars tuberalis (PT) of the adenohypophysis which displays a very high number of melatonin binding sites in mammals and is implicated in the seasonal regulation of prolactin secretion. To gain insight into the mechanism by which the melatonin signal is decoded in the PT, we studied the effect of photoperiod on the PT cells expressing the MT1 melatonin receptor in a highly photoperiodic species, the European hamster. Recently, we showed that, in the rat, the MT1 receptor mRNA is expressed in PT‐specific cells characterized by their expression of β‐thyroid stimulating hormone (β‐TSH) along with the α‐glycoprotein subunit (α‐GSU). As the cellular composition of the PT shows variability among species, we first identified the cell type expressing the MT1 receptor in the European hamster by combining immunocytochemistry and nonradioactive in situ hybridization for the MT1 receptor mRNA. Our results show that, in the European hamster, as in the rat, the MT1 receptor is only expressed by the PT‐specific‐cells, β‐TSH and α‐GSU positive. In a second step, we analysed the effects of photoperiod on the MT1 mRNA, and on β‐TSH and α‐GSU both at the mRNA and protein levels. Our data show that, compared to long photoperiod, short photoperiod induces a dramatic decrease of MT1, β‐TSH and α‐GSU expression. Protein levels of β‐TSH and α‐GSU were also dramatically reduced in short photoperiod. Together, our data suggest that melatonin exerts its seasonal effects in the PT by signalling to PT specific‐cells through the MT1 receptor subtype.

S U M M A R YThe pars tuberalis (PT) of the pituitary represents an important target site for the time-pacing pineal hormone melatonin because it expresses a large number of mt1 receptors. Functional studies suggest that the PT mediates the seasonal effects of melatonin on prolactin (PRL) secretion. The aim of this study was the characterization of the phenotype of melatonin-responsive cells. Furthermore, we determined whether ROR ␤ , a retinoid orphan receptor present in the PT, was co-expressed in the same cells. We combined nonradioactive in situ hybridization (ISH) with hapten-labeled riboprobes for detection of the receptors and immunocytochemistry (ICC) for detection of ␣ GSU ( ␣ -glycoprotein subunit), ␤ TSH, ␤ FSH, ␤ LH, GH, PRL, and ACTH. Expression of mt1 mRNA was found in small round cells, co-localized with ␣ GSU and ␤ TSH. However, not all ␤ TSH-containing cells expressed mt1 mRNA. The distribution of mt1-and ROR ␤ -positive cells appeared to overlap, although more cells were labeled for ROR ␤ than for mt1. Gonadotrophs, as well as other pars distalis cell types, were never labeled for mt1 melatonin receptor. Therefore, this study identifies the "specific" cells of the PT as the mt1 melatonin receptor-expressing cells.(