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In mammals, melatonin is the hormone responsible for synchronisation of seasonal physiological cycles of physiology to the solar year. Melatonin is secreted by the pineal gland with a profile reflecting the duration of the night and acts via melatonin-responsive cells in the pituitary pars tuberalis (PT), which in turn modulate hypothalamic thyroid hormone status. Recent models suggest that the actions of melatonin in the PT depend critically on day length-dependent changes in the expression of eyes absent 3 (Eya3), which is a coactivator for thyrotrophin β-subunit (Tshβ) gene transcription. According to this model, short photoperiods suppress Eya3 and hence Tshβ expression, whereas long photoperiods produce the inverse effect. Studies underpinning this model have relied on step changes in photoperiod (from 8 to 16 hours of light/24 hours) and have not compared the sensitive ranges of photoperiods for changes in Eya3 and Tshβ expression with those for relevant downstream molecular and endocrine responses. We therefore performed a "critical day length" experiment in Soay sheep, in which animals acclimated to 8 hours of light/24 hours (SP) were exposed to a range of increased photoperiods spanning the range 11.75 to 16 hours (LP) and then responses at the level of the PT, hypothalamus and hormonal output were assessed. Although Eya3 and Tshβ both showed the predicted SP vs LP differences, they responded quite differently to intermediate photoperiods within this range and, at the individual animal level, no clear Eya3-Tshβ relationship could be seen. This result is inconsistent with a simple coactivator model for EYA3 action in the PT. Further downstream layers of nonlinearity were also seen in terms of the Tshβ-dio2 and the dio2-testosterone relationships. We conclude that the transduction of progressive changes in photoperiod into transitions in endocrine output is an emergent property of a multistep signalling cascade within the mammalian neuroendocrine system., (© 2018 British Society for Neuroendocrinology.)

Increased thyrotrophin-stimulating hormone β (TSHβ) expression in the pars tuberalis is assumed to be an early step in the neuroendocrine mechanism transducing photoperiodic information. The present study aimed to determine the relationship between long-photoperiod (LP) and diurnal TSHβ gene expression in the juvenile chicken by comparing LP-photostimulated birds with groups kept on a short photoperiod (SP) for 1 or 12 days. TSHβ expression increased by 3- and 23-fold after 1 and 12 days of LP-photostimulation both during the day and at night. Under both SP and LP conditions, TSHβ expression was between 3- and 14-fold higher at night than in the day, suggesting that TSHβ expression cycles in a diurnal pattern irrespective of photoperiod. The ratio of DIO2/3 was decreased on LPs, consequent to changes in DIO3 expression, although there was no evidence of any diurnal effect on DIO2 or DIO3 expression. Plasma prolactin concentrations revealed both an effect of LPs and time-of-day. Thus, TSHβ expression changes in a dynamic fashion both diurnally and in response to photoperiod., (© 2017 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)

A photoperiodic species initiates fat deposition (in migrants) and gonadal recrudescence in response to a specific duration of natural daylight, called critical day length (CD), when light extends in the inductive phase of the endogenous circadian rhythm of photoinducibility (CRP). The molecular basis of species-specificCD, determined by the entrainment of the CRP, has been poorly understood. To investigate this, we measured expression levels of genes implicated in the photoperiod-induced changes in reproduction (EYA3, TSH beta, DIO2, DIO3, GNRH and GNIH) and metabolism (SIRT1, HMGCR, FASN and PPAR alpha) in photosensitive redheaded buntings subjected to light-dark cycles of varying period lengths (T-photocycles). Buntings were exposed to six T22, T24 or T26 photocycles, with 1h additional light at night falling at different phases of the entrained CRP (T2211L=6L:4D:1L:11D; T2411L=6L:4D:1L:13D,T2412L=6L:5D:1L:12D, T2413L=6L:6D:1L:11D; T2612L=6L:5D:1L:14D). Photoinduction at genetic and phenotypic levels in T2412L and T2413L, not T2411L, groups confirmed CD being close to 12h in buntings under T24. Compared to T24, exposure to T22 advanced CD by 1h, as evidenced by photoinduction in the T2211L, not T226L, group. Similarly, CD appeared to be delayed under T26, with no photoinduction in the T2612L group. Further, to show that induction of response under a T-photocycle was because of the interaction of inductive phase of the CRP with 1h during the dark period in each cycle, not with the 6h main light periods falling 2h earlier each successive 24hday in a T22 paradigm, a group of buntings was exposed to 6L:16D (T226L), to which they did not respond. The mRNA expression of genes, particularly TSH beta, DIO2, DIO3 and PPAR alpha, was significantly correlated with changes in reproductive and metabolic phenotypes. These results suggest CRP-entrainment based genetic regulation of the CD, and extend the idea that synchronization with environment is a critical measure in a seasonal species for its temporal adaptation in the wild., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Three primitive photoreceptors [melanopsin (Opn4), neuropsin/opsin5 (Opn5) and vertebrate ancient opsin (VAOpn)] were reported as possible avian deep-brain photoreceptors (DBPs) involved in the perception of photoperiodic information affecting the onset and development of reproduction. The objective of this study was to determine the effect of long-day photostimulation and/or sulfamethazine treatment (SMZ, a compound known to advance light-induced testes development) on gene expression of DBPs and key hypothalamic and pituitary genes involved in avian reproductive function. Two-week old chicks were randomly selected into four experimental groups: short-day control (SC, LD8:16), short-day+SMZ (SS, LD8:16, 0.2% diet SMZ), long-day control (LC, LD16:8), and long-day+SMZ (LS, LD16:8, 0.2% diet SMZ). Birds were sampled on days 3, 7, and 28 after initiation of a long-day photoperiod and/or SMZ dietary treatments. Three brain regions [septal-preoptic, anterior hypothalamic (SepPre/Ant-Hypo) region, mid-hypothalamic (Mid-Hypo) region, posterior-hypothalamic (Post-Hypo) region], and anterior pituitary gland were dissected. Using quantitative real-time RT-PCR, we determined changes of expression levels of genes in distinct brain regions; Opn4 and Opn5 in SepPre/Ant-Hypo and Post-Hypo regions and, VAOpn in the Mid-Hypo region. Long-day treatment resulted in a significantly elevated testes weight on days 7 and 28 compared to controls, and SMZ augmented testes weight in both short- and long-day treatment after day 7 (P<0.05). Long-day photoperiodic treatment on the third day unexpectedly induced a large 8.4-fold increase of VAOpn expression in the Mid-Hypo region, a 15.4-fold increase of Opn4 and a 97.8-fold increase of Opn5 gene expression in the Post-Hypo region compared to SC birds (P<0.01). In contrast, on days 7 and 28, gene expression of the three DBPs was barely detectable. LC group showed a significant increase in GnRH-1 and TRH mRNA in the Mid-Hypo compared to SC on day 3. Pituitary LHβ and FSHβ mRNA were significantly elevated in LC and LS groups compared to SC on days 3 and 7 (P<0.05). On days 3 and 7, TSHβ mRNA level was significantly elevated by long-day treatment compared to the SC groups (P<0.05). Results suggest that long-day photoperiodic activation of DBPs is robust, transient, and temporally related with neuroendocrine genes involved in reproductive function. Additionally, results indicate that two subsets of GnRH-1 neurons exist based upon significantly different gene expression from long-day photostimulation and long-day plus SMZ administration. Taken together, the data indicate that within 3 days of a long-day photoperiod, an eminent activation of all three types of DBPs might be involved in priming the neuroendocrine system to activate reproductive function in birds., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A long day response is triggered by the activation of EYA3 (eyes absent 3) and TSH-β (thyroid stimulating hormone beta subunit) genes in the pars tuberalis (PT). However, protein products of these genes are not yet shown in the hypothalamus of a photoperiodic species. Therefore, using the 'first long day paradigm', EYA3 and TSH-β along with c-FOS and GnRH peptides were immunohistochemically localized and measured in the hypothalamus of photoperiodic redheaded buntings that were maintained on short days (SD, LD 8/16) and subjected to one full long day (LD, LD 16/8). Following morning light remained turned off, and birds were sacrificed in the first hour of the day. Brains were collected and processed for immunohistochemistry of peptides. FOS-lir and GnRH-lir cells were significantly higher in the preoptic area (POA) in LD than in SD, which indicated photoperiod induced neuronal activation and downstream effects, respectively, under LD. In LD, EYA3-lir cells were significantly increased in septal lateralis (SL) with fibres extending to sub-septal organ (SSO); EYA3 fibres were very dense in median eminence. Similarly, there were significantly increased TSH-β-lir cells in the ventricular region with much abundance in the PT and TSH-β-lir fibres in the SSO (extending up to SL), inferior hypothalamic nucleus (IH) and infundibular nucleus (IN) in LD birds. Elevated EYA3, TSH-α and TSH-β mRNA levels further confirmed photoperiodic induction at the transcriptional level in buntings on the first long day. These are the first results showing localization of photoperiodically induced peptides in the hypothalamus of a songbird species, the redheaded bunting., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Organisms have seasonal physiological changes in response to day length. Long-day stimulation induces thyroid-stimulating hormone beta subunit (TSHβ) in the pars tuberalis (PT), which mediates photoperiodic reactions like day-length measurement and physiological adaptation. However, the mechanism of TSHβ induction for day-length measurement is largely unknown. To screen candidate upstream molecules of TSHβ, which convey light information to the PT, we generated Luciferase knock-in mice, which quantitatively report the dynamics of TSHβ expression. We cultured brain slices containing the PT region from adult and neonatal mice and measured the bioluminescence activities from each slice over several days. A decrease in the bioluminescence activities was observed after melatonin treatment in adult and neonatal slices. These observations indicate that the experimental system possesses responsiveness of the TSHβ expression to melatonin. Thus, we concluded that our experimental system monitors TSHβ expression dynamics in response to external stimuli., (© 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)

Living organisms detect seasonal changes in day length (photoperiod) [1-3] and alter their physiological functions accordingly to fit seasonal environmental changes. TSHβ, induced in the pars tuberalis (PT), plays a key role in the pathway that regulates vertebrate photoperiodism [4, 5]. However, the upstream inducers of TSHβ expression remain unknown. Here we performed genome-wide expression analysis of the PT under chronic short-day and long-day conditions in melatonin-proficient CBA/N mice, in which the photoperiodic TSHβ expression response is preserved [6]. This analysis identified "short-day" and "long-day" genes, including TSHβ, and further predicted the acute induction of long-day genes by late-night light stimulation. We verified this by advancing and extending the light period by 8 hr, which induced TSHβ expression within one day. In the following genome-wide expression analysis under this acute long-day condition, we searched for candidate upstream genes by looking for expression that preceded TSHβ's, and we identified the Eya3 gene. We demonstrated that Eya3 and its partner Six1 synergistically activate TSHβ expression and that this activation is further enhanced by Tef and Hlf. These results elucidate the comprehensive transcriptional photoperiodic response in the PT, revealing the complex regulation of TSHβ expression and unexpectedly rapid response to light changes in the mammalian photoperiodic system.

Seasonal synchronization based on day length (photoperiod) allows organisms to anticipate environmental change. Photoperiodic decoding relies on circadian clocks, but the underlying molecular pathways have remained elusive [1]. In mammals and birds, photoperiodic responses depend crucially on expression of thyrotrophin β subunit RNA (TSHβ) in the pars tuberalis (PT) of the pituitary gland [2-4]. Now, using our well-characterized Soay sheep model [2], we describe a molecular switch governing TSHβ transcription through the circadian clock. Central to this is a conserved D element in the TSHβ promoter, controlled by the circadian transcription factor thyrotroph embryonic factor (Tef). In the PT, long-day exposure rapidly induces expression of the coactivator eyes absent 3 (Eya3), which synergizes with Tef to maximize TSHβ transcription. The pineal hormone melatonin, secreted nocturnally, sets the phase of rhythmic Eya3 expression in the PT to peak 12 hr after nightfall. Additionally, nocturnal melatonin levels directly suppress Eya3 expression. Together, these effects form a switch triggering a strong morning peak of Eya3 expression under long days. Species variability in the TSHβ D element influences sensitivity to TEF, reflecting species variability in photoperiodic responsiveness. Our findings define a molecular pathway linking the circadian clock to the evolution of seasonal timing in mammals.

Seasonal reproduction depends on photoperiod-regulated activation or suppression of the gonadal axis. Recent studies in quail have identified long-day induced TSH-beta expression in the pars tuberalis (PT) as a rapid trigger of gonadal activation. Thyroid-stimulating hormone (TSH) induces type 2 deiodinase (Dio2) in the ependymal cell layer (EC) of the infundibular recess to stimulate the gonadal axis. A similar mechanism is proposed in sheep and mice, but the experimental data on the temporal patterns of induction and suppression of TSH-beta and Dio2 expression are incomplete. In the present study, we examined the expression of TSH-beta and Dio2 in hamsters transferred from short- to long-day conditions for 9 days, and demonstrate the induction of TSH-beta and Dio2 on day 8 after transition. These data demonstrate the close relationship between TSH-beta and Dio2 expression in the inductive pathway. The temporal expression of TSH-beta and Dio2 in the suppressive pathway was also examined by s.c. melatonin injection, which mimics the transition from long to short days. Importantly, Dio2 expression in the EC is suppressed on day 1 after the onset of injection, whereas TSH-beta expression in the PT was not suppressed until day 10. These data suggest that regulated transcription of TSH-beta is involved in the induction of the gonadal axis in mammals, whereas the suppression of this axis is mediated by different mechanisms.

At temperate latitudes, increases in day length in the spring promote the summer phenotype. In mammals, this long-day response is mediated by decreasing nightly duration of melatonin secretion by the pineal gland. This affects adenylate cyclase signal transduction and clock gene expression in melatonin-responsive cells in the pars tuberalis of the pituitary, which control seasonal prolactin secretion. To define the photoperiodic limits of the mammalian long day response, we transferred short day (8 h light per 24 h) acclimated Soay sheep to various longer photoperiods, simulating those occurring from spring to summer in their northerly habitat (57 degrees N). Locomotor activity and plasma melatonin rhythms remained synchronized to the light-dark cycle in all photoperiods. Surprisingly, transfer to 16-h light/day had a greater effect on prolactin secretion and oestrus activity than shorter (12 h) or longer (20 and 22 h) photoperiods. The 16-h photoperiod also had the largest effect on expression of circadian (per1) and neuroendocrine output (betaTSH) genes in the pars tuberalis and on kisspeptin gene expression in the arcuate nucleus of the hypothalamus, which modulates reproductive activity. This critical photoperiodic window of responsiveness to long days in mammals is predicted by a model wherein adenylate cyclase sensitization and clock gene phasing effects of melatonin combine to control neuroendocrine output. This adaptive mechanism may be related to the latitude of origin and the timing of the seasonal transitions.