A brief history of circannual time.

Innate circannual timing is an ancestral trait that first evolved in free-living eukaryotic cells some 2000 million years ago, with marine algae of the genus Allexandrium providing a living unicellular model. This species shows the primitive trait of 'alternation of generations', where the organism alternates between fast replicating vegetative cells in the summer and a dormant cystic cell over the winter. The resistant cysts sink into the cold ocean sediments. Remarkably, excystment in spring is governed by an endogenous circannual timing mechanism. Thus, a tiny, short-lived unicell can utilise a circannual clock as part of the life-history programme of the species. Innate timing allows for major adjustments in physiology and behaviour in anticipation of the seasons, and provides an internalised sense of seasonal time for the many species where standard environmental cues are weak or ambiguous. This is a highly adaptive strategy irrespective of the size and longevity of an organism. Circannual rhythms are expressed by a diverse range of organisms, from flowering plants to mammals, interwoven into the life-history programme of each species, being a consequence of forever living in a periodic world. In complex vertebrates, the early division of the zygote potentially carries circannual timer genes into all progeny cells and tissues. This supports the concept of a 'clock-shop' where cell-autonomous long-term rhythms are generated in each tissue, orchestrated by a central circannual pacemaker system. This is analogous to the organisation of the circadian timing system. For the circannual time-scale, specialised thyrotroph cells located in the pars tuberalis of the pituitary gland and adjacent tanycyte cells located in the ependymal wall of the third cerebral ventricle of the brain act as putative central circannual pacemakers. At a molecular level, epigenetically regulated, cyclical remodelling of chromatin, which determines whether specific circannual timer genes are transcriptionally active, or not, is considered to drive the oscillation between the summer and winter phenotypes.

Neuroendocrine correlates of the critical day length response in the Soay sheep.

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 ß-subunit (Tshß) gene transcription. According to this model, short photoperiods suppress Eya3 and hence Tshß 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 Tshß 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 Tshß 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-Tshß 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 Tshß-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.

Effects of Photoperiod Extension on Clock Gene and Neuropeptide RNA Expression in the SCN of the Soay Sheep.

In mammals, changing daylength (photoperiod) is the main synchronizer of seasonal functions. The photoperiodic information is transmitted through the retino-hypothalamic tract to the suprachiasmatic nuclei (SCN), site of the master circadian clock. To investigate effects of day length change on the sheep SCN, we used in-situ hybridization to assess the daily temporal organization of expression of circadian clock genes (Per1, Per2, Bmal1 and Fbxl21) and neuropeptides (Vip, Grp and Avp) in animals acclimated to a short photoperiod (SP; 8h of light) and at 3 or 15 days following transfer to a long photoperiod (LP3, LP15, respectively; 16h of light), achieved by an acute 8-h delay of lights off. We found that waveforms of SCN gene expression conformed to those previously seen in LP acclimated animals within 3 days of transfer to LP. Mean levels of expression for Per1-2 and Fbxl21 were nearly 2-fold higher in the LP15 than in the SP group. The expression of Vip was arrhythmic and unaffected by photoperiod, while, in contrast to rodents, Grp expression was not detectable within the sheep SCN. Expression of the circadian output gene Avp cycled robustly in all photoperiod groups with no detectable change in phasing. Overall these data suggest that synchronizing effects of light on SCN circadian organisation proceed similarly in ungulates and in rodents, despite differences in neuropeptide gene expression.

Testes size, testosterone production and reproductive behaviour in a natural mammalian mating system.

1. Testosterone (T) is a key mediator in the expression of numerous morphological and behavioural traits in mammals, but the factors underlying individual variation in circulating T levels are poorly understood. 2. The intimate structural integration of sperm and T production within the testes, alongside the dependency of sperm production on high levels of T, suggests that T requirements for spermatogenesis could be an important driver of individual differences in T. 3. To test this hypothesis, we examine how male capacity for sperm production (as indicated by their testes size) is associated with T levels in a feral population of Soay sheep, resident on St. Kilda, Scotland, during their rutting season. 4. We found a strong positive relationship between an individual's testes size (as measured before their seasonal enlargement) and the levels of circulating T during their rut, suggesting that T requirements for spermatogenesis has a prominent influence on the production of this androgen. 5. In contrast, body condition and competitive ability did not independently predict T levels, findings that are inconsistent with conventional 'condition-dependent' and 'challenge' hypotheses of T production. 6. This influence of male's capacity for sperm production on T appeared to be substantial enough to be biologically relevant, as testes size also predicted male aggression and mate-seeking behaviour. 7. Our results suggest that a male's inherent capacity for sperm and T production is tightly phenotypically integrated, with potential consequences for a wide range of other T-mediated reproductive traits.

Hypothesis: cyclical histogenesis is the basis of circannual timing.

Circannual rhythms are innately timed long-term (tau ≈ 12 months) cycles of physiology and behavior, crucial for life in habitats ranging from the equator to the Poles. Here the authors propose that circannual rhythm generation depends on tissue-autonomous, reiterated cycles of cell division, functional differentiation, and cell death. They see the feedback control influencing localized stem cell niches as crucial to this cyclical histogenesis hypothesis. Analogous to multi-oscillator circadian organization, circannual rhythm generation occurs in multiple tissues with hypothalamic and pituitary sites serving as central pacemakers. Signals including day length, nutrition, and social factors can synchronize circannual rhythms through hormonal influences, notably via the thyroid and glucocorticoid axes, which have profound effects on histogenesis. The authors offer 4 arguments in support of this hypothesis: (1) Cyclical histogenesis is a prevalent process in seasonal remodeling of physiology. It operates over long time domains and exhibits tissue autonomy in its regulation. (2) Experiments in which selected peripheral endocrine signals are held constant indicate that circannual rhythms are not primarily the product of interacting hormonal feedback loops. (3) Hormones known to control cell proliferation, differentiation, and organogenesis profoundly affect circannual rhythm expression. (4) The convergence point between photoperiodic input pathways and circannual rhythm expression occurs in histogenic regions of the hypothalamus and pituitary. In this review, the authors discuss how testing this hypothesis will depend on the use of cellular/molecular tools and animal models borrowed from developmental biology and neural stem cell research.

A molecular switch for photoperiod responsiveness in mammals.

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 ß subunit RNA (TSHß) 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 TSHß transcription through the circadian clock. Central to this is a conserved D element in the TSHß 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 TSHß 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 TSHß 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.

Identification of Eya3 and TAC1 as long-day signals in the sheep pituitary.

Seasonally breeding mammals such as sheep use photoperiod, encoded by the nocturnal secretion of the pineal hormone melatonin, as a critical cue to drive hormone rhythms and synchronize reproduction to the most optimal time of year. Melatonin acts directly on the pars tuberalis (PT) of the pituitary, regulating expression of thyrotropin, which then relays messages back to the hypothalamus to control reproductive circuits. In addition, a second local intrapituitary circuit controls seasonal prolactin (PRL) release via one or more currently uncharacterized low-molecular-weight peptides, termed "tuberalins," of PT origin. Studies in birds have identified the transcription factor Eya3 as the first molecular response activated by long photoperiod (LP). Using arrays and in situ hybridization studies, we demonstrate here that Eya3 is the strongest LP-activated gene in sheep, revealing a common photoperiodic molecular response in birds and mammals. We also demonstrate TAC1 (encoding the tachykinins substance P and neurokinin A) to be strongly activated by LP within the sheep PT. We show that these PRL secretagogues act on primary pituitary cells and thus are candidates for the elusive PT-expressed tuberalin seasonal hormone regulator.

Ancestral TSH mechanism signals summer in a photoperiodic mammal.

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.

Identification of melatonin-regulated genes in the ovine pituitary pars tuberalis, a target site for seasonal hormone control.

The pars tuberalis (PT) of the pituitary gland expresses a high density of melatonin (MEL) receptors and is believed to regulate seasonal physiology by decoding changes in nocturnal melatonin secretion. Circadian clock genes are known to be expressed in the PT in response to the decline (Per1) and onset (Cry1) of MEL secretion, but to date little is known of other molecular changes in this key MEL target site. To identify transcriptional pathways that may be involved in the diurnal and photoperiod-transduction mechanism, we performed a whole genome transcriptome analysis using PT RNA isolated from sheep culled at three time points over the 24-h cycle under either long or short photoperiods. Our results reveal 153 transcripts where expression differs between photoperiods at the light-dark transition and 54 transcripts where expression level was more globally altered by photoperiod (all time points combined). Cry1 induction at night was associated with up-regulation of genes coding for NeuroD1 (neurogenic differentiation factor 1), Pbef / Nampt (nicotinamide phosphoribosyltransferase), Hif1alpha (hypoxia-inducible factor-1alpha), and Kcnq5 (K+ channel) and down-regulation of Rorbeta, a key clock gene regulator. Using in situ hybridization, we confirmed day-night differences in expression for Pbef / Nampt, NeuroD1, and Rorbeta in the PT. Treatment of sheep with MEL increased PT expression for Cry1, Pbef / Nampt, NeuroD1, and Hif1alpha, but not Kcnq5. Our data thus reveal a cluster of Cry1-associated genes that are acutely responsive to MEL and novel transcriptional pathways involved in MEL action in the PT.

A physiological model of a circannual oscillator.

Recent evidence based on studies in hypothalamo-pituitary disconnected Soay sheep suggests that the generation of circannual rhythms may be local to specific tissues or physiological systems. Now, the authors present a physiological model of a circannual rhythm generator centered in the pituitary gland based on the interaction between melatonin-responsive cells in the pars tuberalis that act to decode photoperiod, and lactotroph cells of the adjacent pars distalis that secrete prolactin. The model produces a self-sustained, circannual rhythm in endocrine output that the authors explore by mathematical modeling. The circannual oscillation requires a delayed negative feedback mechanism. The authors highlight specific features of the pituitary dynamics as a guide to future research on circannual rhythms.

Redefining the limits of day length responsiveness in a seasonal mammal.

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.

Influence of photoperiod and gonadal status on food intake, adiposity, and gene expression of hypothalamic appetite regulators in a seasonal mammal.

We studied the effects of photoperiod on metabolic profiles, adiposity, and gene expression of hypothalamic appetite-regulating peptides in gonad-intact and castrated Soay rams. Groups of five to six animals were studied 6, 18, or 30 wk after switching from long photoperiod (LP: 16 h of light) to short photoperiod (SP: 8 h of light). Reproductive and metabolic indexes were measured in blood plasma. Expression of neuropeptide Y (NPY), proopiomelanocortin (POMC), and leptin receptor (ObRb) in the arcuate nucleus was measured using in situ hybridization. Testosterone levels of intact animals were low under LP, increased to a peak at 16 wk under SP, and then declined. Voluntary food intake (VFI) was high under LP in both intact and castrated animals, decreased to a nadir at 12-16 wk under SP, and then recovered, but only in intact rams as the reproductive axis became photorefractory to SP. NPY gene expression varied positively and POMC expression varied negatively with the cycle in VFI, with differences between intact and castrate rams in the refractory phase. ObRb expression decreased under SP, unrelated to changes in VFI. Visceral fat weight also varied between the intact and castrated animals across the cycle. We conclude that 1) photoperiodic changes in VFI reflect changes in NPY and POMC gene expression, 2) changes in ObRb gene expression are not necessarily determinants of changes in VFI, 3) gonadal status affects the pattern of VFI that changes with photoperiod, and 4) in the absence of gonadal factors, animals can eat less but gain adiposity.

Characterizing a mammalian circannual pacemaker.

Many species express endogenous cycles in physiology and behavior that allow anticipation of the seasons. The anatomical and cellular bases of these circannual rhythms have not been defined. Here, we provide strong evidence using an in vivo Soay sheep model that the circannual regulation of prolactin secretion, and its associated biology, derive from a pituitary-based timing mechanism. Circannual rhythm generation is seen as the product of the interaction between melatonin-regulated timer cells and adjacent prolactin-secreting cells, which together function as an intrapituitary "pacemaker-slave" timer system. These new insights open the way for a molecular analysis of long-term timing mechanisms.

Melatonin entrainment of circannual rhythms.

A melatonin-based photoperiod timing mechanism and a circannual rhythm-generating system interact to govern seasonal cycles in physiology and behavior in many vertebrates. This paper focuses on the pars tuberalis (PT) of the mammalian pituitary gland as a model melatonin-responsive tissue to investigate the molecular basis of these two basic long-term timing processes.

Decoding the nightly melatonin signal through circadian clockwork.

Photoperiod regulates the timing of seasonal cycles in reproduction, energy metabolism, moult and other seasonal characteristics, and the effects are transduced through changes in the duration of nocturnal melatonin secretion from the pineal gland. Short daily melatonin signals (4-8 h/day) activate a summer physiology, while long signals (> 10 h/day) produce a winter phenotype. Decoding signal duration occurs in specific target cells in the brain and pituitary gland, each governing a different component of the seasonal adaptation. The pars tuberalis (PT) of the pituitary regulates prolactin release and provides a tractable model system to investigate the molecular decoding mechanism. In the PT, melatonin onset at dusk activates cryptochrone (Cry1) gene expression and melatonin offset at dawn activates period (Per1) gene expression, thus the Cry/Per interval varies directly with nightlength, and inverse to daylength. It is proposed that photoperiod-induced changes in this phase-relationship dictates the level of CRY/PER protein heterodimer formation, and in turn, the level of transcriptional drive to the genes that control PT output--up-regulated under long days stimulating prolactin secretion and a summer physiology, and--down-regulated by short days in winter. The melatonin signal is thus decoded through circadian clock genes.

Multiple effects of melatonin on rhythmic clock gene expression in the mammalian pars tuberalis.

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.

Long-day suppressed expression of type 2 deiodinase gene in the mediobasal hypothalamus of the Saanen goat, a short-day breeder: implication for seasonal window of thyroid hormone action on reproductive neuroendocrine axis.

In most animals that live in temperate regions, reproduction is under photoperiodic control. In long-day breeders such as Japanese quail and Djungarian hamsters, type 2 deiodinase (Dio2) plays an important role in the mediobasal hypothalamus, catalyzing the conversion of prohormone T4 to bioactive T3 to regulate the photoperiodic response of the gonads. However, the molecular basis for seasonal reproduction in short-day breeders remains unclear. Because thyroid hormones are also known to be involved in short-day breeders, we examined the effect of an artificial long-day stimulus on Dio2 expression in the male Saanen goat (Capra hircus), a short-day breeder. Dio2 expression was observed in the caudal continuation of the arcuate nucleus, known as the target site for both melatonin and T4 action. In addition, expression of Dio2 and T3 content in the mediobasal hypothalamus was suppressed by artificial long-day conditions, which is the opposite of the results of long-day breeders. Thyroid hormone action on the development of neuroendocrine anestrus is known to be limited to a specific seasonal window. This long-day suppression of Dio2 may provide a mechanism that accounts for the lack of responsiveness to thyroxine during the mid to late anestrus.

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.

Photorefractoriness in mammals: dissociating a seasonal timer from the circadian-based photoperiod response.

In seasonal animals, prolonged exposure to constant photoperiod induces photorefractoriness, causing spontaneous reversion in physiology to that of the previous photoperiodic state. This study tested the hypothesis that the onset of photorefractoriness is correlated with a change in circadian expression of clock genes in the suprachiasmatic nucleus (circadian pacemaker) and the pars tuberalis (PT, a melatonin target tissue). Soay sheep were exposed to summer photoperiod (16-h light) for either 6 or 30 wk to produce a photostimulated and photorefractory physiology, and seasonal changes were tracked by measuring the long-term prolactin cycles. Animals were killed at 4-h intervals throughout 24 h. Contrary to the hypothesis, the 24-h rhythmic expression of clock genes (Rev-erbalpha, Per1, Per2, Bmal1, Cry1) in the suprachiasmatic nucleus and PT reflected the ambient photoperiod/melatonin signal and not the changing physiology. Contrastingly, the PT expression of alpha-glycoprotein hormone subunit (alphaGSU) and betaTSH declined in photorefractory animals toward a short day-like endocrinology. We conclude that the generation of long-term endocrine cycles depends on the interaction between a circadian-based, melatonin-dependent timer that drives the initial photoperiodic response and a non-circadian-based timer that drives circannual rhythmicity in long-lived species. Under constant photoperiod the two timers can dissociate, leading to the apparent refractory state.