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 CK1 epsilon ) 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 CK1 epsilon, 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.     

Clock genes in calendar cells as the basis of annual timekeeping in mammals--a unifying hypothesis

Melatonin-based photoperiod time-measurement and circannual rhythm generation are long-term time-keeping systems used to regulate seasonal cycles in physiology and behaviour in a wide range of mammals including man. We summarise recent evidence that temporal, melatonin-controlled expression of clock genes in specific calendar cells may provide a molecular mechanism for long-term timing. The agranular secretory cells of the pars tuberalis (PT) of the pituitary gland provide a model cell-type because they express a high density of melatonin (mt1) receptors and are implicated in photoperiod/circannual regulation of prolactin secretion and the associated seasonal biological responses. Studies of seasonal breeding hamsters and sheep indicate that circadian clock gene expression in the PT is modulated by photoperiod via the melatonin signal. In the Syrian and Siberian hamster PT, the high amplitude Per1 rhythm associated with dawn is suppressed under short photoperiods, an effect that is mimicked by melatonin treatment. More extensive studies in sheep show that many clock genes (e.g. Bmal1, Clock, Per1, Per2, Cry1 and Cry2) are expressed in the PT, and their expression oscillates through the 24-h light/darkness cycle in a temporal sequence distinct from that in the hypothalamic suprachiasmatic nucleus (central circadian pacemaker). Activation of Per1 occurs in the early light phase (dawn), while activation of Cry1 occurs in the dark phase (dusk), thus photoperiod-induced changes in the relative phase of Per and Cry gene expression acting through PER/CRY protein/protein interaction provide a potential mechanism for decoding the melatonin signal and generating a long-term photoperiodic response. The current challenge is to identify other calendar cells in the central nervous system regulating long-term cycles in reproduction, body weight and other seasonal characteristics and to establish whether clock genes provide a conserved molecular mechanism for long-term timekeeping.     

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.     

Decoding photoperiodic time through Per1 and ICER gene amplitude

The mammalian Per1 gene is expressed in the suprachiasmatic nucleus of the hypothalamus, where it is thought to play a critical role in the generation of circadian rhythms. Per1 mRNA also is expressed in other tissues. Its expression in the pars tuberalis (PT) of the pituitary is noteworthy because, like the suprachiasmatic nucleus, it is a known site of action of melatonin. The duration of the nocturnal melatonin signal encodes photoperiodic time, and many species use this to coordinate physiological adaptations with the yearly climatic cycle. This study reveals how the duration of photoperiodic time, conveyed through melatonin, is decoded as amplitude of Per1 and ICER (inducible cAMP early repressor) gene expression in the PT. Syrian hamsters display a robust and transient peak of Per1 and ICER gene expression 3 h after lights-on (Zeitgeber time 3) in the PT, under both long (16 h light/8 h dark) and short (8 h light/16 h dark) photoperiods. However, the amplitude of these peaks is greatly attenuated under a short photoperiod. The data show how amplitude of these genes may be important to the long-term measurement of photoperiodic time intervals.     

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.     

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.     

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.     

The pineal gland and the photoperiodic control of luteinizing hormone secretion in intact and castrated Japanese quail

The effects of pinealectomy on a range of photoperiodic responses were investigated in male Japanese quail by measuring plasma LH concentrations in intact, sham-operated and pinealectomized birds in the following four experiments: (1) transfer of sexually quiescent birds from a short photoperiod of 8 h light: 16 h darkness (8L:16D) to a photostimulatory daylength of 16L:8D; (2) transfer of sexually mature birds from 16L:8D to 8L:16D; (3) castration in 16L:8D and exposure to 13L:11D; (4) castration in 8L:16D and exposure to 13L:11D. There was no evidence of effects of the pineal gland on the photoperiodically induced changes in LH secretion, the quantitative relationship between LH secretion and photoperiod in intact and castrated birds, or the induction of relative photorefractoriness by prolonged exposure to 16L:8D. This suggests that there is no pineal influence on the photoperiodic clock or its effectors in this bird.     

Detection of neural connection to the infundibular complex by partial or complete hypothalamic deafferentation in male quail

To elucidate the stimulatory and inhibitory neural systems for photoperiodic control of avian reproduction, immature male Japanese quail were subjected to partial or complete hypothalamic deafferentation, followed by exposure to long and short photoperiods. The results indicated that when the encephalic photosensitive area (infundibular complex, INF) was preserved after hypothalamic deafferentation, birds were able to respond to long days and their gonads eventually recrudesced, and that testicular atrophy under short days was prevented by the semicircular cuts posterior to INF or by orbital enucleation. It is concluded that in male Japanese quail, INF plays the pivotal role in photoperiodic gonadostimulation and regulatory neurons in the retina and anterior hypothalamus may have neural connection to the posterior side of INF.     

Separate oscillating cell groups in mouse suprachiasmatic nucleus couple photoperiodically to the onset and end of daily activity

The pattern of circadian behavioral rhythms is photoperiod-dependent, highlighted by the conservation of a phase relation between the behavioral rhythm and photoperiod. A model of two separate, but mutually coupled, circadian oscillators has been proposed to explain photoperiodic responses of behavioral rhythm in nocturnal rodents: an evening oscillator, which drives the activity onset and entrains to dusk, and a morning oscillator, which drives the end of activity and entrains to dawn. Continuous measurement of circadian rhythms in clock gene Per1 expression by a bioluminescence reporter enabled us to identify the separate oscillating cell groups in the mouse suprachiasmatic nucleus (SCN), which composed circadian oscillations of different phases and responded to photoperiods differentially. The circadian oscillation in the posterior SCN was phase-locked to the end of activity under three photoperiods examined. On the other hand, the oscillation in the anterior SCN was phase-locked to the onset of activity but showed a bimodal pattern under a long photoperiod [light-dark cycle (LD)18:6]. The bimodality in the anterior SCN reflected two circadian oscillatory cell groups of early and late phases. The anterior oscillation was unimodal under intermediate (LD12:12) and short (LD6:18) photoperiods, which was always phase-lagged behind the posterior oscillation when the late phase in LD18:6 was taken. The phase difference was largest in LD18:6 and smallest in LD6:18. These findings indicate that three oscillating cell groups in the SCN constitute regionally specific circadian oscillations, and at least two of them are involved in photoperiodic response of behavioral rhythm.     

The stimulation of luteinizing hormone and follicle-stimulating hormone secretion in quail with complete and skeleton photoperiods

Photostimulation of quail by long daylengths stimulates LH and FSH release, the earliest increases in these hormones being detectable after 1 long day. Reproductive maturity is complete within 1 month. Steroid feedback becomes important during the second week of photostimulation and restricts LH and FSH secretion to a fraction of the concentrations observed in gonadectomized birds. The photoperiodic response is not all-or-none, and above a certain “critical daylength” the rate of gonadal growth, primarily controlled by the levels of FSH and testosterone, is proportional to daylength. Photoperiodic responses are qualitatively identical in gonadectomized and intact quail, arguing that stimulatory daylengths act directly on the hypothalamo-pituitary axis to alter LH and FSH secretion, and do not act by altering steroid feedback sensitivity and thereby gonadotrophin release. Any changes in feedback sensitivity are a consequence not a cause of the photoperiodic drive on the system. The use of “skeleton” photoperiods to mimic complete photoperiods is discussed. Whether or not one or two peaks of photoperiodic induction appear in asymmetric skeleton experiments depends both upon the duration of the first light period and of the night break itself. The rate of induction is less when quail are photostimulated with night breaks than with “complete” daylengths. The temporal position of the circadian rhythm(s) involved in photoperiodic time measurement in quail is controlled by both “dawn” and “dusk,” and so the position of the rhythm is phase-delayed as the days lengthen, occurring later and later in the night. This arrangement might have some bearing on the seasonal shift in critical daylength that occurs in quail exposed to natural photoperiods. The situation is compared with that in insects and plants.     

A possible role for the eyes in the photoperiodic response of quail

The effects of blinding on the photoperiodic responses of male Japanese quail were investigated by measuring plasma luteinizing-hormone (LH) concentrations in intact and castrated birds. Blinded birds were still able to respond to short and long days with appropriate changes in LH levels, suggesting that the basic photoperiodic mechanisms do not require retinal photoreception. However, there were clear-cut differences between blinded and sighted birds with the LH levels being higher in blinded quail. This difference between blinded and sighted was greater in short than in long days, and was also enhanced by castration. In conclusion we propose that short day information transmitted by the eyes has an inhibitory effect for LH secretion independent from sex steroid negative feedback effects in quail.     

Encoding and decoding photoperiod in the mammalian pars tuberalis

In mammals, the nocturnal melatonin signal is well established as a key hormonal indicator of seasonal changes in day-length, providing the brain with an internal representation of the external photoperiod. The pars tuberalis (PT) of the pituitary gland is the major site of expression of the G-coupled receptor MT1 in the brain and is considered as the main site of integration of the photoperiodic melatonin signal. Recent studies have revealed how the photoperiodic melatonin signal is encoded and conveyed by the PT to the brain and the pituitary, but much remains to be resolved. The development of new animal models and techniques such as cDNA arrays or high throughput sequencing has recently shed the light onto the regulatory networks that might be involved. This review considers the current understanding of the mechanisms driving photoperiodism in the mammalian PT with a particular focus on the seasonal prolactin secretion.     

The rat suprachiasmatic nucleus is a clock for all seasons.

Seasonal changes of daylength (photoperiod) affect the expression of hormonal and behavioral circadian rhythms in a variety of organisms. In mammals, such effects might reflect photoperiodic changes in the circadian pace-making system [located in the suprachiasmatic nucleus (SCN) of the hypothalamus] that governs these rhythms, but to date no functionally relevant, intrinsic property of the SCN has been shown to be photoperiod dependent. We have analyzed the temporal regulation of light-induced c-fos gene expression in the SCN of rats maintained in long or short photoperiods. Both in situ hybridization and immunohistochemical assays show that the endogenous circadian rhythm of light responsiveness in the SCN is altered by photoperiod, with the duration of the photosensitive subjective night under the short photoperiod 5-6 h longer than under the long photoperiod. Our results provide evidence that a functional property of the SCN is altered by photoperiod and suggest that the nucleus is involved in photoperiodic time measurement.     

Hypothalamic secretion of thyrotrophin releasing hormone is decreased in male Japanese quail exposed to long daily photoperiods

Adult male Japanese quail held under short daily photoperiods (8 h light: 16 h darkness; 8L: 16D) had significantly higher plasma concentrations of thyroid-stimulating hormone (TSH), tri-iodothyronine (T3) and thyroxine (T4) than did those kept under long days (16L:8D). When given a single s.c. injection of 50 microgram thyrotrophin releasing hormone (TRH) the birds held under both the 8L: 16D and 16L: 8D photoperiods showed rapid increases in their blood concentrations of TSH, T4 and T3, the amplitude of the TSH response of the birds exposed to 16L: 8D being particularly marked. These results suggest that, in the male quail, long daily photoperiods produce a hypothyroid state as a result of diminished TRH secretion. The synthetic and secretory capacities of the thyroid gland and pituitary thyrotrophs are apparently unimpaired by long days.     

Photoperiodic response of rain quail

The photoperiodic response of the migratory sexually dimorphic Rain quail were studied. Birds were subjected to constant long (18L : 6D) and constant short (6L : 18D) photoperiods. In addition birds receiving 6L : 18D treatment were given additional light pulse of an hour duration at 0,2, 4 & 6 hours after the end of 6 hour photophase. It is obvious that the photoinducible phase occurs after 12 hours of Sun rise or "Light on" phase. This is based on the fact that the testis remain small and inactive under short photoperiods and alson in birds receiving light pulse at 0,2 & 4 hours in which the photophase occured at 12 hour or earlier. The full development was observed in groups of birds in which only when the light was available at 13 hour. It also seems that the light inducible phase in this species probably like in weaver bird but similar to in temperate zone birds may be quite long. The results it is suggested confirm the concept of Bunning's hypothesis.     

Involvement of thyrotropin in photoperiodic signal transduction in mice

Local thyroid hormone catabolism within the mediobasal hypothalamus (MBH) by thyroid hormone-activating (DIO2) and -inactivating (DIO3) enzymes regulates seasonal reproduction in birds and mammals. Recent functional genomics analysis in birds has shown that long days induce thyroid-stimulating hormone production in the pars tuberalis (PT) of the pituitary gland, which triggers DIO2 expression in the ependymal cells (EC) of the MBH. In mammals, nocturnal melatonin secretion provides an endocrine signal of the photoperiod to the PT that contains melatonin receptors in high density, but the interface between the melatonin signal perceived in the PT and the thyroid hormone levels in the MBH remains unclear. Here we provide evidence in mice that TSH participates in this photoperiodic signal transduction. Although most mouse strains are considered to be nonseasonal, a robust photoperiodic response comprising induced expression of TSHB (TSH β subunit), CGA (TSH α subunit), and DIO2, and reduced expression of DIO3, was observed in melatonin-proficient CBA/N mice. These responses could not be elicited in melatonin-deficient C57BL/6J, but treatment of C57BL/6J mice with exogenous melatonin elicited similar effects on the expression of the above-mentioned genes as observed in CBA/N after transfer to short-day conditions. The EC was found to express TSH receptor (TSHR), and ICV injection of TSH induced DIO2 expression. Finally, we show that melatonin administration did not affect the expression of TSHB, DIO2, and DIO3 in TSHR-null mice. Taken together, our findings suggest that melatonin-dependent regulation of thyroid hormone levels in the MBH appears to involve TSH in mammals.     

Interaction of surgical deafening and photoperiod on cloacal gland and testes size in Japanese quail

Two experiments were conducted to further examine the interaction of social stimuli and photoperiod on cloacal and gonadal responses in male Japanese quail. In experiment 1, adult males kept on a maximal (15L:9D) stimulatory photoperiod were surgically deafened and/or visually isolated from roommates. After 6 weeks, there were no differences in cloacal gland or testes size among visually isolated birds, deafened birds, visually isolated and deafened birds, and control birds. It would appear that neither auditory nor visual contact with other males is necessary to maintain reproductive activity under a maximal stimulatory photoperiod. In Experiment 2, 5-week-old males were surgically deafened and kept on either a maximal (15L:9D) or minimal (12L:12D) stimulatory photoperiod for about 10 weeks. The rates of cloacal gland growth were significantly slower in birds kept on the shorter photoperiod compared to the longer photoperiod and in deafened quail compared to intact quail. The difference in cloacal gland size between deafened and intact birds was markedly greater on the minimal compared to the maximal stimulatory photoperiod. These findings suggest that under a minimal stimulatory photoperiod auditory information supplements the photoperiod to accelerate reproductive maturation. At a maximal stimulatory photoperiod, however, the effects of auditory cues on cloacal gland growth appear masked.     

Effects of photoperiod and temperature on testicular and thyroid activity of the Japanese quail

The testes of Japanese quail (Coturnix coturnix japonica) grow during a short photoperiod (8L:16D) and reach maturation at 10 to 12 weeks after hatching. When the ambient temperature was lowered from 23 to 10° in this photoperiod, the size of the cloacal gland, a secondary sex character in Japanese quail, was significantly reduced. These involuted cloacal glands attained mature size after elevation of the ambient temperature to 23°. Mature cloacal glands during long photoperiods (16L:8D) remained unchanged by transferring birds to low temperature. These results indicate that ambient temperature as well as the length of photoperiod affect the testicular activity of Japanese quail and that the effect of the length of the photoperiod is predominant.Triiodothyronine (T₃) levels in serum were significantly increased by low temperature and short photoperiod. Thyroxine (T₄) levels were not different among the environmental conditions tested. The result suggests the existence of an inverse relationship between gonadal activity and T₃ activity.