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Endocrinology, doi:10.1210/en.2007-0658
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Endocrinology Vol. 149, No. 1 32-39
Copyright © 2008 by The Endocrine Society

Redefining the Limits of Day Length Responsiveness in a Seasonal Mammal

Gabriela C. Wagner, Jonathan D. Johnston, Iain J. Clarke, Gerald A. Lincoln and David G. Hazlerigg

School of Biological Sciences (G.C.W., D.G.H.), University of Aberdeen, Aberdeen AB24 2TZ, United Kingdom; School of Biomedical and Molecular Sciences (J.D.J.), University of Surrey, Guildford GU2 7XH, United Kingdom; Department of Physiology (I.J.C.), Monash University, Victoria 3880, Australia; and School of Biomedical Sciences (G.A.L.), University of Edinburgh, The Queen’s Medical Research Institute, Edinburgh EH8 9JZ, United Kingdom

Address all correspondence and requests for reprints to: Dr. David G. Hazlerigg, School of Biological Sciences, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, United Kingdom. E-mail: d.hazlerigg{at}abdn.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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°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 (βTSH) 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STUDIES IN A WIDE range of organisms have led to the concept that transition from winter to spring/summer phenotypes occurs when day length (photoperiod) exceeds a minimum threshold, termed the critical day length (1). This threshold varies within individuals as a function of prior photoperiodic exposure and between individuals in a manner reflecting latitude of origin (2, 3). This implies that a genetically controlled mechanism determines the working range of environmental photoperiods over which an organism can precisely time seasonal transitions in physiology and behavior.

In mammals, the effect of changes in photoperiod is relayed via changes in nocturnal melatonin secretion by the pineal gland (3). Melatonin is synthesized at night in proportion to night length and progressive changes in the duration of the melatonin signal are transduced into seasonal cycles of physiology, often showing sharp changes in state around the critical day length. Decoding of the melatonin signal is thus believed to depend on durational discrimination at the molecular level in melatonin receptor expressing cells (4).

The cellular mechanisms by which melatonin signal duration is decoded have been studied principally in the cells in the pars tuberalis (PT) of the pituitary gland (5). The thyrotroph-like cells of the PT express a notably high density of melatonin receptors and regulate seasonal prolactin secretion (6, 7, 8). Several studies suggest that melatonin acts in the PT through effects on the rhythmical expression of the canonical circadian clock genes (9, 10, 11). These are widely expressed in mammalian tissues and are required for circadian rhythm generation, produced by a sequence of rhythmical transcriptional activation and repression over 24 h (12, 13). The PT expresses circadian rhythms in a broad complement of known clock genes including period (per), cryptochrome (cry), and the basic-helic-loop-helix (bHLH)/PER-ARNT-SIM (PAS) domain transcriptional activators clock and brain and muscle ARNT-like protein-1 (bmal1). The phase-relationships between these rhythms and the sensitivity of clock gene expression in the PT to melatonin treatment are consistent with the PT operating as a melatonin-dependent circadian oscillator (5, 10, 11).

The hypothesis that clock gene expression in the PT is crucial for photoperiodic time measurement was first advanced after the finding that Syrian hamsters express a higher amplitude rhythm of per1 RNA expression under long days (16 h light), compared with short days (8 h light) (9). Peak expression occurs in the early light phase after withdrawal of melatonin (9). This photoperiodic effect is also seen in Siberian hamsters, and to a lesser extent in sheep (10, 14), and in Siberian hamsters, it carries through to changes in PER1 protein expression (15). Studies in mice indicate that per1 rhythmicity depends on expression of melatonin receptors in the PT. These are coupled to the adenylate cyclase signaling pathway and the regulation of per1 is known to be cAMP dependent in PT cells (16). In mouse PT explants, prolonged exposure to melatonin followed by withdrawal causes increased PER1 responsiveness to secretagogues of adenylate cyclase (17). Collectively, these data support the hypothesis that melatonin signal duration is decoded in part through changes in the amplitude of the PER1 rhythm in melatonin-responsive cells.

This model leads to an interesting and so-far-untested prediction: very long photoperiods, such as occur at high temperate latitudes in summer (20 h light) should fail to activate a long-day response. This is because in vitro data (18) predict that the melatonin signal would be too short to sensitize adenylate cyclase for per1 gene induction. Hence, a change from short to very long photoperiods (20 h and above) should elicit weaker photoinduction than transfer to an intermediate-long photoperiod of 16 h light, most commonly used in studies of photoperiodism. Such a mechanism could define the range of photoperiods over which melatonin acts to synchronize seasonal rhythms.

In the present study, we therefore exposed Soay sheep to lighting regimens spanning the range of spring-summer photoperiods experienced in their natural habitat (North Atlantic archipelago of St. Kilda, 57°N). The sheep breed was used because of its highly seasonal physiology (19). We assayed behavioral and endocrine markers of photoperiodic responses and gene expression in the PT and arcuate nucleus at the end of the study. The data confirm the predicted biphasic response to photoperiod. We suggest a model for the molecular basis of this window of responsiveness that sets the timing of seasonal transitions according to the latitude of origin in a natural environment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Animal experiments were conducted in accordance with the U.K. Animals (Scientific Procedures) Act of 1986. Soay sheep were selected as a model because of their seasonal wild-type characteristics (19). The animals were penned in light-sealed rooms and fed a standardized diet of grass pellets (500 g/animal; Vitagrass, Cumbria, UK) given 1 h into the light phase. There was free access to hay and water and temperature was regulated within the range 10–25 C by a ventilator/heater system. White florescent strip lights provided approximately 160 lux at the sheep’s’ eye level during the light phase. Dim red light (<5 lux) was provided during the dark phase. Daily locomotor activity was recorded continuously for individuals using infrared sensors coupled to a MiniMitter VitalView system (Sunriver, OR) (19). The long-term changes in physiology in each animal were tracked by measuring the circulating concentrations of prolactin in blood samples collected from the jugular twice a week. The animals were habituated to handling and care was taken to avoid any stressor that might activate prolactin secretion. Samples were placed in heparinized tubes and the blood plasma separated by centrifugation within 30 min and stored at –20 C until assayed. Diurnal rhythms in melatonin secretion were characterized after 4 wk in the set photoperiod. Blood samples were collected hourly for 24 h using an indwelling jugular cannula inserted the previous day to avoid interference with normal behavior. The blood samples (3 ml) were separated and the plasma stored as for the routine weekly samples.

Experimental design
Fifty-five Soay ewes were brought indoors in winter (November) and acclimatized to short days [8 h light, 16 h darkness (LD 8:16)] for 6 wk. Zeitgeber time (ZT) 0 was taken as time of lights on throughout. The short photoperiod (SP) treatment group (n = 11) was killed after acclimatization; four other treatment groups were exposed to photoperiods LD 12:12 (n = 11), LD 16:8 (n = 12), LD 20:4 (n = 12), and LD 22:2 (n = 9) for 6 wk. In each treatment group, six animals were marked for routine plasma sampling to monitor the long-term changes in prolactin secretion. At the end of the study, animals were killed by an overdose of barbiturate (Euthatal; Rhone Merieux, Essex, UK) in groups of three, 3 h after lights off and 1, 3, and 5 h after lights on the following morning. These times were selected based on our earlier work (10) to allow assessment of evening peak values for cry1 and morning peak values for per1. In the case of the LD 22:2 group, the first two sampling points coincide. Blood samples were collected before death for a subsequent RIA. The hypothalamus and upper pituitary gland were dissected as a single block from the skull within 10 min of death, frozen in isopentane at –30 C, and stored at –80 C. Both ovaries of each animal were examined and scored for presence of corpora lutea.

Prolactin/melatonin RIA
Prolactin concentrations in the twice-weekly blood samples and melatonin concentrations in the hourly samples were measured by standardized RIAs validated for ovine plasma (20). The prolactin assay had a lower limit of detection of 0.5 µg/liter plasma and intra- and interassay coefficients of variation (CVs) were less than 10%. Melatonin concentrations were measured by RIA (21) using a commercial antibody (PF-1288; SPI-BIO, France). The assay had a lower limit of detection of 5.0 pg/ml plasma and CVs were less than 12%. Melatonin peak duration was defined as the period when melatonin concentration is continuously above mean baseline value (first three samples collected in light phase) + 2 x intraassay CV. Melatonin amplitude was defined as the mean melatonin concentration during the dark phase for each animal producing a group mean ± SEM.

In situ hybridization
Twenty-micrometer coronal sections were cut at the level of the PT of the pituitary, thaw mounted onto 0.008% poly-L-lysine per 0.5% gelatin-coated glass slides in sequential order, and stored at –80 C. Radioactive in situ hybridization was performed as described previously (9).

The expression of a range of genes was studied by in situ hybridization using homologous RNA probes for the ovine cry1 and per1 as described previously (10). The βTSH probe was based on the rat cDNA sequence and generously provided by Dr. P. Klosen (22) and had been previously validated for use in sheep (23). The probe for KiSS-1 consists of the 357 bp of the ovine sequence (GenBank accession no. DQ059506).

Statistical analysis
Quantitative data are expressed as mean ± SEM and were analyzed with GraphPad Prism 4.03 software (GraphPad Software, San Diego, CA), using one- or two-way ANOVA with Bonferroni’s post hoc analysis, as appropriate. The effect of photoperiod on the frequency of animals showing corpora lutea was analyzed by {chi}2 test. Statistical significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Female Soay sheep were brought indoors at approximately 6 months age and acclimated to an SP of LD 8:16 for a period of 6 wk. A subset of the animals was killed after acclimatization. The remainder were transferred to one of four experimental photoperiods (LD 12:12, LD 16:8, LD 20:4, or LD 22:2) for a further 6 wk.

At the end of the treatments, the animals showed a diurnal pattern of locomotor activity, and the duration of activity corresponded to the length of the light phase (Fig. 1Go). A clearly defined nocturnal pattern of melatonin secretion was seen in all groups (P < 0.0001, one-way ANOVA, Figs. 1Go, 2AGo, and 3CGo); duration of increased melatonin concentrations was proportional to the length of the dark phase (P < 0.001, r2 = 0.99, linear regression, data not shown). The amplitude of the nocturnal peak was significantly lower on LD 22:2, compared with LD 12:12 or LD 16:8 (P < 0.05, one-way ANOVA, Fig. 2AGo). These data demonstrate that Soay sheep synchronize daily patterns of physiology and behavior to the external light dark cycle, even when exposed to 22 h light per day.


Figure 1
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  		FIG. 1. Soay sheep entrain to 20-h photoperiods. Double-plot of group activity recording and melatonin secretion data for animals transferred from 8- to 20-h light per day. ZT0, Time of lights on. Note the decompression of diurnal activity into the extended light phase (top), whereas melatonin secretion is limited to the 4-h night (bottom).

 

Figure 2
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  		FIG. 2. The biphasic endocrine response of Soay sheep to increasing photoperiods. A, Duration and amplitude of melatonin secretion across the five photoperiod treatments; melatonin signal duration declines proportional to declining night length (P < 0.001, one-way ANOVA, n = 6); amplitude does not vary significantly, except on the longest, 22-h photoperiod (P < 0.01, one-way ANOVA). B, Blood prolactin levels sampled 6 wk after the switch to a new photoperiod. Bars with different superscripts differ significantly (P < 0.0001, one-way ANOVA, n ≤ 6). C, Proportion of animals bearing corpora lutea at the end of the study; there was a significant overall effect of photoperiod (P < 0.05 by {chi}2 test, n ≤ 9).

 

Figure 3
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  		FIG. 3. Effect of photoperiod on clock gene expression in the sheep pars tuberalis. A, Anatomy of the sheep brain showing positions of elements of the photoneuroendocrine system (arrows) and the level at which coronal sections were taken to assay PT gene expression (vertical line). SCN, Suprachiasmatic nucleus. B, Representative autoradiographs showing maximum and minimum levels of cry1 and per1 expression at selected time points under 16-h photoperiods. ZT0 = time of lights on. C, Mean OD measurements at the time points specified under each photoperiod. Background line graphs show melatonin (MEL) secretory profiles assayed by serial bleed 1 wk before tissue collection. Under all photoperiods external time 12 = midlight phase. Data are mean ± SEM, n = 6. Sample points for clock gene expression were 3 h after lights off and 1, 3, and 5 h after lights on in each photoperiod group. Data points are mean ± SEM, n = 3.

 
The treatments had a biphasic effect on prolactin secretion and reproductive activation. Plasma prolactin concentrations (Fig. 2BGo) were low after 6 wk exposure to SP, and secretion was increased 4- to 5-fold after exposure to LD 16:8 (P < 0.01). Secretion was intermediate in animals switched to either shorter (LD 12:12) or longer (LD 20:4 and LD 22:2) photoperiods. A similar, but inverted, biphasic response was seen in the proportion of animals showing ovarian cyclicity (P < 0.05, Fig. 2CGo). Under SP, the majority of animals were reproductively active with one to two corpora lutea present in the ovaries, whereas fewer than 20% were active after transfer to LD 16:8 for 6 wk. Again, shorter or longer photoperiods produced intermediate levels of ovarian activity. To our knowledge, this is the first description of a biphasic response to increased day length in SP acclimated animals.

To analyze gene expression, animals were killed at specific time points designed to reveal evening (3 h after lights out) and morning (1, 3, and 5 h after lights on) patterns of gene expression. We used multiple morning sampling points to account for documented variation in the rate of decline in melatonin secretion at the end of the dark phase. In situ hybridization was performed in coronal sections at the level of the pituitary stalk containing the PT and more caudally to reveal expression in the medial arcuate nuclei (ARC) of the hypothalamus.

In the PT, the temporal patterns of expression of the clock genes per1 and cry1 were highly photoperiod dependent (Fig. 3Go) (P < 0.0001 for time x photoperiod interaction in both cases). Significant temporal variation in per1 expression was only seen under the 8-, 12-, and 16-h photoperiods (P < 0.05 by one-way ANOVA), and in each of these peak per1 expressions was seen 1 to 3 h into the light phase. Cry1 expression was always increased 3 h after lights off, even under LD 22:2, when this occurs 1 h into the light phase (Fig. 3CGo). The amplitude of maximal per1 expression showed a biphasic response to photoperiod (Fig. 4AGo, middle panel; P < 0.0001, one-way ANOVA): values were high under LD 16:8 and low under shorter or longer photoperiods. In contrast, the amplitude of the cry1 signal did not vary with photoperiod (Figs. 3CGo and 4AGo, top panel; P = 0.1196, one-way ANOVA),


Figure 4
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  		FIG. 4. Biphasic responses of per1 and βTSH but not cry1 gene expression in the sheep pars tuberalis. A, Bars show maximal recorded expression of cry1 (3 h after lights off) and per1 (1 or 3 h after lights on) and average βTSH expression across all sampling times. Data are mean ± SEM, n ≤ 3; P values indicate effect of photoperiod under one-way ANOVA; bars with different superscripts are significantly different by Bonferroni’s test (P < 0.05). B, Scatter diagram showing the intraindividual relationship between βTSH gene expression in the PT and prolactin levels in postmortem blood samples.

 
βTSH is a hormonal output gene expressed by the PT and suppressed by SP exposure in the European hamster (22). Here we found that the expression of mRNAs for βTSH, and the glycoprotein hormone {alpha}-subunit, showed a pronounced biphasic response to photoperiod in the sheep PT, with maximal expression on LD 16:8 (P < 0.001, Fig. 4AGo, bottom panel, and data not shown). Across the five treatment groups, there was a strong positive correlation between prolactin secretion at time of death and PT expression of βTSH (P < 0.0001, Fig. 4BGo). In the hypothalamus, the expression of the Kiss-1 gene that encodes kisspeptin, known to be crucial for reproductive activation in hamsters and sheep (24, 25, 26), declined with transfer to long photoperiod in the sheep. Maximal KiSS-1 expression was seen in the LD8:16 animals, and transfer to LD 16:8 had the strongest suppressive effect on KiSS-1 expression (Fig. 5Go).


Figure 5
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  		FIG. 5. KiSS-1 mRNA expression in the ARC of the female Soay sheep brain. A, Top left panel shows a diagrammatic representation of KiSS-1 mRNA localization in the hypothalamus. PVN, Periventricular nucleus. The bottom left panel is a representative autoradiograph of a section labeled with the sense riboprobe. Scale bar, 1 mm. The right panels represent autoradiographs showing KiSS-1 expression at the level of the ARC from sheep kept under a SP (top right) or LP (bottom right). B, Integrated OD measurements of KiSS-1 expression at the level of the ARC. Data are mean ± SEM, n ≤ 8.

 
Overall, the differential responses to the long-day treatments could be traced from clock gene expression in the PT to output gene expression in the PT and hypothalamus and, in turn, to the seasonal regulation of prolactin secretion and the reproductive axis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Photoperiodic responses in mammals depend on light effects on the circadian system. Unlike ungulates living within the Arctic Circle, which undergo a breakdown of circadian rhythmicity during the polar summer (27), our sheep clearly showed synchronization of both, locomotor activity and melatonin secretion to the light-dark cycle, even on the 22-h photoperiod. The melatonin signal was also sufficient to maintain rhythmic cry1 gene expression in the PT under all the photoperiods studied. Hence, a general breakdown of circadian coordination, or loss of the melatonin signal, cannot explain the biphasic response in Soay sheep exposed to the long day treatments. We therefore believe that the explanation for the observed physiological responses lies in mechanisms that decode the duration of the melatonin signal in the melatonin-target tissues.

The amplitude of the 24-h rhythm in per1 expression in the PT varies markedly with photoperiod. This is decreased under SP and increased under long days, as reported in seasonal rodents. Peak per1 expression occurs in the early light phase and is thought to depend on the duration of the melatonin signal the previous night. Melatonin sensitizes the cAMP generating enzyme adenylate cyclase during the night, leading to dawn induction of cAMP-regulated genes, including per1, through a derepression mechanism (16, 17, 18, 28). The prediction for the current study was that per1 induction would be diminished on photoperiods that limited the melatonin signal duration so far that the adenylate cyclase system was not sensitized; our latest results confirm this prediction.

Additional to the effect of photoperiods greater than 16 h, per1 amplitude is also reduced in the SP group (LD 8:16). Hence, based on the known dynamics in cultures of sheep PT cells (18), adenylate cyclase sensitization alone is not sufficient to produce the variation in the amplitude of morning peak of cAMP-dependent gene expression observed in this study. The sampling window used to assay per1 was based on two previous experiments in which per1 consistently peaked round ZT3 (10, 23, 28), and this was also the case in LD 8:16 animals in the present study; therefore, reduced amplitude of per1 expression in this group was not an artefact of sampling time. This effect may be related to the rate at which melatonin levels decline at the end of the night: On 12- and 16-h photoperiods lights on masks melatonin secretion causing precipitous decline in melatonin levels, whereas on 8-h photoperiods, melatonin starts to decline some 4 h before lights on and does so progressively. We suspect that rebound increases in cAMP levels after melatonin withdrawal are most marked when melatonin levels decline abruptly. It is interesting to note that there are pronounced seasonal variations in the duration of twilight at high-temperate latitudes, with the light-dark transition being most rapid around the equinoxes, possibly favoring pronounced melatonin-dependent effects on gene expression during spring and autumn.

Whereas per1 induction is highest on 12-h and 16-h photoperiods, with no clear distinction between them, the prolactin and βTSH responses are both significantly higher in LD 16:8 animals than all other groups. This suggests that a model based purely on variation in per1 amplitude is insufficient to account for the biphasic long day response described. In marked contrast to the per1 amplitude phenomenon, cry1 induction in response to rising melatonin levels in the evening occurs on all photoperiods examined. Because PER and CRY proteins form transcription modulating complexes crucial for circadian oscillator function, we hypothesized previously that regulation of the cry-per interval by the melatonin signal may govern the output response by influencing levels of PER-CRY complex formation (10).

We now propose a modified model combining the adenylate cyclase sensitization and per-cry coincidence effects to account for the photoinductive effects of different long photoperiods (Fig. 6Go). According to this model, high levels of daytime PER accumulation require a melatonin signal that will adequately sensitize adenylate cyclase. For PER1 to be incorporated into a transcriptional complex, it must become bound to CRY. Hence, maximal complex formation will be favored by melatonin signals that reduce the interval between cry1 and per1 induction, subject to the criteria above. This AND gate requirement defines a critical photoperiodic window for induction of the spring/summer phenotype. Our model hinges on the hypothesized causal link between melatonin-induced changes in clock gene expression and photoperiodic output. In the mouse, Jilg et al. (29) provided some evidence that nuclear accumulation of PER1 and CRY1 follows the Per1 RNA rhythm, but a definitive test, involving genetic manipulation in the PT of a photoperiodic species remains an outstanding technical challenge.


Figure 6
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  		FIG. 6. Coincidence to amplitude model to account for the biphasic photoperiodic response. The model proposes that per1 amplitude in melatonin-responsive PT cells is determined by the interaction between sensitization of the adenylate cyclase signaling pathway (dashed lines) and temporal coincidence between times of peak expression of the per and cry genes (dotted lines). These two components vary with melatonin signal duration in different ways; adenylate cyclase sensitization is permissive on short to intermediate long day length photoperiods but disappears when the nocturnal melatonin signal duration becomes too short. Cry1-per1 coincidence increases as photoperiod increases, disappearing only when adenylate cyclase sensitization becomes insufficient to support a morning per1 peak. The vernal response (apparent in per1 amplitude and attendant downstream phenomena, solid line) is determined by the net effect of these two opposing photoperiod effects. This leads to a critical photoperiodic window on intermediate to long days (bounded by shading in lower panel), which determines expression of the specific long day phenotype.

 
There are limited data on the relative importance of melatonin signals experienced at different times of the year for overall synchronization of the seasonal cycle. Woodfill et al. (30) infused pinealectomized ewes with sequences of melatonin signals to mimic melatonin exposure in different segments of the year. It was found that spring/summer melatonin infusion patterns were more potent synchronizers of the circannual breeding cycle than autumn/winter infusion patterns. Because only the spring and summer infusion patterns produced melatonin signals corresponding to photoperiods of around 16 h, this earlier result can be rationalized based on our molecular definition of the critical spring photoperiodic window. This suggests that high amplitude PER1 rhythms are a key component of circannual entrainment.

Distinct anatomical areas mediate the effects of melatonin on seasonal lactotrophic and gonadotrophic responses. Whereas the PT is known to mediate melatonin effects on the prolactin axis (5), the mediobasal hypothalamus appears to be the site for melatonin action on reproduction (31, 32, 33). In the ARC the KiSS-1 gene encodes kisspeptin, which is regulated by steroid levels (34, 35) and photoperiod (24, 25, 26). KiSS-1 has been shown to be crucial for reproductive activation at puberty (36, 37), and exogenous kisspeptin treatment overcomes the suppressive effects of SP exposure on reproduction in the hamsters (24). Here we found that transfer to a 16-h photoperiod had the strongest inhibitory effect on estrus activity and ARC KiSS-1 expression; melatonin-dependent control of hypothalamic kisspeptin neurons may therefore account for the differential effects of photoperiod in spring (hamsters) vs. autumn (sheep) breeders. The pronounced biphasic response seen for prolactin secretion and gene expression in the PT was not evident in the case of KiSS-1 expression. This may reflect the involvement of additional factors in photoperiodic control of the reproductive axis, or possible differences in hypothalamic as opposed to pituitary melatonin readout mechanisms.

In conclusion, we have shown that a clock gene based molecular model for melatonin action yields novel predictions for the photoperiodic response of a seasonal mammal and that these are upheld by an in vivo test. A critical photoperiodic window defines the range of photoperiods that will promote a long day phenotype in sheep previously exposed to short days. Earlier work in the Turkish hamster also showed a biphasic photoperiodic response, suggesting an underlying similarity in the readout mechanisms of rodents and ungulates (38). The relationship between melatonin signal duration and night length varies between species (39), and we predict similar variability in duration-dependent signaling effects of melatonin. Jointly, this provides a basis for evolutionary tuning of the photoperiodic timing system to life history and latitude.


   Acknowledgments
 
We thank Paul Klosen for the generous gift of the βTSH plasmid. We are grateful to Marjorie Thomson, Ian Swanston, Irene Cooper, and the late Norah Anderson for their expert care of the animals. We also thank Mr. M. Birnie for excellent laboratory technical assistance.


   Footnotes
 
This work was supported by a grant from the Biotechnology and Biological Sciences Research Council (to D.G.H.) and the Medical Research Council (to G.A.L.).

Disclosure Statement: The authors declare that they have nothing to disclose.

First Published Online September 27, 2007

Abbreviations: ARC, Arcuate nuclei; cry, cryptochrome gene; CV, coefficient of variation; LD light-darkness; per, period gene; PT, pars tuberalis; SP, short photoperiod; ZT, Zeitgeber time.

Received May 17, 2007.

Accepted for publication September 18, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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