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Endocrinology, doi:10.1210/en.2008-0834
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Endocrinology Vol. 149, No. 11 5527-5539
Copyright © 2008 by The Endocrine Society

Identification of Melatonin-Regulated Genes in the Ovine Pituitary Pars Tuberalis, a Target Site for Seasonal Hormone Control

Sandrine M. Dupré, Dave W. Burt, Richard Talbot, Alison Downing, Daphne Mouzaki, David Waddington, Benoit Malpaux, Julian R. E. Davis, Gerald A. Lincoln and Andrew S. I. Loudon

Faculty of Life Sciences and Faculty of Medical and Human Sciences (S.M.D., J.R.E.D., A.S.I.L.), University of Manchester, Manchester M13 9PT, United Kingdom; Roslin Institute (D.W.B., R.T., A.D., D.M., D.W.), University of Edinburgh, Midlothian EH25 9PS, United Kingdom; Physiologie de la Reproduction et des Comportements (B.M.), Unité Mixte de Recherche Institut National de la Recherche Agronomique-Centre National de la Recherche Scientifique-University of Tours-Haras Nationaux, 37380 Nouzilly, France; and Centre for Reproductive Biology (G.A.L.), Queen’s Medical Research Institute, University of Edinburgh, Little France Crescent, Edinburgh EH16 4SB, United Kingdom

Address all correspondence and requests for reprints to: Andrew Loudon, Faculty of Life Sciences, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, United Kingdom. E-mail: Andrew.loudon{at}manchester.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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), Hif1{alpha} (hypoxia-inducible factor-1{alpha}), and Kcnq5 (K+ channel) and down-regulation of Rorβ, a key clock gene regulator. Using in situ hybridization, we confirmed day-night differences in expression for Pbef / Nampt, NeuroD1, and Rorβ in the PT. Treatment of sheep with MEL increased PT expression for Cry1, Pbef / Nampt, NeuroD1, and Hif1{alpha}, 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEASONALLY BREEDING animals have to anticipate environmental events to optimize their survival and reproductive success. The annual photoperiod cycle provides a critical environmental signal, which entrains seasonal physiology. Nocturnal secretion of the pineal hormone melatonin reflects these seasonal changes in photoperiod and thereby provides the brain with an internal hormonal representation of external photoperiod changes. Seasonal cycles in melatonin modulates multiple physiological systems including reproduction, food intake, adiposity, body temperature regulation, and many neuroendocrine pathways (reviewed in Ref. 1). One prominent seasonal neuroendocrine output is the release of prolactin. This axis is exquisitely sensitive to photoperiod in all seasonally breeding mammals, with marked activation of prolactin secretion by long summer-like photoperiods (2).

The pars distalis (PD) of the pituitary is thought to be regulated by paracrine signals from the pars tuberalis (PT), a melatonin receptor-rich tissue lying on the ventral surface of the median eminence (ME) and creating an interface between the hypothalamus and the PD (3). PT cells are known to secrete an uncharacterized low-molecular-weight peptide (termed tuberalin), which is capable of eliciting prolactin secretion from distal lactotroph cells in culture (4, 5, 6, 7). These studies have led to the proposal that melatonin acts on PT cells to elicit a local paracrine secretion of a prolactin-releasing peptide. In vitro studies of seasonal hamster tissue has revealed that the activity of this secretagogue is under photoperiodic control, with higher levels of activity observed in PT tissue derived from long photoperiod (LP)- compared with short photoperiod (SP)-housed animals (6). Surgically hypothalamo-pituitary disconnected sheep, in which the PT has been disconnected from the hypothalamus, retain both robust photoperiodically driven cycles of prolactin secretion and sensitivity to melatonin (8). Furthermore, hypothalamo-pituitary disconnected sheep are also capable of generating long-term circannual rhythms of prolactin secretion when maintained in unchanging photoperiod conditions. This result suggests that this tissue may operate both as a seasonal calendar, responding to seasonal changes in the melatonin signal, and as a circannual oscillator (9). Collectively, these studies support a hypothesis that melatonin regulation of the seasonal prolactin axis is dependent upon melatonin acting on PT cells without input from the hypothalamus or other neural structures and regulating seasonal prolactin secretion via a direct intrapituitary paracrine mechanism.

Here, we investigated seasonal transcriptional changes occurring in the PT, using RNA extracted from sheep kept under SP or LP and collected at three time points over the light-dark cycle. For whole genome transcriptome analysis, sheep RNAs were cross-hybridized against a bovine 15,000-gene cDNA microarray, allowing comparison of relative expression at each of the three time points for SP- and LP-housed individuals. Of the 15,000 probes spotted on the array, 54 transcripts exhibited a significant, photoperiod-dependent alteration in expression, including Prnp, encoding the prion protein, which was strongly up-regulated on SP. A total of 153 transcripts exhibited significantly altered expression associated with the light-dark transition and onset of melatonin secretion on SP. Among these, the circadian clock gene Cry1 was the most strongly displaced of all genes on the array, with strong up-regulation at the time of melatonin rise on SP. In addition to Cry1, we identified a small cluster of genes at this time point, including Kcnq5; Pbef/Nampt involved in insulin regulation, energy metabolism sensing, and adipose tissue physiology; and NeuroD1 and the hypoxia-inducible factor (Hif1{alpha}), both of which act as basic helix-loop-helix (bHLH) transcription factors. We validated the arrays by quantitative PCR (Q-PCR) for two of these transcripts (NeuroD1 and Pbef/Nampt) and confirmed differential expression at early night in SP. All five transcripts showed strong expression in the PT using in situ hybridization. To determine whether these Cry1-associated transcripts were also directly responsive to melatonin, we tested acute responses of these genes after melatonin treatment in sheep. Four genes (Cry1, NeuroD1, Pbef/Nampt, and Hif1{alpha}) were immediately and strongly induced by melatonin treatment.

Our study identifies new transcriptional pathways and candidate genes regulated by photoperiod and/or at specific phases of the light-dark cycle in the sheep PT and also identifies novel genes that are acutely regulated by melatonin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Array experiments and collection of tissue for validation of outcomes
Animals and experimental design.
Animals were castrated as lambs on the farm as part of normal agricultural practice, and use of this castrate model allowed studies of seasonal changes in gene expression without the complication of altered background levels of sex steroids.

Array experiments.
For comparison using arrays, 40 male Black-face sheep were housed indoors from mid-winter (January 13) under artificial LP (16 h light, 8 h dark). At wk 14, 20 animals were killed at three different time points: zeitgeber time (ZT) 4 (n = 7), ZT12 (n = 7), and ZT20 (n = 6), where ZT0 by convention represents the transition from dark to light of a 24-h cycle. Two weeks later, the remaining cohort were switched to artificial SP (8 h light, 16 h dark) for 10 wk and then killed at the same time points at wk 26. Dim red background lighting was provided throughout (<5 lux). By this means, the LP cohort were collected at two time points in the light (ZT4 and -12) and one in the dark (ZT20), whereas the SP group were culled at one time point in the light (ZT4) and two in the dark (ZT12 and -20). The experimental design is outlined in Fig. 1Go. This allowed subsequent comparisons between photoperiods and also at time of lights off/melatonin rise for LP- and SP-housed animals (see below). Animals used in the array experiment were blood-sampled for prolactin determination (10 animals per photoperiod group) at twice-weekly intervals; plasma was separated by centrifugation and stored at –20 C until assayed. Prolactin samples were assayed in duplicate as previously described (10). Intra- and inter-assay coefficients of variance were less than 10%.


Figure 1
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  		FIG. 1. Blood prolactin concentration in sheep kept under LP and SP. Animals were placed under LP (16 h light, 8 h dark) for 14 wk, half of the cohort was culled throughout the light-dark cycle at three different ZT times (ZT4, ZT12, and ZT20), and 2 wk later (dashed line), the remaining animals were transferred to SP (8 h light, 16 h dark) for an additional 10 wk. After 10 wk, animals were culled throughout the light-dark cycle at the same time points (ZT4, ZT12, and ZT20). Inset, Times of cull for each photoperiod treatment.

 
Collection of tissue for validation of array outcomes.
To provide tissues for the localization of candidate genes identified on the arrays, 12 intact male Soay sheep were kept under SP for 6 wk, and six were culled at ZT3 and six at ZT11 on SP (lights off at ZT8).

For the array studies, brains were rapidly removed, and the PT was removed by microdissection, snapped frozen on dry ice, and kept at –80 C until RNA extraction. For the other in situ hybridization determinations, brains were removed and hypothalamic blocks including the PT were isolated by dissection and kept at –80 C until cryostat sectioning. All animals were killed by overdose of sodium pentobarbital. During the dark phase, there was no use of accessory lighting, and after death, the animal’s head was covered before removal of the brain. All experiments were undertaken in accordance with the Home Office Animals (Scientific Procedures) Act (1986), UK, under a Project License held by G.A.L.

Animals used for melatonin treatment
The experimental procedure reported in this study was carried out in accordance with the Authorization 37801 for Animal Experimentation and Surgery from the French Ministry of Agriculture. Twenty-four adult intact female Ile de France sheep were used in this experiment. They were reared outdoors at Nouzilly, Institut National de la Recherche Agronomique (INRA), France, and moved to an open indoor shed exposed to natural photoperiods (14.5 h light, 9.5 h dark) 2 wk before the study (April 15). On May 2, the animals were exposed to continuous light over the entire nocturnal period to suppress natural melatonin production. At 0730 h the following day, 12 animals were treated with two sc melatonin implants (Mélovine; CEVA Animal Health, Libourne, France) (11) in the ear, and the remainder received no implant but had their ear punctured identically. The animals (four per group) were subsequently culled at +1 h 30 min, +3 h 30 min, and +6 h 30 min for collection of PT and hypothalamic tissue. All animals were blood sampled 30 min after implant insertion and again just before culling for determination of melatonin concentrations in blood plasma using a standardized RIA (12). All samples were included in the same assay, and the intraassay coefficient of variance was 2.7%.

Ovine cDNA
Ovine PT RNA (1.5 µg) was reverse transcribed to undertake 3'- and 5'-rapid amplification of cDNA ends PCR (BD SMART RACE cDNA amplification; BD Biosciences, Oxford, UK) to obtain ovine cDNA sequences for NeuroD1 and Pbef/Nampt, following the manufacturer’s recommendations. Gene-specific primers (GSPs) and nested GSPs (NGSPs) were designed according to mouse NeuroD1 mRNA sequence (NM 010894) (GSP1, 5'-CAG TCA CTG TAC GCA CAG TGG ATT CG-3'; NGSP1, 5'-GGA ATA GTG AAC TGA CGT GCC-3'; GSP2, 5'-GAG CGA GTC ATG AGT GCC CAG C-3'; and NGSP2, 5'-GGC ACG TCA GTT TCA CTA TTC C-3') and human Pbef1 mRNA sequence (NM 005746) (GSP1, 5'-GCC TAA TGA TGT GCT GCT TCC AGT TC-3', and NGSP1, 5'-TCT TCA CCC CAT ATT TTC TCA CAC GC-3'). Amplified product were then ligated into pGEM-T Easy (Promega, Southampton, UK), and selected clones were sequenced using Big Dye Terminator version 1.1 Cycle Sequencing kits (Amersham, Piscataway, NJ), following the manufacturer’s protocol, by the DNA sequencing service at the University of Manchester. Sequences obtained for NeuroD1 and Pbef were submitted to the GenBank database (accession numbers DQ_82274, DQ_822275).

A 350- to 450-bp cDNA fragment was subcloned in pGEM-T Easy for NeuroD1 and Pbef, using 5'-ACT GCC TTT GGT AGA AAC AGG G-3' or 5'-AGA TCC AAG AAG CCA AAG AGG-3', respectively, as forward primer and 5'-GCT AAG GCA ACC CCA ACA AC-3' or 5'-GAA GTT AAC CAA ATG AGC AGA TG respectively as reverse primer. These clones were used to prepare standards for Q-PCR experiments and riboprobes for in situ hybridizations.

Ovine PT tRNA was reverse transcribed and used directly as template to clone a fragment of 350–450 bp in pGEM-T Easy using the following primers: 5'-CCA TGG TGA CCA CGG GT-3' and 5'-AGC TAA GAG CAT CGA GGG G-3' for 18S (AF_176811), 5'-CTG CAC CAC CAA CTG CTT AG-3' and 5'-TG TCG TAC CAG GAA ATG AGC-3' for Gapdh (U_94889), 5'-CCC ACT CAA ATG CAA GAA CCT CC-3' and 5'-CGC TTT CTC TGA GCA TTC TGC A-3' for Hif1{alpha} (NM_174339), and 5'-TGG TCT ACG CGC ACA GCA AG-3' and 5'-CTG CAG GTG TGC AAG GCC TT-3' for Kcnq5 (XM_864552). Sequences obtained for Hif1{alpha} and Kcnq5 were submitted to the GenBank database (accession numbers EU_706449 and EU_706550). Clones for 18S and Gapdh were used to obtain Q-PCR standards, and those obtained for Hif1{alpha} and Kcnq5 were used to obtain riboprobes for in situ hybridizations.

Q-PCR and in situ hybridization
Two micrograms of each RNA sample used in the microarray experiments were reverse transcribed for Q-PCR validation. Genomic DNA was removed using 1 U RQ1 ribonuclease-free deoxyribonuclease (Promega) for 30 min at 37 C, and the reaction was stopped by adding 1 µl of the deoxyribonuclease stop solution (Promega) and incubating the samples 10 min at 65 C and the 1 min on ice. Samples were then processed for cDNA conversion in a final volume of 20 µl containing 0.5 µM oligo-dT (5'-TTT TGT ACA AGC T23-3'), 500 µM each dNTP, 1x first-strand buffer, 10 µM dithiothreitol, and 10 U SuperScript Reverse Transcriptase II (Invitrogen, Paisley, UK). The samples were incubated for 1 h at 42 C, and reverse transcriptase was inactivated by heating at 70 C for 15 min. Negative controls without enzyme were done in parallel. For quantification, standards were cloned as described above and used as template for PCR with the cloning primers and usual PCR methods. Each sample was processed for Q-PCR as duplicates; reactions were done in 25 µl final volume containing 5 µl cDNA (or diethylpyrocarbonate-treated water for negative control, or standard samples), 100 nM TaqMan probe (Eurogentec, Southampton, UK), forward and reverse primers at 300 nM each, and 1x qPCR MasterMix and using the following cycling conditions: 2 min at 50 C, 10 min at 95 C, and 40 cycles of 95 C for 15 sec and 60 C for 1 min. Primers and TaqMan probes were designed using the Primer Express 2.00 softwarep, and sequences are shown in supplemental Table S1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Data were obtained through the ABI Prism 7000 SDS software (version 1.1; Applied Biosystems, Warrington, UK). Values are shown as relative units with the ZT4 value in LP set at 1.

Radiolabeled sense and antisense ovine riboprobes for Cry1, Per1, NeuroD1, Pbef/Nampt, Rorβ, Hif1{alpha}, and Kcnq5 (cloned as described above) were produced using a T7 or Sp6 polymerase as appropriate on linearized templates in the presence of [33P]UTP (MP Radiochemicals, Illkirch, France). The following in situ hybridization procedure was previously described (13). Briefly, probes were hybridized overnight at 60 C on coronal or sagittal hypothalamic sheep brain cryostat sections (20 µm thick for the validation of the array outcome and 18 µm for the melatonin experiment). Hybridization signals were visualized on Kodak Biomax MR autoradiographic films (Sigma-Aldrich, Gillingham, UK) after 1 wk exposure at –80 C. Signal intensity was quantified by densitometry analysis of autoradiographs using the image-Pro Plus 6.0 software (Media Cybernetics, Inc., Marlow, UK) on the coronal sections [four animals per group except the control 1 h 30 min (n = 3) in Fig. 6Go]. Relative OD in the PT was then calculated for each group.


Figure 6
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  		FIG. 6. In situ hybridization and quantification of Cry1, Kcnq5, NeuroD1, Pbef/Nampt, and Hif1{alpha} mRNA expression in the PT sheep treated or not with melatonin. Representative expression of Cry1 (A), Kcnq5 (B), NeuroD1 (C), Pbef/nampt (D), and Hif1{alpha} (E) in the sheep hypothalamus at the level of the PT on coronal 20-µm cryostat sections after 1 h 30 min, 3 h 30 min, or 6 h 30 min on melatonin-treated (Mel) or untreated animals (Ctl). Cry1 (A), NeuroD1 (C), Pbef/nampt (D), and Hif1{alpha} (E) show a significant up-regulation upon melatonin treatment (black bars) in the sheep PT. Kcnq5 (B) does not show any significant change in treated animals (black bars) compared with untreated animals (white bars). Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Analysis of differentially expressed gene sets
The R/Bioconductor analysis, described in the supplemental information, identified genes differentially expressed between LP/SP days at ZT4, ZT12, or ZT20. In addition, a filter of greater than 1.3-fold difference in gene expression was used to limit the number of genes to the most biologically relevant. Up-regulated genes show expression on LP more than SP days, and down-regulated genes expression on LP less than SP days. Because cDNA arrays yield relative ratio-based data rather than absolute values, for purposes of subsequent descriptions, all gene changes are described with reference to long-day mRNA values (i.e. described as up- or down-regulated on LP). The design of the experiment allowed three gene expression patterns to be analyzed (Table 1Go), namely photoperiodically regulated, SP early night regulated, and SP late night regulated.


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  		TABLE 1. Definition of photoperiodic expression gene sets

 
Ontologizer (14) (www.charite.de/ch/medgen/) was used to analyze differentially expressed genes for overrepresentation of Gene Ontology (GO) terms. This parent-child procedure measures overrepresentation conditional on annotations to the parent of any term, whereas previous approaches measure overrepresentation of each term in isolation. Definitions of GO terms (version 5.623) and gene associations were downloaded from the Gene Ontology website (15) (www.geneontology.org). Human orthologs (Uniprot accessions) of cattle proteins were used for GO term analyses. The associations of GO terms with Uniprot accessions of human proteins were provided by GOA (version 1.75) (16). The overrepresentation of GO terms for proteins in a set of differentially expressed genes was compared with a reference set of proteins, which contains all the genes expressed on the 15-kDA cattle cDNA array. The parent-child-union option was used, which is less conservative than the parent-child-intersection method. The P values were adjusted using Westfall-Young single-step multiple testing correction (17).

Pathway-Express (18) (http://vortex.cs.wayne.edu/) was used to detect significant associations between differentially expressed genes and known pathways. In this method, an impact factor is calculated for each pathway incorporating parameters such as the normalized fold change of the differentially expressed genes, the statistical significance of the set of pathway genes, and the topology of the signaling pathway. The impact factor corresponds to the negative logarithm of the global probability of having both a statistically significant number of differentially expressed genes and a large perturbation in the given pathway. The input to Pathway-Express was a set of differentially expressed genes and their fold changes, using the gene symbols of human orthologs of cattle genes. These gene sets were compared with the reference chip, which contains all the gene symbols expressed on the 15K cattle cDNA array.

Potential transcription factor binding sites were predicted using Match/F-Match (19) as overrepresented sites in cattle promoter sequences for a set of coregulated genes. A Perl script takes as input a set of coregulated genes (positive set) and a set of genes not affected by the treatment (negative set, limited to 2000 sequences) to extract cattle promoter sequences for further analysis. These promoter sequences were downloaded using Biomart from Ensembl (version 48) as the 5-kb flanking regions of all known cattle genes. Match was used to detect potential transcription factor binding sites using the positional weight matrices from the Transfac database (version 12.1; a library of 846 positional weight matrices for vertebrate transcription factors) constructed from collections of known binding sites for a given transcription factor or transcription factor family. A built-in matrix profile with cutoff values adjusted to minimize the sum of false-positive and false-negative errors (vertebrate_non_redundant_minFP) was used in the detection of transcription factor binding sites. F-Match was then used with default parameters to detect overrepresented transcription factor binding sites. Raw probability values were extracted and used as input to Qvalue (20) to calculate false discovery rates.

General statistical analysis
Results are shown as means ± SEM. After ANOVA, the Bonferroni test was applied to analyze differences between groups. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Photoperiod response in the animals used in the array analysis
Plasma prolactin concentrations were approximately 50-fold higher in the sheep culled across the three time points (ZT4, ZT12, and ZT20) under LP compared with the cohort culled under SP (Fig. 1Go). Thus, the PT tissues used in the array analysis derived from animals in seasonally distinct physiological states.

Gene transcriptome analysis of the ovine PT
The use of a bovine cDNA microarray relied on cross-species hybridization between two closely related ungulates with approximately 97% sequence identity (21). The nature of the array comparison did not permit absolute values for altered expression to be defined because LP and SP samples were cohybridized to the array. Thus, a transcript may appear as being up-regulated on LP, but equally the same outcome could occur if it were strongly suppressed by SP. By convention, we describe our results below in terms of relative expression in LP-housed sheep, so that transcripts exhibiting relatively greater expression in LP-derived samples are defined as being relatively up-regulated. Single-channel analysis revealed that about 55% of the transcripts on the array were expressed in the sheep PT. The design of the experiment allowed three gene expression patterns to be analyzed, namely genes exhibiting altered expression at all three time points (designated as photoperiodically regulated), genes exhibiting altered expression at ZT12 (designated SP early night) but not at ZT4, and genes showing altered expression at ZT20 only (designated SP late night; Table 1Go).

Photoperiodic genes
Of the transcripts exhibiting significantly altered expression at all three time points, a total of 36 transcripts were relatively up-regulated and 18 down-regulated (TableGo S2). These included up-regulation of a cAMP response element-binding protein-regulated transcription coactivator 1 gene (CRTC1), represented by two different probes, both of which were significantly displaced on the array, and a methyl transferase gene (PMRT5). The gene encoding the prion protein (PRNP) was the most strongly displaced transcript in the LP-repressed cluster. The Pathway-Express analysis revealed overrepresentation of 15 pathways including olfactory transduction, MAPK, and peroxisome proliferator-activated receptor signaling pathways in the LP-repressed gene set (Table 2Go). No significant over represented pathway was found in the LP-induced set. Ontologizer analysis revealed significant overrepresentation of GO terms for carbohydrate and lipopolysaccharide processes for LP-repressed genes (supplemental Fig. S2, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). There was no detectable significant overrepresentation of GO terms in the LP-induced group of genes. Analysis of putative transcription factor binding sites within each gene set included sites for homeobox, paired homeobox, bZIP, and forkhead transcription factors (Table 3GoGo).


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  		TABLE 2. Pathway analysis: overrepresentation of pathways within gene sets

 

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  		TABLE 3. Transcription factor binding sites: overrepresentation of transcription factor binding sites within each gene set

 

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  		TABLE 3A. Continued

 
Genes changing at ZT12 (SP early night)
A total of 147 transcripts exhibited altered expression at ZT12, with 28 up-regulated and 125 down-regulated at this time point (supplemental Table S3). In the latter group, the focal gene Cry1 exhibited the greatest differential expression for any gene on the array (3.38-fold increase on SP). The activation of Cry1 predictably occurred when ZT12 coincided with darkness under SP and light under LP. Genes exhibiting gene expression patterns similar to Cry1 included a general transcription factor (Gtf2a1), the voltage-gated potassium channel Kcnq5, NeuroD1 (an E-box-regulated bHLH transcription factor), Pbef (Visfatin/Nampt) [a key regulator of the nicotinamide adenine dinucleotide (NAD)/reduced NAD (NADH) salvage pathway] and the hypoxia-induced transcription factor 1{alpha} (Hif1{alpha}, a close relative of the circadian clock gene Bmal1) (supplemental Table S3; Fig. 2Go).


Figure 2
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  		FIG. 2. Characterization of a Cry1 coexpression gene cluster induced at ZT12 under SP. Scatter plots show log2 values of the fold change SP/LP for each clone at ZT4 vs. ZT12 (A) and ZT20 vs. ZT12 (B). Cry1 shows the strongest down-regulation at ZT12 on both scatter plots together with c9Orf77, NeuroD1, Pbef/Nampt, and GTF2A1.

 
Both Hif1{alpha} and NeuroD1 were represented twice on the array by different regions of the cDNA, and both clones exhibited significant and similar magnitude changes, providing additional validation. Expression of MEF2A, a transcription factor, was also increased in SP at ZT12, consistent with a subsequent analysis identifying overrepresented transcription factor binding sites (see below). SP early night-repressed genes (28 transcripts) included two genes involved in circadian regulation (the kinase GSK3β and the nuclear hormone receptor Rorβ). Analysis of overrepresentation of pathways within each gene set included pathways involved in cytokine-cytokine receptor interaction [overrepresented in the SP early night (ZT12) repressed gene set] and pathways involved in circadian rhythm (overrepresented in the SP early night-induced gene set; Table 2Go). Ontologizer analysis revealed significant overrepresentation of GO terms for immune function for SP early night repressed genes (supplemental Fig. S2). There was no significant overrepresentation for genes up-regulated on SP at ZT12. Analysis of putative transcription factor binding sites within each gene set included sites for bHLH-ZIP, bZIP, forkhead, and paired homeobox factors for genes down-regulated at ZT12 on SP and bZIP, forkhead, paired homeobox, POU, and MADS (represented by MEF2, myocyte-specific enhancer factor) for the SP early night-induced gene set (Table 3GoGo).

Genes changing at ZT20 (SP late night)
Genes showing significant changes at ZT20 are listed in supplemental Table S4. A total of 11 transcripts were significantly up-regulated on LP at this time point (SP late night repressed), and 35 were down-regulated (SP late night induced). The circadian clock gene Bmal1 (ARNTL) was one of the most strongly down-regulated genes (SP late night induced) and was coexpressed with the IgG receptor precursor FCGRT, a glutamate/aspartate transporter (SLC1A3), and QK1, an RNA-binding protein. Analysis of overrepresentation of pathways included ErbB and Notch signaling pathways, circadian rhythm, and olfactory transduction in the SP late night-induced gene set (Table 2Go), whereas no significant overrepresented pathway was found in the other gene set. GO term enrichment within these SP late night-induced genes included genes involved in oxidoreductase activity and organelle and endoplasmic reticulum membrane.

Identification of a Cry1-associated coexpression gene cluster
The distribution of relative expression changes for ZT12 vs. ZT4 (dark vs. light) and ZT12 vs. ZT20 (early night vs. late night) under SP is shown in Fig. 2Go. At both time comparisons, the target gene Cry1 was the most strongly displaced gene of the 15,000 transcripts detected using the array. For comparison, NeuroD1 and c9orf77 (both represented by two transcripts on the array) and Pbef/Nampt and Gtf2a1 are also significantly displaced with strong down-regulation at ZT12 and closely associated with the Cry1 cluster. For subsequent analyses, we selected Pbef/Nampt and NeuroD1 to confirm time-of-day changes in expression and regulation in the PT and also Rorβ (significantly up-regulated at ZT12 on LP) as an example of a gene expressed at an opposite phase.

Q-PCR and in situ hybridization comparisons
The regulation of NeuroD1 and Pbef/Nampt mRNA in the PT was confirmed using Q-PCR of PT tissue RNA (Fig. 3Go). In confirmation of the array data, both NeuroD1 and Pbef were significantly up-regulated at ZT12 in SP compared with LP, as revealed in the microarray analysis (P < 0.05) and values were higher at SPs at ZT12 compared with SP at ZT4 for both NeuroD1 and Pbef/Nampt (2.69 compared with 5.25 and 1.45 compared with 2.25, respectively). Control genes (18S and GAPDH) did not reveal significantly altered expression in either photoperiod with no significant variation between each condition (ranging from 1–1.40 and 0.97–1.23, respectively). Radioactive in situ hybridization results for sagittal and coronal hypothalamic sections collected from sheep kept under SP and culled at ZT3 and ZT11 are shown in Fig. 4Go. As expected, Cry1 exhibited markedly altered expression, with low levels at ZT3 (day) and strong induction at ZT11 (night), whereas Per1 revealed an opposite pattern of expression with elevated expression at ZT3 (Fig. 4Go, A and B). Both Pbef/Nampt and NeuroD1 were strongly expressed in the PT (Fig. 4Go, C and D), and the quantification of the PT revealed that there was significant up-regulation at ZT11 for both transcripts (Fig. 4Go, E and F). For NeuroD1, the pattern of expression was very similar to Cry1. Pbef/Nampt was also strongly expressed in the PT, but a signal was also observed in the ME (Fig. 5CGo). In contrast to the PT, quantification of relative changes in expression from ZT3 to ZT11 revealed no significant alterations in Pbef/Nampt expression in the ME. Rorβ expression in the PT showed a significant down-regulation at ZT11 on coronal sections as assessed by in situ hybridization (Fig. 5Go).


Figure 3
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  		FIG. 3. Validation of photoperiodic expression of NeuroD1 and Pbef/nampt in the PT. Relative expression of ovine NeuroD1, Pbef/nampt, 18S, and Gapdh mRNA by Q-PCR. The results confirm the microarray data showing a strong down-regulation of NeuroD1 and Pbef in LP compared with SP at ZT12 in the sheep PT. Data are shown as relative to the ZT4 value in LP set at 1. Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

Figure 4
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  		FIG. 4. In situ hybridization and quantification of NeuroD1 and Pbef/Nampt mRNA expression in the PT of SP-housed sheep. Representative expression of Cry1 (A), Per1 (B), Pbef/nampt (C), and NeuroD1 (D) in the sheep hypothalamus at the level of the PT on sagittal and coronal 20-µm cryostat sections at ZT3 and ZT11. NeuroD1 (E) and Pbef/Nampt (F) are specifically up-regulated in the sheep PT in the dark phase (ZT11) under SP. Scale bar, 1 mm. Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

Figure 5
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  		FIG. 5. In situ hybridization and quantification of Rorβ mRNA expression in the PT of SP-housed sheep. Representative expression of Rorβ (A) in the sheep hypothalamus at the level of the PT on coronal 20-µm cryostat sections at ZT3 and ZT11. Rorβ (B) shows a significant down-regulation in the sheep PT in the dark phase (ZT11) under SP. Scale bar, 1 mm. Statistical significance was determined by one-way ANOVA followed by Bonferroni post hoc test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Response of Cry1-assciated genes to acute melatonin treatment
Blood samples from animals treated with melatonin implants (Mélovine) rose to 131 pg/ml melatonin within 30 min of treatment and were 398 ± 215, 142 ± 33, and 301 ± 156 pg/ml at 1 h 30 min, 3 h 30 min, and 6 h 30 min, respectively. Untreated animals had levels of less than 4 pg/ml. Typical reference samples from animals sampled under natural ambient daytime lighting are less than 4 pg/ml and 260 pg/ml for mid-night. We quantified by in situ hybridization genes showing a similar change in expression to Cry1 at ZT12 (Cry1, Kcnq5, NeuroD1, Pbef//Nampt, and Hif1{alpha}; supplemental Table S3; Fig. 2Go). This revealed strong PT expression for all five transcripts (Fig. 6Go). Melatonin treatment resulted in a significant increase in expression for all but Kcnq5, with a peak at 3 h 30 min and subsequent decline by 6 h 30 min in all four cases. At this latter time point, relative expression levels were still approximately twice that of untreated control animals, with the exception of Hif1{alpha}, which had returned to a similar level to that of controls. With the exception of Nampt, expression was confined to the PT for all transcripts.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Use of cDNA array to identify candidate genes and pathways
In our study, exposure of sheep to LP resulted in a robust activation of prolactin secretion over a 2-month period, which was reversed by exposure to SP consistent with earlier publications on this species (8), providing a key physiological endpoint validating subsequent analyses of the PT for transcriptome analysis. The array we used provided extensive genome coverage (~50%), and because sheep PT tissue provided sufficient material from individual animals to be run on a single array slide, errors associated with RNA pooling were reduced and confidence in the statistical power of the data greatly increased. Our data presented here demonstrate that we are able to use a bovine-derived cDNA array to identify altered expression in ovine genes from the PT. Specifically, the sequence homology with bovine genes was more than 97%, allowing a high level of cross-hybridization (further details in the supplemental information). Furthermore, candidate genes were confirmed by Q-PCR and in situ hybridization, and in all cases, we identified the correct ovine homolog expressed within the PT (i.e. there were no false-positive). We identified 53–65% of genes on the array that were expressed in the PT, and this is within the typical range of expression observed in a particular tissue (22). Thus, use of this well characterized cDNA array and sheep PT model offers an excellent insight into global transcriptome changes at a melatonin target site.

Genes exhibiting time-of-day alteration in expression
Of all of the genes exhibiting differential expression on the array, the circadian clock regulator Cry1 exhibited the greatest relative expression change. The relative phasing of arrayed Cry1 and Bmal1 clock genes revealed that Cry1 was strongly down-regulated at ZT12 (SP early night induced) with Bmal1 induced at late night (ZT20) in SP. Our in situ hybridization studies also confirmed that strong expression of Per1 and Cry1 were anchored to dawn and dusk, respectively, matching the putative offset and onset of the melatonin signal. Collectively, these data are in accord with previous studies of clock gene expression in the PT of sheep and seasonal rodents, which show strong phasic expression associated with the rise and fall of the daily melatonin signal (23, 24). Other clock transcripts such as Hif1{alpha}/MOP1, GSK3β, and Rorβ were also identified as significantly altered in expression on the array, and the subsequent pathway analysis revealed a significant general overrepresentation of circadian genes within this data set.

Our Q-PCR and in situ hybridization validation revealed that Pbef/Nampt and NeuroD1 exhibited specific expression in the sheep PT and were strongly expressed at ZT11 (night phase) compared with ZT3 (light phase) in SP-housed animals, matching array outcomes. In contrast, the Rorβ in situ hybridization revealed down-regulation at ZT11, also consistent with the array data. The expression pattern of NeuroD1 and Rorβ in the PT very closely matched that of Cry1. In contrast, Pbef/Nampt exhibited lower levels of expression in the ME, but expression in this structure did not alter with time of day. NeuroD1 (neurogenic differentiation factor 1) is a bHLH transcription factor that binds E-boxes after dimerization with other HLH domain-containing proteins (25). It is widely expressed in the vertebrate developing central nervous system (CNS) and is also expressed in specific areas in the adult brain (cerebellum, hippocampus, and hypothalamic paraventricular and dorsomedial nuclei) (26, 27, 28). NeuroD1 is a key transcription factor involved in corticotroph regulation and pancreatic β-cell differentiation (29, 30), and the knockout in mice is lethal (30). In the adult, it is strongly expressed in the endocrine cells of pancreas, intestine, and pituitary and in these structures is known to regulate insulin, secretin, and proopiomelanocortin, respectively (25, 26, 31, 32). NeuroD1 thus plays a central role in both the development and subsequent regulation of multiple endocrine tissues, and our data now suggest that it may play an important additional role in the PT.

Pbef (pre-B-cell colony enhancing factor) was first isolated from peripheral blood lymphocytes and was characterized as a growth factor of B cell precursors by facilitating development of early-stage B cells (33). As reviewed by Revollo et al. (34), Pbef is also known as Nampt (nicotinamide phosphoribosyltransferase), because it acts as a key rate-limiting enzyme in the NAD salvage cycle of the cell, and more recently as visfatin. Pbef/Nampt has been associated with the development of obesity and insulin resistance and type 2 diabetes (35, 36), but it is currently unclear whether it is directly involved in the development of insulin resistance, or indirectly as a marker of an inflammatory state (37). The gene is strongly expressed in a circadian rhythmical fashion in adipose tissue in mice and, as we have observed in the PT, strongly coassociated with Cry1 (38). Our study shows for the first time that Pbef/Nampt is strongly and rhythmically expressed in the PT.

A number of studies have linked the NAD cycle of the cell to circadian clock function. For instance, investigations by McKnight and co-workers (39, 40) have shown close links between energy sensing and the NAD cycle and the regulation of circadian clock genes Clock and NPAS2 in the CNS. A key transcriptional regulator of Pbef/Nampt is Hif1{alpha}, a close relative of the circadian clock gene Bmal1 (41, 42), and we observed that HIF1{alpha} was also strongly induced on our array in early night, at the same time point as Pbef/Nampt (i.e. Cry1-associated). Both Pbef/Nampt and Hif1{alpha} are strongly induced in conditions of both hypoxia and cell stress (43). Hypoxia-inducible factor-1{alpha} protein cooperates with bHLH circadian transcription factors such as CLOCK and BMAL1, allowing cross talks between hypoxic and circadian pathways (44, 45). Through its action on the NAD/NADH cycle, Pbef/Nampt is the key enzymatic regulator of a sirtuin gene (Sirt1) coding for a NAD-dependent deacetylase with widespread actions on cellular senescence and ageing (reviewed in Ref. 46). Pbef/Nampt is thus ideally poised to serve both as a link between circadian timing complexes and, via its action on the NAD/NADH cycle, as an energy metabolism sensor, regulating through its action on Sirt1 both genomic (i.e. histone modification) and nongenomic pathways (47).

Melatonin regulation of PT gene expression
Recent studies have shown that circadian clock genes are rhythmically expressed in the PT of seasonally breeding mammals and also in strains of mice that secrete melatonin (23, 24, 48, 49, 50, 51). In seasonal rodents and sheep, the circadian clock gene Per1 is expressed in the PT and is activated in the early morning in direct response to the decline in the nocturnal melatonin signal and altered cAMP signaling. In contrast, the clock gene Cry1 is rhythmically expressed in the dark phase, coincident with the onset of pineal melatonin secretion, and in both sheep and hamsters, melatonin treatment has been shown to directly induce Cry1 expression in the PT irrespective of the phase of the light-dark cycle (52). PER and CRY are key components of the feedback loop controlling the master clock in the suprachiasmatic nucleus (SCN). These two proteins are coincidentally expressed within the SCN and heterodimerize to rhythmically modulate circadian gene transcription via E-box motifs on target genes (53). Studies of seasonally breeding sheep have revealed that in contrast to the SCN, within the PT, Per1 and Cry1 mRNA track the offset and onset, respectively, of the melatonin signal such that seasonal changes in the duration of the melatonin signal are reflected in an altered phase relationship of these two core clock components. From this, it has been proposed that melatonin-regulated seasonal changes in the phasing of the PER/CRY interval may operate as an internal coincidence detector, providing a genetic mechanism based on circadian clock genes for the PT to decode the seasonal photoperiodic melatonin signal (9, 54, 55, 56) and drive downstream molecular events via E-box-mediated transcription. To date, Cry1 is the only gene known to be regulated by melatonin.

Our study has confirmed earlier studies that demonstrate that Cry1 is an acutely melatonin-regulated gene and now identifies additional Cry1-associated genes in the array. Remarkably, several of these (Pbef/Nampt, Hif1{alpha}, and NeuroD1) are, like Cry1, also strongly induced by melatonin, and over the same time course. These data now suggest that in addition to Cry1, melatonin may regulate a number of downstream molecular pathways in the PT, although we have no insight yet as to whether these novel melatonin-regulated genes contribute to the photoperiodic response, or simply act as a marker of phase for the onset of melatonin secretion. Little is known of how melatonin may induce such rapid changes in gene expression in a target tissue. Classically, this hormone is thought to act by inhibiting cAMP signaling pathways (57). The speed of transcriptional response in the PT would mitigate against regulation by de novo production of an intermediate factor, and it is possible that these responses represent direct transcriptional activation by a melatonin-regulated signaling system. Cry1 and NeuroD1 are known to be regulated in a circadian manner by E-box acting transcription factors, and we have recently identified a putative E-box site within the presumptive promoter of Nampt (Kim, D. H., and S. M. Dupré, unpublished). However, hypoxia-inducible factor 1{alpha} is an E-box-acting transcription factor and is not known to be regulated itself via an E-box. Because a large number of genes were shown to be significantly expressed on SP at ZT12 and several/many of these may also be melatonin regulated, identification of further melatonin-regulated clusters may allow us to define unique transcription factor binding sites within the regulatory elements of these genes and thus candidate proteins for the immediate downstream pathways on which melatonin acts.

Phenotypes of the PT
To date, no studies have been undertaken of the transcriptome of the mammalian PT, and relatively little is known of the genetic phenotype of these cells. Melatonin receptors are strongly expressed in the PT of seasonal mammals, and studies of Siberian hamsters have revealed that PT secretory granules are more numerous in animals housed under LP (58). The mammalian PT contains three different main cell types, a follicular cell (reviewed in Refs. 3 and 60), PD cells of gonadotroph origin (61) that have migrated from the PD to the PT, and a third cell type that expresses the β-subunit for TSH and the common {alpha}-glycoprotein hormone subunit ({alpha}GSU). These latter cells are known to colocalize with the melatonin receptor in the rat and European hamster (62, 63). In these TSHβ-positive cells, levels of immunostaining are significantly reduced under short photoperiods and are also sensitive to treatment with melatonin or pinealectomy (64, 65). All three cell types are included in any PT dissection; in addition, it is impossible to avoid inclusion of some material from the ME, including GnRH and other nerve terminals and associated glial cells. Therefore, the tissue screened is heterogeneous and not confined solely to melatonin receptor-expressing cells. A remarkable feature of our data is that with the exception of Nampt (which reveals weak ME expression as well as PT), all novel PT transcripts identified from the array revealed strong expression by in situ hybridization to be specific to the PT, validating this combined approach as a means for detecting novel PT pathways.

Genes exhibiting photoperiodic alteration in expression
By comparing three different time points over the light-dark cycle for LP- or SP-housed animals, we were able to identify differentially expressed genes that exhibited a consistent alteration in expression at all three time points (photoperiodic genes) or were significantly altered in expression at a specific phase (time-of-day-regulated genes). Clearly, genes in this latter category may also be photoperiodic if their general waveform of expression over the 24-h cycle is sculpted by photoperiod, but with only three time points assessed, we have adopted the conservative classification of these genes as time-of-day regulated. Generally, our data revealed that relatively few transcripts exhibited significant alterations in expression (of the order of <1%). Within the photoperiodic gene set, the most strongly altered transcript encoded the prion protein (PRNP). Prion proteins are now recognized as serving important biological functions in both the mammalian CNS and lymphoid tissue (66). Within the CNS, the endogenous prion protein is important in neural development, synaptic transmission, and neurite outgrowth, probably by binding to the neural cell adhesion molecule (N-CAM) as a signaling receptor (reviewed in Ref. 67). mRNA levels for prion proteins have been shown to be highly rhythmic both in the SCN and throughout the forebrain of rats, and in mice, severe disruption of circadian activity and sleep-wake cycles is observed in prion protein-deficient transgenic mice (68). Our data suggest that the prion protein may serve an important role in this melatonin target tissue.

We also observed that the potassium channel coded by Kcnq5, not previously identified as being expressed in the PT, was also strongly LP repressed. A different clone of the same gene was also detected in the SP early night-induced gene set, implying that this gene may fall into the category of a transcript in which expression over the day is regulated by photoperiod. Kcnq5 was only recently discovered and exhibits a strong expression specifically within the CNS (69). Recent studies have shown that Kcnq5 is strongly expressed in the arcuate nucleus where it colocalizes to neuropeptide Y and proopiomelanocortin neurons (70). Regulation of the Kcnq family of genes is also known to be important for the regulation of neural plasticity in the hippocampus (71). Thus, photoperiod-responsive expression of Prnp and Kcnq5, two transcripts implicated in cellular communication and plasticity suggests that such processes may play an important role in the PT of a seasonal mammal. A number of genes exhibiting significant up-regulation on LP are associated with histone and nonhistone protein regulation, including two methyltransferases (SUV39H2 and PRMT5).

Homologous structures in birds
A recent paper on Japanese quail has used heterologous chicken arrays to reveal key photoperiodic changes in the basal hypothalamus and PT (72). In this study, the authors defined transcript changes over the first 2 d after an extreme shift from short (4 h light) to long (20 h light) photoperiods. In quail, this results in a significant rise in LH by the end of the first long day. Using arrays, Nakao et al. (72) were able to detect two genes (Tshβ and Eya3) that were elevated at the end of the light phase of the first long day in the avian PT and that thus may act as a signal to the neuroendocrine system of photoperiodic activation. Subsequent studies by this group also revealed that TSHβ acted on ependymal cells to induce expression of deiodinase 2 (Dio2), a thyroid converting enzyme known to be critical to the regulation of the hypothalamic reproductive response in birds and mammals (73). Up-regulation of Dio2 was also associated with a larger number of transcripts showing altered expression in the hypothalamus on d 2. This study therefore indicates the central importance of the PT to photoperiodic responsiveness in birds and also demonstrates that photoperiodic activation is associated with synchronized waves of gene expression that follow a strict temporal pattern. TSHβ and the common subunit ({alpha}GSU) as well as Eya3 were arrayed as transcripts on the cattle microarrays we used. However, none of these genes showed significantly altered expression at either time of day or photoperiod. Our current study was based on a comparison of transcript changes in the PT after chronic (14 wk) exposure to photoperiods, and it is possible that altered TSH signaling represents earlier events in the photoperiodic response. Using in situ hybridization, a recent study of sheep has also failed to identify significant changes in TSH expression on d 1 of LP exposure in the sheep PT, although in this study, the authors collected samples in the early light phase rather than later in the day when expression might be expected to be elevated (74). It is, however, highly likely that birds and mammals use a similar pathway and mechanism, because this recent study (74) has shown that altered Dio2 expression occurs in ovine ependymal cells and that these cells are responsive to TSH, as in the study of Japanese quail. It is possible that our own study and others have missed the activation of TSH by sampling at the wrong time of day, or that in sheep, the latency of response to LP differs from birds. These issues can only be addressed by additional studies over a wider range of time points. Although further resolution of the identity of common pathways used by birds and mammals must await more extensive studies, it is likely that the photoperiodic response involves a complex series of gene expression changes, from the initial acute responses to the chronic changes detected here after many weeks to months (reviewed in Ref. 59).

In summary, we have identified novel PT-expressed transcripts in the sheep, which reveal significant time-of-day alteration in expression. Several of these genes we now show to be directly regulated by melatonin. Identification of the pathways on which these genes act and the manner in which they are regulated will offer key insight into how this critical melatonin target site translates a seasonally variant melatonin signal into a neuroendocrine output.


   Acknowledgments
 
Plasmids containing a partial cDNA sequence of ovine Per1, Cry1, and murine Rorβ were kindly provided by Drs. D. Hazelrigg and P. Barrett. We thank Mark Fell at the ARK-genomics facility, Roslin Institute, for bioinformatics support; David Bechtold and Karl-Arne Stokkan for comments on the manuscript; Joan Docherty, Marjorie Thomson, and Norah Anderson at the Marshall Building sheep research facility, University of Edinburgh, for the animal care; Didier Chesneau, Institut National de la Recherche Agronomique (INRA) Nouzilly, for the melatonin assays; staff of the INRA-Unite Pluri-espèces d’Expérimentation Animale Nouzilly for animal care; and Larisa Logunova for technical assistance in cloning Hif1a and Kcnq5 ovine partial cDNAs.


   Footnotes
 
This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC, Grant BBS/B/08744 to A.S.I.L. and J.R.E.D.) and Medical Research Council for financial support and the BBSRC ARK-Genomics facility at Roslin.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 31, 2008

Abbreviations: bHLH, Basic helix-loop-helix; CNS, central nervous system; GO, Gene Ontology; GSP, gene-specific primer; LP, long photoperiod; ME, median eminence; NAD, nicotinamide dinucleotide; NADH, reduced NAD; NGSP, nested GSP; PD, pars distalis; PT, pars tuberalis; Q-PCR, quantitative PCR; SCN, suprachiasmatic nucleus; SP, short photoperiod; ZT, zeitgeber time.

Received June 4, 2008.

Accepted for publication July 22, 2008.


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 Materials and Methods
 Results
 Discussion
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