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School of Biological Sciences, University of Manchester, UK;
* School of Biological Sciences, University of Liverpool, UK; and
School of Medicine, University of Manchester, UK.
1Correspondence: 3.614 Stopford Building, School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK. E-mail: andrew.loudon{at}man.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: clock gene • melatonin • pars tuberalis • refractoriness • photoperiod • circadian
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Photoperiod is interpreted by the master circadian clock in the suprachiasmatic nuclei (SCN) (2)
. Output from the SCN drives rhythmical pineal melatonin secretion, which varies in proportion to the length of the night. Tissues in the neuroendocrine system then interpret melatonin signal duration to drive physiological change (1
, 3)
.
Loss of responsiveness (refractoriness) to prolonged short days (SD) and melatonin is also a critical feature in the biology of seasonal mammals. In spring-breeding rodents, SD refractoriness initiates the reactivation of physiological systems each year, without the need for direct long day (LD) photoperiodic stimulation (4
, 5)
. Refractoriness is thought to occur at melatonin target sites (6
, 7)
, but the cellular and molecular mechanisms underlying this process are poorly understood.
The pars tuberalis (PT) region of the pituitary gland is an important melatonin target site and is strongly implicated in the regulation of prolactin secretion via endocrine action on distal lactotroph cells (8)
. The PT is rich in melatonin receptors and secretes a low molecular weight prolactin-releasing factor, termed tuberalin (9
10
11)
. In seasonal mammals, tuberalin activity is modified by photoperiod and melatonin (10
, 11)
, whereas the synthesis and secretion of prolactin are maximal in LD and low to undetectable in SD (12)
. Suppression of prolactin secretion by SD persists in rams that bear a surgical disconnection of the hypothalamus and pituitary (13)
. This suggests that uniquely, seasonal prolactin secretion may be regulated independently of hypothalamic input, possibly by direct action of melatonin on the PT.
Endogenous rhythms of circadian clock gene expression are subject to both daily and seasonal variation. In the SCN, transcriptional and posttranslational feedback loops are synchronized to external light-dark cycles by photic input from the retina (14)
. In the PT, a transient melatonin-dependent expression of period1 (per1) and inducible cAMP early repressor (ICER) mRNA occurs in the early light phase and is regulated by photoperiod, such that amplitude of expression is either greatly attenuated or lost after exposure to SD for 2–8 wk (15
16
17
18)
. As both per1 and ICER are early response genes stimulated via the cAMP pathway (15
, 19
, 20)
, their expression provides a marker of intracellular cAMP signaling, which is melatonin-responsive in the PT (21)
. Furthermore, PER1 and ICER proteins can repress transcriptional activity (14
, 19)
and therefore may directly act on target gene(s) relevant to the endocrine activity of the PT.
We have therefore combined the study of circadian clock gene expression with recently developed in vitro assays of PT endocrine activity (11)
to examine refractoriness, using the prolactin neuroendocrine pathway as a model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were raised under an LD (16 h light: 8 h dark) photoschedule. Throughout, photoperiod phase is referred to as Zeitgeber time (ZT), where ZT 0 is lights-on. For experimental manipulations, hamsters were housed in age- and weight-matched groups and exposed to either LD or SD (8 h light: 16 h dark), with ZT 0 set at 03:00 for both photoperiod groups. Temperature and humidity were maintained at 21°C and 80%, respectively. Food and water were available ad libitum throughout each experiment. All procedures were performed in accordance with the Animals (Scientific Procedures) Act, UK, 1986. Two groups of hamsters were housed on LD or SD for 28 wk, and left testis width recorded every 2 wk under light halothane anesthesia using digital calipers. The sensitivity of measurement was designated as 5 mm, since below this value it is difficult to distinguish between testicular tissue and the scrotal wall. Additional groups of hamsters were housed on LD or SD for 12 wk. After exposure to LD (for 12 wk) or SD (for 12 or 28 wk), animals in these groups were killed at mid-light phase. Trunk blood and PT tissue were collected for analysis of plasma prolactin concentration and assay of tuberalin secretion, respectively.
In a second experiment, three groups (n=48/group) of hamsters were maintained on LD for 12 wk or SD for either 12 or 28 wk, then killed in groups of four at 2 h intervals across the light-dark cycle. Trunk blood was collected for analysis of plasma melatonin concentration and brain/pituitary tissue was collected for in situ hybridization analysis of per1 and ICER mRNA expression.
Radioimmunoassays
Plasma prolactin concentrations were determined using a validated radioimmunoassay for hamster prolactin described previously (11)
. Assay sensitivity was 0.78 ng/mL. Intra- and interassay coefficients of variation were 6.7 and 5.1%, respectively. Plasma melatonin concentrations were measured using a commercial radioimmunoassay kit (Stockgrand Ltd., Guildford, Surrey, UK). Extracted plasma samples were assayed as described previously (22)
. Mean assay sensitivity was 10.98 pg/mL. Intra- and interassay coefficients of variance were 13.3 and 13.8%, respectively.
In situ hybridization histochemistry
For analysis of per1 and ICER mRNA expression in the SCN and PT, 33P-labeled riboprobes were transcribed from either Syrian hamster per1 cDNA (bases 620–1164) or mouse ICER cDNA (bases 58–236). Cryosections (20 µm) were hybridized overnight at 60°C with 5 x 105 cpm of probe per section. Posthybridization, sections were washed in 2xSSC-50% formamide (RT, 10 min), 2xSSC-50% formamide (60°C, 2x30 min), RNaseA (20 µg/mL in NTE buffer; 37°C, 30 min), 2xSSC-50% formamide (60°C, 2x15 min), and 0.5xSSC (60°C, 30 min). Sections were then dehydrated in graded ethanol solutions and exposed to autoradiography film (Kodak Biomax) for 6 (per1-SCN), 10 (per1-PT), or 13 (ICER-PT) days. SCN and PT optical densities are expressed relative to the cingulate cortex of the same section. For analysis of prolactin mRNA, cultured pars distalis (PD) cells were hybridized with 35S-labeled riboprobes transcribed from full-length rat prolactin cDNA, as described previously (11)
. Hybridized cells were coated with photographic emulsion (Ilford K5) and stored for 60 h at 4°C before emulsion development. The number of silver grains above background per cell was quantified for at least 20 fields chosen at random per group of PD cells. The resulting data were expressed as frequency of cells expressing hybridization signal within discrete limits (1–15 grains, 16–30 grains, 31+ grains) and analyzed as previously described (11)
. Cells expressing hybridization signal too dense to allow quantification of individual silver grains were assigned to the upper category (31+ grains).
Cell culture and assay of tuberalin secretion
The PD region of the pituitary gland was dissected from hamsters exposed to an appropriate photoperiod treatment and digested with collagenase mix. The dispersed PD cells were then cultured in 8-well glass Falcon Chamberslides (Becton Dickinson Labware, Bedford, MA, USA), as described previously (11)
. Conditioned medium (PT-CM) was prepared by incubating PT fragments obtained from animals culled at mid-light phase for 24 h in serum-free culture medium (150 µL per fragment) at 37°C in 5% CO2. Tuberalin activity in the medium was assayed using cultures of PD cells from hamsters exposed to SDs for 12 wk, followed by single cell prolactin mRNA analysis by in situ hybridization. The PT tissue was subsequently used in a coculture assay. Coculture assays used a rat pituitary (GH3) cell line stably transfected with 5 kb of the human prolactin promoter coupled to the firefly luciferase gene (hPRL-Luc-GH3), which has been characterized (23)
. hPRL-Luc-GH3 cells were seeded in 24-well culture plates at a density of 5 x 104 cells per well and cultured for 3 days in serum-free culture medium. The cells were then cocultured with 3 PT fragments in 200 µL fresh medium per well for 8 h. Finally, cells were washed and lysed before analysis of luciferase activity, as previously described (11)
.
Statistics
Results are expressed as mean ± SE. Statistical comparisons were made by chi-square contingency test, 1-way ANOVA, 2-way ANOVA, or MANOVA as appropriate. Statistical significance was defined as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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a), followed by spontaneous testicular regrowth to an LD-like fertile state (P<0.001, LD vs. SD, 2-way ANOVA). Plasma prolactin declined significantly after 12 wk of SD, but reverted to LD concentrations by 28 wk (Fig. 1b
). Additional animals used to generate the 24 h hormone and gene expression profiles exhibited similar responses (data not shown). Refractoriness after 28 wk of SD was not associated with modulation of nocturnal melatonin secretion, which was prolonged in both SD groups, in contrast to short duration profiles of LD-housed animals (Fig. 1c
) as shown previously (24)
. Profiles for both SD groups were significantly (P<0.001, MANOVA) different from the LD group but not from each other.
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SCN per1 gene expression in refractory animals
There was strong expression of per1 in the SCN of hamsters during the light phase (Fig. 2
a, b), with significant differences in the time-to-peak expression and duration of expression between LD- and SD-housed animals (Fig. 2c, d
). SD-housed animals exhibited a lower amplitude peak and levels declined 4–6 h earlier than for LD-housed animals. Quantification of mRNA levels for animals housed for 12 or 28 wk on SD revealed a similar profile of short duration compared with LD-housed animals (Fig. 2c,d
), such that both SD profiles were significantly different from LD-housed animals (P<0.001, MANOVA) but not from each other.
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Photoperiodic regulation of prolactin gene expression and endocrine signaling from the pars tuberalis
Single cell analysis of dispersed cells revealed that there was no significant change in the overall proportion of PD cells expressing prolactin mRNA in any photoperiod group (1-way ANOVA). However, there was a marked down-regulation of prolactin gene expression after 12 wk of SD (Fig. 3
a). Prolactin mRNA expression in SD refractory animals increased above that observed in SD animals to levels not significantly different from those observed on LD (Fig. 3a
). PT-CM collected from cultured PT tissue derived from LD-housed animals significantly stimulated prolactin gene expression. This contrasted with the effect of PT-CM derived from animals housed for 12 wk in SD, which was not significantly different from control unconditioned medium (Fig. 3b
). PT-CM from refractory hamsters induced LD-like increases in prolactin mRNA (Fig. 3b
). Thus, this primary cell assay revealed a reversion of PT endocrine activity to an LD phenotype in SD refractory animals. To ensure these effects were not a possible artifact of the 15-grain bin size used, the data were further tabulated into 1-grain bins. Here, the median values of prolactin mRNA expression were 14 (control CM), 30 (LD PT-CM), 17 (SD PT-CM), and 31+ (refractory PT-CM) grains per cell. Coculture of PT tissue with the GH3 reporter cell line also revealed a clear effect of photoperiod treatment, as both LD and SD refractory PT tissue activated this reporter construct to a significantly greater extent than SD PT tissue (Fig. 3c
). Together, these two assays demonstrate that secretion from the PT reverts to an LD state in SD refractory animals despite persistent SD-like melatonin secretion (Fig. 1c
) and SCN per1 expression (Fig. 2d
).
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per1 and ICER gene expression in the pars tuberalis
LD-housed animals exhibited high-amplitude peaks in expression for both genes in the PT, with a rapid rise at light onset (Fig. 4
a–d). In marked contrast, SD-housed animals exhibited significantly attenuated expression for per1 and ICER after both 12 and 28 wk of SD exposure (Fig. 4c-f
). per1 and ICER mRNA profiles for both SD groups were significantly different (P<0.001, MANOVA) from the LD group, but not from each other.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Within the SCN, daily rhythms of photosensitivity (25)
, electrical activity (26
, 27)
, and circadian clock gene expression (16
17
18)
are modified by photoperiod. Our data confirm a marked photoperiodic modulation of rhythmic per1 expression in the SCN, but in addition reveal persistent SD-like expression in refractory animals. We also confirm that melatonin secretion remains SD-like in refractory animals. Thus, SCN per1 mRNA rhythms and pineal melatonin secretion reflect ambient photoperiod rather than physiological state. This implies that refractoriness to SD lies downstream of the photoperiod-regulated SCN circadian clock and pineal melatonin signal (6
, 7)
.
The striking similarity of heterogeneous prolactin mRNA expression in individual PD cells taken from LD and SD refractory hamsters may reflect a common transcriptional mechanism underlying increased prolactin secretion. A previous study has implicated intrapituitary timing in this process, as seasonal prolactin cycles persisted in sheep lacking a functional connection between the pituitary, and hypothalamus, exhibiting constant melatonin signal duration (28)
. Pharmacological experiments using the same in vivo model have clearly shown that seasonal changes in prolactin secretion occur independent of hypothalamic dopamine or noradrenaline secretion (29
, 30)
. Studies of seasonal rodents have only implied a partial role for hypothalamic signaling in seasonal prolactin regulation (31
, 32)
and suggest that some neuronal control of prolactin secretion occurs independent of photoperiod (33)
. It has therefore been hypothesized that hypothalamic factors primarily regulate prolactin homeostasis via feedback loops, whereas tuberalin acts within these loops to drive seasonal prolactin secretion and thereby regulate the ‘set point’ of prolactin homeostasis throughout the seasonal cycle (30)
. Together, these findings suggest that endogenous seasonal timing of prolactin secretion occurs predominantly within the pituitary but do not elaborate on the cellular or molecular intrapituitary mechanisms involved.
Evidence from two separate assays now reveals increased endocrine signaling from the PT in refractory hamsters. These assays have used conditioned medium generated from PT fragments that may have contained some median eminence tissue. However, earlier studies have shown that the activity of PT-conditioned medium can be blocked my melatonin, indicating that activity is derived from the PT and not from residual factors of median eminence origin (11)
. Although the current data have used gene expression as a sensitive marker of PT endocrine activity, ovine PT-conditioned medium is able to stimulate both gene expression and prolactin secretion (9
10
11)
, suggesting that hamster tuberalin is also likely to regulate both the synthesis and secretion of prolactin from lactotroph cells. Our data therefore implicate the PT as a key structure in neuroendocrine signaling involved in the generation of a refractory response to melatonin, thereby driving seasonal prolactin synthesis and secretion. Such a conclusion is consistent with a study of Siberian hamsters, in which increased expression of glycoprotein 
-chain in the PT and prolactin in the PD followed a similar time course after long-term SD exposure (34)
.
Transient expression of per1 and ICER mRNA occurs in the PT shortly after lights-on (15
16
17)
. Expression of PT per1 requires a daily melatonin signal and functional melatonin MT1 receptor (35
36
37)
and is regulated by timed melatonin injections (16
, 17)
. Transition from LD to SD greatly attenuates the amplitude of both genes (15
16
17
18)
. Our data now show that in refractory animals, the waveform of gene expression in the PT remains SD-like. It therefore appears that the intracellular signaling pathway(s) that drive expression of these genes does not become refractory to a persistent SD melatonin signal. This is in stark contrast to tuberalin secretion and other aspects of neuroendocrine physiology, which return to an LD-like state in refractory animals.
The expression of ICER (19)
and per1 (15
, 20)
is rapidly induced after activation of the cAMP signal transduction pathway. Within the PT, melatonin inhibits cAMP-mediated gene expression (21)
and sensitizes cAMP stimulation and PER1 protein expression (37
, 38)
. The continued low amplitude expression of per1 and ICER mRNA in the PT of refractory hamsters therefore suggests that increased endocrine secretion from the PT occurs in the absence of increased cAMP-mediated signaling and may therefore use different intracellular signaling pathways from those resulting from LD melatonin signals.
There is strong evidence to support the hypothesis that melatonin uses separate target sites to drive photoperiodic changes in reproduction and prolactin secretion (13
, 39)
. A recent study of seasonal Siberian hamsters demonstrated that neuronal melatonin target sites exhibit reproductive refractoriness independently of each other (7)
. It is therefore probable that in seasonal mammals, reactivation of multiple melatonin target sites represents a well-orchestrated temporal program driving annual physiological cycles of diverse traits in response to the same photoperiodic signal. As the PT may share common rhythm-generating mechanisms with less accessible and poorly defined melatonin target sites in the central nervous system, it now offers an ideal model for further dissection of the mechanisms involved in generating endogenous annual cycles in seasonal mammals at diverse melatonin target sites.
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ACKNOWLEDGMENTS |
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Received for publication October 3, 2002. Accepted for publication January 7, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chain in hypophyseal pars tuberalis-specific cells in spontaneous recrudescent hamsters. Endocrinology 138,4101-4108This article has been cited by other articles:
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  S. M. Dupre, D. W. Burt, R. Talbot, A. Downing, D. Mouzaki, D. Waddington, B. Malpaux, J. R. E. Davis, G. A. Lincoln, and A. S. I. Loudon Identification of Melatonin-Regulated Genes in the Ovine Pituitary Pars Tuberalis, a Target Site for Seasonal Hormone Control Endocrinology, November 1, 2008; 149(11): 5527 - 5539. [Abstract] [Full Text] [PDF]
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 M. P. Butler, M. J. Paul, K. W. Turner, J. H. Park, J. R. Driscoll, L. J. Kriegsfeld, and I. Zucker Circadian Rhythms of Photorefractory Siberian Hamsters Remain Responsive to Melatonin J Biol Rhythms, April 1, 2008; 23(2): 160 - 169. [Abstract] [PDF]
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 G. A. Lincoln, I. J. Clarke, R. A. Hut, and D. G. Hazlerigg Characterizing a Mammalian Circannual Pacemaker Science, December 22, 2006; 314(5807): 1941 - 1944. [Abstract] [Full Text] [PDF]
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J. D. Johnston, B. B. Tournier, H. Andersson, M. Masson-Pevet, G. A. Lincoln, and D. G. Hazlerigg Multiple Effects of Melatonin on Rhythmic Clock Gene Expression in the Mammalian Pars Tuberalis Endocrinology, February 1, 2006; 147(2): 959 - 965. [Abstract] [Full Text] [PDF]
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G. A. Lincoln, J. D. Johnston, H. Andersson, G. Wagner, and D. G. Hazlerigg Photorefractoriness in Mammals: Dissociating a Seasonal Timer from the Circadian-Based Photoperiod Response Endocrinology, September 1, 2005; 146(9): 3782 - 3790. [Abstract] [Full Text] [PDF]
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B. J. Prendergast, R. A. Renstrom, and R. J. Nelson Genetic Analyses of a Seasonal Interval Timer J Biol Rhythms, August 1, 2004; 19(4): 298 - 311. [Abstract] [PDF]
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