Current Research Projects
1. Modelling the circannual clock. The aim is to produce a model of a circannual oscillator based on how the melatonin signal acts in the pituitary pars tuberalis (PT) via cAMP signalling, clock genes and a prolactin-releasing factor to regulate long-term rhythms in prolactin secretion from the adjacent pituitary pars distalis (PD) – modelling a tissue timer. Collaborator: Dr Duncan MacGreggor, University of Edinburgh.

2. Circannual timing through a pituitary-adrenal mechanism. To use the Soay sheep model to test the hypothesis that the expression of a circannual rhythm in prolactin secretion under constant long photoperiod depends to the temporal pattern in cortisol secretion from the adrenal gland. Does cortisol provide a negative feedback signal for the long-term oscillator? Collaborator: Professor Iain Clarke, Monash University, Australia; Edwin Carter, University of Edinburgh.

3. Circannual rhythms modulated by photoperiod. To investigate whether the period, or amplitude, in expression of the circannual prolactin rhythm varies with the length of the photophase of the daily light-dark (LD) cycle. Groups of Soay sheep have been transferred from short photoperiod (LD 8:16) to various long photoperiods (LD 16:8; 20:4; and 2:22) and the long-term biology is being recorded. Collaborators: Professor Iain Clarke, Monash University, Australia; Dr Gabi Wagner and Dr David Hazlerigg, University of Aberdeen.

4. Clock gene rhythms under natural photoperiod. Here we plan to define more precisely the 24-h melatonin rhythms and clock gene expression profiles in the SCN and PT in sheep living outdoors. Groups of animals will be killed at the autumn equinox, winter solstice, spring equinox and summer solstice and the rhythmicity compared to sheep housed under standard indoor photoperiod regimens. Collaborators: Dr Gabi Wagner and Dr David Hazlerigg, University of Aberdeen.

5. Disruption of clock gene expression in the PT. The aim is to demonstrate a functional role of specific clock genes (e.g. cryptochrome 1) by generating transgenic sheep where the selected gene is permanently neutralised within the PT. Photoperiod-induced and circannual cycles in prolactin will be measured in affected and control animals to record the phenotype. Collaborators: Dr David Hazlerigg and Dr Hugues Dardente, University of Aberdeen; Dr Bruce Whitelaw, Roslin BioCentre, Edinburgh.

6. Natural mutations. Mutations in the core circadian clock genes and associated timer-genes are being screened in sheep expressing unusual seasonal phenotypes. This includes individuals that shows delayed onset of melatonin secretion and are unable to respond to photoperiod due to the circadian defect. Collaborators: Professor Josephine Pemberton, University of Edinburgh; Dr Hugues Dardente, University of Aberdeen.

7. Identifying novel timing genes. To isolate novel clock genes, clock-controlled genes and genes related to long-term timing expressed in the ovine PT. This involves analysis of micro-array data of mRNA derived from PTs of sheep exposed to short and long photoperiod, sampled at different times of photoinduction (e.g days 1, 7 and 28 after transfer to long photoperiod) and at different times of day (e.g. ZT 3, 11 and 19). Candidate genes will be sequenced and tissue-typed for confirmation. Other methods will be used to search for potential ‘tuberalin’ candidates - the missing link between the PT and PD in the control of prolactin secretion. Collaborators:  Professors Andrew Loudon, Professor Julian Davis and Dr Sandrine Dupre, School of Biological Sciences, University of Manchester; Dr Richard Talbot, ARC-genomics, Roslin BioCentre, Edinburgh; Dr Steve Hart, University College London.

8. Clock gene expression in the corpus luteum. To establish whether the diurnal pattern of clock genes expression in the ovine corpus luteum (CL) changes across the luteal phase of the oestrous cycle as part of the mechanism that determines the life-span of the CL in the non-pregnant animal. Parallel studies are underway on cultured human luteal cells collected during an IVF programme. Collaborators: Dr Colin Duncan and Cynthia Chen, University of Edinburgh.

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Research Staff/Laboratory Members
PI: Gerald Lincoln
PhD Student: Cynthia Chen
Postdoctoral Fellow: Duncan MacGreggor and empty post
Laboratory technician: Edwin Carter
Animal technician: Marjorie Thomson
Special facilities: University of Edinburgh, Marshall Building Large Animal Facility, Roslin

Grant aid
(i) MRC programme/transfer grant funding research and salaries in the University of Edinburgh 2006 -10 ‘Clock genes and long-term biological timing’ 1130K
(ii) BBSRC joint grant (joint applicant: Dr Hazlerigg, University of Aberdeen; Dr Bruce Whitelaw, Roslin Institute, Edinburgh) 2007-11 ‘Defining the molecular basis of photoperiodism in mammals’ 480K

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Affiliations

Principal Collaborators
Chronobiology Group. This group located within the Queen’s Medical Research Institute, University of Edinburgh, provides a forum for regular discussion http://chronobiology.mvm.ed.ac.uk

Gerald Lincoln has a network of long-standing collaborators:
1. Professor Iain Clarke, Head of Physiology, Monash University, Melbourne. Professor Clarke visits Edinburgh regularly and provides surgical expertise. His group works on the neuroendocrinology of obesity in sheep models using immunocytochemistry, in situ hybridization, neural track tracing, in vivo experimentation and provides research methods and advice. 

2. Dr David Hazlerigg and colleagues at the School of Biological Sciences, University of Aberdeen, collaborate in the study of clock gene expression in the sheep hypothalamus and pituitary gland, They have cloned target sheep genes providing homologous probes and in situ hybridization methodology

3. Dr Bruce Whitelaw at the Roslin Institute, Edinburgh, has pioneered the use of lentivirus in the introduction of transgenes in large animals. We have a new joint BBSRC Grant to knock down cryptochrome expression in the sheep PT.

4. Professor Andrew Loudon, School of Biological Sciences, University of Manchester. Professor Loudon has a major research project to identify novel genes expressed in the sheep PT using material from animals in specific physiological states set up by us in Edinburgh. The results are to be published shortly.

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Markers of Esteem
2007 University of Edinburgh, Personal Chair
2007 British Society for Newoendocrinology, Lecture
1998 University of Surrey, Guilday Award
1995 University of Florida, Henry Barren Medal
1993 Royal Society of Edinburgh, Elected Member
1989 Endocrine Society UK, Scientific Medal
1987 Society for Study of Fertility UK, Hammond Lecture
1979 Lauentian Hormone Conference USA, Pincus Lecture
1979 Zoological Society of London, Scientific Medal
1969 Zoological Society of London, Special Award for Science

Recent Invitation to International Conferences
1. Gordon Conference on Pineal Cell Biology, Il Ciocco, Italy, 24-29 April 2008. Invited speaker ‘Role of the pineal in clocking seasonal adaptation’.

2. Lorentz Centre, Workshop, Leiden, The Netherlands 14-18 January 2008. Invited speaker ‘Keeping track of the seasons’.

3. 2nd World Congress on Chronobiology, Tokyo, Japan 4-6 November 2007. Invited speaker ‘Decoding the melatonin signal in the mammalian pars tuberalis.

4. 10th International Congress on Obesity, 3-8 September 2006, Sydney, Australia. Invited speaker ‘Seasonal effects on appetite; variation in homeostatic set-point.

5. Gordon Conference on Pineal Cell Biology, Buellton, CA, USA. Invited speaker ‘Circannual rhythms revisited’.

6.  4th International Symposium on Signal Transduction in Health & Disease, 26-28 October 2005 Tel Aviv, Israel. Invited speaker ‘Decoding melatonin through circadian clockwork’.

7. 10th Congress of European Biological Rhythms Society 1-5 September 2005 Frankfurt, Germany. Invited speaker ‘Melatonin entrainment of circannual rhythms’.

8.  9th Meeting of the Society for Research on Biological Rhythms 24-26 June 2004, Whistler, BC, Canada.  Invited Speaker ‘Circannual timing’.

Membership of Societies
Royal Society of Edinburgh (Scotland)
Endocrine Society (UK)
Society for the Study of Reproduction (USA)
European Biological Rhythms Society (EU)
British Society for Neuroendocrinology (UK)

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Biographical Profile
Gerald Lincoln was born in Norfolk into a farming family with two older brothers. After the loss of their father at an early age the boys were encouraged towards academic careers in the Biological Sciences. Gerald gained a first class honours degree in Zoology at Imperial College, London in 1970. PhD studies were at the Veterinary School, University of Cambridge under Professor Roger Short studying reproductive physiology and behaviour of red deer living wild on the Isle of Rhum, Scotland. This started a long research career at the MRC Centre for Reproductive Biology in Edinburgh, studying seasonal breeding in mammals. The highly seasonal Soay sheep, native of St Kilda, has been developed as an animal model. Recently Gerald Lincoln joined the University of Edinburgh funded by a MRC Research Grant to continue the work on biological calendars. He was awarded a Personal Chair in the School of Biomedical Sciences in August 2007 (Designation; Professor of Biological timing). He has published over 150 papers in scientific journals - the most quoted “Seasonal breeding: Nature’s contraceptive” co-authored with Roger Short, and the best known “The irritable male syndrome".

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Teaching and Training
1. School of Biomedical Science.  Mammalian Chronobiology Course (organiser Professor Tony Harmer). Lectures, tutorials and appraisal of student presentations.

Lay summary of research
Cycles in biological systems are all pervasive in nature. All mammals express daily rhythms in activity/sleep, body temperature, pituitary hormone secretion and other familiar rhythmic characteristics. Seasonal animals also show long-term cycles in feeding behaviour, fattening, reproduction, hibernation or migration. Our working hypothesis is that clock genes, well characterised for their role in generating daily rhythms, also form the molecular basis for seasonal timekeeping. Internal calendars allow animals to migrate across the world despite ambiguous time cues, to emerge from hibernation having been in the dark all winter, or time their breeding cycles in the deserts on the basis of past experience - all with remarkable precision. Man is thought to have reached the Arctic Circle by 100,000 years ago thus seasonality is likely to be an inherited adaptation.  We are only just beginning to identify the genes that make us more, or less, seasonal animals. Overall, it is clear that the molecular revolution, including the discovery of the clock genes, is fast transforming our understanding of biology and medicine.

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Current Research Programme ‘Clock genes and long term biological timing’
In the last 5 years, remarkable progress has been made in defining the molecular basis of the circadian clock in mammals. We now know that the clock is generated endogenously by a cell autonomous mechanism involving a small number of core clock genes (about 12 genes identified currently). These interact to produce molecular, cellular and electrophysiological oscillations with a precise periodicity close to 24 hours – the ‘clock’. The positive-drive is through two transcription factor genes, called Clock and Bmal1. The protein products of these genes control the activity of other clock genes, notably three Period (Per1/Per2/Per3) genes and two Cryptochrome (Cry1/Cry2) genes, which via the formation of protein complexes, provide the negative feedback signal that shuts down the Clock/Bmal drive to complete the circadian cycle. Other, clock genes provide additional negative and positive transcriptional/translational feedback loops to form the rest of the core clockwork, which has been characterised notably in rodents by a transgenic gene-deletion methodology.

In mammals, clock genes are expressed in the 10-20,000 cells of the bilateral suprachiasmatic nuclei (SCN) of the hypothalamus, which function together to provide a central pacemaker. The SCN rhythm is entrained to the daily light dark cycle by light signals perceived through the eyes, and the SCN orchestrates the many overt cycles in physiology and behaviour. This is achieved through links to integrative control centres in the brain (e.g dorsomedial hypothalamus) and through hormones, notably melatonin produced by the pineal gland, that signals ‘night time’ around the body.

Outside the central pacemaker, clock genes are also expressed in many different tissues not initially thought to be part of the circadian system. In various rodent models, liver, kidney and lungs have been shown to express Per1 mRNA with daily rhythms of different timing and greater amplitude than seen in the SCN. Moreover, using a light cycle phase-shifting protocol, to mimic the effects of time-zone travel in man (jet-lag protocol), it has been shown that the circadian rhythms in clock gene expression in the different organs become desynchronised, and each takes a specific time to re-entrain to the new environmental light-dark cycle. To an 8-h phase shift, the brain/SCN clock rhythm adjusts in some 3 days while the liver is notably slow, taking up to 14 days; jet-lag is a state of internal de-synchronisation. Different environmental and physiological cues then appear to act to achieve readjustment. The brain uses light, the lungs respond to exercise signals and the liver feeding/nutrition cues. Thus, body organs have autonomy in circadian timing, but external cues and internal signals generate the optimal phasing.

Clock genes are also implicated in the generation of long-term rhythms in reproduction, appetite, growth, fattening and the pelage moult in seasonal mammals. This is based on the observation that clock genes are expressed in tissues that respond to melatonin to affect seasonal responses. The special role of the clock genes in photoperiodism and circannual rhythm generation has been studied in detail in cells of the pars tuberalis (PT), a tissue located in the stalk region of the pituitary gland at the interface with the hypothalamus [8,15]. The PT cells are believed to regulate the lactotrophs in the main body of the pituitary to dictate the long-term cycle in prolactin secretion. Prolactin itself controls many aspects of seasonal physiology including lactation, pelage growth/moulting, blastocyst implantation, food intake, metabolism and parental behaviours. In the PT, clock genes appear to decode the melatonin signal through changes in the phasing of Period and Cryptochrome gene expression - an internal coincidence timer.

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Illustrations
The research is summarised in the diagrams below (see Figures 1-4)

Figure 1. Soay sheep an a model
For many years, we have used rams of the feral Soay breed of sheep as a model to investigate seasonal physiology, and more recently, to study the roles of clock genes in long-term timing. Soay sheep originate from the island of St Kilda off the west coast of Scotland, and have a highly seasonal physiology similar to the European mouflon – the wild ancestor of all domesticated sheep. The seasonal increase in prolactin secretion in summer governs the wool growth cycle producing a conspicuous moult of the winter coat in spring, as seen in the photograph taken in June. Timed to occur later in the year, the seasonal increase in gonadotrophin (FSH and LH) secretion in autumn drives the reproductive cycle, culminating in the rut in October and November. We maintain groups of Soay rams indoors in special facilities at the Marshall Building near Edinburgh, to provide sheep at precisely defined times across the circadian cycle, or across the photoperiod-induced seasonal cycle, to study the internal clockwork.

Figure 2. Photoperiod-melatonin-relay
Seasonal mammals like the Soay sheep utilise the annual cycle in daylegth (photoperiod) to time their seasonal physiology and behaviour. The light information is relayed from the retina to the central circadian pacemaker system  (SCN) in the anterior hypothalamus, and this re-sets the bodies endogenous circadian rhythmicity to the precise 24h Earthly day. The SCN regulates in turn the nocturnal secretion of melatonin from the pineal gland and this acts as an internal index of the seasons. Long winter nights produce long melatonin signals, while short summer nights produce short signals, and it is the changes in melatonin signal duration that drives the seasonal responses. Certain target tissues that express melatonin receptors are able to decode the changing melatonin signal.  The pars tuberalis (PT) of the pituitary gland is one such tissue that controls seasonal prolactin secretion from the adjacent pituitary (PD). Prolactin regulates many aspects of seasonal biology including the moult cycle.

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Figure 3. Decoding the nocturnal melatonin signal
We use the pituitary PT cell as a model system to investigate the molecular mechanism by which the duration of the nocturnal melatonin signal is decoded to activate either a winter, or summer physiology. The current hypothesis is that the circadian clock genes provide the decoding mechanism.  Accordingly, the onset of melatonin release at dusk induces Cryptochrome (Cry1) gene expression and the offset of melatonin release at dawn induces Period  (Per1) gene expression in the PT cell. The Cry-Per interval varies directly with nightlength and thus daylength, and the degree of co-incidence dictates the level CRY-PER protein complexes dictating the transcriptional drive to clock-controlled genes and the down-stream cell biology in the pituitary gland. The clock genes of the PT-PD relay have been co-opted to produce an internal coincidence cellular timer.

Figure 4. Clock gene rhythms in the pituitary PT
The clock genes Bmal1, Per1, Per2 and Cry1 are rhythmically expressed in the sheep pituitary PT cells, with a very specific and reproducible profile for each gene. The molecular rhythms are dictated by the 24-h nocturnal melatonin signal that reflects the photoperiod.  The PT behaves as a slave oscillator - second order clock - that acts as a seasonal calendar.

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Recent advances (see selected references 1-27):
1.Circannual rhythms. Experiments extending over 5 years have characterised a circannual rhythm generator mechanism within the pituitary gland. This produces a rhythm that free runs under constant conditions with a period close to 10 months, and that can be readily entrained to the Earthly year by the annual cycle in photoperiod. Blocking the melatonin signal that encodes photoperiod disrupts the expression of the circannual prolactin rhythm. These studies support the concept of a circadian-based, pacemaker-slave system for long-term timing [references 1,3].

2. Decoding melatonin signals. The sheep studies provide strong evidence that melatonin acts in the pars tuberalis (PT) of the pituitary gland to relay photoperiodic effects on seasonal prolactin secretion (Fig. 1&2). These effects are thought to be mediated through changes in phasing of clock gene expression rhythms in the PT. Melatonin onset at dusk activates Cryptochrome (Cry1) gene expression, and melatonin offset at dawn activates Period (Per1) expression, and the Cry/Per interval varies with nightlength, inverse to daylength. It is proposed that changes in the level of CRY-PER protein complexes (dictated by relative coincidence in gene expression) differentially drives the transcription of clock-controlled genes and thus summer or winter physiology – an internal coincidence timer (Fig. 3) [4,5,9, 10,15,17].

3. First long day response. Exposure of sheep to an abrupt change from short to long days stimulates prolactin secretion - a spring response. This is initiated within 24-h and continues progressively for many weeks. We have used the first-day-response paradigm to demonstrate rapid adjustment of the circadian clock gene expression rhythms in the PT. The results support the view that the clock mechanisms drive the seasonal response [8,10].

4. Photorefractoriness. Prolonged exposure to constant photoperiod for many weeks leads to a spontaneous reversion in the seasonal phenotype – termed photorefractoriness. Sheep exposed to long photoperiod for 8wk (high prolactin, photoinduced) and 30wk (low prolactin, photorefractory) have very similar 24-h patterns of clock gene expression in the PT, faithfully reflecting the stable melatonin signal. Long-term timing appears to depend on the circadian system, but does not require alterations in phasing of clock gene rhythms to produce the photorefractory/circannual cycle (Fig. 4)[1,7,13].

5. Photoperiodism and homeostasis. Sheep express normal seasonal cycles in prolactin secretion even in the absence of the neural link between the hyopthalamus and pituitary gland. We have investigated the role of dopamine (DA) and noradrenaline (NA) in the hypothalamic control of prolactin release by treating rams with selective antagonists to dopamineric and adrenergic receptor subtypes during short days (low prolactin) and agonists during long days (high prolactin). This has revealed that DA acting through DA-D2 receptors, and to a lesser extent, NA acting through alpha-1/beta-2 adenoceptors provide the homeostatic control of prolactin secretion in the normal animal. This homeostatic mechanism is not required for the photoperiod response because the melatonin signal acts via the blood circulation directly in the pituitary PT  [21].   

6. Multiple targets for melatonin. The melatonin signal that encodes photoperiod appears to act at different sites in the mediobasal hypothalamus to govern seasonal changes in gonadal activity and body weight, separate from the control of prolactin via the PT. The differential control allows species to vary markedly in the timing of the breeding cycle but to have a similar summer activation of the prolactin axis. The PT-PD control of prolactin is seen as a conserved/ancestral timing mechanism (8,22).

7. Appetite and body weight.  Neuropeptide Y (NPY), agouti related protein (AGRP) and pro-opiomealanocortin (POMC) derived peptides synthesised in the hypothalamic arcuate nucleus (ARC) are specifically implicated in the photoperiodic regulation of cycles in food intact and body weight in the sheep model. Orexin expression in the lateral hypothalamus was unaffected by photoperiod, but may function in daily energy homeostasis (13,14).

8. Human clinical studies. Two recent studies are of clinical relevance. The first investigated the role of prolactin in the human testis and accessory sex glands and tested the effectiveness of a 3-month combined treatment with a prolactin inhibitor (quinagolide) and testosterone in suppressing spermatogenesis in normal men - a potential contraceptive [14,18]. The second study described the behavioural syndrome of depression and irritability that occurs following withdrawal of testosterone in the adult male. This is a feature in men following a chronic decline in circulating testosterone concentrations and in seasonal breeding animals after the peak of the sexual cycle – coining the term ‘irritable male syndrome’ (IMS) [23].

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