Thomas Dickmeis
email: thomas.dickmeis@kit.edu

Nicholas S Foulkes
email: nicholas.foulkes@kit.edu

Glucocorticoids and Circadian Clock Control of Cell Proliferation: At the Interface between Three Dynamic Systems

Keywords: adrenal gland, cell proliferation, circadian clock, glucocorticoid, neurogenesis, signalling

The circadian clock, an endogenous timekeeper that regulates daily rhythms of physiology, also influences the dynamic release of glucocorticoids. The release of glucocorticoids is characteristically pulsatile and is further modulated in a circadian fashion. A circadian pacemaker in the brain regulates daily rhythms of hypothalamicpituitary-adrenal axis and autonomic nervous system activity that both influence glucocorticoid release from the adrenal gland. This systemic regulation interacts with rhythms in the adrenal gland itself that are driven by its own circadian clock. One function of glucocorticoids is the regulation of cell proliferation. Depending on the tissue, this can involve both negative and positive regulation of a variety of processes, including cell differentiation and cell death. Cell proliferation is also under circadian control, and recent evidence suggests that this regulation may involve glucocorticoid signalling. Here, we review the dynamic processes participating in the interplay between the circadian clock, glucocorticoids and cell proliferation, and we discuss the potential implications for therapy.

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Introductionthe circadian clock

Among the many challenges that organisms face are the daily environmental changes brought about by the rotation of our planet on its axis. Endogenous timing mechanisms have evolved which allow organisms to anticipate these regular changes in the environment and so adapt to the specific needs of certain times of day in a timely manner. These mechanisms drive periodic changes in physiology even in the absence of environmental timing cues, such as under constant darkness and temperature. Under such conditions, physiological rhythms e.g. in locomotor behaviour run with an endogenous period that is slightly different from 24 hours. The biological clocks driving these rhythms are therefore termed "circadian", from Latin "circa" -"around" and "dies" -"day". Under constant conditions circadian clocks are said to "free-run", as they are not longer restrained by the environmental cues which serve to regularly reset or "entrain" them under natural conditions. Such cues, also called "Zeitgeber" (German for "time-givers"), include for example light and temperature, but also certain chemical compounds and social behaviour can entrain the clock [START_REF] Dunlap | Chronobiology[END_REF].

The central clock in the brain

In mammals, a small hypothalamic nucleus has been demonstrated to play a crucial role for the generation of circadian rhythms: The suprachiasmatic nucleus, or SCN [START_REF] Klein | Suprachiasmatic Nucleus -The Mind's Clock[END_REF]. Ablation of this nucleus leads to arrhythmicity of behaviour and of hormonal secretion. Furthermore, elegant transplantation experiments in hamsters showed that this structure was indeed sufficient to drive behavioural rhythms: animals in which the SCN was ablated and that then received a SCN transplant showed restored rhythms [START_REF] Ralph | Transplanted suprachiasmatic nucleus determines circadian period[END_REF]. When the transplant was derived from a mutant hamster strain that had a shorter endogenous period [START_REF] Ralph | A mutation of the circadian system in golden hamsters[END_REF], the transplanted animals showed locomotor A c c e p t e d M a n u s c r i p t activity with the mutant period [START_REF] Ralph | Transplanted suprachiasmatic nucleus determines circadian period[END_REF]. The SCN sends projections to a number of brain targets where its rhythmical electrical activity interacts with neural networks governing various outputs. However, also paracrine factors are involved in SCN output, since even encapsulated transplants, which do not allow neural connections, are able to restore locomotor rhythms in SCN ablated animals [START_REF] Silver | A diffusible coupling signal from the transplanted suprachiasmatic nucleus controlling circadian locomotor rhythms[END_REF]. Interestingly, endocrine rhythms such as rhythmical melatonin and corticosteroid secretion are not restored by SCN transplants, suggesting a specific requirement for neural connections in their circadian control [START_REF] Meyer-Bernstein | Effects of suprachiasmatic transplants on circadian rhythms of neuroendocrine function in golden hamsters[END_REF].

Information about the environmental lighting conditions reaches the SCN via the retinohypothalamic tract of the optic nerve (reviewed in [START_REF] Antle | Physiological responses of the circadian clock to acute light exposure at night[END_REF]). Only a specific subset of retinal ganglion cells projects to the SCN. These cells are intrinsically photosensitive, as they express a photopigment, melanopsin (reviewed in [START_REF] Bailes | Melanopsin and inner retinal photoreception[END_REF][START_REF] Paul | The role of retinal photoreceptors in the regulation of circadian rhythms[END_REF]).

Their function is crucial for entrainment of the SCN, since transgenic animals in which these cells are genetically ablated show free-running rhythms when exposed to light-dark cycles, just as normal animals do under constant darkness [START_REF] Guler | Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision[END_REF]. The photopigments of the rods and cones of the retina and melanopsin are highly redundant in this process, because only animals that lack both functional rods and cones and melanopsin show this "circadian blindness", whereas the presence of one of the two systems is sufficient to maintain circadian entrainment [START_REF] Panda | Melanopsin is required for non-image-forming photic responses in blind mice[END_REF]. These findings may also explain why only certain blind people suffer from free-running circadian rhythms: If only their photoreceptor layer is affected, leaving the photosensitive retinal ganglion cells intact, they retain some light input into the circadian system in spite of visual blindness [START_REF] Zaidi | Short-wavelength light sensitivity of circadian, pupillary, and visual awareness in humans lacking an outer retina[END_REF].

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The cogs of the clock Brain slice cultures of the SCN exhibit circadian rhythms of electrical activity, demonstrating that this structure is able to generate rhythms autonomously ( [START_REF] Herzog | Circadian rhythms in mouse suprachiasmatic nucleus explants on multimicroelectrode plates[END_REF] and references therein). However, this property is not due to a "neural oscillator circuit", since even single dissociated SCN neurons show circadian patterns of activity [START_REF] Welsh | Individual neurons dissociated from rat suprachiasmatic nucleus express independently phased circadian firing rhythms[END_REF]!

The circadian oscillator mechanism must therefore reside at the single cell level, a concept consistent with the observation of circadian rhythms in many unicellular organisms [START_REF] Dong | How a cyanobacterium tells time[END_REF][START_REF] Hastings | The Gonyaulax clock at 50: translational control of circadian expression[END_REF][START_REF] Lakin-Thomas | New models for circadian systems in microorganisms[END_REF]. A wealth of genetic and biochemical studies has revealed the nature of the cellular oscillator mechanism in vertebrates [START_REF] Ko | Molecular components of the mammalian circadian clock[END_REF]: An autoregulatory transcriptional-translational feedback loop leads to circadian oscillations of transcripts and proteins of a set of so-called clock genes (Fig. 1,B). The feedback loop starts when transcription of the period and cryptochrome genes is activated by the binding of a heterodimer of the bHLH-PAS domain transcription factors CLOCK and BMAL1 to so-called E-box enhancer elements in their promoters. PER and CRY proteins then accumulate in the cytoplasm, where they are modified by posttranslational mechanisms that modulate their entry into the nucleus. Once in the nucleus, they inhibit the activity of the CLOCK-BMAL1 dimer and thereby inhibit the transcription of their own genes. Consequently, per and cry transcript and protein levels decline, and the repression of CLOCK-BMAL1 activity is released, allowing the cycle to start again. The various delays in this process, notably those brought about by posttranslational modifications [START_REF] Gallego | Post-translational modifications regulate the ticking of the circadian clock[END_REF], confer a cycle duration of roughly 24 hours. Such modifications include phosphorylation of core clock factors, e.g. of PER2, which is phosphorylated on several residues by the kinases CK1, CK1 and CK2, [START_REF] Lowrey | Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau[END_REF][START_REF] Xu | Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome[END_REF][START_REF] Meng | Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins[END_REF][START_REF] Maier | A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock[END_REF] or of CRY1 by AMPK [START_REF] Lamia | AMPK regulates the circadian clock by cryptochrome phosphorylation and degradation[END_REF]. Other modifications include the ubiquitination of CRYs by F-box type ubiquitin ligases that targets them for A c c e p t e d M a n u s c r i p t degradation by the proteasome [START_REF] Busino | SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins[END_REF][START_REF] Godinho | The after-hours mutant reveals a role for Fbxl3 in determining mammalian circadian period[END_REF][START_REF] Siepka | Circadian mutant Overtime reveals F-box protein FBXL3 regulation of cryptochrome and period gene expression[END_REF]. Furthermore, acetylation of BMAL1 and PER2 has been reported [START_REF] Hirayama | CLOCK-mediated acetylation of BMAL1 controls circadian function[END_REF][START_REF] Asher | SIRT1 regulates circadian clock gene expression through PER2 deacetylation[END_REF].

The transcriptional-translational feedback loop described above defines the socalled "core loop" of the clock. Other feedback loops are thought to interact with this loop and to contribute to its robustness and fine tuning. The "accessory loop" consists of nuclear receptor transcription factors of the ROR and RevERB type, the transcription of which are again under control of the CLOCK-BMAL1 dimer [START_REF] Duez | The nuclear receptors Rev-erbs and RORs integrate circadian rhythms and metabolism[END_REF].

RORs activate transcription of the bmal1 gene via a RORE (retinoic acid related orphan receptor response element) in its promoter, while the RevERBs, which bind this element after a certain delay, repress its transcription. The resulting oscillatory expression of BMAL1 is believed to reinforce the core loop oscillations.

Clocks are everywhere

Clock genes are not only expressed in the neurons of the SCN or in the other central pacemaker structures found in non-mammalian vertebrates (the retina and the pineal gland). Instead, they are expressed in nearly all tissues of the body and also show oscillating expression there [START_REF] Balsalobre | Clock genes in mammalian peripheral tissues[END_REF]. Indeed, organ explants show circadian oscillations in some of their functions and in gene expression, demonstrating that they contain autonomous pacemakers [START_REF] Andrews | Circadian rhythms in adrenal organ cultures[END_REF][START_REF] Whitmore | Zebrafish Clock rhythmic expression reveals independent peripheral circadian oscillators[END_REF][START_REF] Yamazaki | Resetting central and peripheral circadian oscillators in transgenic rats[END_REF][START_REF] Yoo | PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues[END_REF][START_REF] Kaneko | Diversity of zebrafish peripheral oscillators revealed by luciferase reporting[END_REF]. Even the individual cells in fibroblast cell lines exhibit circadian rhythms in gene expression [START_REF] Nagoshi | Circadian gene expression in individual fibroblasts: cellautonomous and self-sustained oscillators pass time to daughter cells[END_REF][START_REF] Welsh | Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression[END_REF][START_REF] Carr | Imaging of single light-responsive clock cells reveals fluctuating free-running periods[END_REF]. Thus, circadian clocks are not an exclusive property of SCN neurons. The "peripheral" oscillators appear to be coordinated by the "central" oscillator, the SCN, since they drift out of phase with respect to each other in animals in which the SCN has been ablated [START_REF] Yoo | PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues[END_REF]. How exactly this coordination is achieved is still a field of active research, and cues proposed to mediate the synchronisation include body temperature changes [START_REF] Brown | Rhythms of mammalian body temperature can sustain peripheral circadian clocks[END_REF], food derived signals [START_REF] Damiola | Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus[END_REF][START_REF] Hara | Restricted feeding entrains liver clock without participation of the suprachiasmatic nucleus[END_REF][START_REF] Stokkan | Entrainment of the circadian clock in the liver by feeding[END_REF] and also glucocorticoids ( [START_REF] Balsalobre | Resetting of circadian time in peripheral tissues by glucocorticoid signaling[END_REF][START_REF] Minh | Glucocorticoid hormones inhibit food-induced phaseshifting of peripheral circadian oscillators[END_REF], see below). The importance of the A c c e p t e d M a n u s c r i p t peripheral oscillators for circadian organisation in vivo becomes apparent when the clock is stopped only in specific tissues by tissue-specific gene knock-out or knockdown techniques. For example, in mice that carry a liver specific deletion of the bmal1 gene which stops the clock in this tissue, one observes a deregulation of the circadian gene expression of many metabolic genes as well as a fasting glycemia [START_REF] Lamia | Physiological significance of a peripheral tissue circadian clock[END_REF]. However, circadian expression of certain genes in the liver is driven by systemic mechanisms, as they continue to cycle when the liver clock is stopped [START_REF] Kornmann | System-driven and oscillator-dependent circadian transcription in mice with a conditionally active liver clock[END_REF]. Systemic circadian changes more directly controlled by the SCN, e.g. in autonomic nervous system activity, also contribute to the daily regulation of glucose levels by the liver [START_REF] Kalsbeek | Circadian control of the daily plasma glucose rhythm: an interplay of GABA and glutamate[END_REF].

The circadian timing of metabolism

As shown by the example above, circadian clocks are involved in various aspects of metabolic regulation, both systemically and locally. Many rate-limiting enzymes of metabolic pathways show circadian expression [START_REF] Panda | Coordinated transcription of key pathways in the mouse by the circadian clock[END_REF], and disturbance of the circadian clock can lead to phenotypes resembling human metabolic diseases [START_REF] Turek | Obesity and metabolic syndrome in circadian Clock mutant mice[END_REF].

It becomes increasingly apparent that metabolism in turn can also feed back on the clock: as already mentioned above, the nutrient sensing kinase AMPK phosphorylates CRY1 and thereby destabilizes it [START_REF] Lamia | AMPK regulates the circadian clock by cryptochrome phosphorylation and degradation[END_REF]. The histone deacetylase SIRT1 deacetylates not only histones, but also the core clock components BMAL1 and PER2 (most likely previously acetylated by CLOCK itself, which has HAT activity [START_REF] Hirayama | CLOCK-mediated acetylation of BMAL1 controls circadian function[END_REF]), and it requires NAD+ as a cofactor [START_REF] Asher | SIRT1 regulates circadian clock gene expression through PER2 deacetylation[END_REF][START_REF] Nakahata | The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control[END_REF][START_REF] Nakahata | Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1[END_REF][START_REF] Ramsey | Circadian clock feedback cycle through NAMPTmediated NAD+ biosynthesis[END_REF]. NAD+ levels show circadian oscillation, partially mediated by clock control of the rate limiting enzyme in NAD+ biosynthesis, nicotinamide phosphoribosyltransferase. This complicated interplay of central and peripheral clocks, of cell autonomous and systemic regulation and of feedback from clock outputs to the clocks themselves is still poorly understood.

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Clock control of cell proliferation

Another highly temporally regulated event is cell proliferation, and it is perhaps not too surprising that the timing of cell proliferation and the circadian clock may be coupled (Fig. 1,C). Indeed, circadian rhythms in cell proliferation are observed across a wide range of organisms, from cyanobacteria and unicellular algae to humans, suggesting an early evolutionary origin and strong adaptive value for this cell cycle control [START_REF] Bjarnason | Circadian variation of cell proliferation and cell cycle protein expression in man: clinical implications[END_REF][START_REF] Mori | Circadian control of cell division in unicellular organisms[END_REF][START_REF] Reddy | Circadian clocks: neural and peripheral pacemakers that impact upon the cell division cycle[END_REF][START_REF] Borgs | Cell "circadian" cycle: new role for mammalian core clock genes[END_REF]. Since the circadian timing of the cell cycle shows a tendency to place S phase and mitosis in the night period, it has been suggested that the main adaptive pressure for circadian cell cycle control might stem from environmental UV light and its harmful effects on DNA replication and mitosis [START_REF] Nikaido | Daily and circadian variation in survival from ultraviolet radiation in Chlamydomonas reinhardtii[END_REF][START_REF] Pittendrigh | Temporal organization: reflections of a Darwinian clockwatcher[END_REF].

Cell cycle and circadian cycle

Various genes involved in cell proliferation are under transcriptional control of the core clock loop, including cell cycle components (cyclin D1, cdc2), regulators of the cell cycle and components of cell cycle checkpoints (wee1, mdm2, p21) as well as growth factors (vegf, kitlg) (reviewed in [START_REF] Borgs | Cell "circadian" cycle: new role for mammalian core clock genes[END_REF][START_REF] Sahar | Metabolism and cancer: the circadian clock connection[END_REF]). When circadian gene expression control is disturbed, e.g. in animals carrying clock gene mutations, defects in cell proliferation are observed. For example, in wild type mice, liver cell proliferation in response to partial removal of the liver (partial hepatectomy) is gated to certain times of day. This gating is absent in cry1/2 double mutant animals, leading to reduced efficiency of liver regeneration [START_REF] Matsuo | Control mechanism of the circadian clock for timing of cell division in vivo[END_REF]. Most interestingly, per2 mutants show an increased sensitivity to radiation induced cancer [START_REF] Fu | The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo[END_REF]. Furthermore, per1 has been implicated in the regulation of the DNA damage checkpoint of the cell cycle and shown to be deregulated in human cancers [START_REF] Gery | The circadian gene per1 plays an important role in cell growth and DNA damage control in human cancer cells[END_REF]. In contrast, clock mutant animals do not have any cancer-related phenotype [START_REF] Antoch | Disruption of the circadian clock due to the Clock mutation has discrete effects on aging and carcinogenesis[END_REF], and also cry1/2 double mutant mice do not exhibit a higher tendency to develop cancer [START_REF] Gauger | Cryptochrome, circadian cycle, cell cycle checkpoints, and cancer[END_REF]. On the contrary, they even show A c c e p t e d M a n u s c r i p t reduced cancer incidence in a p53 mutant background [START_REF] Ozturk | Loss of cryptochrome reduces cancer risk in p53 mutant mice[END_REF]. Cells derived from these triple knock-out (cry1/cry2/p53 -/-) mice are more sensitive to radiation induced apoptosis and thus die before the induced mutations can lead to oncogenic transformation. Since previous work had shown that DNA damage repair may also be under circadian clock control [START_REF] Kang | Circadian oscillation of nucleotide excision repair in mammalian brain[END_REF], it was hypothesized that the increased sensitivity to irradiation induced apoptosis was dependent on impaired DNA damage repair.

However, the triple mutant cells showed no significant reduction of DNA repair capacity. All these diverse findings indicate that the circadian clock intervenes at various levels in cancer prevention, and that different clock genes might target different aspects of cell proliferation control. In addition, cell proliferation and metabolism are closely intertwined, with proliferating cells and cancer cells showing characteristic changes in metabolic pathways, such as a switch from oxidative phosphorylation to glycolysis (the Warburg effect [START_REF] Deberardinis | The biology of cancer: metabolic reprogramming fuels cell growth and proliferation[END_REF][START_REF] Vander Heiden | Understanding the Warburg effect: the metabolic requirements of cell proliferation[END_REF]). One might therefore also expect a contribution of clock regulation of metabolism to the circadian control of cell proliferation [START_REF] Sahar | Metabolism and cancer: the circadian clock connection[END_REF].

Treating in time: chronotherapy

The discovery of a clock control of cell proliferation and metabolism has already led to improvements in cancer therapy. So-called "chronotherapeutical" approaches increase the efficiency and reduce side effects of anti-cancer drugs. Cell proliferation rhythms are deregulated in many cancer types [START_REF] Canaple | The days and nights of cancer cells[END_REF][START_REF] Levi | Implications of circadian clocks for the rhythmic delivery of cancer therapeutics[END_REF][START_REF] Levi | Circadian timing in cancer treatments[END_REF], and so delivering drugs that kill proliferating cells at a time when fewer normal cells divide will preferentially target the cancer cells that have escaped clock control. Another rationale for chronotherapy is linked to the metabolism of xenobiotics (such as cancer drugs): Degradation and modification of these substances is subject to circadian control. Thus, giving a drug at the trough point of degradation activity will allow the A c c e p t e d M a n u s c r i p t use of overall lower doses, thereby reducing the potential side effects [START_REF] Levi | Circadian timing in cancer treatments[END_REF][START_REF] Levi | Circadian rhythms: mechanisms and therapeutic implications[END_REF][START_REF] Levi | Chronotherapeutics: the relevance of timing in cancer therapy[END_REF]. The beneficial effects of chronotherapeutics have now been demonstrated in various clinical trials (reviewed in [START_REF] Levi | Circadian timing in cancer treatments[END_REF]).

Central rhythms and cell proliferation: a role for hormones?

As discussed above, there is good evidence that the circadian clock can regulate circadian rhythms of cell proliferation at least partially in a cell-autonomous manner.

Nevertheless, a wealth of non-cell-autonomous signals, such as growth factors, nutrients or hormones, also contribute to controlling cell proliferation (see e.g. [START_REF] Dechant | Nutrient signals driving cell growth[END_REF][START_REF] Puzianowska-Kuznicka | Thyroid hormones and their receptors in the regulation of cell proliferation[END_REF][START_REF] Nilsson | Endocrine regulation of the growth plate[END_REF][START_REF] Pollak | Insulin and insulin-like growth factor signalling in neoplasia[END_REF][START_REF] Ulloa | Morphogens and the control of cell proliferation and patterning in the spinal cord[END_REF]), and many of these signals are under circadian clock control [START_REF] Hastings | Circadian clocks: regulators of endocrine and metabolic rhythms[END_REF][START_REF] Haus | Chronobiology in the endocrine system[END_REF]. An influence of systemic circadian signals is also indicated by experiments in which tumors were transplanted into animals with SCN ablations: the tumors grew faster in the SCN ablated animals than in normal controls [START_REF] Filipski | Host circadian clock as a control point in tumor progression[END_REF]. These systemic signals might interact with peripheral clocks or function independently.

Rhythms of glucocorticoids

One such systemic signal might be the glucocorticoids, steroid hormones produced by the adrenal gland. They constitute an important part of the stress response and affect numerous physiological processes, such as carbohydrate and lipid metabolism as well as immune system function [START_REF] Norris | Vertebrate Endocrinology[END_REF]. They exert their effects on the target tissues via glucocorticoid receptors (GRs). These receptors of the nuclear receptor type reside in the cytoplasm as a complex with heat shock proteins, and translocate to the nucleus when glucocorticoids bind to their ligand binding domain.

In the nucleus, they act either by binding to specific DNA sequences, the Glucocorticoid Response Elements (GREs), or via interactions with other transcription factors that do not involve direct DNA binding of the GRs (reviewed in [START_REF] Kassel | Crosstalk between the glucocorticoid receptor and other transcription factors: molecular aspects[END_REF]). In both cases they can function as activators or repressors. A second nuclear A c c e p t e d M a n u s c r i p t receptor, the mineralocorticoid receptor (MR), binds the endogenous glucocorticoids cortisol and corticosterone with even greater affinity than the glucocorticoid receptor itself (and with a similar affinity as its other natural ligand, the mineralocorticoid aldosterone). It is therefore already activated by the low baseline levels of these glucocorticoids. Aldosterone, which circulates at even lower levels than the glucocorticoids, only becomes its prominent ligand in cells that express the enzyme 11-hydoxysteroid dehydrogenase 2, which converts glucocorticoids to an inactive form (reviewed in [START_REF] Joels | The coming out of the brain mineralocorticoid receptor[END_REF]). Finally, glucocorticoids may also act via mechanisms that do not involve transcriptional regulation. These so-called non-genomic effects are still poorly understood (reviewed in [START_REF] Haller | The effects of non-genomic glucocorticoid mechanisms on bodily functions and the central neural system. A critical evaluation of findings[END_REF]).

Glucocorticoids have long been recognised to show diurnal rhythms of release (reviewed in [START_REF] Haus | Chronobiology in the endocrine system[END_REF][START_REF] Chrousos | Ultradian, circadian, and stress-related hypothalamicpituitary-adrenal axis activity--a dynamic digital-to-analog modulation[END_REF][START_REF] Lightman | The significance of glucocorticoid pulsatility[END_REF][START_REF] Dickmeis | Glucocorticoids and the circadian clock[END_REF]). In diurnal animals, peak levels of glucocorticoids are encountered in the early morning, whereas nocturnal animals show a peak in the early night. This diurnal rhythm is superimposed on a pulsatile ("ultradian" = shorter than a day) rhythm of release, which consists of 1-2 pulses per hour (Fig 1,A). Similar less robust rhythms, of lower amplitude, are also observed for the adrenocorticotropic hormone ACTH. This peptide hormone, which is produced by the corticotrope cells of the pituitary gland, stimulates adrenal corticosteroid synthesis. Its release in turn is controlled by hypothalamus-derived peptides, the "secretagogues" CRH, AVP and oxytocin, produced by neurons of the paraventricular nucleus (PVN). They are released into the portal blood stream of the median eminence of the pituitary, which transports them to the corticotrope cells in the anterior part of the pituitary. The entire system, linking the hypothalamus with the adrenal gland via the pituitary gland, is referred to as the hypothalamic-pituitary-adrenal (HPA) axis.

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Glucocorticoids also feed back on the HPA axis and their own release, acting through GRs and MRs in the brain (e.g. the hippocampus and the PVN) and the pituitary (reviewed in [START_REF] Jacobson | Hypothalamic-pituitary-adrenocortical axis regulation[END_REF][START_REF] Kolber | Hypothalamic-pituitary-adrenal axis dysregulation and behavioral analysis of mouse mutants with altered glucocorticoid or mineralocorticoid receptor function[END_REF]). Data from mutant mouse models indicate a major role for the GR in this feedback, whereas pharmacological analysis also support an important role for the MR. Feedback sensitivity of the system varies diurnally and appears to involve MR mediated effects at the trough point of the cycle, whereas both MR and GR would mediate feedback at the peak point (see e.g. [START_REF] Ratka | On the role of brain mineralocorticoid (type I) and glucocorticoid (type II) receptors in neuroendocrine regulation[END_REF][START_REF] Spencer | Evidence for mineralocorticoid receptor facilitation of glucocorticoid receptor-dependent regulation of hypothalamic-pituitaryadrenal axis activity[END_REF][START_REF] Young | The role of mineralocorticoid receptors in hypothalamicpituitary-adrenal axis regulation in humans[END_REF]). Based on computer simulations, it has been proposed recently that the glucocorticoid-ACTH feedback loop alone could explain the ultradian pulsatility of glucocorticoid release [START_REF] Walker | Origin of ultradian pulsatility in the hypothalamic-pituitary-adrenal axis[END_REF]. According to this model, the delays generated by the transport of ACTH via the blood stream to the adrenal gland and the subsequent inhibition of ACTH release from the pituitary by the rising blood glucocorticoid levels would be sufficient to generate the hourly ultradian pulses. This would exclude the need for an ultradian pacemaker in the brain. This interesting hypothesis awaits testing in vivo.

Central pacemaker control of glucocorticoid rhythms

Circadian aspects of HPA activity are under control of the SCN (Fig. 2, reviewed in [START_REF] Dickmeis | Glucocorticoids and the circadian clock[END_REF]). Thus, lesions of the SCN abolish both glucocorticoid and ACTH rhythms. Only very few projections from the SCN contact the secretagogue-producing neurons of the PVN directly, therefore most of the SCN influence involves indirect connections. These are mediated via interneurons in the PVN itself, but also through relay stations in the subparaventricular zone (subPVZ) and the dorsomedial nucleus of the hypothalamus (DMH) [START_REF] Buijs | The biological clock tunes the organs of the body: timing by hormones and the autonomic nervous system[END_REF][START_REF] Engeland | Neural circuitry in the regulation of adrenal corticosterone rhythmicity[END_REF].

Interestingly, changes in glucocorticoid release do not always appear to be dependent on changes in ACTH levels (reviewed in [START_REF] Bornstein | Dissociation of ACTH and glucocorticoids[END_REF]). Thus, rhythms of Furthermore, it was shown that nocturnal light exposure can lead to changes in corticosterone release from the adrenal gland without accompanying changes in ACTH [101,102]. This effect of light upon corticosterone release was dependent on the SCN, since ablation of the SCN abolished it. Apparently, in addition to the HPA axis pathway, an alternative neural pathway exists from the SCN to the adrenal gland (Fig. 2). Transneural retrograde virus tracing showed that this pathway runs via the autonomic nervous system [START_REF] Engeland | Neural circuitry in the regulation of adrenal corticosterone rhythmicity[END_REF]101,103]. SCN neurons contact neurons of the preautonomic PVN, which project to the sympathetic preganglionic neurons of the spinal cord. These neurons in turn innervate the adrenal gland via the splanchnic nerve.

Consistent with the functionality of this pathway, denervation of the adrenal gland blocks the light induced corticosterone changes [102]. The splanchnic nerve also seems to mediate changes in the sensitivity of the adrenal gland to ACTH, which modulate corticosterone production and release in a circadian fashion even when ACTH levels do not change (reviewed in [START_REF] Dickmeis | Glucocorticoids and the circadian clock[END_REF][START_REF] Engeland | Neural circuitry in the regulation of adrenal corticosterone rhythmicity[END_REF]104]). In summary, a view emerges in which circadian regulation of the HPA axis is not alone in directing circadian glucocorticoid rhythms. Autonomic nervous control mechanisms and the peripheral clock of the adrenal gland itself also appear to contribute substantially to these rhythms. However, ACTH-dependent regulation of glucocorticoid release might be more prominent in acute stress responses [108,109].

A clock in the adrenal gland

Glucocorticoids talk back to the clock?

Not only does the circadian clock regulate production and release of glucocorticoids, but these also influence the circadian clock itself. Furthermore, glucocorticoids interact with other circadian outputs, one of which is the cell cycle [START_REF] Dickmeis | Glucocorticoids and the circadian clock[END_REF].

Glucocorticoids have been implicated in the entrainment of peripheral clocks. This conclusion is based on the observation that injection of the glucocorticoid receptor agonist dexamethasone can phase shift rhythmic gene expression in the liver in vivo. Furthermore, glucocorticoid pulses are able to synchronise clocks in mammalian cell culture cells [START_REF] Balsalobre | Resetting of circadian time in peripheral tissues by glucocorticoid signaling[END_REF]110]. However, mice lacking the glucocorticoid receptor in the liver still exhibit phases of clock gene expression identical to those of their wild type siblings, arguing against a strict requirement for glucocorticoids in liver clock entrainment [START_REF] Balsalobre | Resetting of circadian time in peripheral tissues by glucocorticoid signaling[END_REF]. Nevertheless, glucocorticoids do appear to be required for normal circadian expression of per2 in some areas of the brain, since cycling While a potential direct contribution of glucocorticoids to peripheral clock entrainment in the liver still remains unclear, glucocorticoids have been shown to modulate another pathway for peripheral clocks: food entrainment. Under daytime restricted feeding, which inverts the feeding habits of nocturnal rodents, peripheral pacemakers become decoupled from the SCN pacemaker. The timing of rhythmic clock gene expression in the periphery adjusts to that of the feeding regime, while the phase of the SCN clock still matches that of the light-dark cycle [START_REF] Damiola | Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus[END_REF][START_REF] Hara | Restricted feeding entrains liver clock without participation of the suprachiasmatic nucleus[END_REF][START_REF] Stokkan | Entrainment of the circadian clock in the liver by feeding[END_REF].

Glucocorticoids appear to retard the food induced phase shift of peripheral clocks, since phase shifts induced by daytime feeding occur faster in adrenalectomised or GR mutant animals [START_REF] Minh | Glucocorticoid hormones inhibit food-induced phaseshifting of peripheral circadian oscillators[END_REF]. Precisely how this interaction of food entrainment and glucocorticoids is mediated remains to be established.

Glucocorticoids also interact with transcriptional clock outputs in peripheral tissues. Thus, part of the cycling liver transcriptome is dependent on the adrenal gland [114]. SCN lesions also result in desynchronisation of the circadian liver

Glucocorticoids and cell proliferation

Among the many effects of glucocorticoids on physiology is the regulation of cell proliferation. Glucocorticoids have been described to act both as stimulators and inhibitors of cell proliferation, depending on the cell type and the concentrations used (reviewed in [117]). In general, it appears that a lower concentration of dexamethasone can stimulate cell growth, whereas higher doses inhibit proliferation.

In many cell lines, the antiproliferative effects of glucocorticoids seem to stem from a reversible block at the G1 phase of the cell cycle. Some of the mechanisms proposed for this cell cycle arrest are glucocorticoid mediated repression of cyclin D3 and cmyc, negative crosstalk of the GR with p53, and glucocorticoid mediated induction of the cyclin dependent kinase inhibitors p21 WAF1/CIP1 and p27 kip (reviewed in [117,118]). As mentioned above, both p21 and c-myc are also targets of circadian clock control, thus providing potential candidates for a direct cross talk of circadian clock regulation with glucocorticoid signalling.

A c c e p t e d M a n u s c r i p t

The effects of glucocorticoids upon cell proliferation are also revealed in animals that are deficient for glucocorticoids or glucocorticoid signalling.

Interestingly, cell proliferation appears disturbed only in a rather limited set of tissues (Table 1). Adrenalectomy has been reported to result in reduced cell proliferation, for example in the small intestine [119-121], and in increased proliferation in regions of the brain ([122, 123] and see the section on neurogenesis below). Also lung cell proliferation is influenced by glucocorticoids, as revealed by studies of mice lacking the CRH peptide [124,125]. These mice show glucocorticoid deficiency, but develop normally and are fertile. However, homozygous mutant pups which are the offspring of homozygous mutant mothers die from pulmonary insufficiency. This mortality is at least partially caused by continued lung cell proliferation during gestation and an overall delay in pulmonary maturation.

Cell proliferation defects in glucocorticoid receptor mutants

Interestingly, mice in which the glucocorticoid receptor has been disrupted die due to defects in lung function (atelactasis = collapse of lung tissue) shortly after birth. Other defects seen in these mice include impaired maturation of the skin, reduced gluconeogenic gene expression in the liver, a lack of in vitro T cell apoptosis and upregulation of POMC expression [126,127]. However, no major tissue defects due to impaired cell proliferation are observed, suggesting that the GR is not strictly required for the regulation of cell proliferation during development in many tissues.

Nevertheless, certain tissues are significantly affected. Thus, the lack of glucocorticoid receptor function abolishes stress induced (but not normal) erythropoiesis in vivo, as shown by transplantation studies using erythroid progenitor A c c e p t e d M a n u s c r i p t cells derived from GR -/-fetal livers [128]. Stress erythropoiesis appears to require binding of the receptor to DNA, since stress induced proliferation is also severely impaired in mice carrying a receptor dimerisation mutant (GR dim/dim ). In these mutants, the GR gene has been engineered to disrupt a residue required for dimerisation and hence the binding to GRE elements, but which still allows the GR to interact with other transcription factors [129].

Interestingly, the impaired maturation of the skin observed in GR -/-mice, resulting in compromised skin barrier competence, can be linked to a defective switch between differentiation and proliferation. Although overall proliferation in the epidermis is not increased, abnormally proliferating suprabasal keratinocytes are observed in these fetuses. Additionally, primary keratinocytes from GR -/-animals show an increased proliferation rate in culture compared to wild type cells. This indicates that the GR participates in the decision between proliferation and differentiation in a cell autonomous manner [130]. In contrast to what was seen in the case of stress erythropoiesis, this role seems to be independent of the DNA binding of the GR [130,131].

Glucocorticoids and neurogenesis

Another proliferative process affected by loss of glucocorticoids is neurogenesis (Table 2). Adrenalectomy leads to an increase in proliferation of the granule cells of the dentate gyrus in the hippocampus, one of the regions of adult neurogenesis [132,133]. Conversely, treatment with exogenous corticosterone inhibits granule cell proliferation [133]. Therefore, it has been proposed that glucocorticoids might at least in part mediate the decrease of neurogenesis observed in the dentate gyrus under stress conditions, during which glucocorticoids are elevated (reviewed in [134], see Apparently paradoxically however, the number of proliferating granule cells is reduced in MR knock-out mice, in contrast to the increase observed upon adrenalectomy. One potential explanation for this comes from the increased plasma levels of corticosterone in the MR knock-out mice, which might potentially inhibit proliferation via the GR. Importantly, no degeneration is observed in mice with a nervous system specific GR knock-out (driven by nestin regulatory elements [143]), reinforcing the notion of a MR dependent cell death protection mechanism [144].

A c c e p t e d M a n u s c r i p t

Interestingly however, mice with a brain specific MR knock-out (driven by CAMKII regulatory elements) do not exhibit the increased glucocorticoid levels observed in the mice completely lacking the MR, and also they do not show conspicuous changes in cell number and density in the dentate gyrus of the hippocampus [145]. Thus, the emerging picture of how glucocorticoids regulate neurogenesis is complex and may involve mechanisms combining the direct and indirect effects of both GR and MR.

Neurogenesis and circadian rhythmsare glucocorticoids involved?

Interestingly, some recent studies implicate a role for the circadian changes of glucocorticoid levels in hippocampal neurogenesis. Thus, artificially dampening the circadian corticosterone rhythm prevents fluoxetine, a serotonin uptake inhibitor, from stimulating neurogenesis. Restoring the corticosterone rhythm also restores the action of fluoxetine [146]. Similar results were obtained with L-NAME, a nitric oxide synthase (NOS) inhibitor that also stimulates hippocampal neurogenesis [147,148].

Thus, it appears that the circadian rhythm of glucocorticoids interacts with other signalling pathways in regulating neurogenesis. It remains to be seen to what extent these interactions contribute to normal neurogenesis. Do the highly dynamic changes in glucocorticoid levels, together with the associated differential GR and MR signalling activity, contribute to the timing of proliferation and apoptosis? It appears that the lower glucocorticoid affinity of the GR, together with the rapid clearance of the activated receptor by a proteasomedependent mechanism, leads to a signalling activity that closely follows changes in glucocorticoid levels. In contrast, the high affinity MR already responds to low glucocorticoid levels and its signalling activity appears to be more sustained (reviewed in [START_REF] Lightman | The significance of glucocorticoid pulsatility[END_REF]). These dynamics have consequences for gene expression: For the Furthermore, per2 mutant animals show a higher number of neural progenitor cells and newborn neurons, but also increased apoptosis. These observations may in part be related to the dampened glucocorticoid rhythms also observed in these animals [155].

However, in vitro studies also suggest that per2 exerts more direct effects on neural progenitors [154], which seem to be independent of its role in circadian clock regulation. Thus, the precise links between the circadian clock mechanism, glucocorticoid dynamics and neurogenesis remain unclear.

Glucocorticoids and rhythms of cell proliferation

In contrast to the hippocampus, cell proliferation rhythms have been described in other tissues in which proliferation is also affected by glucocorticoids, (e. g. blood and bone marrow [156], gastrointestinal mucosa [157], and skin [157,158]). Some However, much of the data presented has limitations, and so for example, the effects of the treatments upon the circadian clock itself were frequently not examined.

Our own studies have revealed a role for glucocorticoids in the regulation of circadian rhythms of cell proliferation in zebrafish larvae [161]. Circadian cell cycle rhythms can be observed both in larvae and in zebrafish-derived cell lines, implying that cell-autonomous clock regulation also exists in this species [162]. However, in vivo, systemic input is also required: in mutants lacking a pituitary gland, cell cycle rhythms are severely attenuated [161]. Subsequent analysis of mutants that lack subsets of the pituitary cell types identified the corticotrope lineage as the source for the required signal. Thus, larvae homozygous for a truncated allele of the homeobox The interaction of glucocorticoid and circadian clock control of cell proliferation may not be limited to one single mechanism, given the diverse effects of glucocorticoids on cell proliferation in different tissues. As mentioned above, a similar situation exists for the contribution of glucocorticoids to circadian clock gene cycling in different tissues: While circadian per2 expression in some regions of the brain depends on glucocorticoids [111,112], this is not the case in other brain regions [112] or the liver [START_REF] Balsalobre | Resetting of circadian time in peripheral tissues by glucocorticoid signaling[END_REF]114]. Clearly, similar tissue specific differences might also be at work in the control of circadian cell cycle rhythms by glucocorticoids, with rhythmic glucocorticoid presence required in some tissues and tonic levels being sufficient in others.

Timing of glucocorticoidspotential in cancer therapy?

Glucocorticoid mediated induction of apoptosis and cell cycle arrest have long been exploited for therapy (reviewed in [117,165,166]). Thus, the glucocorticoid mediated induction of apoptosis in the blood cell lineage is exploited for the treatment of leukaemia. On the other hand, glucocorticoids can also confer resistance to apoptosis induced by various other stimuli. This is the case for example in cells of epithelial origin (e.g. cells of the mammary gland or lung epithelial cells, see [167])

A c c e p t e d M a n u s c r i p t and in solid malignant tumours [168,169]. Interestingly, the anti-proliferative effect of glucocorticoids mentioned above might also negatively influence cancer therapy.

The pretreatment of patients with glucocorticoids to alleviate nausea or to treat tumour-associated oedema and inflammatory responses can lead to cell cycle arrest in the G1 phase. Thus, in glucocorticoid sensitive tumours, can be negatively affected, because non-proliferating cells are less vulnerable to chemotherapy. Also radiotherapy can be impaired by the G1 arrest, since the sensitivity of cells to radiotherapy is highest at M phase [117]. In this scenario a chronotherapeutic approach might prove particulary useful: One could administer glucocorticoids and chemo-or radiotherapy with a distinct time profile that would retain at least some of the beneficial effects of glucocorticoids while avoiding their effects on the cell cycle

Where are we heading?

This survey illustrates the impressive diversity of effects that glucocorticoid hormones have upon cell proliferation. Depending on the cell type and tissue, they can either promote or inhibit cell proliferation, and they do this by targeting different aspects of tissue homeostasis. Thus, they influence proliferation itself, but are also involved in One major unexplored area is how the dynamic changes of glucocorticoid ligand concentrations translate into signalling activity and how this affects the temporal regulation of gene expression. This fundamental question is of direct relevance for all cell signalling pathways. The glucocorticoid pathway promises to be a particularly attractive model system to study such temporal aspects of signal transduction in both the cellular and systemic context, given the characteristic circadian and ultradian rhythms of glucocorticoid release. The circadian clock represents probably the best understood biological timing system from the physiological to the molecular level.

One of its regulatory targets is the cell cycle, which constitutes yet another cellular timing system. Thus, links between the circadian clock and glucocorticoid control of cell proliferation form an ideal framework in which to examine the temporal integration of signalling pathways and of their physiological output. Future research in this field will need to develop experimental tools to specifically manipulate the dynamics of glucocorticoid signalling. In the long term, knowledge gained from such

  A c c e p t e d M a n u s c r i p t corticosteroid secretion can still be observed in animals in which the pituitary gland has been removed and which do not show any changing ACTH levels[START_REF] Meier | Daily variation in concentration of plasma corticosteroid in hypophysectomized rats[END_REF] 100].

  Circadian glucocorticoid rhythms are not only controlled by systemic mechanisms. Recent experiments have also revealed a role for the circadian clock present in the adrenal gland in the circadian gating of ACTH sensitivity. Adrenal slice cultures from mice show a stronger response to ACTH stimulation in the evening, at the time of the natural glucocorticoid peak, than in the morning. Furthermore, wildtype adrenal glands transplanted into a host with a non-functional clock are still able to secrete corticosterone in a rhythmic fashion [105]. Finally, tissue specific antisense mediated knock-down of the Bmal1 protein in the adrenal gland abolishes

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  c c e p t e d M a n u s c r i p t circadian glucocorticoid production under constant conditions (however, under a light dark cycle rhythmic production continues, potentially owing to the neural mechanisms mentioned above)[106]. Another facet of circadian regulation of glucocorticoid production seems to involve clock control of gene transcription in the glucocorticoid synthesis pathway, in addition to that of genes participating in the ACTH signalling pathway [105-107].

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  c c e p t e d M a n u s c r i p t expression of this gene is abolished in adrenalectomised animals (animals in which the adrenal gland has been removed)[111, 112]. Interestingly, the per2 rhythms can resume when diurnal changes in corticosterone are restored in adrenalectomised animals by delivering corticosterone with the drinking water. This is not the case when the animals are supplemented with constant release pellets, indicating that rhythmic glucocorticoid signalling might contribute to rhythm generation[113]. An attenuation of the amplitude (but no effect on the phase) of circadian per1 expression has also been reported in livers of the animals with a clock-less adrenal gland mentioned above, whereas rhythmic per2 expression was unaffected [106]. Together, these findings point to the possibility that tissue specific mechanisms might operate in clock gene regulation via glucocorticoids.

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  c c e p t e d M a n u s c r i p t transcriptome, and a single dexamethasone treatment can synchronise about 60% of this desynchronised gene transcription [115]. The glucocorticoid-sensitive circadian gene HNF4A may in part mediate this synchronisation: The promoter analysis of a subset of the synchronised genes reveals an enrichment of binding sites for HNF4A, together with glucocorticoid response elements (GREs) and E-boxes [115]. This suggests that glucocorticoids may affect circadian transcription at various levels. They may act directly via GREs in target gene promoters, indirectly via glucocorticoid target genes such as HNF4A, via direct effects on clock gene expression and by combinations of these mechanisms. Recent work also implicates clock-glucocorticoid interactions at the protein level: The HAT activity of CLOCK also targets the glucocorticoid receptor, potentially affecting its DNA binding capacity [116].

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  c c e p t e d M a n u s c r i p t also [135] for a review of recent studies supporting mechanisms independent of glucocorticoids).Adrenalectomy also leads to an increase in granule cell death, suggesting that while proliferation increases, survival of the cells decreases[136, 137]. The cell death can be rescued by adding corticosterone to the drinking water[137]. Importantly, treatment with aldosterone, which activates the MR but not the GR, can also prevent adrenalectomy induced cell death, implicating a protective role for this receptor[138].Conversely, activation of the GR using its specific agonist dexamethasone does not rescue this cell death[139, 140]. Interestingly, in intact animals, glucocorticoid receptor agonists can induce apoptosis in granule cells, and mineralocorticoid receptor activation can protect against this effect[141, 142]. It thus appears that a balance between MR and GR activation, affecting both cell proliferation and survival, is required for normal neurogenesis in the dentate gyrus.Examination of mutant mice lacking MRs or GRs in the brain has brought additional insight into the role of these two receptors in neurogenesis. Mice completely lacking the MR can be rescued from death by renal salt and water loss via exogenous NaCl administration and thereby can be studied as adults. These mice show granule cell degeneration similar to that observed with adrenalectomy.

A

  c c e p t e d M a n u s c r i p t GR, it has been shown that pulsatile glucocorticoid signals can lead to different induction levels and expression dynamics of GR target genes compared with those observed under continuous stimulation [149]. It will be interesting to examine links between the circadian and ultradian changes in glucocorticoid levels, the different dynamic signalling properties of GR and MR, as well as the regulation of cell proliferation and survival. If the daily dynamics of glucocorticoids are important in the regulation of proliferation, they might at least in part exert their effect by regulating circadian rhythms of cell proliferation. However, in the case of hippocampal neurogenesis, while some studies did observe diurnal rhythms of granule cell layer proliferation in the dentate gyrus, several other studies failed to detect such rhythms ([150-153], reviewed in [135]). Thus, the effects of glucocorticoids on neurogenesis might not involve changes in rhythmic cell proliferation per se. Interestingly, expression of the clock gene per2 does not show circadian regulation in the adult dentate gyrus [154].

A

  c c e p t e d M a n u s c r i p t early reports also suggested changes in these rhythms upon manipulation of glucocorticoids, e.g. in the cornea epithelium [159] or during liver regeneration [160].

  transcription factor rx3 [163], which possess reduced numbers of corticotrope cells, have severely attenuated cell cycle rhythms. In contrast, pit1 mutants [164], which lack the somatotrope, thyrotrope and lactotrope lineages but still possess gonadotrope, melanotrope and corticotrope cells, show rhythms indistinguishable from their wildtype siblings. As a consequence of their strongly reduced corticotrope cell number, rx3 mutants contain lower cortisol levels than wildtype larvae, and also the diurnal cortisol changes observed in the wildtype siblings are not present. Interestingly, overall clock gene rhythms in these animals are unchanged, excluding a dominant role for glucocorticoids in peripheral clock synchronisation. Importantly, normal cell cycle rhythms can be rescued in the rx3 mutants by tonic treatment with dexamethasone. Hence, glucocorticoids cooperate with the peripheral circadian clock in the regulation of circadian cell cycle rhythms. They might act as permissive signals that enable the peripheral clocks to control the cell cycle, or alternatively the A c c e p t e d M a n u s c r i p t peripheral clocks might gate glucocorticoid input into the cell cycle machinery. The dexamethasone rescue experiments indicate that circadian changes of glucocorticoid levels are not required for their action on the circadian cell cycle rhythms. However, cycling glucocorticoid levels may still contribute by ensuring correct signalling input to the cell cycle machinery. It will be of great interest to determine whether similar interactions between peripheral clocks and systemic signals regulate circadian rhythms of cell proliferation in the mammalian system, e.g. those observed in liver regeneration after partial hepatectomy [61].

  at the time of therapy. Conversely, arresting normal cell proliferation via glucocorticoids might allow chemo-or radiotherapy of glucocorticoid resistant tumors with less toxic side effects. Glucocorticoid chronotherapy has already been employed successfully to treat rheumatoid arthritis and asthma [170, 171]. Importantly, glucocorticoid rhythms are deregulated in many cancer patients [172] and have been correlated with survival [173]. Thus, a better understanding of the molecular mechanisms underlying the involvement of glucocorticoids in the control of cell cycle timing should also ultimately help to design more efficient treatment schemes in cancer therapy.

A

  c c e p t e d M a n u s c r i p t switches between differentiation and proliferation, and they can regulate cell death and survival. Their effects can be systemic or local, direct or indirect, and involve cross talk with other signalling pathways. Glucocorticoid signalling can require DNA binding of the glucocorticoid receptors, or involve DNA-binding independent interactions with other transcription factors. Importantly, the signalling outcome can also depend on the balanced activation of the two main types of glucocorticoid receptors, GR and MR. Clearly, one major goal for future research will be to better understand the precise mechanisms underlying the glucocorticoid control of cell proliferation in various tissues and to convert this knowledge into more efficient therapies.

A

  c c e p t e d M a n u s c r i p t studies should pave the way towards novel, more specific therapeutic strategies entailing fewer harmful side effects. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 173. Sephton, S.E., et al., Diurnal cortisol rhythm as a predictor of breast cancer survival. J Natl Cancer Inst, 2000. 92(12): p. 994-1000. 174. Loudon, A.S., et al., Ultradian endocrine rhythms are altered by a circadian mutation in the Syrian hamster. Endocrinology, 1994. 135(2): p. 712-8.

Fig. 1 Three

 1 Fig.1

Fig. 2 ModelFigure 2

 22 Fig.2

Table 1

 1 Overview of glucocorticoid signalling deficiency effects on various tissue types observed in the studies cited in the text. ADX, adrenalectomy; CRH, corticotropin releasing hormone; CORT, corticosteroids; GR, glucocorticoid receptor; dim/dim, dimerisation mutant; -/-, knock out mutant; ↓ downregulation; ↑, upregulation.

Table 2

 2 Overview of the effects of glucocorticoid manipulation on adult hippocampal neurogenesis observed in the studies cited in the text. For details see text. ADX,

adrenalectomy; CORT, corticosteroid; CRH, corticotropin releasing hormone; GC, glucocorticoid receptor agonist; GR, glucocorticoid receptor; GR dim/dim, dimerisation mutant of the GR; MC, mineralocorticoid receptor agonist; MR, mineralocorticoid receptor; -/-, knock out mutant; n.e., not examined; ↓ downregulation; ↑, upregulation; ↔, no change.

Acknowledgements

We thank Meltem Sahinbas and Benjamin Weger for a critical reading of the manuscript and help with figure preparation.


Corvec
email: stephane.corvec@chu-nantes.fr

Semen cultures analysis : retrospective study during a six-year period and interest in the management of infertility

Semen cultures analysis: retrospective study during a 6-year period and interest in the management of infertility

Introduction

In France, over the past decade, an increasing number of couples consulted an Assisted Reproductive Techniques (ART) center for the management of infertility [START_REF]Bilan des activités de procréation et de génétique humaines en France en[END_REF]. In this framework, a clinical and biological assessment of infertility is realized for each partner. For men, this assessment systematically requires a semen analysis including a microbiological semen culture [START_REF]Laboratory manual for the examination of human semen and semen-cervical mucus interaction[END_REF][START_REF] Kvist | Manual on basic semen analysis[END_REF]. This bacteriological analysis is indicated for the safety of the partner and to optimize the preparation of the semen before insemination [START_REF] Askienazy-Elbhar | Male genital tract infection : the point of view of the bacteriologist[END_REF]. The purpose of the semen culture is to identify a possible bacteriospermia in order to eradicate it and to prevent contamination of embryonic culture media. A positive bacterial culture could result in fertilization failure and altered embryonic development, leading to lowered pregnancy rates, making it an essential consideration before in vitro fertilization (IVF) cycle. This analysis has to be performed in a systematic fashion, as most men are asymptomatic. Indeed, chronic bacterial infection of semen is rare, but may be a potential cause of male infertility [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF].

Bacteria involved in that case could act on semen quality either directly on spermatozoa (spermatozoa motility and viability), or indirectly on the seminal plasma or by forming anti-semen antibodies [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF]. Semen bacterial analysis can recover various bacterial species, including potential pathogens (i.e. Enterobacteriaceae or Staphylococcus aureus), but also bacterial species usually considered as contaminants (coagulase-negative staphylococci, alpha-hemolytic streptococci…) originating from the urethral or cutaneous flora [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF][START_REF] Virecoulon | Bacterial flora of the low male genital tract in patients consulting for infertility[END_REF].

The aim of this study was to evaluate the results of semen analysis in the bacteriology laboratory at Nantes University hospital and to assess the relevance of the procedure in the global management of infertile patients.

Materials and methods

This retrospective study has been conducted at Nantes University Hospital for a six-year period, between 2003 and 2008.

Patient selection

During the study period, each bacterial semen analysis performed was included. Most of the men were referred for semen analysis before an IVF cycle (approximately 2000 per year ; semen analysis was performed after a first infertility consultation or 6 weeks before each IVF cycle ; control semen analysis was performed after antibiotherapy). Others were semen donor or men referred for semen cryopreservation before potentially gonadotoxic treatment.

Bacteriological analysis of semen

Sample collection procedure

Before semen analysis, men were previously instructed to drink abundantly (generally, up to 1.5 liter) the day before semen collection in order to increase diuresis and to assure a cleaning of the urethra, as previously reported [START_REF] Virecoulon | Bacterial flora of the low male genital tract in patients consulting for infertility[END_REF]. Semen samples were collected at the ART center after 2 to 3 days of sexual abstinence. Just before semen collection, both verbal and written advices were given by the medical team to the patient to follow a drastic hygiene procedure : first, men were asked to wash hands, then to urinate and finally to wash their penis with chlorhexidine (aqueous chlorhexidine, 0.05 %, one dose recipient, Gilbert laboratory, France). Semen was collected in an aseptic recipient (MT245, JCD, La Mulatiere, France) after masturbation and analyzed after 30 minutes of liquefaction at 37°C.

Bacteriological culture procedure

After collection of the ejaculate into a sterile plastic receptacle, samples were aliquoted. Semen aliquots (150 µL) were transferred within three hours to the bacteriology laboratory to quantify aerobic rods only [START_REF] Keck | Seminal tract infections: impact on male fertility and treatment options[END_REF]. Semen samples were plated pure (10 µL) and diluted with sterile saline solution at 1:10 (100 µL) on horse blood agar plates (bioMérieux, Marcy-l'Etoile, France). Media were incubated for 24 to 48 hours in atmosphere supplemented with 5 % of CO 2 at 37 °C.

Diagnostic procedure

Each colony from the plate inoculated with diluted semen represented 10 2 CFU mL -1 . The bacterial enumeration was categorized as follows : enumeration less than 10 3 CFU mL -1 was considered as negative cultures according to WHO criteria [START_REF]Laboratory manual for the examination of human semen and semen-cervical mucus interaction[END_REF] ; enumeration from 10 3 CFU mL -1 to 3 10 3 CFU mL -1 or enumeration up to 3 10 3 CFU mL -1 were considered as positive cultures, in the case of pure or predominant flora culture. According to the World Health Organization guidelines [START_REF]Laboratory manual for the examination of human semen and semen-cervical mucus interaction[END_REF], a bacterial concentration in the ejaculate up to 10 3 bacteria mL -1 is a significant bacteriospermia. Therefore, no qualitative analysis was realized for samples with enumeration less than to 10 3 CFU mL -1 .

If necessary, bacteria were identified by fast and simple tests such as Lancefield's group determination for streptococci (Streptex ® , DiaMondial, Sees, France) and agglutination test for staphylococci (Statest ® , Biorad, Marnes-la-Coquette, France). For Enterobacteriaceae biochemical identification was performed using the Vitek2 IDGN card (bioMérieux) and Api Coryne ® (bioMérieux) for Corynebacterium seminale.

Susceptibility testing was systematically performed on bacterial strains potentially pathogenic or resistant to antibiotics such as Enterobacteriaceae (VITEK2 ® AST-N052, bioMérieux),

Staphylococcus aureus (VITEK2 ® AST-P551, bioMérieux) and C. seminale (disc diffusion

technique on horse blood agar plates at 37 °C in an atmosphere supplemented with 5 % of CO 2 ).

An antibiotic treatment was indicated in the following cases :

When an Enterobacteriaceae or a S. aureus was detected, an antibiotic treatment with good urogenital diffusion was recommended. Antibiotic regimen was in first intention, nitrofurantoine (150 mg per day, 8 days except for Proteus-Providencia-Morganella) or in second intention, ofloxacine (400 mg per day, 10 days). Antibiotherapy was adapted according to susceptibility testing and a control semen culture was systematically performed 10 days after the end of the treatment in order to check the efficiency of the antibiotherapy.

When C. seminale was detected associated with leucospermia, an antibiotic treatment with good uro-genital diffusion was also recommended.

On the opposite, no antibiotic treatment was established for the patient (i) when a monomicrobial culture of Enterococcus (enumeration up to 3 10 3 CFU mL -1 ) was detected, but a control semen culture was systematically performed or (ii) when a plurimicrobial culture (i.e. up to 3 10 3 CFU mL -1 ) with cutaneous flora bacteria or Enterococcus was observed, suggesting a contamination during semen collection. The results have been interpreted according to bacterial enumeration and number of species recovered (Table 1). Very few samples were classified as non-interpretable due to the invasion of the culture by Proteus species. Furthermore, for a limited number of samples, the volume was insufficient for bacterial enumeration. The percentage of positive semen cultures was relatively stable during the 6 years with an average of 44.8 % (39.9 to 46.6 %), respectively 11 % with a bacterial enumeration from 10 3 CFU mL -1 to 3 10 3 CFU mL -1 and 33.8 % with an enumeration up to 3 10 3 CFU mL -1 . Whatever the enumeration for positive semen cultures, a majority of these samples was polymicrobial (77 %).

Results

Quantitative analysis

Qualitative analysis : bacterial ecology of semen samples

The different types of bacteria recovered from semen cultures are summarized in Table 2. In most cases, they corresponded to bacteria considered as contaminants or "belonging" in the male genital tract (average : 50.3 %). These results were confirmed all over the study period. Enterococci were found from 10.5 to 24.4% of the isolated species and Enterobacteriaceae from 11.7 to 27.5 %, mostly Escherichia coli (57.1 % to 65.6 %).

Resistance pattern of the bacteria recovered

Between 2003 and 2008, out of the 14,119 semen cultures performed, 1,376 antibiotic susceptibility tests were realized (9.75 %). In 2003, according to our criteria, 16.7 % of the requests required an antibiotic susceptibility test compared with 7.2 % in 2008 (Table 3). In most cases, regardless of the identified species, a susceptible phenotype was generally recovered. Isolation of multi-drugresistant bacteria (MDR) remained an exceptional phenomenon as shown in table 3. Six of these 10 cases concerned Enterobacteriaceae resistant to third-generation cephalosporins. Most of the corresponding patients had spinal cord injury resulting in paraplegia, with a permanent urinary catheter.

Discussion

This retrospective study during a six-year period revealed a 45% increase in the annual number of semen cultures requests. Over a 10 year period (1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008) this increase was 137 % with a 72 % increase of the number of patients consulting the ART center and a doubling of the number of medical consultations from 2003 to 2008 (data not shown). These observations are part of the national trend of increasing number of couples consulting for infertility [START_REF]Bilan des activités de procréation et de génétique humaines en France en[END_REF].

It could be partly due to the increasing maternal age at the time of the first pregnancy in most European countries (30 years in France) [START_REF] Auger | Decline in semen quality among fertile men in Paris during the past 20 years[END_REF]. Moreover, improved and extensive medical and media information about infertility allows faster and more efficient referral of couples experiencing difficulties to conceive to ART centers.

During this study, the proportion of positive semen cultures remained stable during the study period with an average of 44.8 %. Previously reported data appeared to be highly variable from one study to another. Indeed, values ranging from 20 to a surprising 100 % of positive semen cultures were reported [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF][START_REF] Virecoulon | Bacterial flora of the low male genital tract in patients consulting for infertility[END_REF][START_REF] Swenson | Asymptomatic bacteriosemenia in infertile men[END_REF][START_REF] Levy | Bacterial risk and semen cryopreservation[END_REF][START_REF] Cottell | Processing of semen in an antibiotic-rich culture medium to minimize microbial presence during in vitro fertilization[END_REF][START_REF] Moretti | The presence of bacteria species in semen and semen quality[END_REF]. For example, Virecoulon et al worked with a threshold of 10 3 CFU mL -1 [START_REF] Virecoulon | Bacterial flora of the low male genital tract in patients consulting for infertility[END_REF].

Stovall et al reported 69 % of positive cultures with a threshold of 10 2 CFU mL -1 [START_REF] Stovall | The role of aerobic and anaerobic semen cultures in asymptomatic couples undergoing in vitro fertilization: effects on fertilization and pregnancy rates[END_REF]. Moretti et al worked with a threshold of 10 4 CFU mL -1 in case of Gram positive cocci but a 10 5 CFU mL -1 in case of Gram negative rods [START_REF] Moretti | The presence of bacteria species in semen and semen quality[END_REF]. Finally, comparison between different studies is difficult since some authors do not mention a specific positivity threshold [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF].

An interesting parameter to consider is the influence of hand washing on the number of positive semen cultures. Indeed, a study among couples followed for IVF has shown that the number of positive semen cultures could fall from 92.9 to 39 % with instructions for washing hands before sampling [START_REF] Krissi | Effect of contaminated preprocessed semen on fertilization rate and embryo quality in assisted reproductive techniques[END_REF]. Furthermore, Boucher et al. have shown that direct verbal counseling of patients by a member of the medical staff improved the bacterial quality of the sample compared with short written instructions [START_REF] Boucher | Semenoculture : improvement of the bacteriological quality of samples by direct verbal counseling before semen collection[END_REF]. In our study, there was no change in the procedure of the ART center for semen sampling (verbal instructions) and the bacterial ecology of semen sample was relatively stable during the 6 years.

According to the results, a large proportion of species can be considered as contaminants, resulting from bad asepsis during sampling in agreement with the literature [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF][START_REF] Virecoulon | Bacterial flora of the low male genital tract in patients consulting for infertility[END_REF][START_REF] Moretti | The presence of bacteria species in semen and semen quality[END_REF]. High-prevalences for coagulase-negative staphylococci (CNS) and for alpha-haemolytic streptococci were previously reported [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF][START_REF] Stovall | The role of aerobic and anaerobic semen cultures in asymptomatic couples undergoing in vitro fertilization: effects on fertilization and pregnancy rates[END_REF]. CNS usually predominate in the distal urethra and reflect the aerobic and facultative bacterial ecosystem of the male genital tract.

In our cohort, there was also a large proportion of enterococci which is controversial in the literature. For example, Rodin et al. found a low proportion of 7.4 % [START_REF] Rodin | Relationship between semen cultures, leukosemenia, and semen analysis in men undergoing fertility evaluation[END_REF]. Different teams have worked on the role of enterococci in infertility and as far as we know, only one study suggested an high incidence of oligozoospermia and teratozoospermia in human semen infected with E. faecalis [START_REF] Mehta | High incidence of oligozoosemenia and teratozoosemenia in human semen infected with the aerobic bacterium Steptococcus faecalis[END_REF]. Although the French guidelines in Microbiology recommended systematic search for anaerobes [START_REF] Sfm | Examen des sécrétions et exsudats anogénitaux[END_REF], due to contradictory data in the literature, these bacteria were not searched. Indeed, anaerobes normally colonize the male urethral orifice and therefore are not considered to be a major cause of male infertility [START_REF] Keck | Seminal tract infections: impact on male fertility and treatment options[END_REF]. Urogenital Mycoplasma spp. were not systematically searched in agreement with the clinicians and biologists of the ART center except for specific request [START_REF] Rosemond | Systematic screening tests for Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in urogenital specimens of infertile couples[END_REF].

For Enterobacteriaceae, different data from the literature found a proportion of around 15 to 20 % [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF][START_REF] Sanocka-Maciejewska | Bacterial infection and semen quality[END_REF], which is in agreement with our results. A recent study has shown their potential role in semen motility alteration [START_REF] Berktas | Semen motility changes after coincubation with various uropathogenic microorganisms : an in vitro experimental study[END_REF]. Presence of Enterobacteriaceae in semen involves the prescription of antibiotic treatment, with a significant potential impact on the digestive flora [START_REF] Andremont | Consequences of antibiotic therapy to the intestinal ecosystem[END_REF]. Here, the presence of Enterobacteriaceae in semen involved a delay in the IVF procedure but had no influence on the assisted reproductive technique outcome. The management of patients was shifted : the IVF procedure was not started before negative semen culture was obtained, confirming that the semen was free of infection.

The impact of bacteriospermia on semen parameters remains controversial [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF][START_REF] Stovall | The role of aerobic and anaerobic semen cultures in asymptomatic couples undergoing in vitro fertilization: effects on fertilization and pregnancy rates[END_REF][START_REF] Krissi | Effect of contaminated preprocessed semen on fertilization rate and embryo quality in assisted reproductive techniques[END_REF][START_REF] Sanocka-Maciejewska | Bacterial infection and semen quality[END_REF][START_REF] Bussen | Relationship of bacteriological characteristics to semen indices and its influence on fertilization and pregnancy rates after IVF[END_REF].

Berktas et al. have shown the negative impact of certain species such as Enterobacteriaceae on semen motility in vitro [START_REF] Berktas | Semen motility changes after coincubation with various uropathogenic microorganisms : an in vitro experimental study[END_REF]. Merino et al. found that bacterial infection can cause alteration in seminal characteristics, such as volume, motility and viability [START_REF] Merino | Bacterial infection and semen characteristics in infertile men[END_REF]. Nevertheless, the lack of association between bacteriospermia and abnormal semen parameters or the occurrence of adverse events in IVF was also reported [START_REF] Stovall | The role of aerobic and anaerobic semen cultures in asymptomatic couples undergoing in vitro fertilization: effects on fertilization and pregnancy rates[END_REF][START_REF] Krissi | Effect of contaminated preprocessed semen on fertilization rate and embryo quality in assisted reproductive techniques[END_REF][START_REF] Bussen | Relationship of bacteriological characteristics to semen indices and its influence on fertilization and pregnancy rates after IVF[END_REF]. However, bacterial examination of semen remains necessary before IVF to avoid contamination of culture media, especially for those who do not contain antibiotics, which is the case in our IVF center.

Regarding antibiotic resistance, whatever the species studied, antibiotic susceptibility testing revealed mostly a susceptible phenotype. Only ten multi-drug-resistant (MDR) bacteria isolated were recovered during the six-year period. Clinical data analysis revealed that most patients with MDR bacteria had spinal chord injury and consequent paraplegia, leading to permanent urinary catheter use with potential repeated antibiotic regimens.

From

  January 2003 1 st to December 2008 31 th , 14,119 samples from 7,723 men were included in this study, representing an average of 1,287 patients per year. The activity increased from 1,843 samples in 2003 to 2,664 in 2008, (+ 45 % -Figure 1). Although the ratio of requests per patient was stable (ranging from 1.74 to 1.86 between 2003 and 2008), there was a 35 % increase in the number of patients consulting and then included in the ART program (1,060 in 2003 to 1,435 patients in 2008), reflecting the increasing activity of the ART center during the study period.

Figure 1 :

 1 Figure 1 : Evolution of the activity (number of semen analysis versus number of medical consultations) between 2003 and 2008

Table 2 :

 2 Distribution of bacterial species isolated from semen cultures, at Nantes University hospital, between 2003 and 2008 340

			2003		2004		2005		2006		2007	2008
		N	%	N	%	N	%	N	%	N	%	N	%
	alpha-haemolytic Streptococci	288	34.3	187	32.6	126	28.9	144	27.1	115	21.9	33.7
	coagulase negative Staphylcocci	235	28	107	18.7	78	17.9	111	20.9	93	17.8	20.2
	Enterococcus	159	19	60	10.5	82	18.8	105	19.8	128	24.4	17.7
	Group B Streptococcus	16	1.9	27	4.7	24	5.5	20	3.8	22	4.2		3.3
	Staphylococcus aureus	1	0.1	5	0.9	4	0.9	8	1.5	3	0.6		0.8
	β-haemolytic Stretococcus	10	1.2	4	0.7	4	0.9	7	1.3	7	1.3		0.1
	Escherichia coli	56	6.7	101	17.6	63	14.4	75	14.1	92	17.6	14.5
	Proteus/Providencia/Morganella	13	1.5	22	3.8	15	3.5	16	3	20	3.8		5.9
	Other Enterobacteria	29	3.5	31	5.4	27	6.2	26	4.9	32	6.1		2.9
	Pseudomonas aeruginosa	1	0.1	2	0.4	4	0.9	2	0.4	1	0.2		0.5
	Corynebacterium seminale	17	2	21	3.7	3	0.7	2	0.4	1	0.2		0.4
	Others	14	1.7	6	1	6	1.4	15	2.8	10	1.9		0
	Total	839	100	573	100	436	100	531	100	524	100	100