Gonadotropic regulation of circadian clockwork in rat granulosa cells

The circadian clock is responsible for the generation of circadian rhythms in hormonal secretion and metabolism. These peripheral clocks could be reset by various cues in order to adapt to environmental variations. The ovary can be characterized as having highly dynamic physiology regulated by gonadotropins. Here, we aimed to address the status of circadian clock in the ovary, and to explore how gonadotropins could regulate clockwork in granulosa cells (GCs). To this end, we mainly utilized the immunohistochemistry, RT-PCR, and real-time monitoring of gene expression methods. PER1 protein was constantly abundant across the daily cycle in the GCs of immature ovaries. In contrast, PER1 protein level was obviously cyclic through the circadian cycle in the luteal cells of pubertal ovaries. In addition, both FSH and LH induced Per1 expression in cultured immature and mature GCs, respectively. The promoter analysis revealed that the Per1 expression was mediated by the cAMP response element binding protein. In cultured transgenic GCs, both FSH and LH also induced the circadian oscillation of Per2. However, the Per2 oscillation promoted by FSH quickly dampened within only one cycle, whereas the Per2 oscillation promoted by LH was persistently maintained. Collectively, these findings strongly suggest that both FSH and LH play an important role in regulating circadian clock in the ovary; however, they might exert differential actions on the clockwork in vivo due to each specific role within ovarian physiology.     

The molecular clockwork of a protein-based circadian oscillator

The circadian clock of the cyanobacterium Synechococcuselongatus PCC 7942 is governed by a core oscillator consisting of the proteins KaiA, KaiB, and KaiC. Remarkably, circadian oscillations in the phosphorylation state of KaiC can be reconstituted in a test tube by mixing the three Kai proteins and adenosine triphosphate. The in vitro oscillator provides a well-defined system in which experiments can be combined with mathematical analysis to understand the mechanism of a highly robust biological oscillator. In this Review, we summarize the biochemistry of the Kai proteins and examine models that have been proposed to explain how oscillations emerge from the properties of the oscillator’s constituents.     

The photoperiodic entrainment and induction of the circadian clock regulating seasonal responses in the migratory blackheaded bunting (Emberiza melanocephala)

The properties of the circadian photoperiodic oscillator have been investigated in detail only in the Japanese quail. While the study of the quail is clearly very important, one cannot simply assume that other species, especially passerines that seem to have a different circadian organization than quail, function the same way. The current set of experiments was conducted to understand the entrainment and photoinduction of the circadian photoperiodic oscillator in a passerine species, the blackheaded bunting (Emberiza melanocephala). The experimental paradigm used skeleton photoperiods with two light periods, the first called the “entraining light pulse” (E-pulse) and the second called the “inducing light pulse” (I-pulse). Three experiments were performed on photosensitive male birds (N=6-8/group). Experiment 1 investigated the effects of the temporal relationship between E- and I-pulses on photoperiodic induction. Buntings entrained to 8h:16h L:D for 4 wk were released into constant dim light (LL_dim, ∼1 lux). Beginning on subjective day 8, they received for 8 wk, E- and I-pulses only at alternate cycles. While I-pulse was 1 h and always began at zt 11.5, E-pulse varied in duration and timing (the 1h E-pulse beginning either at zt 0, zt 5, or zt 9, the 4h one beginning at zt 0 or zt 6, and the 10h one at zt 0; zeitgeber time 0=time of lights-on under 8h:16h L:D prior to release into LL_dim). A photoperiodic response was induced only when the E-pulse began at zt 0, and thus the beginning of E- and I-pulses were separated by 11.5 h. Experiment 2 determined whether the duration of the E-pulse influences the position of the photoinducible phase (φi) of the circadian photoperiodic oscillator. Birds were entrained to 1h:23h L:D or 10h:14h L:D for 2 wk, and then exposed to 1h I-pulse at zt 11.5, zt 15, or zt 18.5 for another 8 wk. Photoperiodic induction occurred at all 3 zts in birds entrained to 10 h but only at zt 11.5 in birds entrained to 1 h, which infers the circadian rhythm of photoinducibility (CRP) in buntings was re-entrained when I-pulse fell at zt 15 and after. The last experiment examined the possibility of the re-entrainment of the CRP to light pulses falling at zt 15 and after. Birds received 1h I-pulse for 8 wk at zt 15 following 2 wk of 2.5h:21.5h L:D or 3.5h:20.5h L:D, or at zt 21.5 or zt 22.5 following 2 wk of 10h:14h LD. Photoperiodic induction was consistent with the hypothesis of the re-entrainment of the CRP under these light-dark cycles. The I-pulse appeared to be interpreted as a “new dawn”, and so the photoperiodic induction was determined by the coincidence of φi with the E-pulse. These results suggest a phase-dependent action of light on the circadian oscillator regulating photoperiodic responses in the blackheaded bunting. This could be a useful strategy for a photoperiodic species to regulate its seasonal responses in nature.     

A circadian system is involved in photoperiodic entrainment of the circannual rhythm of Anthrenus verbasci

In the circannual pupation rhythm of the varied carpet beetle, Anthrenus verbasci, entrainment to annual cycles is achieved by phase resetting of the circannual oscillator in response to photoperiodic changes. In order to examine whether a circadian system is involved in expression of the periodic pattern and phase resetting of the circannual rhythm as photoperiodic responses, we exposed larvae to light-dark cycles with a short photophase followed by a variable scotophase (the Nanda-Hamner protocol). When the cycle length (T) was a multiple of 24 h, i.e., 24, 48, or 72 h, short-day effects were clearer than when T was far from a multiple of 24 h, i.e., 36 or 60 h. Exposure to light-dark cycles of T = 36 h had effects similar to exposure to long-day cycles of T = 24 h. The magnitude of phase shifts depended on the duration and the phase of exposure to the cycles of T = 36 or 60 h. It was therefore concluded that a circadian system is involved in photoperiodic time measurement for phase resetting of the circannual oscillator of A. verbasci.     

The hormonal and circadian basis for insect photoperiodic timing

Daylength perception in temperate zones is a critical feature of insect life histories, and leads to developmental changes for resisting unfavourable seasons. The role of the neuroendocrine axis in the photoperiodic response of insects is discussed in relation to the key organs and molecules that are involved. We also discuss the controversial issue of the possible involvement of the circadian clock in photoperiodicity. Drosophila melanogaster has a shallow photoperiodic response that leads to reproductive arrest in adults, yet the unrivalled molecular genetic toolkit available for this model insect should allow the systematic molecular and neurobiological dissection of this complex phenotype.     

Period responses to Zeitgeber signals stabilize circadian clocks during entrainment

Circadian clocks with characteristic period (τ) can be entrained to light/dark (LD) cycles by means of (i) phase shifts which are due to D/L “dawn” and/or L/D “dusk” transitions, (ii) period changes associated with long-term light exposure, or (iii) by combinations of the above possibilities. Based on stability analysis of a model circadian clock it was predicted that nocturnal burrowing mammals would benefit less from period responses than their diurnal counterparts. The model further predicted that maximal stability of circadian clock is reached when the clock slightly changes both its phase and period in response to light stimuli. Analyses of empirical phase response curve (PRC) and period response curve (τRC) of some diurnal and nocturnal mammals revealed that PRCs of both diurnal and nocturnal mammals have similar waveform while τRCs of nocturnal mammals are of smaller amplitude than those of diurnal mammals. The shape of the τRC also changes with age and with increasing strength of light stimuli. During erratic fluctuations in light intensity under different weather conditions, the stability of phase of entrainment of circadian clocks appears to be achieved by an interplay between phase and period responses and the strength of light stimuli.     

Dose-dependent entrainment of rat circadian rhythms by daily injection of melatonin

Previous work in our laboratory has shown that daily injection of large doses of the pineal hormone melatonin entrains the free-running locomotor rhythms of rats held in constant darkness and synchronizes the disrupted patterns of rats maintained in constant bright light. The present experiments determined the dose-response characteristics of entrainment to daily melatonin injections and made preliminary biochemical estimates of blood melatonin levels and half-lives after two critical doses of the hormone. The data indicated that the median effective dose for melatonin as an entraining agent in free-running rats was 5.45 +/- 1.33 micrograms/kg, considerably lower than doses previously employed and lower than doses employed in reproductive and metabolic studies in rats and hamsters. The data further indicated that the response to melatonin was quantal; rats either entrained to melatonin or they did not. No "partial entrainment" was evident, nor were there differences in phase angle, activity, or period among all effective doses. Biochemical estimates of blood melatonin after either 1 mg/kg or 1 microgram/kg of melatonin indicated that all effective doses resulted in supraphysiological levels of blood melatonin, although doses of 1 microgram/kg resulted in blood levels that were within one order of magnitude of normal nighttime values. Together, the data suggest that the rat circadian system is sensitive to the pineal hormone melatonin at or below doses required to effect rodent reproduction. Whether this sensitivity reflects a role for the pineal gland in rat circadian organization, however, still remains to be determined.     

Range of entrainment of rat circadian rhythms to sinusoidal light-intensity cycles

The range of entrainment of the circadian behavioral rhythm was compared between two groups of Sprague-Dawley rats (each n = 10) exposed to daily cycles of rectangular light-dark alternation (LD) and sinusoidal fluctuations of light intensity (SINE), respectively. The maximum illuminance (20 lx), the minimum illuminance (0.01 lx), and the total amount of light exposure per cycle were the same under the two lighting conditions. The periods (Ts) of both lighting cycles were lengthened stepwise from 24 through 25, 26, 26.5, 27, 27. 5, and 28 h to 28.5 h in experiment 1 and were shortened stepwise from 24 through 23.5, 23, and 22.5 h to 22 h in experiment 2. Each T cycle lasted for 30 cycles. In experiment 1, 60% of rats under the LD condition entrained up to T = 28.5 h, whereas 50% of rats under the SINE condition entrained up to T = 28.5 h. In experiment 2, no animal under the LD condition entrained to T < 23.5 h, whereas 40% of rats under the SINE condition entrained down to T = 23 h and 20% of rats remained to entrain down to T = 22 h cycles. The phase angle of entrainment was systematically changed, depending on T under both conditions. These results suggest that the lower limit of entrainment is expanded under the SINE condition compared with the LD condition.     

Dynamics of discrete entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity during transient cycles

To elucidate entrainment of a pacemaker controlling the N-acetyltransferase (NAT) rhythm in the rat pineal gland, we studied the phase response curves (PRCs) of this rhythm. We exposed 50- to 60-day-old male Wistar rats maintained in a light-dark cycle (LD 12:12) to a 1-min light pulse at different times before midnight or at various times throughout the whole night. We then released them into constant darkness and studied the morning NAT decline during the night when rats were pulsed before midnight, as well as the evening NAT rise and the morning decline after 4 days following the pulses. The PRC for the first NAT decline and the PRCs for the NAT rise and decline after 4 days were compared with published transient PRCs (Illnerová and Van?cek, 1982b), in order to obtain a complete picture of the dynamics of the NAT rhythm entrainment during the transient cycles. Phase delays in the NAT rise due to a pulse before midnight were complete (i.e., identical to those of day 4) on day 1. Phase delays in the NAT decline were almost complete on day 1, while incomplete phase delays were observed on day 0. Phase advances in the NAT rise and decline due to a pulse past midnight had different dynamics: Advances in the decline were complete on day 1, while advances in the rise were absent on day 1 and much smaller than in the decline on day 4. The results are discussed in terms of a two-component (E-M) pacemaker controlling the NAT rhythm. The NAT rise may reflect the phase of the E-component, while the decline reflects the M-component. Phase delays of the E-component are accomplished within one cycle, and so are phase advances of the M-component. However, although delays of E already result in delays of M one cycle after the pulse, it takes several transient cycles before advances of M begin to induce advances of E.     

The effects of chronic marijuana use on circadian entrainment

Animal literature suggests a connection between marijuana use and altered circadian rhythms. However, the effect has not yet been demonstrated in humans. The present study examined the effect of chronic marijuana use on human circadian function. Participants consisted of current users who reported smoking marijuana daily for at least a year and non-marijuana user controls. Participants took a neurocognitive assessment, wore actigraphs and maintained sleep diaries for three weeks. While no significant cognitive changes were found between groups, data revealed that chronic marijuana use may act as an additional zeitgeber and lead to increased entrainment in human users.     

The de-ubiquitinylating enzyme, USP2, is associated with the circadian clockwork and regulates its sensitivity to light

We have identified a novel component of the circadian clock that regulates its sensitivity to light at the evening light to dark transition. USP2 (Ubiquitin Specific Protease 2), which de-ubiquitinylates and stabilizes target proteins, is rhythmically expressed in multiple tissues including the SCN. We have developed a knockout model of USP2 and found that exposure to low irradiance light at ZT12 increases phase delays of USP2(-/-) mice compared to wildtype. We additionally show that USP2b is in a complex with several clock components and regulates the stability and turnover of BMAL1, which in turn alters the expression of several CLOCK/BMAL1 controlled genes. Rhythmic expression of USP2 in the SCN and other tissues offers a new level of control of the clock machinery through de-ubiqutinylation and suggests a role for USP2 during circadian adaptation to environmental day length changes.     

Extraocular photoreception and circadian entrainment in nonmammalian vertebrates

In mammals both the regulation of circadian rhythms and photoperiodic responses depend exclusively upon photic information provided by the lateral eyes; however, nonmammalian vertebrates can also rely on multiple extraocular photoreceptors to perform the same tasks. Extraocular photoreceptors include deep brain photoreceptors located in several distinct brain sites and the pineal complex, involving intracranial (pineal and parapineal) and extracranial (frontal organ and parietal eye) components. This review updates the research field of the most recent acquisitions concerning the roles of extraocular photoreceptors on circadian physiology and behavior, particularly photic entrainment and sun compass orientation.     

The role of Clock in the plasticity of circadian entrainment

The mammalian circadian clock lying in suprachiasmatic nucleus (SCN) is synchronized to about 24 h by the environmental light-dark cycle (LD). The circadian clock exhibits limits of entrainment above and below 24 h, beyond which it will not entrain. Little is known about the mechanisms regulating the limits of entrainment. In this study, we show that wild-type mice entrain to only an LD 24 h cycle, whereas Clock mutant mice can entrain to an LD 24, 28, and 32 h except for LD 20 h and LD 36 h cycle. Under an LD 28 h cycle, Clock mutant mice showed a clear rhythm in Per2 mRNA expression in the SCN and behavior. Light response was also increased. This is the first report to show that the Clock mutation makes it possible to adapt the circadian oscillator to a long period cycle and indicates that the clock gene may have an important role for the limits of entrainment of the SCN to LD cycle.     

The Paramecium circadian behavioral rhythm: light phase response curves and entrainment

The population of a ciliate protozoan, Paramecium multimicronucleatum, exhibits a circadian rhythm as measured by the number of the cells traversing an observation point ("traverse frequency," or TF). The present study examined phase shifting of the TF rhythm by administering 2-hr light pulses at different phases of the circadian cycle to cultures free-running in constant darkness (DD). The results were summarized in a phase response curve (PRC), categorized as Type 1. This PRC indicated a relatively narrow phase zone insensitive to the light pulse ("dead zone"). Entrainment of the rhythm to light pulses repeated at 24-hr intervals was also examined, and it was found that the rhythm gradually reached a steady state, following several transient cycles, with the pulses falling at a phase corresponding to the narrow dead zone. Such a steady-state rhythm, with a minimum at approximately 3 hr after the pulse and a maximum at approximately 12 hr after the pulse, was mathematically simulated by superimposing a response function to the pulse on a sinusoidal function representative of the free-running rhythm in DD.     

Integration of light signaling with photoperiodic flowering and circadian rhythm

PLANT DE-ETIOLATION IS TRIGGERED BY LIGHT SIGNALS Light is arguably the most important resource for plants, and plants have evolved an array of photosensory pig- ments enabling them to develop optimally in a broad range of ambient light conditions. The ph…     

Influence of the mode of daily melatonin administration on entrainment of rat circadian rhythms.

In previous entrainment studies, melatonin (MEL) was administered by handling the animal, but because such handling may act as a confounding variable, the results from these studies are equivocal. The authors used MEL administration techniques that do not involve direct handling of the animal. Long Evans rats were used, and core body temperature (CBT) and wheel-running activity were recorded. One group of rats received a daily 1-h time-fixed infusion of MEL or the vehicle via a subcutaneous catheter. Animals in a second group had timed access to drinking water involving daily presence of drinking water containing MEL or the vehicle for 2 h at a fixed time of the day. Following entrainment to LD 12:12, both groups were transferred to constant darkness to free-run under vehicle administration. MEL was then administered, and entrainment occurred when activity onset coincided with MEL onset. Under both regimens, entrainment of wheel-running and CBT rhythms showed equal phase-relation to the onset of MEL administration, and free-running reoccurred when MEL was withdrawn. The authors concluded that MEL administration via drinking water and via infusion represent efficient ways to synchronize free-running rhythms in rats.