Chicken Pineal Gland Research Articles

Fresh insights into the light‐induced pineal gland circadian rhythm transmission mechanism derived from mRNA and miRNA profiling

AbstractThe circadian clock significantly impacts animal health and productivity, with light playing a crucial role in regulating circadian rhythms. However, the mechanisms behind light‐induced circadian transmission remain unclear, particularly in light‐sensitive avian species. The pineal gland is a key component acting as the photosensitive master oscillator in the avian clock system. Using transcriptome sequencing and small RNA sequencing technologies, we identified circadian genes and miRNAs in the chick pineal gland under light–dark and sudden constant‐light conditions. We observed rhythmic oscillations in up to 1299 genes during the light–dark cycle, with 400 genes maintaining rhythms under constant light. Our findings highlight the light‐sensitive temporal organization in birds as the phase distribution of circadian genes in the pineal gland correlates with light exposure changes. A novel regulatory mechanism involving light, cyclic adenosine monophosphate, cyclic guanosine monophosphate, light‐sensitive miRNAs, such as gga‐miR‐34b‐5p, and light‐sensitive circadian genes, such as CRY2, was discovered to participate in the light input system of the chick pineal clock, through which light regulates the oscillators and outputs of the circadian clock system. Additionally, transcriptomic analysis, liquid chromatography–mass spectrometry, and Oil Red O staining revealed cyclic changes in lipid synthesis and metabolism throughout the circadian day, which may be a key mechanism through which the circadian clock influences pineal physiology. Our results enhance the understanding of light‐induced circadian transmission mechanisms and identify potential targets for optimizing the circadian clock through light.

Embryonic development of the chick pineal gland throughout the incubation periods.

Birds have a very different pineal gland structure morphologically and cytologically. The structure of the organ shows significant changes during the incubation periods. This study, which follows the embryological development of the pineal gland and makes histomorphometric measurements of the cellular elements that make up the gland parenchyma, is a current reference for studies in these areas. These brains were taken from 24 Babcock White Leghorn chick embryos on the 10th, 13th, 16th and 21st days of incubation. At 10th embryonic day, the pineal recess was in the structure of an elongated pineal canal. Solid rosette-shaped cell clusters were transforming into round vesicles with a small lumen. These vesicles had developed into larger, oval-shaped follicles with a well-defined central lumen. On 13th day, it was observed that the number and development of follicles increased considerably. The pineal gland showed a follicular-solid structure in 16th day embryos. While the mean follicle diameter was determined as 123.46 ± 13.28 μm on the 10th embryonic day, the highest value was measured as 187.62 ± 7.37 μm on the hatching day (p < 0.05). While the mean follicle area had the lowest value in the 10th day embryos, it was determined that this value gradually increased compared to the advancing embryonic days (p < 0.05). As conclusion, it is thought that this study provides new data to the literature about pineal gland development by monitoring the histological and histomorphometric developments of chick pineal gland in different incubation periods.

A negative regulatory element required for light-dependent pinopsin gene expression.

In vertebrates, a variety of light-stimulated genes are distributed in the retina, the pineal gland, and the suprachiasmatic nucleus, but a cis-element(s) responsible for the light-dependent transcriptional regulation is left unexplored. Focusing on the pinopsin gene, a light-stimulated gene in the chick pineal gland, we performed a transcriptional analysis in the primary culture of the chick pineal cells that were transiently transfected with a luciferase reporter gene fused with various lengths of the 5' upstream region of the pinopsin gene. Light-dependent enhancer activity was detectable in the construct with the upstream region between -1156 and +31. Introduction of mutations within the 18 bp sequence at positions -1103 to -1086 (TGGCACGTGGGGTTCCTC), including a CACGTG E-box sequence, elevated the transcriptional activity in the dark and thereby abrogated the light dependency, suggesting that the 18 bp sequence is essential for a reduction of the transcriptional activity in the dark. In an electrophoretic mobility-shift assay, we identified a pineal nuclear factor(s) capable of binding to the 18 bp element in a sequence-specific manner. When a 49 bp fragment (-1122 to -1074) including the 18 bp sequence was placed upstream of the simian virus 40 promoter, the transcriptional activity was dramatically suppressed regardless of light conditions in the chick pineal cells, and a more pronounced repression was observed in nonpineal/nonphotosensory LMH and NIH 3T3 cells. These results suggest that the 18 bp element in the pinopsin promoter constitutes the binding site of a ubiquitous factor that serves for the transcriptional repression that is required, although not sufficient, for the light-dependent expression of pinopsin gene in the chick pinealocytes.

Daily Profiles of Neuropeptides, Catecholamines, and Neurotransmitter Receptors in the Chicken Pineal Gland.

The avian pineal gland is one of three central biological clocks that contain all the components of a circadian system: a photoreceptive input, oscillator, and rhythmically secreted melatonin (MEL) as an effector. The biosynthesis of MEL is regulated by the neurotransmitters noradrenaline (NA), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP). The aim of the present study was to characterize the daily profile of neurotransmitters and their receptors in the pineal gland of male Hy-Line chickens housed under controlled light (12:12 light:dark) conditions. The pineal glands were isolated from 16-day-old birds every 2 h over a 24-h period, immediately after decapitation. The catecholamine content was measured using HPLC with electrochemical detection, whereas expression of VIP and PACAP was measured using quantitative real-time PCR (RT-qPCR) assays and Western blotting. Expression of the neurotransmitter receptors was also measured using RT-qPCR. We found daily changes in NA content, with elevated nocturnal levels, whereas the NA receptor was expressed in antiphase. Although we did not observe daily changes in VIP and PACAP protein levels, we found prominent diurnal changes in the expression of the Vip and Pacap genes. We also detected precursors of NA, 3,4-dihydroxy-L-phenylalanine (DOPA), and dopamine (DA) in the pineal glands, in addition to the DA metabolites. Our results provide the first evidence that the pineal gland itself may synthetize the neurotransmitters needed to regulate MEL biosynthesis.

Daily profiles of melatonin synthesis-related indoles in the pineal glands of young chickens (Gallus gallus domesticus L.)

The aim of the present study was to characterize the daily profiles of melatonin synthesis-related indoles in the pineal glands of male Hy-Line chickens hatched in the winter and reared under controlled light (L:D 12:12) conditions. The pineal glands were isolated from 16-day-old birds immediately after decapitation every 2h over a 24-h period. The indole contents were measured using HPLC with fluorescence detection. According to the obtained data, in chicken pineal glands tryptophan occurred at the highest level among the investigated compounds, showing rhythmical, daily changes with decreased levels during the scotophase. Rhythmical fluctuations were also observed for all studied tryptophan derivatives, except serotonin, 5-hydroxyindole acetic acid and 5-hydroxytryptophol. The highest levels of 5-hydroxytryptophan, N-acetylserotonin and melatonin were observed during the night, whereas the highest levels of 5-methoxytryptophol and 5-methoxyindoleacetic acid were observed during the day. The most interesting results concerned serotonin and the associated oxidative deamination products. The serotonin content did not exceed 5% of all investigated indoles, suggesting that there is no reserve pool of this indoleamine in the chicken pineal gland and that the activity of aromatic L-amino acid decarboxylase might be an important factor of melatonin synthesis regulation. In contrast to serotonin, 5-hydroxyindole acetic acid occurred at high levels in the investigated pineal gland, suggesting intensive oxidative deamination and explaining the observed low content of this indoleamine.

Role of monochromatic light on daily variation of clock gene expression in the pineal gland of chick

The avian pineal gland is a master clock that can receive external photic cues and translate them into output rhythms. To clarify whether a shift in light wavelength can influence the circadian expression in chick pineal gland, a total of 240 Arbor Acre male broilers were exposed to white light (WL), red light (RL), green light (GL) or blue light (BL). After 2weeks light illumination, circadian expressions of seven core clock genes in pineal gland and the level of melatonin in plasma were examined. The results showed after illumination with monochromatic light, 24h profiles of all clock gene mRNAs retained circadian oscillation, except that RL tended to disrupt the rhythm of cCry2. Compared to WL, BL advanced the acrophases of the negative elements (cCry1, cCry2, cPer2 and cPer3) by 0.1–1.5h and delayed those of positive elements (cClock, cBmal1 and cBmal2) by 0.2–0.8h. And, RL advanced all clock genes except cClock and cPer2 by 0.3–2.1h, while GL delayed all clock genes by 0.5–1.5h except cBmal2. Meanwhile, GL increased the amplitude and mesor of positive and reduced both parameters of negative clock genes, but RL showed the opposite pattern. Although the acrophase of plasma melatonin was advanced by both GL and RL, the melatonin level was significantly increased in GL and decreased in RL. This tendency was consistent with the variations in the positive clock gene mRNA levels under monochromatic light and contrasted with those of negative clock genes. Therefore, we speculate that GL may enhance positive clock genes expression, leading to melatonin synthesis, whereas RL may enhance negative genes expression, suppressing melatonin synthesis.

Avian biological clock – Immune system relationship

Biological rhythms in birds are driven by the master clock, which includes the suprachiasmatic nucleus, the pineal gland and the retina. Light/dark cycles are the cues that synchronize the rhythmic changes in physiological processes, including immunity. This review summarizes our investigations on the bidirectional relationships between the chicken pineal gland and the immune system. We demonstrated that, in the chicken, the main pineal hormone, melatonin, regulates innate immunity, maintains the rhythmicity of immune reactions and is involved in the seasonal changes in immunity. Using thioglycollate-induced peritonitis as a model, we showed that the activated immune system regulates the pineal gland by inhibition of melatonin production at the level of the key enzyme in its biosynthetic pathway, arylalkylamine-N-acetyltransferase (AANAT). Interleukin 6 and interleukin 18 seem to be the immune mediators influencing the pineal gland, directly inhibiting Aanat gene transcription and modulating expression of the clock genes Bmal1 and Per3, which in turn regulate Aanat.

Light-dependent activation of G proteins by two isoforms of chicken melanopsins.

In the chicken pineal gland, light stimuli trigger signaling pathways mediated by two different subtypes, Gt and G11. These G proteins may be activated by any of the three major pineal opsins, pinopsin, OPN4-1 and OPN4-2, but biochemical evidence for the coupling has been missing except for functional coupling between pinopsin and Gt. Here we investigated the relative expression levels and the functional difference among the three pineal opsins. In the chicken pineal gland, the pinopsin mRNA level was significantly more abundant than the others, of which the OPN4-2 mRNA level was higher than that of OPN4-1. In G protein activation assays, Gt was strongly activated by pinopsin in a light-dependent manner, being consistent with previous studies, and weakly activated by OPN4-2. Unexpectedly, illuminated OPN4-2 more efficiently activated G protein(s) that was endogenously expressed in HEK293T cells in culture. On the other hand, Gq, the closest analogue of G11, was activated only by OPN4-1 although the activity was relatively weak under these conditions. These results suggest that OPN4-1 and OPN4-2 couple with Gq and Gt, respectively. Two melanopsins, OPN4-1 and OPN4-2, appear to have acquired mutually different functions through the evolution.

Clock-Controlled Regulation of the Acute Effects of Norepinephrine on Chick Pineal Melatonin Rhythms.

The chicken pineal gland synthesizes and releases melatonin rhythmically in light/dark (LD) cycles, with high melatonin levels during the dark phase, and in constant darkness (DD) for several cycles before it gradually damps to arrhythmicity in DD. Daily administration of norepinephrine (NE) in vivo and in vitro prevents the damping and restores the melatonin rhythm. To investigate the role of the circadian clock on melatonin rhythm damping and of its restoration by NE, the effects of NE administration at different phases of the melatonin cycle revealed a robust rhythm in NE sensitivity in which NE efficacy in increasing melatonin amplitude peaked in late subjective night and early subjective day, suggesting a clock underlying NE sensitivity. However, NE itself had no effect on circadian phase or period of the melatonin rhythms. Transcriptional analyses indicated that even though the rhythm of melatonin output damped to arrhythmicity, messenger RNA (mRNA) encoding clock genes gper2, gper3, gBmal1, gclock, gcry1, and gcry2; enzymes associated with melatonin biosynthesis; and enzymes involved in cyclic nucleotide signaling remained robustly rhythmic. Of these, only gADCY1 (adenylate cyclase 1) and gPDE4D (cAMP-specific 3',5'-cyclic phosphodiesterase 4D) were affected by NE administration at the mRNA levels, and only ADCY1 was affected at the protein level. The data strongly suggest that damping of the melatonin rhythm in the chick pineal gland occurs at the posttranscriptional level and that a major role of the clock is to regulate pinealocytes' sensitivity to neuronal input from the brain.

Seasonality of inflammation in the chicken: clock vs. melatonin control over the pro-inflammatory cytokine gene transcription

Seasonality of immunity in birds has been confirmed, but underlying mechanisms are still not fully explained. Present study was undertaken to verify hypothesis on the role of melatonin in the season-related development of inflammation in chickens. Chickens hatched in summer and winter were kept for 12 days in the season-corresponding LD conditions 16:8 or 8:16, respectively, in continuous illumination (LL) and in LL with melatonin supplementation during subjective night. Peritonitis evoked at two time points (subjective “day” or “night”) by thioglycollate injection stimulated serum lysozyme concentration regardless of lighting conditions and melatonin availability. Pro-inflammatory cytokine IL-6 and IL-18 gene transcription in the spleen exhibited day–night differences, was augmented in inflamed birds in a season-related way, and partially modified by melatonin. Transcription of the core clock genes in the pineal gland was affected by peritonitis confirming a cross-talk between the inflammatory mediators and master clock present in the chicken pineal gland.

The embryonic pineal gland of the chicken as a model for experimental jet lag

The circadian clock in the chicken pineal model develops before hatching, at around the 17th embryonic day (ED17). By this stage, it runs in synchrony with environmental cues. To address if phase resetting mechanisms are comparable to those of post-hatched chicken, we investigated ED19 stage chicken embryos under 12h light:12h dark (LD), under constant darkness (DD), or under acute 4h phase delay of the LD condition (LD+4). The 24h mRNA-expression patterns of clock gene clock and of clock controlled genes Aanat and hiomt were analyzed with qRT-PCR. Under DD the rhythm of Aanat did not change significantly, however the 24h pattern of hiomt was altered. Clock shows a delayed response to DD with a phase-shift in its rhythm. After the first cycle under LD+4 conditions, the 24h patterns of Aa-nat and hiomt mRNA-s were phase delayed. Clock showed both acute and delayed changes in response to LD+4. These results show that the embryonic chicken pineal gland has a fully functioning clock mechanism, and that it is a good model for phase-change experiments. In addition it demonstrates that only one cycle of altered light schedule is sufficient to trigger changes within the molecular clock mechanisms of the chicken embryonic pineal model.

Season-dependent Postembryonic Maturation of the Diurnal Rhythm of Melatonin Biosynthesis in the Chicken Pineal Gland

Previously, we have demonstrated that the timing of the nocturnal peak of activity of the pineal arylalkylamine-N-acetyltransferase – a key enzyme in the melatonin biosynthesis pathway – in 3-wk-old chickens kept from the day of hatch under controlled laboratory conditions (L:D 12:12) varies depending on the season of hatch (summer vs. winter). The present study was undertaken to answer the following questions: (1) are season-related differences seen in the level of transcription of genes encoding enzymes of the melatonin biosynthesis pathway? (2) Does the pineal content of the main precursor (serotonin) and the final product (melatonin) exhibit age- and season-related changes? (3) At which step in postembryonic development are these season-related variations in pineal gland function most pronounced? Male Hy-line chickens hatched in the summer or winter, from eggs laid by hens held on L:D 16:8, were kept from the day of hatch under L:D 12:12 conditions. At the age of 2, 9, or 16 d, chickens were sacrificed every 2 h over a 24-h period and their pineal glands, isolated under dim red light, were processed for the measurement of (i) the level of Aanat and Asmt (acetylserotonin O-methyltransferase) mRNAs encoding the two last enzymes involved in melatonin biosynthesis, (ii) the activity of these enzymes, and (iii) the pineal content of serotonin and melatonin. Circadian rhythmicity of all the measured parameters was evaluated by the cosinor method. The levels of Aanat mRNA, AANAT enzymatic activity, and the pineal melatonin content changed during postembryonic development in a manner that was dependent on the season of hatch. Furthermore, the diurnal profile of Asmt mRNA was elevated during the light phase. In “winter” birds, the pattern and amplitude of the diurnal rhythm of accumulation of this transcript did not change with age, while in “summer” birds it increased in an age-related way. In contrast, the enzymatic activity of hydroxyindole-O-methyltransferase (HIOMT; encoded by the Asmt gene) did not change rhythmically, although it increased with age in a season-related way. In “winter” chickens, the pineal serotonin content was low, regardless of age, and did not change rhythmically, whereas in “summer” individuals the serotonin rhythm was already well established by day 2, with the amplitude increasing with age. These results confirm the existence of a “seasonal memory” operating within the chicken pineal gland, although the mechanism(s) underlying this phenomenon have yet to be characterized.

Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways

The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by >2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF)1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7α-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7α-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-day-dependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland.

Clock mRNA expression patterns in the chick pineal gland under experimental jet lag

Clock mRNA expression patterns in the chick pineal gland under experimental jet lag

Season-Related Differences in the Biosynthetic Activity of the Neonatal Chicken Pineal Gland

Influence of the season of hatch on the functional characteristics of the pineal gland was examined in neonatal Hi-Line male chickens. The pineal glands from 2-day-old birds hatched in and winter, and kept from the day of hatch in artificial lighting conditions (12L:12D), were isolated under dim red light in the middle of the day or night. The pineal glands were analyzed to characterize their melatonin biosynthetic activity: (1) expression of the Arylalkylamine- N-acetyltransferase and Hydroxyindole-O-methyltransferase genes, encoding the final two enzymes of the melatonin biosynthesis pathway; (2) the activity of AA-NAT and HIOMT; and (3) the content of the main substrates of this pathway, tryptophan (TRY) and serotonin (5-HT). Daily changes in pineal AA-NAT activity were observed in chickens hatched in both seasons, with a more pronounced nocturnal increase in summer. In contrast, the level of Aa-nat gene expression, although exhibited the same nocturnal/diurnal pattern in both seasons, was much lower in the summer. The activity of HIOMT was season- and daytime-independent. In winter chickens the pineal content of 5-HT was low and stable, while in summer birds it was correlated with levels of AA-NAT activity and Aa-nat gene expression. TRY content was very high and exhibited neither daily nor seasonal changes. The pineal gland of newly hatched chickens kept in controlled 12L:12D conditions exhibits daily variations in melatonin biosynthetic activity influenced by the season. This suggests a maternal effect on the perinatal/postnatal development of the circadian clock residing in the chicken pineal gland.

Expression of Cry2 in the Chicken Pineal Gland

Pineal expression of Cry2 mRNA has been examined in chickens under normal (LD) and reversed (DL) light-dark conditions. In vivo the peak of Cry2 mRNA content at late subjective day under LD diminished after switching to a DL schedule. In vitro, Cry2 mRNA levels showed a steady decrease during light exposure at subjective night. Our data show that light-sensitive clock components in the pinealocytes may be involved in the repression of Cry2 transcription at night, which may contribute to resetting the phase of the clock within 24 h.

The in Vitro and in Ovo Effects of Environmental Illumination and Temperature on the Melatonin Secretion from the Embryonic Chicken Pineal Gland

Pineal glands of chicken embryos were placed into a perifusion system for 4 days. The pineal glands were illuminated or exposed to elevated temperature for 8 or 12 h during the in vitro experiment and/or in ovo. Both daily illumination and repeated elevations of environmental temperature transitionally inhibited melatonin release before, and controlled the phase of melatonin rhythm after, the 17th day of embryonic life (E17). In addition, the in ovo rhythmic illumination applied before E13 advances the development of the circadian hormone synthesis.

Circadian Expression of Clock Genes Clock and Cry1 in the Embryonic Chicken Pineal Gland

Clock and Cry1 expression were examined in the pineal gland of chicken embryos incubated under constant darkness from embryonic day (ED) 0. From ED13, Clock and Cry1 mRNA levels showed episodic alterations. After ED17, circadian pattern of clock gene expression was seen both in vivo and in vitro. Our results support the idea that rhythmic environmental factors are not necessary for the generation of circadian patterns of clock gene expression during development.

Distribution and Cytoarchitecture of Sympathetic Neurons Innervating the Pineal Gland in Chick: A CTB‐HRP Study

The neurons in bilateral superior cervical ganglia (SCG) innervating the chick pineal gland were labelled by using the technique of retrograde axonal labelling with cholera toxin B subunit linked to horseradish peroxidase (CTB-HRP). To our results, perikarya of these sympathetic neurons distributed from rostral to caudal in the SCG, and mainly localized in the opposite side of the paravertebral trunk. The fibres of these neurons were collected by the cephalic carotid nerve. According to the sizes of somal area and dendritic field, these sympathetic neurons projecting to the pineal gland were classified into four major groups: group I cells (52.4%) with a small somal area (303.5 microm(2) on average) and narrow dendritic field (3767.8 microm(2) on average), group II cells (39.0%) with a middle-sized somal area (473.3 microm(2)) and middle-sized dendritic field (7522.2 microm(2)), group III cells (6.4%) with a middle-sized somal area (473.4 microm(2)) and wide dendritic field (13 104.4 microm(2)), and group IV cells (2.2%) with a large somal area (940.7 microm(2)) and wide dendritic field (14 553.2 microm(2)). Of these pineal projecting neurons, most took on a lesser dendritic field. The neurons with small or middle-sized dendritic field from group I and II were about 91.4% of the total neurons labelled with CTB-HRP, and the neurons with wide dendritic field from group III and IV were less with 8.6%.

Circadian genomics of the chick pineal gland in vitro

BackgroundChick pinealocytes exhibit all the characteristics of a complete circadian system, comprising photoreceptive inputs, molecular clockworks and an easily measured rhythmic output, melatonin biosynthesis. These properties make the in vitro pineal a particularly useful model for exploring circadian control of gene transcription in a pacemaker tissue, as well as regulation of the transcriptome by primary inputs to the clock (both photic and noradrenergic).ResultsWe used microarray analysis to investigate the expression of approximately 8000 genes within cultured pinealocytes subjected to both LD and DD. We report that a reduced subset of genes was rhythmically expressed in vitro compared to those previously published in vivo, and that gene expression rhythms were lower in amplitude, although the functional distribution of the rhythmic transcriptome was largely similar. We also investigated the effects of 6-hour pulses of light or of norepinephrine on gene expression in free-running cultures during both subjective day and night. As expected, both light and norepinephrine inhibited melatonin production; however, the two treatments differentially enhanced or suppressed specific sets of genes in a fashion that was dependent upon time of day.ConclusionOur combined approach of utilizing a temporal, photic and pharmacological microarray experiment allowed us to identify novel genes linking clock input to clock function within the pineal. We identified approximately 30 rhythmic, light-responsive, NE-insensitive genes with no previously known clock function, which may play a role in circadian regulation of the pineal. These are candidates for future functional genomics experiments to elucidate their potential role in circadian physiology. Further, we hypothesize that the pineal circadian transcriptome is reduced but functionally conserved in vitro, and supports an endogenous role for the pineal in regulating local rhythms in metabolism, immune function, and other conserved pathways.