- Email: [email protected]
Clock Genes Expression in Peripheral Leukocytes and Plasma Melatonin Daily Rhythm in Horses
Journal Pre-proof Clock genes expression in peripheral leucokytes and plasma melatonin daily rhythm in horses. Claudia Giannetto, Francesco Fazio, Daniela Alberghina, Elisabetta Giudice, Giuseppe Piccione PII:
S0737-0806(19)30605-7
DOI:
https://doi.org/10.1016/j.jevs.2019.102856
Reference:
YJEVS 102856
To appear in:
Journal of Equine Veterinary Science
Received Date: 4 July 2019 Revised Date:
1 August 2019
Accepted Date: 8 November 2019
Please cite this article as: Giannetto C, Fazio F, Alberghina D, Giudice E, Piccione G, Clock genes expression in peripheral leucokytes and plasma melatonin daily rhythm in horses., Journal of Equine Veterinary Science (2019), doi: https://doi.org/10.1016/j.jevs.2019.102856. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Clock genes expression in peripheral leucokytes and plasma melatonin daily rhythm in horses.
Claudia Giannetto, Francesco Fazio, Daniela Alberghina, Elisabetta Giudice, Giuseppe Piccione*
Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, 98168, Messina, Italy.
*Corresponding autor: Giuseppe Piccione, Department of Veterinary Sciences, University of Messina,
Polo
Universitario
[email protected]
dell’Annunziata,
98168,
Messina,
Italy.
Email
address:
Abstract In mammals, behavioral and physiological processes display 24 hour rhythms that are regulated by the circadian system. In the present study we investigated clock gene expression in peripheral leukocytes in horses. At this purpose, ten Italian Saddle gelding horses (9-11 years old; 475±28 Kg) were housed in individual boxes under natural photoperiod and natural environmental temperature. Blood samples were collected at 4 hour intervals over a 48 hour period. The day before the start of sampling, left jugular furrow of each horse was cannulated for the blood sample collection performed in heparinized tubes, for the assessment of melatonin concentration by means of radioimmunoassay; and into PAX gene Blood RNA Tube for the assessment of clock genes by realtime RT-quantitative polymerase chain reaction (RTqPCR). How well establish, melatonin showed a daily rhythm with nocturnal acrophase (day 1-21:30; day 2-21:40). All genes tested (Bmal1, Cry 1, Per 1, Per 2 and Per 3) except Clock, showed daily rhythmicity of their expression in peripheral blood. Oscillations of Bmal1 and Per 2 were correlated with the oscillation of melatonin, which acrophase anticipated the acrophase of Bmal1 (day 1-01:29; day 2-01:00) and Per 2 (day 1-01:00; day 2-00:32) of about 3 hours. Our results support the presence of a cyclic transcription of clock genes in peripheral leucocytes in horses.
Keywords: Melatonin, clock genes, leucocytes, horses
1.0 Introduction Circadian rhythms throughout the body are coordinate by suprachiasmatic nucleus (SCN) of the hypothalamus. It processes external cues, such as light and temperature, and internal cues to set circadian rhythm [1]. All this information is communicate to the rest of the body by neural and hormonal signals [2]. The main mediator used by the central master clock is melatonin. It is an internal synchronizer able to act on peripheral oscillator regulating their phase and period, mainly by controlling the transcription/translation circadian cycle of the peripheral clock genes [3]. Melatonin is involved in the organization of the circadian system and plays a central role in the control of photoperiodic responses and can be considered as an endogenous synchronizer [4-5]. Daily oscillation in blood levels of melatonin has been documented in various species of birds [6-8] and mammals [9-12], including humans [13-15]. Microdialysis studies have confirmed that secretion from the pineal is indeed rhythmic and responsible for the oscillation in blood levels [16]. These robust and predictable rhythms in circulating melatonin can be considered as an endogenous synchronizer for the expression of numerous physiological processes in photoperiodic species, including reproduction, feed intake, adiposity and body temperature [17-18]. Eight genes essential for mammalian circadian clock function have been identified: the homologues of the Drosophila gene period (Per 1, Per 2 and Per 3), cryptochrome isoforms (Cry 1 and Cry 2), PAS helix-loophelix transcription factors (Clock and Bmal1), and Casein kinase 1 ε (CK1 ε). The products of these genes constitute a model of a molecular oscillator, based on interconnected positive and negative transcriptional-translational feedback loops [19]. In peripheral blood circadian rhythmicity is manifested in terms of clock gene expression [20-21]. Clock genes in blood cells have been studied in humans [21], rats [22], dogs [23], cow [2, 24] and horses [25]. Following identification of key clock genes, demand for a higher-order understanding of the design principles of the mammalian circadian clock has increased. we want to improve the knowledge on the expression of clock genes investigating the clock genes expression in peripheral leukocytes in horse, compared with the rhythm of plasma melatonin, already known in this species [12].
2.0 Materials and methods 2.1 Animal and experimental design Ten Italian Saddle gelding horses (9-11 years old) with a mean body weight of 475±28 Kg were enrolled in the study carried out in Sicily, Italy (38°00′49″N 15°25′18″E, 80 m above sea level). All animals were clinically healthy with no evidence of disease and free from internal and external parasites. All animals were kept in individual boxes under natural photoperiod (sunrise at 6:10 h, sunset at 18:10 h over the study period) and natural environmental conditions. The horses were fed three times a day (06.30, 12.00, and 19.30) with good-quality hay and concentrate. Water was available ad libitum. Thermal and hygrometric records were carried out inside the box for the whole study by means of a data logger (Gemini, UK), and they followed the normal seasonal pattern for the location (mean ambient temperature and mean relative humidity of 25°C and 65%, respectively). The day before the start of sampling, left jugular furrow of each horse was clipped and surgically prepared for placement of indwelling jugular catheters (Terumo, Roma, Italy). The jugular catheter was secured in place with suture (Vicryl, Ethicon, Somerville, USA). All data collections were performed by the same technician. General animal care was carried out by professional staff not associated with the research team. Dim red light (<3 lux, 15 W Safelight lamp filter 1A, Kodak Spa) was used for sample collections during the scotophase. Blood samples were collected at 4 hour intervals over a 48 hour period (starting at 00:00 on day 1 and finishing at 00:00 on day 3) in heparinized tubes, centrifuged at 2,000 g for 10 minutes, and frozen at -20 °C until determination of melatonin concentration by means of radioimmunoassay (Melatonin Direct RIA, Labor Diagnostika Nord GmbH, Nordhorn, Germany) with resolution of 1.5 pg/mL; and into PAX gene Blood RNA Tube (Qiagen) and stored at -80 °C until determination of clock genes.
All treatments, housing, and animal care reported previously were carried out in accordance with the standards recommended by the European Directive 2010/63/EU for animal experiments.
2.2 Real-time RT-quantitative polymerase chain reaction (RTqPCR) Total RNA was purified directly from whole blood samples collected from healthy horses, using a PAX Gene Blood RNA kit (Qiagen), according to the manufacturer's instructions and resuspended in 80 µl of Elution Buffer. Reverse Transcription was carried out immediately, using the Superscript VilocDNA Synthesis Kit (Invitrogen), in a final volume of 20 µl, containing 3 µl of total RNA, a 5X Vilo Reaction mix (including random hexamers, MgCl2, and dNTPs) and a 10X SuperScript Enzyme mix. An initial step at 25 °C for 10 min was followed by a reverse transcription step at 42 °C for 1 h. The resulting cDNA was stored at -20°C prior to further analysis by RT-qPCR. Gene specific primers (Table 1) were designed using Primer3 software to amplify fragments of Equus caballus clock genes (Per1; Per2; Cry1). All reactions (in triplicate) were performed in a 20 µl of final volume, containing 2 µl of cDNA product, 1X Buffer Sybr green (Fast Sybr green master mix - Applied Biosystems) and 1 mM of each primer. The thermal profile was: 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 seconds, 60 °C for 1 min. Melting curve cycles were set as follows: 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. We verified the efficiency of the primers by doing standard curves for all genes investigated. Moreover, the dissociation curve was used to confirm the specificity of the amplicon. Gene expression levels of selected equine clock genes were tested together at GADPH, a gene previously used as reference for ruminant species [26]. The relative levels of each RNA were calculated by the 2-∆∆CT method (CT standing for the cycle number at which the signal reaches the threshold of detection) [27]. Each CT value used for these calculations is the mean of three replicates of the same reaction.
2.3 Statistical analysis
Data were normally distributed (p>0.05, Kolmogorov-Smirnov test). We applied a trigonometric statistical model to each subject values of each time series, so as to describe the periodic phenomenon analytically, by characterizing the main rhythmic parameters according to the single cosinor procedure [28]. Four rhythmic parameters were determined: mesor, amplitude (the difference between the peak, or trough, and the mean value of a wave), acrophase (the time at which the peak of a rhythm occurs), and robustness (strength of rhythmicity). Also, we performed a correlation analyses between variation in the expression of clock genes and that of plasma melatonin secretion. P<0.05 was considered statistically significant. The data were analysed with Statistica 7 (StatSofts, Inc, USA).
3.0 Results Melatonin showed a nocturnal daily rhythm with a high percentage of robustness of rhythm; all clock genes tested showed a daily rhythm except Clock (Table 2). Figure 1 shows the daily oscillation of all parameters tested during the whole experimental period. Bmal1 and Per 2 expression was significantly correlate with plasma melatonin; in a positive way Bmal1 (p<0.01; r=0.36) and in a negative way Per 2 (p<0.01; r=-0.36). Figure 2 shows the mean of 24 hour modification of plasma melatonin and its correlation with clock genes expression tested.
4.0 Discussion Few studies have been conducted to investigate the clock genes in peripheral tissue. The expression profiles of clock genes in the peripheral blood considerably changed from different species. Boivin et al. [21] demonstrate the feasibility of studying peripheral blood mononuclear cells (PBMCs) as an accessible surrogate for the identification of rhythmic clock gene expression in humans. Temporal variations of clock gene in peripheral blood have also been observed in rats [22], dogs [23], and cows [24]. Study involved steers identified a 24 hour pattern of clock gene in neutrophils
and lymphocytes [2]. Study conducted in horses reported the absence of rhythmic expression of clock genes in whole blood of healthy subjects [25]. The blood is not a homogeneous tissue, and the failure to detect the circadian expression of some core clock genes could be due to the different cell types, which were not synchronized among them [29]. We observed a daily rhythm of the expression of clock genes in peripheral leucocytes, except for Clock. All rhythmic genes showed a nocturnal acrophase; Cry 1 some hours before sunrise, Per 1 and Per 3 at about 4 hour after sunset, and Bmal1 and Per 2 around midnight. Expression of Bmal1 and Per 2 was found to correlate significantly with the secretion of plasma melatonin. Peak clock gene expression followed of about 3 hours the peak of melatonin concentration. These results are consistent with study in humans [21] since the oscillation of clock genes in peripheral tissues often lags several hours behind that of the SCN of the central nervous system. Our study want to be a contribution in the study of circadian physiology in horses, identifying the presence of a cyclic transcription of clock genes in peripheral leucocytes in healthy horses housed under natural photoperiod and environmental conditions, in box housing management.
Declaration This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Reference [1] Morin LP, Shivers KY, Blanchard JH, Muscat L. Complex organization of mouse and rat suprachiasmatic nucleus. Neurosci 2006; 137:1285-1297. [2] Nebzydoski SJ, Pozzo S, Nemec L, Rankin MK, Greessley TF. The effect of dexamethasone on clock gene mRNA levels in bovine neutrophils and lymphocytes. Vet Immunol Immunopathol 2010; 138:183-192.
[3] Cipolla-Neto J, Amaral FG, Afeche SC, Tan DX, Reiter RJ. Melatonin, energy metabolism, and obesity: a review. J Pineal Res 2014; 56: 371-381. [4] Gorman MR, Borman BD, Zucker I. Mammalian photoperiodism. In: Takahashi JS, Turek FW, Moore RY (eds) Circadian clocks. Kluwer/Plenum, New York, 2001. pp. 481-508 [5] Corbalán-Tutau D., Madrid JA, Nicolás F, Garaulet M. Daily profile in two circadian markers “melatonin and cortisol” and associations with metabolic sindrome components. Physiol Behav 2014; 123: 231-235 [6] Hasegawa M, Ebihara S. Circadian rhythms of pineal melatonin release in the pigeon measured by in vivo microdialysis. Neurosci Lett 1992; 148: 89-92 [7] Brandstätter R, Kumar V, Abraham U, Gwinner E. Photoperiodic information acquired and stored in vivo retained in vitro by a circadian oscillator, the avian pineal gland. Proc Nat Acad Sci USA 2000; 97: 12324-12328 [8] Zawilska JB, Lorenc A, Berezinska M, Vivien-Roels B, Pévet P, Skene DJ. Diurnal and circadian rhythms in melatonin synthesis in the turkey pineal gland and retina. Gen Comp Endocrinol 2006; 145: 162-168 [9] Ebling FJP, Lincoln GA, Wollnik F, Anderson N. Effects of constant darkness and constant light on circadian organization and reproductive responses in the ram. J Biol Rhythms 1998; 3: 365-384 [10] Maywood ES, Hastings MH, Max M, Ampleford E, Menaker M, Loudon ASI. Circadian and daily rhythms of melatonin in the blood and pineal gland of free-running and entrained Syrian hamsters. J Endocrinol 1993; 136: 65-73 [11] Aarseth JJ, Van't Hof TJ, Stokkan KA. Melatonin is rhythmic in newborn seals exposed to continuous light. J Comp Physiol B 2003; 173: 37-42 [12] Piccione G, Giannetto C, Bertolucci C, Refinetti R. Daily rhythmicity of circulating melatonin is not endogenously generated in the horse. Biol. Rhythm Res 2013; 44(1): 143-149
[13] Voultsios A, Kennaway DJ, Dawson D. Salivary melatonin as a circadian phase marker: validation and comparison to plasma melatonin. J Biol Rhythms 1997; 12: 457-466 [14] Wright KP, Hughes RJ, Kronauer RE, Dijk DJ, Czeisler CA. Intrinsic near-24-h pacemaker period determines limits of circadian entrainment to a weak synchronizer in humans. Proc Nat Acad Sci USA 2001; 98: 14027-14032 [15] Selmaoui B, Touitou Y. Reproducibility of the circadian rhythms of serum cortisol and melatonin in healthy subjects: a study of three different 24-h cycles over six weeks. Life Sci 2003; 73: 3339-3349 [16] Sun X, Liu T, Deng J, Borjigin J. Long-term in vivo pineal microdialysis. J Pineal Res 2003; 35: 118-124 [17] Bartness T, Bittman EL, Hastings MH, Powers JB, Goldman B. Timed melatonin infusion paradigm for melatonin delivery: what has it taught us about the melatonin signal, its reception, and the photoperiodic control of seasonal responses? J Pineal Res 1993; 15:161-190. [18] Pévet P. Mélatonine et rythmes biologiques. Thérapie 1998; 53:411-420. [19] Fukuya H, Emoto N, Nonaka H, Yagita K, Okamura H, Yokoyama M. Circadian expression of clock genes in human peripheral leucocytes. Biochem Bioph Res Comm 2007; 354:924-928. [20] Takimoto M, Hamad A., Tomoda A, Ohdo S, Ohmura T, Sakato H, et al. Daily expression of clock genes in whole blood cell in healthy subjects and a patient with circadian rhythm sleep disorders. Am J Physiol Regul Integr Comp Physiol 2005; 289: R1273-R1279. [21] Boivin DB, James FO, Wu A, Cho-Park PF, Xiong H, Sun ZS. Circadian clock genes oscillate in human peripheral blood mononuclear cells. Blood 2003; 102: 4143-4145. [22] Oishi K, Sakamoto K, Okada T, Nagase T, Ishida N. Antiphase circadian expression between Bmal1 and period homologue mRNA in the suprachiasmatic nucleus and peripheral tissue of rats. Biochem Buiophys Res Commun 1998; 253:199-203.
[23] Ohmori K, Nishikawa S, Oku K, Oida K, Amagai Y Kajiwara N, et al. Circadian rhythms and the effec of glucocorticoids on expression of the clock period 1 in canine peripheral blood mononuclear cells. Vet J 2013; 196: 402-407. [24] Piccione G, Cannella V, Monteverde V, Bertolucci C, Frigato E, Congiu F, et al. Circadian gene expression in peripheral blood of Bos taurus under different experimental period. J Appl Biomed 2014; 12: 271-275. [25] Murphy BA, Vick MM, Sessions DR, Cook RF, Fitzgerald BP. Evidence of an oscillating peripheral clock in an equine fibroblast cell line and adipose tissue but not in peripheral blood. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 2006; 192: 743-751. [26] Robinson T, Sutherland I, Sutherland J. Validation of candidate bovine reference genes for use with RT-qPCR. Vet. Immunol. Immunopathol 2007; 115; 160–165. [27] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method. Methods 2001; 25: 402–408. [28] Nelson W, Tong YL, Lee JK., Halberg F. Methods for cosinor rhythmometry, Chronobiol 1979; 6; 305-323. [29] Gachon F, Nagoshi E, Brown SA, Ripperger J, Schibler U. The mammalian circadian timing system: from gene expression to physiology. Cromosoma 2004; 113:103-112
Figure captions Figure 1. Trend of plasma melatonin and clock gene expression in peripheral blood during the experimental period (48 hours). White and black bars indicate the light and dark phases of the natural photoperiod.
Figura 2. Plasma melatonin daily oscillation and its correlation with the peripheral gene expression.
Table 1. Nucleotide sequences and positions of primers used in RT-qPCR. Gene
Genbank Number
Clock
XM_023636853.1
Sequence (5’→3’)
Length (bp)
for: 3’-GGACACGGATGATAGAGGCA-5’
1 99
rev: 3’-ACATCTGCAGACCTTGACCA-5’ for: 3’-ATGGGGCTGGATGAAGACAA-5’ Bmal1
DQ988038.1
1 1 85
rev: 3’-CATGAGAATGCAGTCGTCCG-5’ for: 3’-AAGGCCTCGCATGAATGC-5’ CRY1
DQ 988039.1
1 1 69
rev: 3’-AAACCGGAGATAAGGACTGA-5’ for: 3’-CAGGCCGCATCGTCTACAT-5’ PER1
XM_001503185.4
1 1 123
rev: 3’-AACCATAGAAGACGCCCACATC-5’ for: 3’-TGGCCCTCATCATCTTTGTG-5’ PER2
XM_012755704.1
1 1 78
rev: 3’-GACCTGAAAGTTCCGGTGATACTG-5’
1
for: 3’- GCTCGCCCCTCGAGATG-5’ Per3
XM_005607561.1
1 72
rev: 3’- CAGTTGTTCCAGAAGGGAAGCT-5’
1
for: 3’-GGTGGAGCCAAAAGGGTCAT -5’ GAPDH
Primer (µM)
NM_001163856.1
1 68
rev: 3’-TTCACGCCCATCACAAACAT -5’
1
Table 2: Mean values of circadian parameters (mesor, amplitude, acrophase and robustness) expressed in their conventional unit, of all parameters studied, during the two days of monitoring.
Parameter Melatonin (pg/ml) Clock (arbitrary unit) Bmal 1 (arbitrary unit) Cry 1 (arbitrary unit) Per 1 (arbitrary unit) Per 2 (arbitrary unit) Per 3 (arbitrary unit)
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
Mesor 7.40 7.50
Amplitude 5.20 5.10
Acrophse (hh:mm) 21:30 21:40
Robustness (%) 74.30 86.50
No rhythmicity 31530.31 36488.79 7589.29 5035.83 3602.98 2274.94 3281.74 3108.31 16404.00 45448.00
30034.78 27866.68 7676.16 1796.34 1206.28 1105.17 801.07 1270.32 20788.00 64094.44
01:29 01:00 03:45 06:18 22:12 22:54 01:00 00:32 20:49 22:14
76.10 77.40 63.55 60.20 76.60 81.20 73.40 66.20 59.60 62.70
S0737-0806(19)30605-7
DOI:
https://doi.org/10.1016/j.jevs.2019.102856
Reference:
YJEVS 102856
To appear in:
Journal of Equine Veterinary Science
Received Date: 4 July 2019 Revised Date:
1 August 2019
Accepted Date: 8 November 2019
Please cite this article as: Giannetto C, Fazio F, Alberghina D, Giudice E, Piccione G, Clock genes expression in peripheral leucokytes and plasma melatonin daily rhythm in horses., Journal of Equine Veterinary Science (2019), doi: https://doi.org/10.1016/j.jevs.2019.102856. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Clock genes expression in peripheral leucokytes and plasma melatonin daily rhythm in horses.
Claudia Giannetto, Francesco Fazio, Daniela Alberghina, Elisabetta Giudice, Giuseppe Piccione*
Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata, 98168, Messina, Italy.
*Corresponding autor: Giuseppe Piccione, Department of Veterinary Sciences, University of Messina,
Polo
Universitario
[email protected]
dell’Annunziata,
98168,
Messina,
Italy.
address:
Abstract In mammals, behavioral and physiological processes display 24 hour rhythms that are regulated by the circadian system. In the present study we investigated clock gene expression in peripheral leukocytes in horses. At this purpose, ten Italian Saddle gelding horses (9-11 years old; 475±28 Kg) were housed in individual boxes under natural photoperiod and natural environmental temperature. Blood samples were collected at 4 hour intervals over a 48 hour period. The day before the start of sampling, left jugular furrow of each horse was cannulated for the blood sample collection performed in heparinized tubes, for the assessment of melatonin concentration by means of radioimmunoassay; and into PAX gene Blood RNA Tube for the assessment of clock genes by realtime RT-quantitative polymerase chain reaction (RTqPCR). How well establish, melatonin showed a daily rhythm with nocturnal acrophase (day 1-21:30; day 2-21:40). All genes tested (Bmal1, Cry 1, Per 1, Per 2 and Per 3) except Clock, showed daily rhythmicity of their expression in peripheral blood. Oscillations of Bmal1 and Per 2 were correlated with the oscillation of melatonin, which acrophase anticipated the acrophase of Bmal1 (day 1-01:29; day 2-01:00) and Per 2 (day 1-01:00; day 2-00:32) of about 3 hours. Our results support the presence of a cyclic transcription of clock genes in peripheral leucocytes in horses.
Keywords: Melatonin, clock genes, leucocytes, horses
1.0 Introduction Circadian rhythms throughout the body are coordinate by suprachiasmatic nucleus (SCN) of the hypothalamus. It processes external cues, such as light and temperature, and internal cues to set circadian rhythm [1]. All this information is communicate to the rest of the body by neural and hormonal signals [2]. The main mediator used by the central master clock is melatonin. It is an internal synchronizer able to act on peripheral oscillator regulating their phase and period, mainly by controlling the transcription/translation circadian cycle of the peripheral clock genes [3]. Melatonin is involved in the organization of the circadian system and plays a central role in the control of photoperiodic responses and can be considered as an endogenous synchronizer [4-5]. Daily oscillation in blood levels of melatonin has been documented in various species of birds [6-8] and mammals [9-12], including humans [13-15]. Microdialysis studies have confirmed that secretion from the pineal is indeed rhythmic and responsible for the oscillation in blood levels [16]. These robust and predictable rhythms in circulating melatonin can be considered as an endogenous synchronizer for the expression of numerous physiological processes in photoperiodic species, including reproduction, feed intake, adiposity and body temperature [17-18]. Eight genes essential for mammalian circadian clock function have been identified: the homologues of the Drosophila gene period (Per 1, Per 2 and Per 3), cryptochrome isoforms (Cry 1 and Cry 2), PAS helix-loophelix transcription factors (Clock and Bmal1), and Casein kinase 1 ε (CK1 ε). The products of these genes constitute a model of a molecular oscillator, based on interconnected positive and negative transcriptional-translational feedback loops [19]. In peripheral blood circadian rhythmicity is manifested in terms of clock gene expression [20-21]. Clock genes in blood cells have been studied in humans [21], rats [22], dogs [23], cow [2, 24] and horses [25]. Following identification of key clock genes, demand for a higher-order understanding of the design principles of the mammalian circadian clock has increased. we want to improve the knowledge on the expression of clock genes investigating the clock genes expression in peripheral leukocytes in horse, compared with the rhythm of plasma melatonin, already known in this species [12].
2.0 Materials and methods 2.1 Animal and experimental design Ten Italian Saddle gelding horses (9-11 years old) with a mean body weight of 475±28 Kg were enrolled in the study carried out in Sicily, Italy (38°00′49″N 15°25′18″E, 80 m above sea level). All animals were clinically healthy with no evidence of disease and free from internal and external parasites. All animals were kept in individual boxes under natural photoperiod (sunrise at 6:10 h, sunset at 18:10 h over the study period) and natural environmental conditions. The horses were fed three times a day (06.30, 12.00, and 19.30) with good-quality hay and concentrate. Water was available ad libitum. Thermal and hygrometric records were carried out inside the box for the whole study by means of a data logger (Gemini, UK), and they followed the normal seasonal pattern for the location (mean ambient temperature and mean relative humidity of 25°C and 65%, respectively). The day before the start of sampling, left jugular furrow of each horse was clipped and surgically prepared for placement of indwelling jugular catheters (Terumo, Roma, Italy). The jugular catheter was secured in place with suture (Vicryl, Ethicon, Somerville, USA). All data collections were performed by the same technician. General animal care was carried out by professional staff not associated with the research team. Dim red light (<3 lux, 15 W Safelight lamp filter 1A, Kodak Spa) was used for sample collections during the scotophase. Blood samples were collected at 4 hour intervals over a 48 hour period (starting at 00:00 on day 1 and finishing at 00:00 on day 3) in heparinized tubes, centrifuged at 2,000 g for 10 minutes, and frozen at -20 °C until determination of melatonin concentration by means of radioimmunoassay (Melatonin Direct RIA, Labor Diagnostika Nord GmbH, Nordhorn, Germany) with resolution of 1.5 pg/mL; and into PAX gene Blood RNA Tube (Qiagen) and stored at -80 °C until determination of clock genes.
All treatments, housing, and animal care reported previously were carried out in accordance with the standards recommended by the European Directive 2010/63/EU for animal experiments.
2.2 Real-time RT-quantitative polymerase chain reaction (RTqPCR) Total RNA was purified directly from whole blood samples collected from healthy horses, using a PAX Gene Blood RNA kit (Qiagen), according to the manufacturer's instructions and resuspended in 80 µl of Elution Buffer. Reverse Transcription was carried out immediately, using the Superscript VilocDNA Synthesis Kit (Invitrogen), in a final volume of 20 µl, containing 3 µl of total RNA, a 5X Vilo Reaction mix (including random hexamers, MgCl2, and dNTPs) and a 10X SuperScript Enzyme mix. An initial step at 25 °C for 10 min was followed by a reverse transcription step at 42 °C for 1 h. The resulting cDNA was stored at -20°C prior to further analysis by RT-qPCR. Gene specific primers (Table 1) were designed using Primer3 software to amplify fragments of Equus caballus clock genes (Per1; Per2; Cry1). All reactions (in triplicate) were performed in a 20 µl of final volume, containing 2 µl of cDNA product, 1X Buffer Sybr green (Fast Sybr green master mix - Applied Biosystems) and 1 mM of each primer. The thermal profile was: 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 seconds, 60 °C for 1 min. Melting curve cycles were set as follows: 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s. We verified the efficiency of the primers by doing standard curves for all genes investigated. Moreover, the dissociation curve was used to confirm the specificity of the amplicon. Gene expression levels of selected equine clock genes were tested together at GADPH, a gene previously used as reference for ruminant species [26]. The relative levels of each RNA were calculated by the 2-∆∆CT method (CT standing for the cycle number at which the signal reaches the threshold of detection) [27]. Each CT value used for these calculations is the mean of three replicates of the same reaction.
2.3 Statistical analysis
Data were normally distributed (p>0.05, Kolmogorov-Smirnov test). We applied a trigonometric statistical model to each subject values of each time series, so as to describe the periodic phenomenon analytically, by characterizing the main rhythmic parameters according to the single cosinor procedure [28]. Four rhythmic parameters were determined: mesor, amplitude (the difference between the peak, or trough, and the mean value of a wave), acrophase (the time at which the peak of a rhythm occurs), and robustness (strength of rhythmicity). Also, we performed a correlation analyses between variation in the expression of clock genes and that of plasma melatonin secretion. P<0.05 was considered statistically significant. The data were analysed with Statistica 7 (StatSofts, Inc, USA).
3.0 Results Melatonin showed a nocturnal daily rhythm with a high percentage of robustness of rhythm; all clock genes tested showed a daily rhythm except Clock (Table 2). Figure 1 shows the daily oscillation of all parameters tested during the whole experimental period. Bmal1 and Per 2 expression was significantly correlate with plasma melatonin; in a positive way Bmal1 (p<0.01; r=0.36) and in a negative way Per 2 (p<0.01; r=-0.36). Figure 2 shows the mean of 24 hour modification of plasma melatonin and its correlation with clock genes expression tested.
4.0 Discussion Few studies have been conducted to investigate the clock genes in peripheral tissue. The expression profiles of clock genes in the peripheral blood considerably changed from different species. Boivin et al. [21] demonstrate the feasibility of studying peripheral blood mononuclear cells (PBMCs) as an accessible surrogate for the identification of rhythmic clock gene expression in humans. Temporal variations of clock gene in peripheral blood have also been observed in rats [22], dogs [23], and cows [24]. Study involved steers identified a 24 hour pattern of clock gene in neutrophils
and lymphocytes [2]. Study conducted in horses reported the absence of rhythmic expression of clock genes in whole blood of healthy subjects [25]. The blood is not a homogeneous tissue, and the failure to detect the circadian expression of some core clock genes could be due to the different cell types, which were not synchronized among them [29]. We observed a daily rhythm of the expression of clock genes in peripheral leucocytes, except for Clock. All rhythmic genes showed a nocturnal acrophase; Cry 1 some hours before sunrise, Per 1 and Per 3 at about 4 hour after sunset, and Bmal1 and Per 2 around midnight. Expression of Bmal1 and Per 2 was found to correlate significantly with the secretion of plasma melatonin. Peak clock gene expression followed of about 3 hours the peak of melatonin concentration. These results are consistent with study in humans [21] since the oscillation of clock genes in peripheral tissues often lags several hours behind that of the SCN of the central nervous system. Our study want to be a contribution in the study of circadian physiology in horses, identifying the presence of a cyclic transcription of clock genes in peripheral leucocytes in healthy horses housed under natural photoperiod and environmental conditions, in box housing management.
Declaration This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Figure captions Figure 1. Trend of plasma melatonin and clock gene expression in peripheral blood during the experimental period (48 hours). White and black bars indicate the light and dark phases of the natural photoperiod.
Figura 2. Plasma melatonin daily oscillation and its correlation with the peripheral gene expression.
Table 1. Nucleotide sequences and positions of primers used in RT-qPCR. Gene
Genbank Number
Clock
XM_023636853.1
Sequence (5’→3’)
Length (bp)
for: 3’-GGACACGGATGATAGAGGCA-5’
1 99
rev: 3’-ACATCTGCAGACCTTGACCA-5’ for: 3’-ATGGGGCTGGATGAAGACAA-5’ Bmal1
DQ988038.1
1 1 85
rev: 3’-CATGAGAATGCAGTCGTCCG-5’ for: 3’-AAGGCCTCGCATGAATGC-5’ CRY1
DQ 988039.1
1 1 69
rev: 3’-AAACCGGAGATAAGGACTGA-5’ for: 3’-CAGGCCGCATCGTCTACAT-5’ PER1
XM_001503185.4
1 1 123
rev: 3’-AACCATAGAAGACGCCCACATC-5’ for: 3’-TGGCCCTCATCATCTTTGTG-5’ PER2
XM_012755704.1
1 1 78
rev: 3’-GACCTGAAAGTTCCGGTGATACTG-5’
1
for: 3’- GCTCGCCCCTCGAGATG-5’ Per3
XM_005607561.1
1 72
rev: 3’- CAGTTGTTCCAGAAGGGAAGCT-5’
1
for: 3’-GGTGGAGCCAAAAGGGTCAT -5’ GAPDH
Primer (µM)
NM_001163856.1
1 68
rev: 3’-TTCACGCCCATCACAAACAT -5’
1
Table 2: Mean values of circadian parameters (mesor, amplitude, acrophase and robustness) expressed in their conventional unit, of all parameters studied, during the two days of monitoring.
Parameter Melatonin (pg/ml) Clock (arbitrary unit) Bmal 1 (arbitrary unit) Cry 1 (arbitrary unit) Per 1 (arbitrary unit) Per 2 (arbitrary unit) Per 3 (arbitrary unit)
Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2 Day 1 Day 2
Mesor 7.40 7.50
Amplitude 5.20 5.10
Acrophse (hh:mm) 21:30 21:40
Robustness (%) 74.30 86.50
No rhythmicity 31530.31 36488.79 7589.29 5035.83 3602.98 2274.94 3281.74 3108.31 16404.00 45448.00
30034.78 27866.68 7676.16 1796.34 1206.28 1105.17 801.07 1270.32 20788.00 64094.44
01:29 01:00 03:45 06:18 22:12 22:54 01:00 00:32 20:49 22:14
76.10 77.40 63.55 60.20 76.60 81.20 73.40 66.20 59.60 62.70