234 EXPRESSION OF NUCLEAR AND MEMBRANE MELATONIN RECEPTORS GENES AND THE CLOCK GENES IN RAT OOCYTES: PRELIMINARY RESULTS

2010 ◽  
Vol 22 (1) ◽  
pp. 275
Author(s):  
L. A. Coelho ◽  
R. Peres ◽  
J. Cipolla-Neto
Keyword(s):  
Circadian Rhythms ◽  
Internal Control ◽  
Clock Genes ◽  
Receptor Gene ◽  
Cumulus Cells ◽  
Melatonin Receptors ◽  
Female Rats ◽  
Melatonin Receptor ◽  
Biomedical Sciences ◽  
Preliminary Results

There is evidence that melatonin acts directly on the regulation of ovary function. This action is probably attributed in part to melatonin receptors, which are known to be present in granulosa and cumulus cells (Kang J-T et al. 2009 J. Pineal Res. 46, 22–28). Melatonin is also known to be associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function (Arendt J 1998 Rev. Reprod. 3, 13–22). Circadian rhythms and clock genes appear to be involved in reproductive processes (Dolatshad H et al. 2009 Repro. Fertil. Dev. 21, 1–9). However, the presence of melatonin receptor genes and the clock genes has not been so widely studied or has never been reported to exist in mammalian oocytes.The aim of this study was to investigate the presence of nuclear (Rorα) and membrane (Mt1 and Mt2) melatonin receptors genes and the clock genes (Clock, Bmal1, Cry1, Cry21, Per1, Per2) in rat oocytes by reverse RT-PCR. Twenty-seven-day-old Wistar female rats were treated with 20 UI of pregnant mares serum gonadotropin for induction of follicular development and slaughtered 48 h later. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. The ovaries were removed and placed in TCM-199 supplemented with 100 UI mL-1 penicillin, 100 μg mL-1 streptomycin, and 0.1% polyvinyl alcohol (H-199 medium). Germinal vesicle-intact oocytes, isolated from the ovarian follicles, were denuded from cumulus cells by vortexing for 5 to 8 min. The denuded oocytes were incubated for 5 min in H-199 medium with 0.1% pronase for removal of the zona pellucida. Pools of 80 oocytes per cDNA sample were used.As an internal control for the sample integrity, additional primers for RPL37a were included in each PCR reaction. All the 3 control PCR replicates showed a repeatable amplification. Polymerase chain reaction amplifications of cDNA yielded Rorα, Clock, Bmal1, and Cry1 products in 2 of 3 replicates. No expression of the MT1 and MT2 mRNA was observed. The preliminary results suggest the presence of a nuclear melatonin receptor gene and some clock genes in rat oocytes. However, additional studies are necessary to confirm this hypothesis. This research was supported by FAPESP.

197 EXPRESSION OF MELATONIN-RELATED GENES IN RAT CUMULUS–OOCYTE COMPLEXES

2013 ◽  
Vol 25 (1) ◽  
pp. 247
Author(s):  
L. A. Coelho ◽  
R. Peres ◽  
F. G. Amaral ◽  
J. Cipolla-Neto
Keyword(s):  
Real Time ◽  
Chorionic Gonadotropin ◽  
Real Time Pcr ◽  
Geometric Mean ◽  
Cumulus Cells ◽  
Sybr Green ◽  
Membrane Receptors ◽  
Melatonin Receptors ◽  
Female Rats ◽  
Qrt Pcr

Melatonin is a hormone usually associated with the modulation of circadian rhythms and the regulation of seasonal reproductive function. There is evidence that melatonin acts directly on the regulation of ovary function. The mRNA expression of melatonin membrane receptors genes was detected in mammalian and non-mammalian ovaries. In spite of melatonin receptors and Asmt (limiting enzyme in melatonin biosynthesis) genes being present in cattle cumulus–oocyte complexes (COC), no information regarding rat COC has been reported. The aim of this study was to investigate the expression of melatonin receptor (Mt2) and melatonin synthesis enzyme (Asmt) genes at different meiotic cell cycle stages in COC from 27-day-old female rats. All the procedures involving animals were approved by the Animal Care Committee of the Institute of Biomedical Sciences. To obtain germinal vesicle (GV) immature COC from ovarian follicles, rats were treated with 20 IU equine chorionic gonadotropin (eCG) for induction of follicular development and killed by euthanasia 48 h later. To obtain COC at MII oocyte stage from oviducts, rats were injected with 20 IU of eCG and 48 h later with 20 IU of human chorionic gonadotropin (hCG), and then killed after 14 to 16 h. The oocytes in COC were separated from their cumulus cells by repeated pipetting through a narrow-bore pipette in culture medium. Oocytes and cumulus cells total RNA were isolated using a Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). Pools of 80 COC per cDNA sample were used. Quantitative real-time PCR (qRT-PCR) was performed (7500 Real-Time PCR System; Applied Biosystems Inc., Foster City, CA, USA) with 25-µL reactions containing 2 µL of cDNA (10 ng µL–1), SYBR green (Power SYBR Green; Applied Biosystems Inc.), and 400 nM specific intron-spanning primers. A set of 10-fold serial dilutions of each internal standard (102 to 106 copies/2 µL) was used to generate a standard curve. Transcript numbers were determined by the system software and normalized using the geometric mean calculated from the reference genes: Actb and Rpl37a. All the results were plotted as the mean ± SEM, and 4 replicates were performed. Unpaired t-test was used to evaluate the cell type (oocyte v. cumulus cells) differences. The qRT-PCR analyses revealed the presence of transcripts of the Mt2 gene only in oocytes from immature (GV) and mature (MII) COC. At both maturational stages, the copy numbers for Asmt in oocytes were significantly higher than in cumulus cells. The results confirm the presence of the Asmt gene in rat COC and suggest the possible involvement of these cells on melatonin biosynthesis. The presence of Mt2 transcript in immature and mature oocytes also suggests the potentially important role of melatonin in regulating the rat meiotic cellular cycle. Research supported by FAPESP.

scholarly journals Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora

2021 ◽  
pp. 074873042199994
Author(s):  
Rosa Eskandari ◽  
Lalanthi Ratnayake ◽  
Patricia L. Lakin-Thomas
Keyword(s):  
Circadian Rhythms ◽  
Clock Genes ◽  
Nutrient Sensing ◽  
Mass Spectrometry Analysis ◽  
Growth Responses ◽  
Tor Pathway ◽  
Spectrometry Analysis ◽  
Binding Partner ◽  
Binding Partners ◽  
Free Running

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes ( frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation ( vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δ gtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δ gtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δ gtr2 is defective in entrainment. In all of these assays, Δ gtr2 is similar to Δ vta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

scholarly journals Circadian Rhythm: Potential Therapeutic Target for Atherosclerosis and Thrombosis

2021 ◽  
Vol 22 (2) ◽  
pp. 676
Author(s):  
Andy W. C. Man ◽  
Huige Li ◽  
Ning Xia
Keyword(s):  
Circadian Rhythm ◽  
Cardiovascular Diseases ◽  
Circadian Rhythms ◽  
Circadian Clock ◽  
Therapeutic Target ◽  
Environmental Changes ◽  
Clock Genes ◽  
Vascular System ◽  
Peripheral Tissues ◽  
Inflammatory Processes

Every organism has an intrinsic biological rhythm that orchestrates biological processes in adjusting to daily environmental changes. Circadian rhythms are maintained by networks of molecular clocks throughout the core and peripheral tissues, including immune cells, blood vessels, and perivascular adipose tissues. Recent findings have suggested strong correlations between the circadian clock and cardiovascular diseases. Desynchronization between the circadian rhythm and body metabolism contributes to the development of cardiovascular diseases including arteriosclerosis and thrombosis. Circadian rhythms are involved in controlling inflammatory processes and metabolisms, which can influence the pathology of arteriosclerosis and thrombosis. Circadian clock genes are critical in maintaining the robust relationship between diurnal variation and the cardiovascular system. The circadian machinery in the vascular system may be a novel therapeutic target for the prevention and treatment of cardiovascular diseases. The research on circadian rhythms in cardiovascular diseases is still progressing. In this review, we briefly summarize recent studies on circadian rhythms and cardiovascular homeostasis, focusing on the circadian control of inflammatory processes and metabolisms. Based on the recent findings, we discuss the potential target molecules for future therapeutic strategies against cardiovascular diseases by targeting the circadian clock.

scholarly journals The Increase in Signaling by Kisspeptin Neurons in the Preoptic Area and Associated Changes in Clock Gene Expression That Trigger the LH Surge in Female Rats Are Dependent on the Facilitatory Action of a Noradrenaline Input

Endocrinology ◽  
2016 ◽  
Vol 157 (1) ◽  
pp. 323-335 ◽  
Author(s):  
Bruna Kalil ◽  
Aline B. Ribeiro ◽  
Cristiane M. Leite ◽  
Ernane T. Uchôa ◽  
Ruither O. Carolino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Median Eminence ◽  
Preoptic Area ◽  
Clock Genes ◽  
Third Ventricle ◽  
Female Rats ◽  
Ovariectomized Rats ◽  
Lh Surge ◽  
Clock Gene Expression

Abstract In rodents, kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) of the preoptic area are considered to provide a major stimulatory input to the GnRH neuronal network that is responsible for triggering the preovulatory LH surge. Noradrenaline (NA) is one of the main modulators of GnRH release, and NA fibers are found in close apposition to kisspeptin neurons in the RP3V. Our objective was to interrogate the role of NA signaling in the kisspeptin control of GnRH secretion during the estradiol induced LH surge in ovariectomized rats, using prazosin, an α1-adrenergic receptor antagonist. In control rats, the estradiol-induced LH surge at 17 hours was associated with a significant increase in GnRH and kisspeptin content in the median eminence with the increase in kisspeptin preceding that of GnRH and LH. Prazosin, administered 5 and 3 hours prior to the predicted time of the LH surge truncated the LH surge and abolished the rise in GnRH and kisspeptin in the median eminence. In the preoptic area, prazosin blocked the increases in Kiss1 gene expression and kisspeptin content in association with a disruption in the expression of the clock genes, Per1 and Bmal1. Together these findings demonstrate for the first time that NA modulates kisspeptin synthesis in the RP3V through the activation of α1-adrenergic receptors prior to the initiation of the LH surge and indicate a potential role of α1-adrenergic signaling in the circadian-controlled pathway timing of the preovulatory LH surge.

scholarly journals Co-expression of the calcitonin receptor gene in the hypothalamic kisspeptin neurons in female rats

2018 ◽  
Vol 17 (2) ◽  
pp. 164-172 ◽  
Author(s):  
Assadullah ◽  
Nahoko Ieda ◽  
Narumi Kawai ◽  
Hirotaka Ishii ◽  
Kunio Ihara ◽  
...  
Keyword(s):  
Receptor Gene ◽  
Female Rats ◽  
Calcitonin Receptor ◽  
Calcitonin Receptor Gene

scholarly journals Modulatory Effects of Circadian Rhythms in the Lung Adenocarcinoma Tumor Microenvironment

2021 ◽  
Author(s):  
Qianzhun Huang ◽  
Xiaoyang Su ◽  
Ning Fang ◽  
Jian Huang
Keyword(s):  
Tumor Microenvironment ◽  
Circadian Rhythms ◽  
Lung Adenocarcinoma ◽  
Circadian Clock ◽  
Drug Sensitivity ◽  
Molecular Mechanisms ◽  
Clock Genes ◽  
Functional Enrichment ◽  
Expression Levels ◽  
Circadian Clock Genes

Abstract Background: Dysregulated circadian dynamic balance is strongly associated with cancer development. However, biological functions of circadian rhythms in lung adenocarcinoma (LUAD) have not been elucidated. This study aimed at valuating the modulatory effects of circadian rhythms in the LUAD tumor microenvironment.Methods: Multiple open-source bioinformatics research platforms are used to comprehensively elucidate the expression level, prognosis, potential biological function, drug sensitivity, and immune microenvironment of circadian clock genes in LUAD.Results: Most circadian clock genes in LUAD are dysregulated and are strongly correlated with patient prognosis, and missense mutations at splicing sites of these genes. Besides, these genes are closely associated with some well-known cancer-related marker pathways, which are mainly involved in the inhibition of the Apoptosis, Cell cycle, and DNA Damage Response Pathway. Furthermore, functional enrichment analysis revealedthat circadian clock genes regulate transcription factor activities and circadian rhythms in LUAD tissues. As for drug sensitivity, high expression of CLOCK, CRY1, and NR1D2 as well as suppressedPER2 and CRY2 expression levels are associated with drug resistance. The expression levels of circadian clock genes in LUAD correlate with immune infiltration and are involved in the regulation of immunosuppression.Conclusions: Our multi-omics analysis provides a more comprehensive understanding of the molecular mechanisms of the circadian clock genes in LUAD and provides new insights for a more precise screening of prognostic biomarkers and immunotherapy.

scholarly journals Association of alcohol withdrawal severity with MTNR1A (rs34532313) and MTNR1B (rs10830963) genes polymorphisms

2021 ◽  
Vol 1 (2) ◽  
pp. 111-116
Author(s):  
I. S. Efremov ◽  
D. R. Tukhvatullina ◽  
U. S. Efremova ◽  
V. R. Gashkarimov ◽  
N. R. Tulbaeva ◽  
...  
Keyword(s):  
Alcohol Withdrawal ◽  
Alcohol Use Disorder ◽  
Melatonin Receptors ◽  
Research Centre ◽  
Melatonin Receptor ◽  
Saint Petersburg ◽  
Severe Withdrawal ◽  
Final Sample ◽  
Threatening Condition ◽  
Polymorphic Variants

Alcohol withdrawal is the most threatening condition encountered in patients with alcohol use disorder. Our study aimed to investigate the association of alcohol withdrawal severity with polymorphic variants in melatonin receptor genes. Methods. The clinical study was carried out on the basis of the Republican Narcological Dispensary №1 in Ufa and the Republican Narcological Dispensary №2 in Sterlitamak. Genetic analysis was performed at the Department of Personalised Psychiatry and Neurology at the V.M. Bekhterev Research Centre, Saint Petersburg. The final sample consisted of 307 subjects. Results. Carriers of the TT genotype of the MTNR1A gene (rs34532313) were found to have less hypertension during alcohol withdrawal than carriers of the other genotypes. In comparison, carriers of the GG genotype of the MTNR1B gene (rs10830963) experienced more symptoms than other genotypes: paroxysmal sweating, visual hallucinations, anxiety, and overall CIWA-Ar score. Conclusions. Thus, it can be concluded that the TT genotype of MTNR1A gene (rs34532313) is associated with a lower risk of hypertension during alcohol withdrawal compared to carriers of other gene genotypes. The GG genotype of MTNR1B gene (rs10830963) is associated with severe withdrawal. In general, it can be concluded that melatonin receptors are involved in the pathogenesis of alcohol withdrawal and the severe of some of its symptoms. 

scholarly journals Vasopressinergic Activity of the Suprachiasmatic Nucleus and mRNA Expression of Clock Genes in the Hypothalamus-Pituitary-Gonadal Axis in Female Aging

2021 ◽  
Vol 12 ◽  
Author(s):  
Angela Cristina Nicola ◽  
Larissa Brazoloto Ferreira ◽  
Milene Mantovani Mata ◽  
Tatiane Vilhena-Franco ◽  
Cristiane Mota Leite ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Suprachiasmatic Nucleus ◽  
Clock Genes ◽  
Feedback Loops ◽  
Female Rats ◽  
Hpg Axis ◽  
Physiological Processes ◽  
Reproductive Axis ◽  
Reproductive Events ◽  
Vasopressinergic Neurons

The important involvement of the suprachiasmatic nucleus (SCN) and the activity of vasopressinergic neurons in maintaining the rhythmicity of the female reproductive system depends on the mRNA transcription-translation feedback loops. Therefore, circadian clock function, like most physiological processes, is involved in the events that determine reproductive aging. This study describes the change of mRNA expression of clock genes, Per2, Bmal1, and Rev-erbα, in the hypothalamus-pituitary-gonadal axis (HPG) of female rats with regular cycle (RC) and irregular cycle (IC), and the vasopressinergic neurons activity in the SCN and kisspeptin neurons in the arcuate nucleus (ARC) of these animals. Results for gonadotropins and the cFos/AVP-ir neurons in the SCN of IC were higher, but kisspeptin-ir was minor. Change in the temporal synchrony of the clock system in the HPG axis, during the period prior to the cessation of ovulatory cycles, was identified. The analysis of mRNA for Per2, Bmal1, and Rev-erbα in the reproductive axis of adult female rodents shows that the regularity of the estrous cycle is guaranteed by alternation in the amount of expression of Bmal1 and Per2, and Rev-erbα and Bmal1 between light and dark phases, which ceases to occur and contributes to determining reproductive senescence. These results showed that the desynchronization between the central and peripheral circadian clocks contributes to the irregularity of reproductive events. We suggest that the feedback loops of clock genes on the HPG axis modulate the spontaneous transition from regular to irregular cycle and to acyclicity in female rodents.

scholarly journals Modeling and analysis of the impacts of jet lag on circadian rhythm and its role in tumor growth

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4877 ◽  
Author(s):  
Azka Hassan ◽  
Jamil Ahmad ◽  
Hufsah Ashraf ◽  
Amjad Ali
Keyword(s):  
Circadian Rhythms ◽  
Circadian Clock ◽  
Clock Genes ◽  
Damage Control ◽  
Vital Role ◽  
Biochemical Networks ◽  
Jet Lag ◽  
Peripheral Tissues ◽  
Modeling And Analysis ◽  
Circadian Clock Proteins

Circadian rhythms maintain a 24 h oscillation pattern in metabolic, physiological and behavioral processes in all living organisms. Circadian rhythms are organized as biochemical networks located in hypothalamus and peripheral tissues. Rhythmicity in the expression of circadian clock genes plays a vital role in regulating the process of cell division and DNA damage control. The oncogenic protein, MYC and the tumor suppressor, p53 are directly influenced by the circadian clock. Jet lag and altered sleep/wake schedules prominently affect the expression of molecular clock genes. This study is focused on developing a Petri net model to analyze the impacts of long term jet lag on the circadian clock and its probable role in tumor progression. The results depict that jet lag disrupts the normal rhythmic behavior and expression of the circadian clock proteins. This disruption leads to persistent expression of MYC and suppressed expression of p53. Thus, it is inferred that jet lag altered circadian clock negatively affects the expressions of cell cycle regulatory genes and contribute in uncontrolled proliferation of tumor cells.

scholarly journals Chronic Corticosterone Disrupts the Circadian Rhythm of CRH Expression and M6a RNA Methylation in the Chicken Hypothalamus

2021 ◽  
Author(s):  
Yang Yang ◽  
Wanwan Han ◽  
Aijia Zhang ◽  
Mindie Zhao ◽  
Wei Cong ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
Circadian Rhythms ◽  
Chronic Stress ◽  
Hpa Axis ◽  
Clock Genes ◽  
Diurnal Fluctuation ◽  
Opposite Pattern ◽  
Rna Methylation ◽  
Diurnal Patterns ◽  
M6a Rna Methylation

Abstract Corticotropin-releasing hormone (CRH), the major secretagogue of the hypothalamic-pituitary-adrenal (HPA) axis, is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions. N6-methyladenosine (m6A) RNA methylation is involved in the regulation of circadian rhythm, yet it remains unknown whether CRH expression and m6A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress. Here, we show that chronic exposure to corticosterone (CORT) eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken. The circadian rhythms of clock genes in hippocampus, hypothalamus and pituitary are all disturbed to different extent in CORT-treated chickens. The most striking changes occur in hypothalamus in which the diurnal fluctuation of CRH mRNA is flattened, together with mRNA of other feeding-related neuropeptides. Interestingly, hypothalamic m6A level oscillates in an opposite pattern to CRH mRNA, with lowest m6A level after midnight (ZT18) corresponding to the peak of CRH mRNA before dawn (ZT22). CORT diminished the circadian rhythm of m6A methylation with significantly increased level at night. Further site-specific m6A analysis on 3’UTR of CRH mRNA indicates that higher m6A on 3’UTR of CRH mRNA coincides with lower CRH mRNA at night (ZT18 and ZT22). Our results indicate that chronic stress disrupts the circadian rhythms of CRH expression in hypothalamus, leading to dysfunction of HPA axis in the chicken. RNA m6A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions.

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