Melatonin action on luteal - granulosa cells in women with marital infertility undergoing in vitro fertilization

Introduction of the subject: Melatonin is a hormone related to the light-dark cycle and it plays an influential role in the reproductive system. In humans, researchers have shown there is three times more melatonin in follicular fluid than in the blood flow, suggesting the influence of the hormone on follicular maturation. However, little is known about melatonin action on granulosa cells in women with infertility, especially the molecular mechanisms involved. We are, therefore, conducting this study to evaluate melatonin action pathways in granulosa cells in the ovaries of women with infertility. To achieve our aim, we will use molecular biology, involving diverse signaling pathways, such as angiogenesis. Objective(s): to analyze melatonin action on luteal - granulosa cells in women with marital infertility undergoing in vitro fertilization. Methodology: For this, 68 patients, aged between 20 and 35 years, attended at the Human Reproduction Sector of the Federal University of São Paulo, were submitted to in vitro fertilization treatment. After all preparatory procedures were performed, luteal granulosa cells were removed and routed to the cell culture. The cells were divided into four groups: a) control; b) 0.1 μM melatonin; c) 1 μM melatonin; d) melatonin at 10μM. After a period of 10 days, the cells were trypsinized for extraction of the total RNA and later analysis of the gene expression by Real Time PCR of the angiogenesis signaling pathway. Results: Our data evaluated 96 genes, which are related to the angiogenesis pathway. The results of transcriptional expression showed important genes involved in this pathway that are hypo or hyperexpressed after treatment with melatonin. The main hyperexpressed genes were: fibroblast growth factor 1 (FGF1) genes; interleukin 1-beta (IL1B); receptor tyrosine kinase (VEGFR-2); folliculogenesis regulating genes (TGFB1). They act in the dynamics of follicular growth. On the other hand, the genes inducing factor 1-alpha (HIF1A), fibroblast growth factor 2 (FGF2), insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGFA) melatonin decreased its expression. Discussion and conclusion of the results: It is concluded that melatonin in high concentration (above 1 μM) may have dual function in increasing the expression of some growth factors and cytokines, in the decrease of other genes and mechanism of physiological compensation and also modulates negative angiogenesis, mainly in the dose (10 uM) of granulosa cells from women submitted to in vitro fertilization. Perhaps this fact is important for adequate follicular growth, avoiding excessive growth, which could turn the follicle into cyst, making ovulation difficult.Keywords: Melatonin; Granulosa cells; Women; Marital infertility; In vitro fertilization

Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats

BACKGROUND: The p16(INK4A) gene product halts cell proliferation by preventing phosphorylation of the Rb protein. The p16INK4a gene is often deleted in human glioblastoma multiforme, contributing to unchecked Rb phosphorylation and rapid cell division. We show here that transduction of the human p16INK4a cDNA using the pCL retroviral system is an efficient means of stopping the proliferation of the rat-derrived glioma cell line, C6, both in tissue culture and in an animal model. C6 cells were transduced with pCL retrovirus encoding the p16INK4a, p53, or Rb genes. These cells were analyzed by a colony formation assay. Expression of p16INK4a was confirmed by immunohistochemistry and Western blot analysis. The altered morphology of the p16-expressing cells was further characterized by the senescence-associated β-galactosidase assay. C6 cells infected ex vivo were implanted by stereotaxic injection in order to assess tumor formation. RESULTS: The p16INK4a gene arrested C6 cells more efficiently than either p53 or Rb. Continued studies with the p16INK4a gene revealed that a large portion of infected cells expressed the p16INK4a protein and the morphology of these cells was altered. The enlarged, flat, and bi-polar shape indicated a senescence-like state, confirmed by the senescence-associated β-galactosidase assay. The animal model revealed that cells infected with the pCLp16 virus did not form tumors. CONCLUSION: Our results show that retrovirus mediated transfer of p16INK4a halts glioma formation in a rat model. These results corroborate the idea that retrovirus-mediated transfer of the p16INK4a gene may be an effective means to arrest human glioma and glioblastoma

Melatonin and the cardiovascular system in animals: systematic review and meta-analysis

Melatonin, a hormone released by the pineal gland, demonstrates several effects on the cardiovascular system. Herein, we performed a systematic review and meta-analysis to verify the effects of melatonin in an experimental model of myocardial infarction. We performed a systematic review according to PRISMA recommendations and reviewed MEDLINE, Embase, and Cochrane databases. Only articles in English were considered. A systematic review of the literature published between November 2008 and June 2019 was performed. The meta-analysis was conducted using the RevMan 5.3 program provided by the Cochrane Collaboration. In total, 858 articles were identified, of which 13 were included in this review. The main results of this study revealed that melatonin benefits the cardiovascular system by reducing infarct size, improving cardiac function according to echocardiographic and hemodynamic analyses, affords antioxidant effects, improves the rate of apoptosis, decreases lactate dehydrogenase activity, enhances biometric analyses, and improves protein levels, as analyzed by western blotting and quantitative PCR. In the meta-analysis, we observed a statistically significant decrease in infarct size (mean difference [MD], -20.37 [-23.56, -17.18]), no statistical difference in systolic pressure (MD, -1.75 [-5.47, 1.97]), a statistically significant decrease in lactate dehydrogenase in animals in the melatonin group (MD, -4.61 [-6.83, -2.40]), and a statistically significant improvement in the cardiac ejection fraction (MD, -8.12 [-9.56, -6.69]). On analyzing potential bias, we observed that most studies presented a low risk of bias; two parameters were not included in the analysis, and one parameter had a high risk of bias. Melatonin exerts several effects on the cardiovascular system and could be a useful therapeutic target to combat various cardiovascular diseases

Maternal Melatonin Programs the Daily Pattern of Energy Metabolism in Adult Offspring

Background: Shift work was recently described as a factor that increases the risk of Type 2 diabetes mellitus. In addition, rats born to mothers subjected to a phase shift throughout pregnancy are glucose intolerant. However, the mechanism by which a phase shift transmits metabolic information to the offspring has not been determined. Among several endocrine secretions, phase shifts in the light/dark cycle were described as altering the circadian profile of melatonin production by the pineal gland. The present study addresses the importance of maternal melatonin for the metabolic programming of the offspring. Methodology/Principal Findings: Female Wistar rats were submitted to SHAM surgery or pinealectomy (PINX). The PINX rats were divided into two groups and received either melatonin (PM) or vehicle. The SHAM, the PINX vehicle and the PM females were housed with male Wistar rats. Rats were allowed to mate and after weaning, the male and female offspring were subjected to a glucose tolerance test (GTT), a pyruvate tolerance test (PTT) and an insulin tolerance test (ITT). Pancreatic islets were isolated for insulin secretion, and insulin signaling was assessed in the liver and in the skeletal muscle by western blots. We found that male and female rats born to PINX mothers display glucose intolerance at the end of the light phase of the light/dark cycle, but not at the beginning. We further demonstrate that impaired glucose-stimulated insulin secretion and hepatic insulin resistance are mechanisms that may contribute to glucose intolerance in the offspring of PINX mothers. The metabolic programming described here occurs due to an absence of maternal melatonin because the offspring born to PINX mothers treated with melatonin were not glucose intolerant. Conclusions/Significance: The present results support the novel concept that maternal melatonin is responsible for the programming of the daily pattern of energy metabolism in their offspring.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)CNPq (Conselho Nacional de Aperfeicoameno Cientifico)CNPq (Conselho Nacional de Aperfeicoameno Cientifico

Rat retina shows robust circadian expression of clock and clock output genes in explant culture.

PURPOSE: Circadian rhythms are central to vision and retinal physiology. A circadian clock located within the retina controls various rhythmic processes including melatonin synthesis in photoreceptors. In the present study, we evaluated the rhythmic expression of clock genes and clock output genes in retinal explants maintained for several days in darkness. METHODS: Retinas were dissected from Wistar rats, either wild-type or from the Per1-luciferase transgenic line housed under a daily 12 h:12 h light-dark cycle (LD12/12), and put in culture at zeitgeber time (ZT) 12 on semipermeable membranes. Explants from wild-type rats were collected every 4 h over 3 days, and total RNA was extracted, quantified, and reverse transcribed. Gene expression was assessed with quantitative PCR, and the periodicity of the relative mRNA amounts was assessed with nonlinear least squares fitting to sine wave functions. Bioluminescence in explants from Per1-luciferase rats was monitored for several days under three different culture protocols. RESULTS: Rhythmic expression was found for all studied clock genes and for clock downstream targets such as c-fos and arylalkylamine N-acetyltransferase (Aanat) genes. Clock and output genes cycled with relatively similar periods and acrophases (peaks of expression during subjective night, except c-fos, which peaked around the end of the subjective day). Data for Per1 were confirmed with bioluminescence monitoring, which also permitted culture conditions to be optimized to study the retina clock. CONCLUSIONS: Our work shows the free-running expression profile of multiple clock genes and potential clock targets in mammalian retinal explants. This research further strengthens the notion that the retina contains a self-sustained oscillator that can be functionally characterized in organotypic culture.journal articleresearch support, non-u.s. gov't20142014 06 02importe

Food-anticipatory activity in Syrian hamsters: behavioral and molecular responses in the hypothalamus according to photoperiodic conditions.

When food availability is restricted, animals adjust their behavior according to the timing of food access. Most rodents, such as rats and mice, and a wide number of other animals express before timed food access a bout of activity, defined as food-anticipatory activity (FAA). One notable exception amongst rodents is the Syrian hamster, a photoperiodic species that is not prone to express FAA. The present study was designed to understand the reasons for the low FAA in that species. First, we used both wheel-running activity and general cage activity to assess locomotor behavior. Second, the possible effects of photoperiod was tested by challenging hamsters with restricted feeding under long (LP) or short (SP) photoperiods. Third, because daytime light may inhibit voluntary activity, hamsters were also exposed to successive steps of full and skeleton photoperiods (two 1-h light pulses simulating dawn and dusk). When hamsters were exposed to skeleton photoperiods, not full photoperiod, they expressed FAA in the wheel independently of daylength, indicating that FAA in the wheel is masked by daytime light under full photoperiods. During FAA under skeleton photoperiods, c-Fos expression was increased in the arcuate nuclei independently of the photoperiod, but differentially increased in the ventromedial and dorsomedial hypothalamic nuclei according to the photoperiod. FAA in general activity was hardly modulated by daytime light, but was reduced under SP. Together, these findings show that food-restricted Syrian hamsters are not prone to display FAA under common laboratory conditions, because of the presence of light during daytime that suppresses FAA expression in the wheel.journal articleresearch support, non-u.s. gov't20152015 05 13importedFunding: This work was supported by doctoral scholarship from Fundação de Amparo à Pesquisa do Estado do São Paulo (São Paulo State, Brazil) to RFDF, and by Centre National de la Recherche Scientifique and University of Strasbourg (France) to EC, VS and PP

12-week melatonin administration had no effect on diabetes risk markers and fat intake in overweight women night workers

IntroductionInteractions between circadian clocks and key mediators of chronic low-grade inflammation associated with fat consumption may be important in maintaining metabolic homeostasis and may pose a risk for the development of obesity-associated comorbidities, especially type 2 diabetes (T2DM).ObjectiveThe aims of the present study were to evaluate the effects of melatonin administration on diabetes risk markers according to dietary lipid profile (pro-inflammatory versus anti-inflammatory) in excessive weight night workers, and to determine the effect of administration on fat consumption profile.MethodsA randomized, controlled, double-blind, crossover clinical trial involving 27 nursing professionals working permanent night shifts under a 12×36-hour system. The melatonin group (12 weeks) used synthetic melatonin (3 mg) only on days off and between shifts, while the placebo group (12 weeks) was instructed to take a placebo, also on days off and between shifts. For inflammatory characteristics, participants were divided into pro-inflammatory (saturated fats, trans fats and cholesterol) and anti-inflammatory (monounsaturated, polyunsaturated fats and EPA + DHA) groups according to fatty acid determinations. At baseline and at the end of each phase, blood glucose, insulin, glycosylated hemoglobin plasma concentrations were collected, and HOMA-IR was calculated.ConclusionMelatonin administration for 12 weeks had no effect on T2DM risk markers according to dietary lipid profile (pro-inflammatory or anti-inflammatory potential) in excessive weight night workers. Among the limitations of the study include the fact that the low dose may have influenced the results expected in the hypothesis, and individual adaptations to night work were not evaluated. The insights discussed are important for future research investigating the influence of melatonin and fats considered anti- or pro-inflammatory on glucose and insulin homeostasis related to night work

A brief review about melatonin, a pineal hormone

ABSTRACT Melatonin is a ubiquitous molecule in nature, being locally synthesized in several cells and tissues, besides being a hormone that is centrally produced in the pineal gland of vertebrates, particularly in mammals. Its pineal synthesis is timed by the suprachiasmatic nucleus, that is synchronized to the light-dark cycle via the retinohypothalamic tract, placing melatonin synthesis at night, provided its dark. This unique trait turns melatonin into an internal synchronizer that adequately times the organism's physiology to the daily and seasonal demands. Besides being amphiphilic, melatonin presents specific mechanisms and ways of action devoted to its role as a time-giving agent, being widely spread in the organism. The present review aims to focus on melatonin as a pineal hormone with specific mechanisms and ways of action, besides presenting the clinical syndromes related to its synthesis and/or function disruptions