The effect of melatonin on sheep endometrial epithelial cell apoptosis through the receptor and non-receptor pathways.

Melatonin potentially regulates the female animal reproductive function, but its regulatory mechanism in the apoptosis of sheep endometrial epithelial cells (SEECs) remains to be elucidated. In the present study, immunofluorescence staining, western blotting, and quantitative real-time polymerase chain reaction were performed to detect the distribution of melatonin receptors (MT1 and MT2) in the uterus of sheep and the effect of melatonin via the receptor and non-receptor pathways on the apoptosis of SEECs in vitro. The results showed that melatonin inhibits the apoptosis of SEECs to varying degrees to regulate the expression of estrogen receptors (ERs) and progesterone receptors (PGR) via its interaction with MT1 and MT2. In addition, the ER antagonist partially relieved the inhibitory effect of melatonin on the apoptosis of SEECs, while the PGR antagonist did not. Thus, melatonin mediates endometrial epithelial apoptosis through the MT receptors and also by regulating estrogen function. This study provides evidence of the regulatory mechanism of melatonin on the physiological function of the sheep uterus.

Melatonin stimulates the secretion of progesterone along with the expression of cholesterol side-chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR) in corpus luteum of pregnant sows.

The direct effect of melatonin on porcine luteal function during the pregnancy remains unknown. The objective of the study was to analyse the molecular mechanism(s) by which melatonin directly affects progesterone (P4) production in the corpus luteum (CL) of pregnant sows. We evaluated the localization of melatonin membrane receptors (MT1 and MT2) in CL, and investigated the effect of melatonin on P4 secretion along with the expression of P4 synthesis intermediates in luteal cells. Immunohistochemistry analysis showed that MT1 and MT2 were predominantly localized in luteal cells in pregnant luteal tissues. The results of our in vitro experiments showed that melatonin from 5 to 625 pg/mL was able to significantly increase P4 release (P < 0.05) in a dose-dependent manner. And at the dose of 125 pg/mL treatment, the time-dependent effect on P4 secretion was observed. Furthermore, melatonin from 5 to 625 pg/mL up-regulated both P450scc and StAR expression (P < 0.05) in a dose-dependent manner, and the effect was also time-dependent. No difference of 3ß hydroxysteroid dehydrogenase (3ß-HSD) expression was observed between control and treatment groups. In addition, melatonin induced a dose- and time-dependent promotion on cell viability. Additionally, the stimulatory effects of melatonin were blocked by luzindole, a non-selective MT1 and MT2 receptor antagonist, or partially blocked by a selective MT2 ligand, 4-phenyl-2-propionamidotetralin (4P-PDOT). The data support the presence of MT1 and MT2 in porcine CL and a regulatory role for melatonin in luteal function through MT1 and MT2-mediated signal transduction pathways.

Urinary Melatonin in Relation to Postmenopausal Breast Cancer Risk According to Melatonin 1 Receptor Status.

Background: Urinary melatonin levels have been associated with a reduced risk of breast cancer in postmenopausal women, but this association might vary according to tumor melatonin 1 receptor (MT1R) expression.Methods: We conducted a nested case-control study among 1,354 postmenopausal women in the Nurses' Health Study, who were cancer free when they provided first-morning spot urine samples in 2000 to 2002; urine samples were assayed for 6-sulfatoxymelatonin (aMT6s, a major metabolite of melatonin). Five-hundred fifty-five of these women developed breast cancer before May 31, 2012, and were matched to 799 control subjects. In a subset of cases, immunohistochemistry was used to determine MT1R status of tumor tissue. We used multivariable-adjusted conditional logistic regression to estimate the relative risk (RR) of breast cancer [with 95% confidence intervals (CI)] across quartiles of creatinine-standardized urinary aMT6s level, including by MT1R subtype.Results: Higher urinary melatonin levels were suggestively associated with a lower overall risk of breast cancer (multivariable-adjusted RR = 0.78; 95% CI = 0.61-0.99, comparing quartile 4 vs. quartile 1; Ptrend = 0.08); this association was similar for invasive vs. in situ tumors (Pheterogeneity = 0.12). There was no evidence that associations differed according to MT1R status of the tumor (e.g., Pheterogeneity for overall breast cancer = 0.88).Conclusions: Higher urinary melatonin levels were associated with reduced breast cancer risk in this cohort of postmenopausal women, and the association was not modified by MT1R subtype.Impact: Urinary melatonin levels appear to predict the risk of breast cancer in postmenopausal women. However, future research should evaluate these associations with longer-term follow-up and among premenopausal women. Cancer Epidemiol Biomarkers Prev; 26(3); 413-9. ©2016 AACR.

Seasonal modulation of immunity by melatonin and gonadal steroids in a short day breeder goat Capra hircus.

Role of melatonin in regulation of immunity and reproduction has never been studied in detail in goats. The aim of the present study was to explore hormonal regulation of immunity in goats with special reference to melatonin. Plasma of male and female goats (n = 18 per sex per season) was processed for hormonal (estrogen, testostrone, and melatonin) and cytokine (interleukin [IL-2], IL-6, and tumor necrosis factor α) measurements during three seasons, i.e., summer, monsoon, and winter. To assess cell-mediated immune response, percent stimulation ratio of thymocytes was recorded during three seasons. To support and establish the modulation by hormones, Western blot analysis for expressions of melatonin receptors (MT1, MT2), androgen receptor, and estrogen receptor α and estimations of marker enzymes, arylalkylamine N-acetyltransferase for melatonin and 3ß-hydroxysteroid dehydrogenase activities for steroidogenesis were performed in thymus. All the hormones and cytokines were estimated by commercial kits. Biochemical, immunologic, and Western blot analyses were done by standardized protocols. We noted a significant increase in estrogen and testosterone levels (P < 0.05) in circulation during monsoon along with melatonin (P < 0.05) presenting a parallel relationship. Expressions of melatonin receptors (MT1 and MT2) in thymus of both the sexes were significantly high (P < 0.01) during winter. Estrogen receptor α expression in female thymus was significantly high during monsoon (P < 0.05). However, androgen receptor showed almost static expression pattern in male thymus during three seasons. Further, both arylalkylamineN-acetyltransferase and 3ß-hydroxysteroid dehydrogenase enzyme activities were significantly high (P < 0.05; P < 0.01, respectively) during monsoon. These results suggest that there may be a functional parallelism between gonadal steroids and melatonin as melatonin is progonadotrophic in goats. Cell-mediated immune parameters (percent stimulation ratio of thymocytes) and circulatory levels of cytokines (IL-2, IL-6, and tumor necrosis factor α) were significantly high (P < 0.01) during monsoon. In vitro supplementation of gonadal steroids to T-cell culture suppressed immunity but cosupplementation with melatonin restored it. Further, we may also suggest that reproductive and immune seasonality are maintained by variations in circulatory hormones and local synthesis of melatonin and gonadal steroids. These functional interactions between melatonin and gonadal steroid might be of great importance in regulating the goat immunity by developing some hormonal microcircuit (gonadal steroid and melatonin) in lymphatic organs.

Subcellular distribution of melatonin receptors in human parotid glands.

The hormone melatonin influences oral health through a variety of actions, such as anti-inflammatory, anti-oxidant, immunomodulatory and antitumour. Many of these melatonin functions are mediated by a family of membrane receptors expressed in the oral epithelium and salivary glands. Using immunoblotting and immunohistochemistry, recent studies have shown that the melatonin membrane receptors, MT1 and MT2, are present in rat and human salivary glands. To date, no investigation has dealt with the ultrastructural distribution of the melatonin receptors. This was the aim of the present study, using the immunogold method applied to the human parotid gland. Reactivity to MT1 and, with less intensity, to MT2 appeared in the secretory granules of acinar cells and in the cytoplasmic vesicles of both acinar and ductal cells. Plasma membranes were also stained, albeit slightly. The peculiar intracytoplasmic distribution of these receptors may indicate that there is an uptake/transport system for melatonin from the circulation into the saliva.

MTNR1A receptor expression in normal and pathological human salivary glands.

AIM: To analyze and compare the expression of MTNR1A receptor in normal and pathological major and minor salivary glands. MATERIALS AND METHODS: Twenty samples of major and minor salivary glands and 10 with Warthin's tumor were studied. Expression of the MTNR1A receptor (goat polyclonal antibody raised against a peptide mapping at the N-terminus of MEL-1A R of human origin) was analyzed. RESULTS: The excretory ducts of major salivary glands demonstrated intense intracytoplasmic positivity but scant cytoplasmic membrane positivity for MTNR1A. The studied Warthin's tumors showed intense cytoplasmic positivity for MT1 receptor in all cylindrical epithelial cells lining spaces and a less intense positivity in basal cells. The lymphoid component accompanying the tumor was negative for MT1 receptor. CONCLUSION: Intense intracytoplasmic positivity for the MTNR1A receptor in the excretory ducts of human major and minor salivary glands and Warthin's tumor was found. The intense expression of MTNR1A receptors observed in this study in the excretory ducts of major and minor salivary glands may be related to salivary regulation.

Melatonin suppresses apoptosis and stimulates progesterone production by bovine granulosa cells via its receptors (MT1 and MT2).

Melatonin and its receptors have been detected in the ovary of many species, and mediate ovarian functions. The present study was designed to investigate the expression and subcellar location of melatonin receptors in bovine granulosa cells (GCs), using reverse transcription (RT) polymerase chain reaction, Western blot, and immunofluorescence analyses. Furthermore, expression level of melatonin receptors mRNA (real-time polymerase chain reaction) after treatment with various concentrations of melatonin, as well as its effects on cell apoptosis, proliferation, and steroidogenesis (by flow cytometry and RIA), were determined. In bovine GCs, melatonin receptors MT1 and MT2 were differentially located at the cell membrane, the cytoplasm, and nuclear membranes. The expression of MT1 and MT2 mRNA was regulated differently by melatonin in time- and dose-dependent manners. Exogenous melatonin suppressed cell apoptosis (P < 0.05) but not proliferation (P > 0.05). After 72 h, the apoptotic rate was significantly inhibited in all treatment groups. Meanwhile, melatonin supplementation stimulated progesterone production, but inhibited estradiol biosynthesis, in a time-dependent manner. Progesterone production was highest (P < 0.05) at 72 h. Estradiol concentrations were almost unaffected (P > 0.05) at 24 h, but were decreased (P < 0.05) at 48 h. In conclusion, exogenous melatonin acts via receptors and has important roles in regulation of development and function of bovine GCs.

Decreased expression of the melatonin receptor 1 in human colorectal adenocarcinomas.

Melatonin exerts anti-proliferative and pro-apoptotic effects in various cancer cell lines. Furthermore, there is evidence for impaired melatonin secretion in human breast and colorectal cancer. Additionally, several studies revealed a modulated expression of the melatonin receptor 1 (MT1), in human breast cancer specimens. Since melatonin binding sites were already identified in the human intestine, our aim is to identify the expression and to characterize the localization of the MT1 receptor in the human colon and in particular to compare MT1 expression levels between non-malignant and malignant colonic tissue. We assessed MT1 transcript levels with real time RT-PCR in colon adenocarcinomas and the adjacent normal colonic mucosa of 39 patients and observed a significant decrease of MT1 mRNA expression in colorectal cancer compared with the healthy adjacent mucosa tissue (0.67 mean difference, P < 0.0001). The results were confirmed at the protein level by Western blot analysis and by immunohistochemistry. MT1 was localized mainly supranuclear in colonic epithelial cells lining the crypts. We also evaluated mRNA expression of different clock genes in the colon samples and found a significant correlation between MT1 and Cryptochrome 1 (Cry1) expression (P < 0.01 for normal and P < 0.05 for tumour tissue). In conclusion, the decreased expression of MT1 in human colorectal cancer could point to a role of melatonin in this disease.

Pancreatic stellate/myofibroblast cells express G-protein-coupled melatonin receptor 1.

In chronic pancreatitis and pancreatic cancer, progressive fibrosis with the accumulation of extracellular matrix occurs. The main extracellular matrix-producing cell types are retinoid-storing pancreatic stellate cells (PSCs) of mesenchymal origin. Similar to liver stellate cells, quiescent PSCs undergo activation and acquire a myofibroblast-like phenotype in response to pro-fibrogenic mediators (reactive oxygen species, cytokines and toxic metabolites). Activated PSCs differ in their differentiation stage and are characterized by the expression of glial fibrillary-acidic protein, alpha-smooth muscle actin, and nestin. As G-protein-coupled receptors were described to regulate PSC differentiation, we investigated tissue samples from patients with pancreatitis and ductal pancreatic adenocarcinoma for the expression of G-protein-coupled melatonin receptors MT1 and MT2 by double immunofluorescence staining. We show that MT1, but not MT2, is occasionally expressed in PSCs in normal tissue, while in the diseased tissue MT1 is found at high rates in activated PSCs at all stages, and, additionally, in ductal epithelial cells. It is speculated that MT1 activation by its ligand melatonin regulates proliferation and differentiation of PSCs. Prevention of myofibroblast formation by MT1 activation could explain favourable effects of the pineal hormone melatonin on the outcome of pancreatic fibrosis in animal models.

La melatonina reduce la respuesta de cortisol al ACTH en humanos / Melatonin reduces cortisol response to ACTH in humans

Background: Melatonin receptors are widely distributed in human tissues but they have not been reported in human adrenal gland. Aim: To assess if the human adrenal gland expresses melatonin receptors and if melatonin affeets cortisol response to ACTH in dexamethasone suppressed volunteers. Material and methods: Adrenal glands were obtained from 4 patients undergoing unilateral nephrectomy-adrenalectomy for renal cáncer. Expression of mRNA MT1 and MT2 melatonin receptors was measured by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The effect of melatonin on the response to intravenous (i.v.) ACTH was tested (randomized cross-over, double-blind, placebo-controlled tríal) in eight young healthy males pretreated with dexamethasone (1 mg) at 23:00 h. On the next day at 08:00 h, an i.v. Une was inserted, at 08:30 h, and after a blood sample, subjeets ingested 6 mg melatonin or placebo. At 09:00 h, 1-24 ACTH (Cortrosyn, 1µg/1.73 m² body surface área) was injected, drawing samples at 0, 15, 30, 45 and 60 minutes after. Melatonin, cortisol, cortisone, progesterone, aldosterone, DHEA-S, testosterone and prolactin were measured by immunoassay. Results: The four adrenal glands expressed only MT1 receptor mRNA. Melatonin ingestión reduced the cortisol response to ACTH from 14.6+1.45µg/dl at 60 min in the placebo group to 10.8+1.2µg/dl in the melatonin group (p <0.01 mixed model test). It did not affect other steroid hormone levels and abolished the morningphysiological decline of prolactin. Conclusions: The expression ofMTl melatonin receptor in the human adrenal, and the melatonin reduction of ACTH-stimulated cortisol production suggest a direct melatonin action on the adrenal gland .

[Melatonin reduces cortisol response to ACTH in humans]. / La melatonina reduce la respuesta de cortisol al ACTH en humanos.

BACKGROUND: Melatonin receptors are widely distributed in human tissues but they have not been reported in human adrenal gland. AIM: To assess if the human adrenal gland expresses melatonin receptors and if melatonin affects cortisol response to ACTH in dexamethasone suppressed volunteers. MATERIAL AND METHODS: Adrenal glands were obtained from 4 patients undergoing unilateral nephrectomy-adrenalectomy for renal cancer. Expression of mRNA MT1 and MT2 melatonin receptors was measured by Reverse TranscriPtase Polymerase Chain Reaction (RT-PCR). The effect of melatonin on the response to intravenous (i.v.) ACTH was tested (randomized cross-over, double-blind, placebo-controlled trial) in eight young healthy males pretreated with dexamethasone (1 mg) at 23:00 h. On the next day, at 08:00 h, an i.v. line was inserted, at 08:30 h, and after a blood sample, subjects ingested 6 mg melatonin or placebo. At 09:00 h, 1-24 ACTH (Cortrosyn, 1 microg/1.73 m2 body surface area) was injected, drawing samples at 0, 15, 30, 45 and 60 minutes after. Melatonin, cortisol, cortisone, progesterone, aldosterone, DHEA-S, testosterone and prolactin were measured by immunoassay. RESULTS: The four adrenal glands expressed only MT1 receptor mRNA. Melatonin ingestion reduced the cortisol response to ACTH from 14.6 +/- 1.45 microg/dl at 60 min in the placebo group to 10.8 +/- 1.2 microg/dl in the melatonin group (p < 0.01 mixed model test). It did not affect other steroid hormone levels and abolished the morning physiological decline of prolactin. CONCLUSIONS: The expression of MT1 melatonin receptor in the human adrenal, and the melatonin reduction of ACTH-stimulated cortisol production suggest a direct melatonin action on the adrenal gland.

Neural stem cells express melatonin receptors and neurotrophic factors: colocalization of the MT1 receptor with neuronal and glial markers.

BACKGROUND: In order to optimize the potential benefits of neural stem cell (NSC) transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different in vitro conditions. In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT1 and MT2 receptors are expressed in NSCs. RESULTS: RT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT1 receptor mRNA was detected in NSCs maintained for two days in culture, but the MT2 receptor was not seen. An immature MT1 receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 - 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT1 receptor is expressed in both neural (beta-tubulin III positive) and glial (GFAP positive) progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours. CONCLUSIONS: The phenotypic characteristics of C17.2 cells suggest that they are a heterogeneous population of NSCs including both neural and glial progenitors, as observed under the cell culture conditions used in this study. These NSCs have an intrinsic ability to express neurotrophic factors, with an apparent suppression of GDNF expression after several days in culture. The detection of melatonin receptors in neural stem/progenitor cells suggests involvement of this pleiotropic hormone in mammalian neurodevelopment. Moreover, the ability of melatonin to induce GDNF expression in C17.2 cells supports a functional role for the MT1 receptor expressed in these NSCs. In view of the potency of GDNF in promoting the survival of dopaminergic neurons, these novel findings have implications for the utilization of melatonin in neuroprotective strategies, especially in Parkinson's disease.