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Melatonin protects premature ovarian insufficiency induced by tripterygium glycosides: role of SIRT1.

ABSTRACT: Melatonin confers protective effects on premature ovarian insufficiency (POI) induced by tripterygium glycosides (TG) by reducing oxidative stress. Silent information regulator 1 (SIRT1) signaling is found to be associated with the physiology and pathology of ovary. We hypothesize that melatonin could protect POI via activating SIRT1 signaling. The aim of this study was to investigate the protective effect of melatonin on POI and elucidate its potential mechanisms. Mice were assigned to melatonin treatment with or without SIRT1 inhibitor Ex527 or melatonin receptor antagonist luzindole (Luz) and then subjected to POI. Melatonin conferred a protective effect by improving estrous phase, ovarian and uterus mass and index, increasing ovarian follicles, corpus luteum and anti-mullerian hormone (AMH), decreasing atresia follicles and follicle stimulating hormone (FSH). Melatonin treatment also could reduce malondialdehyde (MDA) level, MDA5, Gp91^phox, Caspase3 and Bax expression, and increase total antioxidant activity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and Bcl-2 expression by up-regulating SIRT1 signaling. However, these protective effects were blocked by Ex527 and Luz, indicating that SIRT1 signaling and melatonin receptor might be specially involved in these effects. In summary, these findings suggest that melatonin protects POI by reducing oxidative stress and apoptotic damage via activation of SIRT1 signaling in a receptor-dependent manner.

SUBMITTER: Ma M

PROVIDER: S-EPMC5411910 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Premature ovarian insufficiency (POI) is defined as a primary ovarian defect characterized by absent menarche (primary amenorrhea) or premature depletion of ovarian follicles before the age of 40 (secondary amenorrhea) with hypergonadotropism and hypoestrogenism. Premature ovarian insufficiency has few known genetic causes but in familial cases a genetic link is often suspected. A large consanguineous family with three female affected with POI was investigated. All samples including 3 affected and 5 unaffecd underwent whole genome SNP genotyping using Affymetric Axiom_GW_Hu_SNP array. Linkage analysis was carried out using HomozygosityMapper and Allegro softwares.Linkage analysis mapped the disease phenotype to long arm of chromosome 20. Sequence data analysis of potential candidate genes failed to detect any pathogenic variant. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from peripheral blood samples. DNA of eight individuals including three affected subjects was used for homozygosity mapping. Genotyping was performed using the Affymetrix Axiom_GW_Hu_SNP array. Briefly, 250 ng genomic DNA was digested with Digestion Master Mix containing 2 µl NE buffer 2 (10X), 0.5 µl BSA (100X; 10 mg/ml) and 1 µl Nsp1. Digested DNA sample was ligated to Nsp1 adaptor using T4 DNA ligase and amplified by 2 µl of TITANIUM Taq DNA polymerase (50X) and 100 µM PCR primer. PCR products were purified on a Clean-Up plate (Clontech Lab, Madison, USA) and eluted by RB buffer. Purified PCR products were fragmented using Fragmentation Reagent (0.05U/µl DNase 1) for 35 minutes at 37°C followed by labeling of fragmented samples with Labeling Master Mix (30 mM GeneChip DNA Labeling Reagent, 30 U/µl Terminal Deoxynucleotidyl Transferase) for 4 hours at 37°C. Labeled samples were hybridized to Axiom_GW_Hu_SNP array by mixing the sample with Hybridization Master Mix, denatured on thermoblock and loaded on to Array. Array was then placed in a hybridization oven (GeneChip Hybridization Oven 640, USA) for 16-18 hours. After hybridization, array was washed and stained on an automated Fluidic Station 450 followed by scanning on GeneChip Scanner 3000 7G using GeneChip Operating Software (GCOS).

Project description:BACKGROUND:Premature ovarian insufficiency (POI) is a common disease in reproductive women. The pathogenesis of POI is not clear, although it is known that it involves the disorder of oocyte differentiation and development. The introduction of reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) offers a unique opportunity to study many aspects of POI from cell differentiation in vitro that could ultimately lead to novel drug development and testing to help treat the disorder. METHODS:The fibroblasts from POI patients, including fragile X syndrome, abnormal karyotype (45, X; 45, X/46, XX; 45, XO and 47, XXX), and the gene mutation (FIGLA and GDF9) were reprogrammed to pluripotency status by retroviral transduction using defined factors. The morphology, growth characteristics, gene expression profiles, epigenetic status, and in vitro and in vivo differentiation potential of the POI-1-iPSCs (from fragile X syndrome) were analyzed. Then, POI-1-iPSCs were induced to differentiation into primordial germ cells (PGCs) with DNA methyltransferase inhibitors. RESULTS:The iPSCs were successfully generated from POI patients' fibroblasts. The formed iPS clones have the same characteristics of human ESCs. POI-1-iPSCs were successfully generated with germline competence. The POI-1-iPSCs, with genotypes of fragile X syndrome, can be induced to differentiation into PGCs with high efficiency under our culture system by DNA demethylation. This study proved that disease-specific iPSC lines derived from POI patients could be generated and successfully differentiated into PGCs. CONCLUSIONS:We established some novel, systemic cell models for the studying of the pathogenesis of POI patients. Second, DNA demethylation may accelerate the induction of human PGCs from iPSCs in vitro and the conclusion needs further exploration. This represents an important step in the novel approach for the study of the pathophysiology and potential egg resource for POI patients.

Project description:ObjectiveTo investigate the role of FKBP4 protein in cisplatin-induced premature ovarian insufficiency (POI).ObjectiveWe performed ITRAQ assay of the ovarian tissues from 4 mice with cisplatin-induced POI and 4 control mice, and identified FKBP4 as a significantly down-regulated protein in the oocytes and granulosa cells following cisplatin treatment. TargetScan software was used for target analysis of FKBP4, and qRT-PCR and Western blotting were used to verify the expression levels of miR-483-5p and FKBP4 in the mouse models. Serum samples were collected from patients with POI and healthy women for detecting miR-483-5p level with qRT-PCR. Cell transfection and dual-luciferase assay were performed to determine the relationship between miR-483-5p and FKBP4. In primary granulosa cells and KGN cells, we examined the effect of miR-483-5p alone, miR-483-5p and cisplatin, and miR-483-5p combined with both cisplatin and FKBP4 on cell apoptosis. We also assessed ovarian function in a transgenic mouse model with ovarian miR-483-5p overexpression in comparison wigh wildtype mice using immunofluorescence assay, in situ hybridization and ELISA.ObjectiveOvarian FKBP4 expression was significantly decreased in mice with cisplatin-induced POI. Analysis using TargetScan software indicated that FKBP4 was the potential target of miR-483-5p, which was highly expressed in the ovaries and serum of POI mice and in the serum of patients with POI. In vitro experiments further confirmed that FKBP4 was the target of miR-483-5p. In KGN and primary granulosa cells, FKBP4 overexpression significantly reduced cell apoptosis induced by both cisplatin and miR-483-5p overexpression (P= 0.0045 and 0.0177, respectively). In the transgenic mice with miR-483-5p overexpression in the oocytes, cisplatin induced more severe ovarian damages as compared with those in the wild-type mice.ObjectivemiR-483-5p/FKBP4 is a new and important pathway in cisplatin-induced POI, in which cisplatin increases ovarian miR- 483-5p expression to result in targeted downregulation of FKBP4. Up-regulation of miR-483-5p may increase ovarian sensitivity to cisplatin and cause severe ovarian dysfunction. Detection of serum miR-483-5p level may help to predict the occurrence and development of POI.

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Melatonin protects premature ovarian insufficiency induced by tripterygium glycosides: role of SIRT1.

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