Dataset Information

Melatonin Signaling Pathways Implicated in Metabolic Processes in Human Granulosa Cells (KGN).

ABSTRACT: Female reproduction depends on the metabolic status, especially during the period of folliculogenesis. Even though it is believed that melatonin can improve oocyte competence, there is still limited knowledge of how it can modulate metabolic processes during folliculogenesis and which signaling pathways are involved in regulating gene expression. To investigate the effects of melatonin on metabolic signals during the antral stage of follicular development, human granulosa-like tumor cells (KGN) were treated with melatonin or forskolin, and gene expression was analyzed with RNA-seq technology. Following appropriate normalization and the application of a fold change cut-off of 1.5 (FC 1.5, p ≤ 0.05), 1009 and 922 genes were identified as differentially expressed in response to melatonin and forskolin, respectively. Analysis of major upstream regulators suggested that melatonin may activate PKB/mTOR signaling pathways to program the metabolism of KGN cells to support slower growth and differentiation and to prevent follicular atresia. Similarly, PKA activation through stimulation of cAMP synthesis with FSK seemed to exert the same effects as melatonin in reducing follicular growth and regulating differentiation. This study suggests that melatonin may act through PKA and PKB simultaneously in human granulosa cells to prevent follicular atresia and early luteinization at the antral stage.

SUBMITTER: Asma A

PROVIDER: S-EPMC8950389 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Melatonin Signaling Pathways Implicated in Metabolic Processes in Human Granulosa Cells (KGN).

Asma Arjoune A Marc-André Sirard S

International journal of molecular sciences 20220310 6

Female reproduction depends on the metabolic status, especially during the period of folliculogenesis. Even though it is believed that melatonin can improve oocyte competence, there is still limited knowledge of how it can modulate metabolic processes during folliculogenesis and which signaling pathways are involved in regulating gene expression. To investigate the effects of melatonin on metabolic signals during the antral stage of follicular development, human granulosa-like tumor cells (KGN) ...[more]

PMID: 35328408

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Project description:Maternal metabolic disorders such as obesity and diabetes are detrimental factors that compromise fertility and the success rates of medically assisted procreation procedures. During metabolic stress, adipose tissue is more likely to release free fatty acids (FFA) in the serum resulting in an increase of FFA levels not only in blood, but also in follicular fluid (FF). In humans, high concentrations of palmitic acid and stearic acid reduced granulosa cell survival and were associated with poor cumulus-oocyte complex (COC) morphology. Obesity and high levels of circulating FFA were also causatively linked to hampered insulin sensitivity in cells and compensatory hyperinsulinemia. To provide a global picture of the principal upstream signaling pathways and genomic mechanisms involved in this metabolic context, human granulosa-like tumor cells (KGN) were treated with a combination of palmitic acid, oleic acid, and stearic acid at the higher physiological concentrations found in the follicular fluid of women with a higher body mass index (BMI) (≥ 30.0 kg/m2). We also tested a high concentration of insulin alone and in combination with high concentrations of fatty acids. Transcription analysis by RNA-seq with a cut off for fold change of 1.5 and p-value 0.05 resulted in thousands of differentially expressed genes for each treatment. Using analysis software such as Ingenuity Pathway Analysis (IPA), we were able to establish that high concentrations of FFA affected the expression of genes mainly related to glucose and insulin homoeostasis, fatty acid metabolism, as well as steroidogenesis and granulosa cell differentiation processes. The combination of insulin and high concentrations of FFA affected signaling pathways related to apoptosis, inflammation, and oxidative stress. Taken together, our results provided new information on the mechanisms that might be involved in human granulosa cells exposed to high concentrations of FFA and insulin in the contexts of metabolism disorders.

Project description:The ovarian KGN granulosa-like tumour cell line is commonly used as a model for human granulosa cells, especially since it produces steroid hormones. To explore this further, we identified genes that were differentially expressed by KGN cells compared to primary human granulosa cells using three public RNA sequence datasets. Of significance, we identified that the expression of the antioxidant gene TXNRD1 (thioredoxin reductase 1) was extremely high in KGN cells. This is ominous since cytochrome P450 enzymes leak electrons and produce reactive oxygen species during the biosynthesis of steroid hormones. Gene Ontology (GO) analysis identified steroid biosynthetic and cholesterol metabolic processes were more active in primary granulosa cells, whilst in KGN cells, DNA processing, chromosome segregation and kinetochore pathways were more prominent. Expression of cytochrome P450 cholesterol side-chain cleavage (CYP11A1) and cytochrome P450 aromatase (CYP19A1), which are important for the biosynthesis of the steroid hormones progesterone and oestrogen, plus their electron transport chain members (FDXR, FDX1, POR) were measured in cultured KGN cells. KGN cells were treated with 1 mM dibutyryl cAMP (dbcAMP) or 10 μM forskolin, with or without siRNA knockdown of TXNRD1. We also examined expression of antioxidant genes, H2O2 production by Amplex Red assay and DNA damage by γH2Ax staining. Significant increases in CYP11A1 and CYP19A1 were observed by either dbcAMP or forskolin treatments. However, no significant changes in H2O2 levels or DNA damage were found. Knockdown of expression of TXNRD1 by siRNA blocked the stimulation of expression of CYP11A1 and CYP19A1 by dbcAMP. Thus, with TXNRD1 playing such a pivotal role in steroidogenesis in the KGN cells and it being so highly overexpressed, we conclude that KGN cells might not be the most appropriate model of primary granulosa cells for studying the interplay between ovarian steroidogenesis, reactive oxygen species and antioxidants.

Dataset Information

Melatonin Signaling Pathways Implicated in Metabolic Processes in Human Granulosa Cells (KGN).

Publications

Melatonin Signaling Pathways Implicated in Metabolic Processes in Human Granulosa Cells (KGN).

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