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Melatonin induces progesterone production in human granulosa-lutein cells through upregulation of StAR expression.

ABSTRACT: Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in ovarian steroidogenesis and progesterone (P4) synthesis. Melatonin and its receptors are expressed in human granulosa cells, and have been shown to influence basal P4 production. However, previous studies addressing the regulation of StAR expression by melatonin and its impact on P4 secretion yielded contradictory results. Here, we demonstrate that melatonin upregulates StAR expression in primary cultures of human granulosa-lutein (hGL) cells obtained from women undergoing in vitro fertilization (IVF). Using pharmacological inhibitors, we show that the stimulatory effect of melatonin on StAR expression is mediated via both MT1 and MT2 melatonin receptors. Melatonin exposure activates the PI3K/AKT signaling pathway and its inhibition attenuates the stimulatory effect of melatonin on StAR expression. Moreover, siRNA-mediated knockdown of StAR abolishes melatonin-induced P4 production. Importantly, clinical analyses demonstrate that melatonin levels in human follicular fluid are positively correlated with P4 levels in serum. By illustrating the potential physiological role of melatonin in the regulation of StAR expression and P4 production in hGL cells, our results may serve to improve current strategies used to treat clinical infertility.

SUBMITTER: Fang L

PROVIDER: S-EPMC6834401 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Melatonin induces progesterone production in human granulosa-lutein cells through upregulation of StAR expression.

Fang Lanlan L Li Yiran Y Wang Sijia S Yu Yiping Y Li Yuxi Y Guo Yanjie Y Yan Yang Y Sun Ying-Pu YP

Aging 20191016 20

Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in ovarian steroidogenesis and progesterone (P4) synthesis. Melatonin and its receptors are expressed in human granulosa cells, and have been shown to influence basal P4 production. However, previous studies addressing the regulation of StAR expression by melatonin and its impact on P4 secretion yielded contradictory results. Here, we demonstrate that melatonin upregulates StAR expression in primary cultures of human g ...[more]

PMID: 31619582

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Project description:BACKGROUND:Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels. METHODS:Granulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17beta, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size. RESULTS:4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17beta to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production. CONCLUSION:4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Project description:BackgroundOvarian granulosa cells are the predominant source of estradiol and progesterone biosynthesis in vivo. Rosiglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor gamma (PPAR gamma), is applied as the treatment of insulin resistance including women with PCOS. The aim of the study was to investigate the direct effects of rosiglitazone on steroidogenesis and proinflammatory factor production in human granulosa-lutein cells (GLCs).MethodsPrimary human GLCs were separated during in vitro fertilization and cultured in the presence of rosiglitazone, GW9662 (an antagonist of PPAR gamma) and hCG. The mRNA expression of key steroidogenic factors including 3beta- hydroxysteriod dehydrogenase (3beta-HSD), cytochrome P-450 scc (CYP11A1), cytochrome P-450 aromatase (CYP19A1), and steroidogenic acute regulatory protein (StAR) were detected by quantitative real-time PCR. Estradiol and progesterone levels in GLCs cultures were measured by chemiluminescence immunoassay, and the proinflammtory factors (TNFalpha and IL-6) in conditioned culture media were measured by ELISA.ResultsPPAR gamma mRNA levels increased up to 3.24 fold by rosiglitazone at the concentration of 30 microM compared to control (P<0.05). hCG alone or hCG with rosiglitazone had no significant effects on PPAR gamma mRNA levels. The CYP19A1 mRNA level at exposure to rosiglitazone alone showed a drop, but was not significantly reduced comparing to control. The expression levels of enzymes 3beta-HSD and CYP11A1 in all treatments did not alter significantly. The StAR mRNA expression at exposure to rosiglitazone was significantly increased comparing to control (P<0.05). The media concentrations of E2 and progesterone by rosiglitazone treatment showed a declining trend comparing to control or cotreatment with hCG, which did not reach significance. Most importantly, treatment with rosiglitazone decreased TNFalpha secretion in a statistically significant manner compared with control (P<0.05). The concentration of IL-6 following rosiglitazone exposure did not significantly decrease comparing to control.ConclusionIn cultured GLCs, rosiglitazone stimulated StAR expression, but did not significantly affect steroidogenic enzymes, as well as E2 and progesterone production. Moreover, rosiglitazone significantly decreased the production of TNFalpha in human GLCs, suggesting that PPAR gamma may play a role in the regulation of GLCs functions through inhibiting proinflammatory factors.

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Melatonin induces progesterone production in human granulosa-lutein cells through upregulation of StAR expression.

Publications

Melatonin induces progesterone production in human granulosa-lutein cells through upregulation of StAR expression.

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