Cell Line KGN Research Articles

Impact of vitrification on granulosa cell survival and gene expression

IntroductionCryopreservation of ovarian tissue is an essential step in Ovarian Tissue Banking. In order to prevent the formation of ice crystals, typically the tissue is slowly frozen using a cryoprotectant. As an alternative the method of ultra-fast freezing by vitrification becomes more attention for freezing ovarian tissue because it has successfully been used for oocytes, embryos and sperm. However the impact of vitrification on granulosa cells, which are an essential part of ovarian tissue is uncertain. AimIn this study, we have therefore analysed the influence of vitrification on the survival rates of granulosa cells, the impact of DMSO or ethylenglycol containing vitrification protocols and investigated to what extent the gene expression of apoptosis- and temperature-sensitive genes changes. Material and methodsWe used the human granulosa cell line KGN as a model for human granulosa cells and determined the survival rate and cell cycle stages by FACS analyses. The change in gene expression was determined by quantitative PCR analyses. ResultsOur results show that vitrification is possible in granulosa cells but it reduces cell viability and leads to fluctuations in the cell cycle. The DMSO containing protocol results in a lower amount of dead cells than the ethylenglycol containing protocol. Gene expression analysis reveals that TNF-alpha expression is strongly increased after vitrification, while other apoptosis or temperature-related genes seem to stay unaffected. ConclusionWe conclude that vitrification influences the viability of human granulosa cells. Furthermore, our results suggest that this could be mediated by a change in TNF-alpha gene expression.

NLRP1 inflammasome mediates immuno-metabolic dysfunction in follicular niche of PCOS

Investigating mechanism of chronic inflammation in PCOS ovary regulated by NLRP inflammasome To determine whether NLRP expression is associated with inflammatory status in follicular niche of PCOS patients, we analyzed expression level of NLRP genes in ovarian granulosa cells isolated from 35 women with PCOS and 32 healthy adult female controls undergoing in vitro fertilization and embryo treatment (IVF-ET)at the Reproductive Center of Peking University Third Hospital. PCOS patients were diagnosed by Rotterdam-PCOS criteria. Ovarian Granulosa cells was isolated by Ficoll density gradient centrifugation and gene expression was detected by Real-time RT-PCR analysis. Furthermore, we tested IL18 and IL 1B protein level in follicular fluids from these patients by ELSA.LCMS/MS was applied to quantify long-chain fatty acids in in follicular fluid. Human ovarian granulosa cell line KGN was used for in vitro cell tests. Activation of NLRP inflammasome and MMP-9 production was detected by western blotting. Our results showed that IL18 level in follicular fluid of PCOS patients is significantly higher than female controls, besides, IL18 protein level is positively correlated with BMI, which indicated potential link between inflammation and lipid metabolic aberration in follicular microenvironment of PCOS. We also found that NLRP1 transcription level is higher in PCOS women than healthy women. Inflammasome-activating stimuli induced phosphorylation of caspase-1 in granulosa cell line KGN. Long-chain fatty acids from abnormal lipid metabolism is significantly elevated in follicular fluids from PCOS patients. Based on existing results, we infer that up-regulation of NLRP1 in ovarian granulosa cells may be related to lipid metabolic dysfunction in PCOS and result in elevated IL18 secretion in follicular fluid of PCOS women, possibly through NLRP1 inflammasome assembly and activation.

LncRNA LNC-GULP1-2:1 is involved in human granulosa cell proliferation by regulating COL3A1 gene

Long non-coding RNAs (lncRNAs) are defined as transcripts longer than 200 nucleotides that are not translated into protein. Many studies have identified the association between abnormal expression of lncRNAs and specific disease states, especially in tumor-related studies. However, the precise functions of these lncRNAs are not clear. In our previous study, both lnc-GULP1-2:1 and its potential target COL3A1 were significantly downregulated in ovarian cortical tissues of POI patients compared with normal control patients. The results of GO and KEGG analysis also showed that changes in extracellular matrix-related genes play an important role in the development of POI. Therefore, we speculate that lnc-GULP1-2:1 may participate in the development of ovarian follicles by regulating the COL3A1 gene. Basic research study. The lentiviral vectors were used to construct a KGN cell line with stable high expression of lnc-GULP1-2:1. Real-time PCR and western blot were used to analyze the expression of COL3A1 and lnc-GULP1-2:1. Cell proliferation was analyzed by using CCK-8 kits. The stable over-expression of lnc-GULP1-2:1 in KGN cells could significantly inhibit granulosa cell proliferation. Treatment of KGN cells with chemotherapeutic agents (cisplatin and paclitaxel) dose-dependently inhibited granulosa cell proliferation and promoted the expression of lnc-GULP1-2:1, whereas the cytokine TGFα, which promoted granulosa cell proliferation, inhibited the expression of lnc-GULP1- 2:1. These results suggest that lnc-GULP1-2:1 may be involved in proliferation regulation of granulosa cells. Further studies found that the protein levels of COL3A1 were significantly increased in KGN cells with stable over-expression of lnc-GULP1-2:1, indicating that lnc-GULP1-2:1 regulates the expression of COL3A1. Levels of lnc-GULP1-2:1 can be down-regulated by siRNAs (5 nM, 10 nM , and 20 nM) in different concentrations of COL3A1 in KGN cells, and silencing COL3A1 could also significantly inhibit KGN cell proliferation. The results of this study suggest that the involvement of lnc-GULP1-2:1 in the regulation of KGN granulosa cells may partly be mediated through the regulation of its target gene COL3A1. The regulation effect of lnc-GULP1-2:1 on granulosa cell proliferation was consistent with the occurrence of POI in clinical chemotherapeutic agents, suggesting that lnc-GULP1-2:1 may be involved in the regulation of granulosa cell function in POI patients. This is the first study to discover the regulation of lnc-GULP1-2:1 in ovarian granulosa cells, which is of great significance for the further investigation of lncRNA regulation in ovarian follicle development.

Oocyte-Derived Factors (GDF9 and BMP15) and FSH Regulate AMH Expression Via Modulation of H3K27AC in Granulosa Cells.

Anti-Müllerian hormone (AMH) produced by ovarian granulosa cells (GCs) plays a crucial role in ovarian function. It is used as a diagnostic and/or prognostic marker of fertility as well as for pathophysiological conditions in women. In this study, we investigated the underlying mechanism for regulation of AMH expression in GCs using primary mouse GCs and a human GC tumor-derived KGN cell line. We find that growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15) together (GDF9 + BMP15), but not when tested separately, significantly induce AMH expression in vitro and in vivo (serum AMH). Our results show that GDF9 + BMP15 through the PI3K/Akt and Smad2/3 pathways synergistically recruit the coactivator p300 on the AMH promoter region that promotes acetylation of histone 3 lysine 27 (H3K27ac), facilitating AMH/Amh expression. Intriguingly, we also find that FSH inhibits GDF9 + BMP15-induced increase of AMH/Amh expression. This inhibition occurs through FSH-induced protein kinase A/SF1-mediated expression of gonadotropin inducible ovarian transcription factor 1, a transcriptional repressor, that recruits histone deacetylase 2 to deacetylate H3K27ac, resulting in the suppression of AMH/Amh expression. Furthermore, we report that ovarian Amh mRNA levels are significantly higher in Fshβ-null mice (Fshβ-/-) compared with those in wild-type (WT) mice. In addition, ovarian Amh mRNA levels are restored in Fshβ-null mice expressing a human WT FSHβ transgene (FSHβ-/-hFSHβWT). Our study provides a mechanistic insight into the regulation of AMH expression that has many implications in female reproduction/fertility.

LncRNA-LET inhibits cell viability, migration and EMT while induces apoptosis by up-regulation of TIMP2 in human granulosa-like tumor cell line KGN

BackgroundPolycystic ovary syndrome (PCOS) is a common endocrine disease characterized by hyperandrogenism, irregular menses, and polycystic ovaries. Several long non-coding RNAs (lncRNAs) are aberrantly expressed in PCOS patients; however, little is known about the effects of the lncRNA-low expression in tumor (lncRNA-LET) on PCOS. We aimed to explore the effects of lncRNA-LET on human granulosa-like tumor cell line, KGN. MethodsExpression of lncRNA-LET in normal IOSE80 cells and granulosa cells was determined by qRT-PCR. KGN cell viability, apoptosis and migration were measured by trypan blue exclusion method, flow cytometry assay and wound healing assay, respectively. TGF-β1 was used to induce epithelial-mesenchymal transition (EMT) process. LncRNA-LET expression and mRNA expressions of TIMP2 and EMT-related proteins were measured by qRT-PCR. Western blot analysis was used to measure the protein expression of apoptosis-related proteins, EMT-related proteins, TIMP2, and the proteins in the Wnt/β-catenin and Notch signaling pathways. ResultslncRNA-LET was down-regulated in KGN cells, and its overexpression inhibited cell viability and migration, and promoted apoptosis in KGN cells. Overexpression of lncRNA-LET increased the expression of E-cadherin and decreased the expressions of N-cadherin and vimentin in KGN cells. These effects of lncRNA-LET on KGN cells were reversed by TIMP2 suppression. Overexpression of TIMP2 inhibited cell viability, migration and EMT process, and increased apoptosis by activating the Wnt/β-catenin and Notch pathways. ConclusionOverexpression of lncRNA-LET inhibits cell viability, migration and EMT process, and increases apoptosis in KGN cells by up-regulating the expression of TIMP2 and activating the Wnt/β-catenin and notch signaling pathways.

Role of A-kinase anchoring protein 95 in the regulation of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) in human ovarian granulosa cells.

Irregular expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) is involved in the development of polycystic ovary syndrome (PCOS). Activation of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway plays a crucial role in FSH regulation of CYP19A1 in human ovarian granulosa cells. A-Kinase anchor protein 95 (AKAP95) is known to confine PKA to the nucleus. However, it is unclear whether anchoring PKA to the nucleus is essential for the induction of CYP19A1 by FSH in human ovarian granulosa cells. Using the human granulosa cell line KGN and primary cultured human luteinised granulosa cells (hLGCs), we found that knockdown of AKAP8, the gene encoding AKAP95, or inhibition of AKAP95 reduced the amount of PKA anchored in the nucleus and attenuated the phosphorylation of CREB by either FSH or activation of the cAMP/PKA pathway. Moreover, knockdown of AKAP8 or inhibition of AKAP95 also significantly attenuated FSH-induced CYP19A1 expression and oestrogen synthesis. Furthermore, significant decreases in AKAP95 and CYP19A1 were observed in hLGCs obtained from PCOS patients. The results of the present study demonstrate a crucial role for AKAP95 in CYP19A1 expression and oestrogen synthesis in hLGCs, which implies that AKAP95 may be involved in the pathogenesis of PCOS.

Advanced glycation end products interfere in luteinizing hormone and follicle stimulating hormone signaling in human granulosa KGN cells

Advanced glycation end products accumulate in the ovarian granulosa-cell layer of women with polycystic ovarian syndrome. Taken that the MAPK/ERK-pathway is a key regulator of oocyte maturation and function, consisting the main pathway used by the gonadotrophic hormones (luteinizing hormone, follicle stimulating hormone) to control ovulation, the present study aims to assess advanced glycation end products' interference into luteinizing hormone-and follicle stimulating hormone-signaling via the MAPK/ERK-pathway in the human granulosa KGN cell line. KGN cells were treated with luteinizing hormone or follicle stimulating hormone in the absence or presence of human glycated albumin. The specific activation of the main components of the MAPK/ERK1/2-pathway (namely c-Raf, MEK and ERK1/2) was assessed. Treatment of KGN cells with an MEK1/2-inhibitor or a blocking anti-RAGE-antibody was also performed to shed further light on the mechanism of the involvement of advanced glycation end products in luteinizing hormone and/or follicle stimulating hormone-related signaling pathways. Luteinizing hormone treatment increased p-ERK1/2 levels in human granulosa cells, while the combined treatment of luteinizing hormone and human glycated albumin provoked a decrease of p-ERK1/2 levels. A similar reducing effect was also observed for the upstream molecule phospho-cRaf upon combined treatment, while treatment with an MEK-inhibitor confirmed that the phenomenon is MAPK/ERK-pathway-dependent. Similarly, follicle stimulating hormone treatment increased p-ERK1/2 and p-MEK1/2 levels, while the combined treatment of follicle stimulating hormone and human glycated albumin downregulated their levels. Advanced glycation end products reduce the luteinizing hormone- and follicle stimulating hormone-induced ERK1/2 activation that is critical for granulosa cell mitogenesis and proliferation. Inappropriate activation of ERK1/2 in granulosa cells may block the granulosa cell differentiation pathway and/or impair follicular responses to hormones, potentially leading to ovulation failure that characterizes polycystic ovarian syndrome.

Long Noncoding RNAs: Potential Regulators Involved in the Pathogenesis of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility in women of reproductive age, and its etiology remains poorly understood. Altered activities of long noncoding RNAs (lncRNAs) have been associated with human diseases and development. However, the roles of lncRNAs are unknown in reproductive medicine. We investigated the potential role of lncRNAs in the pathogenesis of PCOS, using human granulosa cells (GCs) and the KGN cell line. We used microarrays to compare lncRNA expression profiles in GCs from seven patients with PCOS and seven matched women. GC samples were collected during 2014 to 2016 from infertile women in Guangzhou, China. Quantitative real-time polymerase chain reaction was used to measure levels of the lncRNA HCG26 in GCs from 53 patients with PCOS and 50 controls. HCG26 was knocked down with locked nucleic acid GapmeRs in KGN cells to examine its role in cell proliferation, aromatase and follicle-stimulating hormone receptor gene expression, and estradiol production. A total of 862 lncRNA transcripts and 998 messenger RNA transcripts were differentially expressed (greater than or equal to twofold change; P < 0.05) in PCOS GCs compared with those of controls. HCG26 levels were upregulated in patients with PCOS and were associated with antral follicle count. HCG26 knockdown in KGN cells inhibited cell proliferation and cell-cycle progression and increased aromatase gene expression and estradiol production. Our study reports the lncRNA profiles in GCs from patients who have PCOS and those from healthy women and suggests that dysregulated lncRNAs may play vital roles in GC proliferation and steroidogenesis, providing insights into the pathogenesis of PCOS.

Carbon Black Nanoparticles Inhibit Aromatase Expression and Estradiol Secretion in Human Granulosa Cells Through the ERK1/2 Pathway.

Secretion of 17-β-estradiol (E2) by human granulosa cells can be disrupted by various environmental toxicants. In the current study, we investigated whether carbon black nanoparticles (CB NPs) affect the steroidogenic activity of cultured human granulosa cells. The human granulosa cell line KGN and granulosa cells from patients undergoing in vitro fertilization were treated with increasing concentrations of CB NPs (1 to 100 µg/mL) together or not with follicle-stimulating hormone (FSH). We observed that CB NPs are internalized in KGN cells without affecting cell viability. CB NPs could be localized in the cytoplasm, within mitochondria and in association with the outer face of the endoplasmic reticulum membrane. In both cell types, CB NPs reduced in a dose-dependent manner the activity of aromatase enzyme, as reflected by a decrease in E2 secretion. A significant decrease was observed in response to CB NPs concentrations from 25 and 50 µg/mL in KGN cell line and primary cultures, respectively. Furthermore, CB NPs decreased aromatase protein levels in both cells and reduced aromatase transcript levels in KGN cells. CB NPs rapidly activated extracellular signal-regulated kinase 1 and 2 in KGN cells and pharmacological inhibition of this signaling pathway using PD 98059 significantly attenuated the inhibitory effects of CB NPs on CYP19A1 gene expression and aromatase activity. CB NPs also inhibited the stimulatory effect of FSH on aromatase expression and activity. Altogether, our study on cultured ovarian granulosa cells reveals that CB NPs decrease estrogens production and highlights possible detrimental effect of these common NPs on female reproductive health.

Abstract NTOC-086: PAC–1 COMBINATION WITH TRAIL ENHANCES APOPTOSIS IN CELL–LINE AND PRIMARY CULTURED ADULT GRANULOSA CELL TUMOUR CELLS

Abstract BACKGROUND: Granulosa cell tumour (GCT) constitutes ~5% of ovarian neoplasms. Surgery remains the primary treatment modality due to a generally poor response to chemotherapy in GCT patients. Procaspase activating compound-1 (PAC1) is a small-molecule drug shown in vitro to sequester inhibitory zinc ions from the Caspase-3 (CASP3) zymogen allowing CASP3 to automature and execute apoptosis. PAC1 has demonstrated safety and efficacy in vivo against several cancers and is currently in Phase I trials for advanced malignancies. TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptosis ligand that can bind membrane-bound death receptors. This binding triggers the extrinsic apoptotic pathway resulting in activation of CASP3 to execute its proteolytic role and programmed cell death. TRAIL has been found to induce apoptosis selectively in cancer cells in vitro and in vivo, and has been well tolerated in human patients. We hypothesise that combining PAC1 activation of CASP3 with induction of apoptotic signaling by exogenous TRAIL will significantly heighten biologic effect, reduction of disease, and that these effects will be obtained at doses lower than those required by either agent alone. Here we report on in vitro experiments in support of this hypothesis, suggesting that combining PAC1 with TRAIL may be effective therapy for treatment of GCT. METHODS: The GCT cell line KGN was treated in vitro with a 6-log range of PAC1 concentration for 48 hours to establish a dose-response curve (using a real-time cell analyzer, RTCA). In parallel, KGN cells were treated with a 6-log range of TRAIL concentration to establish its dose-response curve. Calculated EC50 values were then used for both PAC1 (20 μM) and TRAIL (10 ng/mL) to evaluate the biologic response of simultaneous treatment with PAC1 and TRAIL, and TRAIL delayed 24 hours after PAC1 treatment (using resazurin viability assay and RTCA). Separately, cells from fresh primary and recurrent tumour samples were cultured in vitro for 5 days, then treated with PAC1 (20 μM), TRAIL (10 ng/mL), or the combination, and finally assayed for viability and caspase 3/7 activity 48 and 72 hours later. RESULTS: Dose-response assays indicate treatment with PAC1 strongly reduces viability of KGN cells compared to untreated control (p&amp;lt;0.05) in a dose-dependent manner and treatment with PAC1 alone significantly reduced viability compared to untreated control (p&amp;lt;0.05). Similar assays with TRAIL only reduced viability of KGN cells at the highest concentration tested (1 µg/mL). The assays also suggest a ~24 hour delay in PAC1 reduction of GCT viability while TRAIL appears to display a time-limited response. Combination treatment was assessed using calculated EC50 concentrations for both PAC1 (20 µM) and TRAIL (10 ng/mL). Assays for both KGN and patient-derived primary GCT cells tested each drug alone, both drugs applied concurrently and TRAIL applied 24 hours after PAC1. Combination of PAC1 with TRAIL was dramatically more cytotoxic than TRAIL or PAC1 treatment alone (p&amp;lt;0.05) CONCLUSION: Combining CASP3 activator PAC1 with apoptosis-inducing agents may be an effective strategy for treatment of GCT and warrants preclinical assessment. Citation Format: Powel Crosley; Kate Agopsowicz; Marjut Pihlajoki; Markku Heikinheimo; Anniina Färkkilä; Mary Hitt. PAC–1 COMBINATION WITH TRAIL ENHANCES APOPTOSIS IN CELL–LINE AND PRIMARY CULTURED ADULT GRANULOSA CELL TUMOUR CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-086.

Syndecan 1 represses cell growth and FSH responsiveness in human granulosa cells.

Albeit devoid of intrinsic catalytic activity, the transmembrane heparan sulphate proteoglycan syndecan 1 plays critical roles in cellular processes such as extracellular matrix crosstalk, cytoskeletal organization, cell spreading, proliferation and differentiation. During the ovarian cycle, the expression of syndecan 1 in granulosa cells shows cyclic variation suggesting that it might fulfil specific roles in follicle development. To investigate its physiological roles on granulosa cells, syndecan 1 was overexpressed in human granulosa cell line KGN which retains features of granulosa cells from small antral follicle such as estradiol (E2) synthesis and low expression of functional FSH receptor (FSHR). We demonstrated that overexpression of syndecan 1 in immature granulosa cells (KGN-SDC1) induces a profound alteration in their intrinsic characteristics including enhanced spreading and attachment, both associated with a reduced growth rate. Flow cytometry analysis revealed that syndecan 1 overexpression increases the percentage of KGN cells in quiescent phase. This partial cell cycle exit is concordant with downregulated levels of CCND1 and CDK4 and upregulated expression of CDK inhibitor CDKN1A In parallel both unstimulated and FSH-induced E2 synthesis are reduced in KGN-SDC1 through both repression of CYP19A1 and FSHR mRNA associated with decreased levels of potential regulators NR5A1 and ESR2 Additionally, we provide evidence that transient cAMP accumulation reduction in cells overexpressing syndecan 1 is accompanied by an increase in cAMP-hydrolysing PDE activity. Our results demonstrated that syndecan 1 might regulate differentiation of granulosa cells and follicular development by means of various mechanisms involving morphological changes, control of signalling pathways and alterations in gene expressions.Free French abstract: A French translation of this abstract is freely available at http://www.reproduction-online.org/content/153/6/797/suppl/DC1.Reproduction.

Geniposide and geniposidic acid, modified forms of genipin, attenuate genipin-induced mitochondrial apoptosis without altering the anti-inflammatory ability in KGN cell line

Genipin has been demonstrated to induce apoptosis in various cell lines, but in this paper, the apoptosis effect was found attenuated significantly in KGN cell line after when its functional groups was modified, which is a reliable tumor cell line of granulosa cell. Furthermore, geniposide and geniposidic acid, chemicals modified from genipin, were discovered to retain the anti-inflammatory effect of genipin. The apoptosis induced by genipin was considered related to mitochondrial pathway with altered expression of Bcl-2 family, P53, elevated activities of caspase 3 and caspase 9, andactivated mitogen-activated protein kinase members. To evaluate the anti-inflammatory effect and anti-oxidant effect of chemicals modified from genipin, expression of iNOS, IL members, and activities of superoxide dismutase, catalase were investigated. Genipin, geniposide, and geniposidic acid all showed efficient anti-inflammatory effect by inhibiting expression of iNOS and most IL members. Different from genipin, geniposide and geniposidic acid could not inhibit superoxide dismutase activity, showing better antioxidant effects than genipin. In conclusion, genipin was suggested applied after structural modification, retaining anti-inflammatory ability without inducing apoptosis or oxidative stress, and the apoptosis induced by genipin was considered derived from the activated mitogen-activated protein kinase members and mitochondrial pathway.

Iridoids with Genipin Stem Nucleus Inhibit Lipopolysaccharide-Induced Inflammation and Oxidative Stress by Blocking the NF-κB Pathway in Polycystic Ovary Syndrome

Background/Aims: Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women, and it is usually characterized by chronic inflammation, oxidative stress, and altered microRNA expression. The aim of this study is to investigate how the effects of iridoids with genipin stem nucleus inhibit PCOS complications. The interactions between iridoids were investigated, as well. Methods: The chronic inflammation cell model was induced using lipopolysaccharide (LPS) in the RAW 264.7 and KGN cell lines. Levels of mRNA and protein expression were quantified using real time-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The target of the iridoids was identified using the drug affinity responsive target stability (DARTS) method. The ability to scavenge free radicals was evaluated using the DPPH radical scavenging method and the ultra oxygen anion (O₂^-) radical scavenging method. Results: The cells recovered from the inflammatory conditions and showed significantly decreased levels of interleukins after treatment with iridoids. The iridoids were demonstrated to target NF-κB, inhibit the phosphorylation and degradation of IκB, inhibit the nuclear entry of NF-κB, and inhibit the expression of inflammatory factors. Though only genipin showed an efficient ability to scavenge O₂^-, the iridoids, IκB inhibitor (BAY 11-7085), and NF-κB inhibitor (PDTC) could inhibit LPS-induced oxidative stress on the cells, indicating that the iridoids exert their anti-oxidant effects via the NF-κB pathway. The expression levels of microRNAs (miRNAs) were also altered by LPS, but the iridoids could scarcely rescue the abnormal condition. Conclusion: Chronic inflammation may be an important incentive for oxidative stress and abnormal microRNA expression in PCOS, and iridoids can protect patients from inflammatory damage by regulating the NF-κB pathway.

1142 Protein kinase A directly phosphorylates GSK3β, and regulates β-catenin via phosphorylation in granulosa cells

Estradiol serves an important role in female fertility and FSH drives estradiol production. Beta-catenin is a transcriptional co-factor that is required for FSH-induced estradiol production. Beta-catenin is activated via phosphorylation at Ser552 and Ser675 by protein kinase A (PKA) dependent event. Wingless-type mouse mammary tumor virus (WNT) also regulates the β-catenin pathway. Glycogen Synthase Kinase-3 β (GSK3β) is a component of the β-catenin degradation complex; canonical WNT signaling pathway phosphorylates protein kinase B (AKT) and inhibits GSK3β resulting in β-catenin accumulation in the cytoplasm. Work in our laboratory demonstrated that WNT downregulates steroidogenesis. Furthermore, AKT is required for β-catenin accumulation and FSH-induced estradiol production, suggesting convergence of the WNT and FSH pathways. We hypothesize that WNT inhibition of FSH signaling occurs through modulation of phosphorylation patterns on β-catenin. The objective of these experiments was to evaluate the phosphorylation pattern of β-catenin in response to PKA, AKT, and WNT signaling pathways. Granulosa cells (KGN cell line) were cultured and treated with vehicle control, phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (LY) (30 μ M; 30 min), Forskolin (FSK) (10 μ M; 1.5 h), WNT (50 ng/mL, 30 min) and the combination of these treatments. Western blot was used to detect total and phosphorylated β-catenin and phosphorylated GSK3β. Protein abundances were analyzed using densitometry software and densitometry values for treatments were analyzed using the GLM procedure of SAS. When significant model interactions were detected, means were separated using PDIFF. Treatment of FSK combined with WNT and LY enhanced GSK3β phosphorylation compared to control (P < 0.05). Similarly, FSK alone or in combination with WNT and LY enhanced (P < 0.05) phosphorylation of β-catenin at Ser675 and Ser552, but WNT did not alter β-catenin phosphorylation after FSK stimulation (P > 0.10). However, when the AKT pathway was blocked with LY, FSK, and WNT treatment increased phosphorylation of GSK3β and subsequently β-catenin at Ser552 suggesting that PKA can directly phosphorylate GSK3β. Results from these experiments demonstrate that WNT does not alter FSH-stimulated phosphorylation patterns of β-catenin indicating that WNT inhibits FSH signaling via other unknown mechanisms.

MiR-483 is Down-Regulated in Polycystic Ovarian Syndrome and Inhibits KGN Cell Proliferation via Targeting Insulin-Like Growth Factor 1 (IGF1).

BackgroundPolycystic ovarian syndrome (PCOS) is a common metabolic disorder in premenopausal woman, characterized by hyperandrogenism, oligoanovulation, and insulin resistance. microRNAs play pivotal roles in regulating key factors of PCOS. However, relevant research remains limited. This study aimed to reveal the role and potential mechanism of miR-483 in PCOS.Material/MethodsPCOS patients (n=20) were recruited for detecting miR-483 expression in lesion and normal ovary cortex. Human granulosa-like tumor cell line KGN was used to alter miR-483 expression by cell transfection. Cell viability and proliferation were analyzed by MTT assay and colony formation assay, and cell cycle was detected by flow cytometry. Interaction between miR-483 and IGF1 was verified by luciferase reporter assay. KGN cells were further treated by insulin to investigate the relationship between miR-483 and insulin.ResultsmiR-483 was significantly down-regulated in lesion ovary cortex from PCOS patients (P<0.001). In KGN cells, overexpression of miR-483 inhibited cell viability and proliferation, and induced cell cycle arrest. miR-483 also inhibited CCNB1, CCND1, and CDK2. miR-483 sponge induced the opposite effects. miR-483 directly targeted IGF1 3′UTR, and IGF1 promoted KGN cell proliferation and reversed miR-483-inhibited cell viability. Insulin treatment in KGN cells inhibited miR-483, and promoted IGF1 and cell proliferation.ConclusionsThese results suggest that miR-483 is a PCOS suppressor inhibiting cell proliferation, possibly via targeting IGF1, and that it is involved in insulin-induced cell proliferation. miR-483 is a potential alternative for diagnosing and treating PCOS.

A Mixture Reflecting Polybrominated Diphenyl Ether (PBDE) Profiles Detected in Human Follicular Fluid Significantly Affects Steroidogenesis and Induces Oxidative Stress in a Female Human Granulosa Cell Line.

Brominated flame retardants are incorporated into consumer products to prevent flame propagation. These compounds leach into the domestic environment, resulting in chronic exposure. Pregnancy failure is associated with high levels of polybrominated diphenyl ethers (PBDEs), a major class of brominated flame retardants, in human follicular fluid, raising serious questions regarding their impact on female fertility. Our goal was to elucidate the effects of a mixture of PBDEs, similar to the profile found in human follicular fluid, on an immortalized human granulosa cell line, the KGN cell line. We showed that cell viability was altered and oxidative stress was induced as reflected by increased reactive oxygen species formation at 100 μM of the PBDE mixture. Transcriptomic analysis revealed that PBDE treatments of 1, 5, and 20 μM altered the expression of several genes involved in the reactive oxygen species signaling pathway. Significant dose-dependent reductions in progesterone and estradiol levels in the culture medium were measured after PBDE treatment; in parallel, the expression of genes involved in estradiol metabolism, namely CYP1A1, was up-regulated by 5 and 20 μM of the PBDE mixture. Treatment with 20 μM PBDE also increased the expression and secretion of the proinflammatory factor, IL-6, into the KGN cell culture medium. Our results demonstrate that PBDEs can alter human granulosa cell functions by inducing oxidative stress and disrupting steroidogenesis. These results indicate that PBDEs may be detrimental to ovarian functions and thus may adversely affect female reproductive health after chronic exposure.

Effects of BMAL1-SIRT1-positive cycle on estrogen synthesis in human ovarian granulosa cells: an implicative role of BMAL1 in PCOS.

Brain and muscle ARNT-like protein 1 (BMAL1) is necessary for fertility and has been found to be essential to follicle growth and steroidogenesis. Sirtuin1 (SIRT1) has been reported to interact with BMAL1 and function in a circadian manner. Evidence has shown that SIRT1 regulates aromatase expression in estrogen-producing cells. We aimed to ascertain if there is a relationship between polycystic ovary syndrome (PCOS) and BMAL1, and whether and how BMAL1 takes part in estrogen synthesis in human granulosa cells (hGCs). Twenty-four women diagnosed with PCOS and 24 healthy individuals undergoing assisted reproduction were studied. BMAL1 expression in their granulosa cells (GCs) was observed by quantitative real-time polymerase chain reaction (qRT-PCR). The level of expression in the PCOS group was lower than that of the group without PCOS (p<0.05). We also analyzed estrogen synthesis and aromatase expression in KGN cell lines. Both were downregulated after BMAL1 and SIRT1 knock-down and, conversely, upregulated after overexpression treatments of these two genes in KGN cells. Both BMAL1 and SIRT1 had a mutually positive regulation, as did the phosphorylation of JNK. Furthermore, JNK overexpression increased estrogen synthesis activity and the expression levels of aromatase, BMAL1, and SIRT1. In KGN and hGCs, estrogen synthesis and aromatase expression were downregulated after treatment with JNK and SIRT1 inhibitors. In addition, BMAL1, SIRT1, and JNK expression levels were all downregulated. Our results demonstrate the effects of BMAL1 on estrogen synthesis in hGCs and suggest a BMAL1-SIRT1-JNK positive feedback cycle in this process, which points out an important role of BMAL1 in the development of PCOS.

Circulating levels of TNF-related apoptosis inducing-ligand are decreased in patients with large adult-type granulosa cell tumors-implications for therapeutic potential.

Targeted treatments are needed for advanced adult-type granulosa cell tumors (AGCTs). We set out to assess tumor tissue and circulating levels of TNF-related apoptosis-inducing ligand (TRAIL), a promising anti-cancer cytokine, in patients affected by AGCT. We analyzed tissue expression of TRAIL in 127 AGCTs using immunohistochemistry or RT-PCR. Soluble TRAIL was measured by means of ELISA from 141 AGCT patient serum samples, as well as the conditioned media of 15 AGCT patient-derived primary cell cultures, and the KGN cell line. Tissue and serum TRAIL levels were analyzed in relationship with clinical parameters, and serum estradiol, FSH, and LH levels. We found that AGCT samples expressed TRAIL mRNA and protein at levels comparable to normal granulosa cells. AGCT cells did not release soluble TRAIL. TRAIL protein levels were decreased in tumors over 10cm in diameter (p = 0.04). Consistently, circulating TRAIL levels correlated negatively to tumor dimension (p = 0.01). Circulating TRAIL levels negatively associated with serum estradiol levels. In multiple regression analysis, tumor size was an independent factor contributing to the decreased levels of soluble TRAIL in AGCT patients. AGCTs associate with significantly decreased tumor tissue and serum TRAIL levels in patients with a large tumor mass. These findings encourage further study of agonistic TRAIL treatments in patients with advanced or recurrent AGCT.

The endocannabinoid system in the human granulosa cell line KGN

Ovarian steroidogenesis is embedded in a sensitive network of regulatory mechanisms crucial for human fertility. The endocannabinoid system (ECS) represents an intrinsic modulating system involved in the regulation of endocrine functions. In the present study we characterized the ECS in the human granulosa cell line KGN and its impact on gonadotropin sensitivity and steroid hormone synthesis under basal and FSH-stimulated conditions.Expression studies were performed and estradiol was measured. CB1, CB2, DAGL, FAAH, GPR55, MAGL, NAPE-PLD and TRPV1 were expressed without FSH-dependent effects. Treatment with selective cannabinoid receptor agonists reduced basal but not FSH-stimulated estradiol and CYP19. Progesterone was not altered by ECS manipulation. CB1 agonist changed the expression of miRNAs associated with granulosa cell function, e.g. miR-23a, miR-24, miR-181a and miR-320a. Present data indicate a modulating role of the intrinsic ovarian ECS in the regulation of estradiol synthesis.

MiRNA-592 is downregulated and may target LHCGR in polycystic ovary syndrome patients

The polycystic ovary syndrome (PCOS) is an endocrine disorder mainly associated with infertility. Abnormal regulation of relevant genes is required for follicular development in PCOS. In the current study, the expression of serum miRNAs of PCOS patients was explored using miRNA array followed by qRT-PCR assays. The circulating level of miR-592 was significantly down-regulated in PCOS patients in comparison with healthy controls. Furthermore, we found that miR-592 was inversely correlated with the level of luteinizing hormone/chorionic gonadotropin receptor (LHCGR). Computational analysis predicted that miR-592 interacts with the LHCGR mRNA via binding to a site located in the 3′UTR region. Using a luciferase-based reporter assay we found that miR-592 directly targeted the LHCGR. In KGN cell line, miR-592 overexpression inhibited cell viability and the transition of phase G1 to phase S. Knocking down of LHCGR inhibited cell viability and cell cycle progression in KGN cells, and LHCGR co-transfection reversed the inhibitory effect of miR-592. These results shed new light on the diagnosis and treatment of PCOS syndrome.