Human Granulosa Cell Line KGN Research Articles

Lycopene mitigates DHT-induced apoptosis and oxidative stress in human granulosa cell line KGN by regulating the Nrf2 pathway

Lycopene mitigates DHT-induced apoptosis and oxidative stress in human granulosa cell line KGN by regulating the Nrf2 pathway

Perfluorooctane sulfonate (PFOS) induces apoptosis and autophagy by inhibition of PI3K/AKT/mTOR pathway in human granulosa cell line KGN

Perfluorooctane sulfonate (PFOS) is recognized as an environmental endocrine disruptor with widespread use in industrial manufacturing and daily life, contributing to various public health concerns. However, the precise impacts of PFOS on the ovary and its regulatory mechanisms remain unclear. This study aims to delineate the ovarian toxicity of PFOS and scrutinize its effects on apoptosis and autophagy through modulation of the PI3K/AKT/mTOR pathway in the human granulosa cell line (KGN). Cell viability, assessed via the Cell Counting Kit-8 (CCK8), revealed a dose-dependent reduction in cell viability upon PFOS exposure. Flow cytometry analysis demonstrated an elevated proportion of apoptotic cells following PFOS treatment. Western blot analyses unveiled increased expression of Bax, Cyt c, cleaved caspase-9, and LC3-II/I, coupled with decreased expression of Bcl-2 and p62. Transmission electron microscopy (TEM) observations illustrated a heightened number of autophagosomes induced by PFOS. Molecular docking investigations, in conjunction with Western blot experiments, substantiated PFOS's significant inhibition of the PI3K/AKT/mTOR signaling pathway. These findings collectively underscore that PFOS induces apoptosis and autophagy in KGN cells through modulation of the PI3K/AKT/mTOR pathway, providing experimental evidence for PFOS-induced ovarian toxicity and elucidating the underlying regulatory mechanisms in KGN cells.

Human Bone Marrow Derived-Mesenchymal Stem Cells Treatment for Autoimmune Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI) is a relatively common gynecologic endocrine disorder, which is hypogonadism associated with amenorrhea, increased levels of gonadotropins, and hypoestrogenism. POI resulting from ovarian autoimmunity is a poorly understood clinical condition lacking effective treatments. This study is aimed to investigate the therapeutic effect of mesenchymal stem cells (MSCs) on autoimmune premature ovarian insufficiency. In this study, in vivo and in vitro experiments were conducted to clarify the therapeutic effects and possible mechanisms of human bone marrow-derived MSCs (hBMSCs) on autoimmune POI, and to provide an experimental evidence for the treatment of autoimmune POI by hBMSCs. Noteworthy, in this study, we used interferon-gamma (IFN-γ) to induce autoimmune inflammation in human granulosa cell line KGN, simulating the pathophysiological changes of granulosa cells in autoimmune POI, and therefore sought to establish an in vitro cell model of autoimmune POI, which is still lacking in experimental methodology. And we found that, in vitro, co-culture of hBMSCs could promote granulosa cell proliferation, inhibit apoptosis, improve hormone synthesis capacity, and reduce the occurrence of pyroptosis; and in vivo, hBMSCs resulted in improved estrous cycle disorders in autoimmune POI mice, increased serum estradiol, decreased follicle-stimulating hormone, improved ovarian morphology, increased number of primordial and primary follicles, decreased number of atretic follicles, and decreased ovarian granulosa cell apoptosis. hBMSCs have therapeutic effects on autoimmune POI both in vitro and in vivo.

Yangjing Zhongyu decoction facilitates mitochondrial activity, estrogenesis, and energy metabolism in H2O2-induced human granulosa cell line KGN

Ethnopharmacological relevantYangjing Zhongyu decoction (YJZYD) is a recipe from a Chinese classic medical work and has been empirically used in female infertility for hundreds of years, but the mechanisms of YJZYD on facilitating ovarian granulosa cells remain unfold. Aim of the researchThe purpose of the study is to determine the rewarding effects of YJZYD on H2O2-induced KGN cells, involving mitochondrial activity, estradiol biosynthesis, and energy metabolism. Materials and methodsThe ingredients of YJZYD were investigated by UPLC-ESI-MS/MS analysis. The effects of YJZYD and H2O2 on cell viability were determined by CCK-8. Intracellular ROS were assessed by DCFH-DA. Intracellular Ca2+ was detected using Fura-4 AM. Mitochondrial membrane potential (MMP) was measured by JC-1. The production of energy was assessed by ATP. Apoptosis rate was analyzed by Annexin V-FITC/PI. Western blotting was used to evaluate the expression of proteins related to energy metabolism, apoptosis, mitochondrial mitophagy, and estrogen biosynthesis. E2 levels were measured by ELISA. Results121 compounds were identified in YJZYD by UPLC-ESI-MS/MS analysis. YJZYD could enhance mitochondrial activity by suppressing intracellular ROS and Ca2+, and increasing MMP and ATP content. YJZYD stimulated the expression of anti-apoptosis protein Bcl-2 and lowered the early apoptosis rate and the expression of Bax. Besides, YJZYD rescued E2 secretion and improved the expression of FSHR, CYP19A1, and the ratio of p-CREB/CREB. In addition, YJZYD weakened H2O2-induced mitophagy by compromising the expression of PINK1, Parkin, Beclin1 and P62. Moreover, YJZYD strengthened energy metabolism by increasing ATP generation and the expression of SIRT1, PGC1α, NRF1, and COX IV. The combination of YJZYD and autophagy inhibitor had a stronger protective effect on energy metabolism. ConclusionThis study evaluated the protective effects of YJZYD on H2O2-induced KGN cells. YJZYD could enhance mitochondrial activity, E2 biosynthesis, and energy metabolism. These results strongly indicated that YJZYD might play a role in preserving ovarian granulosa cells and female fecundity.

Salidroside alleviates oxidative stress and apoptosis via AMPK/Nrf2 pathway in DHT-induced human granulosa cell line KGN

In the past few years, emerging evidence established persistent oxidative stress to be a key player in the pathogenesis of polycystic ovary syndrome (PCOS). Particularly, it damages the function of granulosa cells, and thus hinders the development of follicles. The present study aimed to explore and establish the protective effects of salidroside on dihydrotestosterone (DHT)‐induced Granulosa‐like tumor cell line (KGN), mediated via antioxidant mechanisms. The study assessed the positive effects of salidroside on DHT‐induced apoptosis, reactive oxygen species (ROS) accumulation, damage of antioxidant capacity, and mitochondrial membrane potential depolarization. Interestingly, salidroside partly reversed DHT mediated effects, via stimulation of nuclear factor erythroid 2‐related factor 2 (Nrf2) signaling pathway and the downstream antioxidant proteins heme oxygenase‐1(HO‐1) and quinine oxidoreductase 1(NQO1). Additionally, the knockdown of Nrf2 partly moderated the antioxidant and anti-apoptosis effects of salidroside in DHT-treated KGN cells. Mechanistically, AMP‐activated protein kinase (AMPK) was identified to be the upstream signaling involved in salidroside‐induced Nrf2 activation, as silencing of AMPK partly prevented the upregulation of Nrf2 and the downstream proteins HO‐1 and NQO1. Altogether, the present study is the first to effectively demonstrate the inhibitory effect of salidroside on DHT‐stimulated oxidative stress and apoptosis in KGN cells, which was dependent on Nrf2 activation that involved AMPK.

Bisphenol A and bisphenol AF co-exposure induces apoptosis in human granulosa cell line KGN through intracellular stress-dependent mechanisms

Bisphenol A (BPA) is a chemical substance used in large amounts in the production of epoxy resins, phenolic resins and polycarbonate plastics. Although its toxicity to the female reproductive system has been extensively studied, little is known about the combined toxicity of BPA and its analogues. In this study, the human granulosa cell line KGN was co-exposed to BPA and bisphenol AF (BPAF), and we subsequently examined the effect of these chemicals on cell apoptosis and their mechanism. We found that co-exposure to BPA and BPAF induced intracellular stress in KGN cells, including significantly elevated levels of reactive oxygen species (ROS) and calcium ions (Ca 2+ ). Then, apoptosis and its associated biological events, including increased caspase-3 activity, and decreased Bcl-2/Bax protein expression ratio, were measured under the combined exposure. Notably, we confirmed that the intracellular stress and activation of the signaling axis of the downstream ASK1-JNK signaling pathway involved in the apoptosis of KGN cells was induced by the mixture of BPA and BPAF. In addition, we verified with pretreatment inhibitors that the BPA and BPAF mixture promoted the co-induction of KGN cell apoptosis via this stress pathway. Our work provides novel insights into the combined cytotoxicity and molecular toxicity mechanism of bisphenol mixtures.

Transcriptional regulation of CYP19 by cohesin-mediated chromosome tethering in human granulosa cells

Human CYP19 spans a region of chromosome 15 of approximately 130 kb and encodes aromatase, an enzyme required for estrogen synthesis. In the human granulosa cell-line KGN, there are seven open chromatin regions within the CYP19 locus. In this study, we demonstrate that two of these regions ~40 kb upstream and ~15 kb downstream of the CYP19 promoter are cohesin-loading sites, physically interacting with the promoter to negatively and positively regulate transcription, respectively. These observations suggest that CYP19 expression is controlled by a balance between the upstream silencer and downstream enhancer. When cohesin is depleted, CYP19 expression is elevated since the silencer is 2.5-fold further from the promoter than the enhancer and most likely depends on cohesin-mediated tethering to influence expression.

Melatonin ameliorates ovarian dysfunction by regulating autophagy in PCOS via the PI3K-Akt pathway.

Emerging evidence has demonstrated that melatonin (MT) plays a crucial role in regulating mammalian reproductive functions. It has been reported that MT has a protective effect on polycystic ovary syndrome (PCOS). However, the protective mechanisms of MT remain poorly understood. This study aims to explore the effect of MT on ovarian function in PCOS and to elucidate the relevant molecular mechanisms in vivo and in vitro. We first analysed MT expression levels in the follicular fluid of PCOS patients. A significant reduction in MT expression levels was noted in PCOS patients. Intriguingly, reduced MT levels correlated with serum testosterone and inflammatory cytokine levels in follicular fluid. Moreover, we confirmed the protective function of MT through regulating autophagy in a DHEA-induced PCOS rat model. Autophagy was activated in the ovarian tissue of the PCOS rat model, whereas additional MT inhibited autophagy by increasing PI3K--Akt pathway expression. In addition, serum-free testosterone, inflammatory and apoptosis indexes were reduced after MT supplementation. Furthermore, we also found that MT suppressed autophagy and apoptosis by activating the PI3K-Akt pathway in the DHEA-exposed human granulosa cell line KGN. Our study showed that MT ameliorated ovarian dysfunction by regulating autophagy in DHEA-induced PCOS via the PI3K-Akt pathway, revealing a potential therapeutic drug target for PCOS.

Bisphenol A and Its Analogues Exhibit Different Cytotoxic and Mitochondrial Dysfunction Potential in Human Granulosa Cells

Bisphenol A (BPA) is an environmental endocrine disruptor and has been strongly associated with the development of numerous diseases, including ovarian follicle development disorders. BPA is being replaced by structurally similar chemicals, such as bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF). However, the toxicity of these analogues in female reproduction is unclear. Here, we investigated the induction of cytotoxicity and mitochondrial dysfunction in the human granulosa cell line KGN by BPA and its selected analogues. We found that BPA and its analogues, especially BPAF, significantly reduced cell viability and caused cytotoxicity. Furthermore, we observed that BPA and BPAF significantly reduced mitochondrial function, including decreasing ATP generation, promoting ROS production and increasing intracellular Ca2+ levels. An oxidative-antioxidant imbalance was also detected after exposure to these chemicals. In contrast, the total antioxidant capacity was significantly reduced. To our knowledge, this is the first report on the evaluation of the potential of BPA and its analogues to induce cytotoxicity and mitochondrial dysfunction in ovarian granulosa cells. Our study revealed the possible mechanism of BPA and its analogues inducing granulosa cell damage and suggested that mitochondrial dysfunction may play an important regulatory role in bisphenol-induced follicular development disorders.

Bisphenol A induces apoptosis through GPER-dependent activation of the ROS/Ca2+-ASK1-JNK pathway in human granulosa cell line KGN

Bisphenol A (BPA) is widely distributed in the environment and human surroundings and is closely related to the occurrence of many chronic diseases including female infertility. Although BPA-induced granulosa cell apoptosis has been widely reported, the underlying mechanisms remain unknown. In this study, we evaluated the induction effect of BPA exposure on apoptosis and mechanisms of regulation in KGN cells (a human granulosa-like tumor cell line). Our results indicated that BPA induced apoptosis of KGN cells in a dose- and time-dependent manner. BPA exposure significantly promoted the expression of pro-apoptotic proteins and decreased mitochondrial membrane potential. We also observed that high concentrations of BPA significantly promoted the generation of reactive oxygen species (ROS) and calcium ion (Ca2+) accumulation. The involvement of ROS and Ca2+ in BPA-induced KGN cell apoptosis was confirmed by pretreatment with NAC (an antioxidant) and BAPTA-AM (a calcium chelator). After inhibitors pretreatment to block the corresponding signaling pathways, it was found that BPA-induced phosphorylation of JNK and ASK1 proteins and apoptosis of KGN cells were significantly inhibited. We pretreated with G15 (a GPER inhibitor) and found that BPA-induced ROS generation and Ca2+ accumulation and apoptosis were significantly inhibited. These results suggest that BPA exposure induces KGN cell apoptosis through GPER-dependent activation of the ROS/Ca2+-ASK1-JNK signaling pathway. Our study provides mechanisms by which BPA induced apoptosis of granulosa cells and ovarian dysfunction.

Long Noncoding RNA HUPCOS Promotes Follicular Fluid Androgen Excess in PCOS Patients via Aromatase Inhibition.

Androgen excess is a key feature of polycystic ovary syndrome (PCOS), but the underlying molecular mechanism remains unclear. To determine the role and mechanism of novel long noncoding RNA (lncRNA) highly up-regulated in PCOS (HUPCOS) in the androgen excess of PCOS patients. The lncRNA expression profile in granulosa cells derived from PCOS and non-PCOS women were analyzed by using microarray assay. Human granulosa cell line KGN was used for mechanism investigation. This was a university-based study. Thirty-eight PCOS and 38 control patients were recruited: 8 PCOS and 8 control samples used for microarray discovery, the remaining 30 PCOS cases and 30 controls for quantitative RT-PCR validation. The aberrant expression lncRNA profile of PCOS patients was measured using microarray. The relationship of HUPCOS and follicular fluid testosterone was measured. Aromatase expression were analyzed after HUPCOS downregulation. HUPCOS interaction protein was confirmed by RNA pull-down. The significantly elevated lncRNA in PCOS granulosa cells was named HUPCOS, which was positively correlated with follicular fluid testosterone of PCOS patients. HUPCOS downregulation increased aromatase expression and promoted conversion of androgen to estrogen. RNA-binding protein with multiple splicing (RBPMS) was the most likely protein that combined with HUPCOS. Our findings suggested that HUPCOS mediated androgen excess in follicular fluid of PCOS patients by suppressing aromatase expression via interaction with RBPMS.

Prenatal ethanol exposure induces susceptibility to premature ovarian insufficiency.

Prenatal ethanol exposure (PEE) adversely affects the offspring reproductive system. We aimed to confirm the susceptibility to premature ovarian insufficiency (POI) in female PEE offspring and elucidate its intrauterine programming mechanism. The pregnant Wistar female rats were intragastrically administered with 4 g/kg×d of ethanol from gestational day (GD) 9 to 20. Offspring reproductive parameters were detected on GD20, postnatal week (PW) 6, and PW12. The PEE foetuses showed a decreased number of oocytes, increased ovarian cell apoptosis, and upregulated expression levels of ovarian insulin-like growth factor 1 (IGF1) signaling pathway and steroidogenic enzymes. The proportion of atretic follicles in adult rats was increased, while the number of anti-Müllerian hormone-positive antral follicles was decreased. The serum oestradiol (E2) levels were decreased, but the follicle stimulation hormone levels were elevated. The ovarian Igf1 signaling pathway was transformed from activation during puberty to relative inhibition in adulthood, and the expression levels of ovarian steroidogenic enzymes were inhibited in adulthood. Furthermore, we treated the human granulosa cell line KGN with different ethanol concentrations (15, 30, 60, 120 mM) and found that the expression of IGF1 signaling pathway components, 3β-HSD, and P450arom, as well as the production of E2, was increased. After IGF1 siRNA transfection, P450arom expression and E2 production were downregulated. These results suggest that PEE induces POI susceptibility in adult females, which may be caused by over-activation of the foetal ovarian Igf1 signaling pathway and steroidogenesis under PEE, resulting in accelerated early development of folliculogenesis and depletion of primordial follicles.

Decreased levels of H3K9ac and H3K27ac in the promotor region of ovarian P450 aromatase mediated low estradiol synthesis in female offspring rats induced by prenatal nicotine exposure as well as in human granulosa cells after nicotine treatment

Prenatal nicotine exposure (PNE) could induce ovarian dysplasia in offspring. This study aimed to confirm its intrauterine origin and explore a programming mechanism of ovarian dysplasia caused by PNE. Pregnant Wistar rats were injected subcutaneously with nicotine (2 mg/kg.d) from gestation day (GD) 9 to GD20. Serum of female offspring was obtained for hormone assays and ovarian tissues were collected. The results showed that PNE impaired ovarian development, and inhibited estradiol production and cytochrome P450 aromatase (P450arom) expression before and after birth. Moreover, the nicotinic acetylcholine receptors (nAChRs) expression was increased in utero, while histone 3 lysine 9 acetylation (H3K9ac) and H3K27ac levels in the P450arom promoter region were decreased persistently in PNE group before and after birth. In vitro, nicotine decreased P450arom expression and estradiol production in human granulosa cell line KGN. Furthermore, nicotine treatment up-regulated nAChRα6 and α9 expression and down-regulated the H3K9ac and H3K27ac levels of the P450arom promoter region. Non-specific nAChRs inhibitor vecuronium bromide reversed these effects. These results suggest that PNE could induce ovarian dysplasia and inhibit estradiol synthesis in the female offspring rats, which was related to the decreased H3K9ac and H3K27ac levels in the promotor region of the P450arom via the nAChRs.

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.

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.

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.

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.

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.

The AhR is constitutively activated and affects granulosa cell features in the human cell line KGN

A well-balanced activity of the aryl hydrocarbon receptor (AhR) is necessary for normal ovarian function. As known from murine AhR knock-out (KO) models, the AhR is involved in folliculogenesis, gonadotrophin receptor expression, proliferation of granulosa cells and intraovarian estrogen signalling. Highly potent, non-physiological ligands such as the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) lead to a blockade of ovulation, estrogen receptor degradation and reduction of estrogen levels. Estrogen synthesis is a typical function of granulosa cells and essential for normal cyclicity and fertility. We employed the human granulosa cell line KGN to further characterize AhR signalling and AhR function in granulosa cell physiology. Real-time PCR quantification of the target genes Cyp1a1 and Cyp1b1 and reporter gene assays after stimulation with TCDD or beta-naphthoflavone (BNF) or inhibition with alpha-naphthoflavone (ANF) or 3'-methoxy-4'-nitroflavone (3,4-MNF) of the AhR demonstrated constitutive activity and functionality of AhR pathway in KGN granulosa cells. In untreated KGN cells, AhR protein was exclusively detected in the nuclear fraction. TCDD stimulation affected the gonadotrophin receptor but not estrogen receptor β (ERβ) protein expression. Additionally, the constitutively activated AhR suppressed aromatase expression and estrogen synthesis (enzyme-linked immunoassay, ELISA) and enhanced proliferation [Bromodeoxyuridine (BrdU) ELISA] of KGN cells. Activation of the AhR by BNF did not override this inhibitory effect on estrogen synthesis or proliferation. In conclusion, the AhR pathway is constitutively activated and functional in human KGN granulosa cells. It is a potential target for endocrine disruption by exogenous ligands and subsequent dysfunction of granulosa cells.

Studies of Human FSH Receptor Mutant K376A Which Does Not Bind APPL1 when Expressed in Human Granulosa Cell Line KGN.

Studies of Human FSH Receptor Mutant K376A Which Does Not Bind APPL1 when Expressed in Human Granulosa Cell Line KGN.