Human Granulosa-lutein Research Articles

Cordycepin suppresses steroidogenic acute regulatory protein expression by reducing SP1 in human granulosa-lutein cells.

Cordycepin (COR), a compound derived from Cordyceps, is recognized as an adenosine analog with numerous beneficial effects on human health. However, its impact on steroidogenic acute regulatory protein (STAR) expression in ovarian granulosa cells is not well understood. This study demonstrates that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. Cordycepin (COR), a pure compound of Cordyceps, is known as an adenosine analog that exerts many beneficial effects on human health. The steroidogenesis mediated by ovarian granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (STAR) regulates the rate-limiting step in steroidogenesis. COR has been shown to stimulate STAR expression in mouse Leydig cells, the steroidogenic cells in the testes. However, the effect of COR on STAR expression in ovarian granulosa cells remains undetermined. In the present study, we show that treatment with COR downregulates STAR expression in a steroidogenic human granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization. We used specific adenosine receptor (AR) antagonists, and our results reveal that the inhibitory effect of COR on STAR expression is mediated by AR-A1, AR-A2A, and AR-A3. In both KGN and primary hGL cells, COR activates ERK1/2 and AKT signaling pathways, but only activation of ERK1/2 is required for the COR-induced downregulation of STAR expression. In addition, our results demonstrate that COR downregulates STAR expression by reducing the expression of the SP1 transcription factor. These results provide a better understanding of the biological function of COR on STAR expression in the ovary, which may lead to the development of alternative therapeutic approaches for female reproductive disorders.

Interleukin-1 increases SERPINE1 expression in human granulosa-lutein cell via P50/P52 signaling pathways

It has been reported that immune factors are associated with the occurrence of polycystic ovary syndrome (PCOS). Interleukin-1 (IL-1) is a member of the interleukin family that widely participates in the regulation of the inflammatory response in the immune system. In addition, it has been reported that aberrant IL-1 accumulation in serum is associated with the occurrence of PCOS. However, little is known about how IL-1 participates in the pathogenesis of PCOS. In the present study, we demonstrated that the immune microenvironment was altered in follicular fluid from PCOS patients and that the expression levels of two IL-1 cytokines, IL-1α and IL-1β were increased. Transcriptome analysis revealed that IL-1α and IL-1β treatment induced primary human granulosa-lutein (hGL) cell inflammatory response and increased the expression of serpin family E member 1 (SERPINE1). Mechanistically, we demonstrated that IL-1α and IL-1β upregulated SERPINE1 expression through IL-1R1-mediated activation of downstream P50 and P52 signaling pathways in human granulosa cells. Our study highlighted the role of immune state changes in the occurrence of PCOS and provided new insight into the treatment of patients with IL-1-induced ovarian function disorders.

EGF-like growth factors upregulate pentraxin 3 expression in human granulosa-lutein cells

The epidermal growth factor (EGF)-like factors, comprising amphiregulin (AREG), betacellulin (BTC), and epiregulin (EREG), play a critical role in regulating the ovulatory process. Pentraxin 3 (PTX3), an essential ovulatory protein, is necessary for maintaining extracellular matrix (ECM) stability during cumulus expansion. The aim of this study was to investigate the impact of EGF-like factors, AREG, BTC, and EREG on the expression and production of PTX3 in human granulosa-lutein (hGL) cells and the molecular mechanisms involved. Our results demonstrated that AREG, BTC, and EREG could regulate follicular function by upregulating the expression and increasing the production of PTX3 in both primary (obtained from 20 consenting patients undergoing IVF treatment) and immortalized hGL cells. The upregulation of PTX3 expression was primarily facilitated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway, induced by these EGF-like factors. In addition, we found that the upregulation of PTX3 expression triggered by the EGF-like factors was completely reversed by either pretreatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, or knockdown of EGFR, suggesting that EGFR is crucial for activating the ERK1/2 signaling pathway in hGL cells. Overall, our findings indicate that AREG, BTC, and EREG may modulate human cumulus expansion during the periovulatory stage through the upregulation of PTX3.

Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 μM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

#285 : Amphiregulin Increases Pentraxin 3 Production via the EGFR-Mediated Erk1/2 Signaling in Human Granulosa Cells

Background and Aims: Pentraxin 3 (PTX3) plays a key role in the assembly of the extracellular matrix, which is critical for cumulus expansion, ovulation, and in vivo fertilization. As a member of the epidermal growth factor family, amphiregulin (AREG) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus expansion in mammalian follicles. The aim of this study was to investigate whether AREG regulates the expression and production of PTX3 in human granulosa cells and to examine its underlying molecular determinants. Method: Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein (hGL) cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression and accumulated levels of PTX3 were examined following cell incubation with AREG at different time points using the RT-qPCR and ELISA, respectively. Two kinase inhibitors (AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) were used to verify the specificity of the effects and to investigate the molecular mechanisms. The results were analyzed by one-way analysis of variance. Results: The results showed that AREG upregulated the expression and production of PTX3 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR receptor. Furthermore, we revealed that the Erk1/2 signaling is the downstream signaling pathway that mediates the increase in PTX expression and production in response to AREG treatment in hGL cells. Conclusion: AREG upregulated the expression of PTX3, leading to increased production of PTX3 in hGL cells. The AREG-induced cellular activities were most likely driven by the EGFR-mediated Erk1/2-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which AREG regulates the process of cumulus expansion and subsequent ovulation in humans.

#236 : Alleviating Effects of EGCG on Ovarian Hyperstimulation Syndrome and the Potential Modulating Mechanism

Background and Aims: Ovarian hyperstimulation syndrome (OHSS) is one of the most severe complications of COH during IVF treatment. Increased capillary permeability is a hallmark of the pathophysiology of OHSS, vascular endothelial growth factor (VEGF) is an important mediator in OHSS, and studies have established a correlation between serum VEGF levels and the severity of the OHSS. (-)-epigallocatechin-3-gallate (EGCG), the most prevalent and biologically active polyphenolic catechin in green tea, has been reported to produce a number of beneficial effects on humans. Numerous studies have shown that in many pathological processes, EGCG could inhibit VEGF and its receptor expression and have an angiogenesis effect. Herein, EGCG might have a therapeutic effect on OHSS. Methods: We investigated the role of EGCG in OHSS both in vitro and in vivo. Primary human granulosa-lutein (hGL) cells and a human granulosa-like tumor (KGN) cell line were cultured and treated for 24 hours with various concentrations of EGCG. In SD rats, an animal OHSS model was established by injecting pregnant mare serum gonadotropin (PMSG) and randomly assigning them to receive vehicle only or EGCG for three days. For comparison, all experiments were repeated 3-8 times. The effect of EGCG on KGN and hGL cells was determined by MTT assay. The body weight of rats was measured every day, and the ovary size and weight were measured after removal. Evans-blue was used to determine permeability. ELISA was used to measure serum estrogen and VEGF levels in rats. RNA and protein expressions of VEGF, VEGFR-2, TGF-[Formula: see text]1, and T[Formula: see text]R II were detected by qPCR and Western-blotting and immunostaining in vitro and in vivo experiments. Results: Our research found that giving rats EGCG prevented the development of OHSS, as evidenced by histological examination, ovarian weight, and morphology. When compared to the OHSS group, the EGCG treatment group had significantly lower ovary weight (147.9 vs 206.5 mg) (Fig. 1). Furthermore, when compared to OHSS rats, EGCG-treated rats have lower ovarian VEGF expression as measured by IHC and RT-qPCR. VEGF and E2 protein levels in the serum of the EGCG treatment group are significantly lower. EGCG exerted inhibitory effects on cell growth only in high dose (50 uM) and longtime (48 h) treatment in KGN and hGL cells. In KGN cells and hGL cells, EGCG significantly reduces the expression of VEGF and TGF-[Formula: see text] at the RNA and protein levels. Furthermore, EGCG inhibits TGF-[Formula: see text]1-induced VEGF production and secretion in KGN cells by suppressing TGF-[Formula: see text] expression and its traditional Smad signaling pathway. EGCG also downregulates VEGF expression through the 67-kDa laminin receptor-mediated PKA-CREB pathway (Fig. 2). Conclusions: EGCG inhibited VEGF via the TGF-[Formula: see text]1 classical-SMAD pathway and the 67LR-mediated CREB pathway, reducing ovarian inflammation and slowing the progression of OHSS in a rat model.

Bone morphogenetic protein 6 induces downregulation of pentraxin 3 expression in human granulosa lutein cells in women with polycystic ovary syndrome

PurposeTo evaluate whether PTX3 is differentially expressed in the granulosa lutein cells derived from women with PCOS and whether BMP6 can regulate the expression of PTX3 in hGL cells.MethodsThe expression levels of BMP6 and PTX3 in granulosa lutein cells were evaluated by RT-qPCR. The correlation between the expression levels of BMP6 /PTX3 and oocyte quality indexes were analyzed using clinical samples. The cells were incubated with BMP6 at different concentrations and times to check the expression of PTX3 in KGN cells. TGF-β type I inhibitors and small interfering RNA targeting ALK2/3/6,SMAD1/5/8 and SMAD4 were used to study the involvement of SMAD dependent pathways in KGN cells.ResultsThe levels of BMP6 in hGL cells were negatively correlated with the corresponding oocyte maturation rate and high-quality embryo rate, whereas the levels of PTX3 were positively correlated with the corresponding oocyte maturation rate in PCOS. Additionally, the in vitro cell cultured results showed BMP6 significantly inhibited the expression of PTX3 in KGN cells. Furthermore, using a dual inhibition approach (kinase inhibitors and small interfering RNAs), we identified the ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors and the downstream SMAD1/SMAD5-SMAD4 signaling pathway were responsible for the BMP6-induced cellular activities in KGN cells.ConclusionsThe suppressive effect of BMP6 on PTX3 was mediated by ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors in granulosa cells through the SMAD1/5-SMAD4 dependent signaling pathway in PCOS.Our findings provides new insights into the understanding of the pathogenesis of PCOS-related ovulatory disorders.

Oral follicle-stimulating hormone receptor agonist affects granulosa cells differently than recombinant human FSH

To determine whether TOP5300, a novel oral follicle-stimulating hormone (FSH) receptor (FSHR) allosteric agonist, elicits a different cellular response than recombinant human FSH (rh-FSH) in human granulosa cells from patients undergoing invitro fertilization. Basic science research with a preclinical allosteric FSHR agonist. University hospital. Patients with infertility at a single academic fertility clinic were recruited under an Institutional Review Board-approved protocol. Primary granulosa cell cultures were established for 41 patients, of whom 8 had normal ovarian reserve (NOR), 17 were of advanced reproductive age (ARA), 12 had a diagnosis of polycystic ovary syndrome (PCOS), and 4 had a combination of diagnoses, such as ARA and PCOS. Primary granulosa-lutein (GL) cell cultures were treated with rh-FSH, TOP5300, or vehicle. Estradiol (E2) production using enzyme-linked immunosorbent assay, steroid pathway gene expression of StAR and aromatase using quantitative polymerase chain reaction, and FSHR membrane localization using immunofluorescence were measured in human GL cells. TOP5300 consistently stimulated E2 production among patients with NOR, ARA, and PCOS. Recombinant FSH was the more potent ligand in GL cells from patients with NOR but was ineffective in cells from patients with ARA or PCOS. The lowest level of FSHR plasma membrane localization was seen in patients with ARA, although FSHR localization was more abundant in cells from patients with PCOS; the highest levels were present in cells from patients with NOR. The localization of FSHR was not affected by TOP5300 relative to rh-FSH in any patient group. TOP5300 stimulated greater expression of StAR and CYP19A1 across cells from all patients with NOR, ARA, and PCOS combined, although rh-FSH was unable to stimulate StAR and aromatase (CYP19A1) expression in cells from patients with PCOS. TOP5300-induced expression of StAR and CYP19A1 mRNA among patients with ARA and NOR was consistently lower than that observed in cells from patients with PCOS. TOP5300 appears to stimulate E2 production and steroidogenic gene expression from GL cells more than rh-FSH in PCOS, relative to patients with ARA and NOR. It does not appear that localization of FSHR at cell membranes is a limiting step for TOP5300 or rh-FSH stimulation of steroidogenic gene expression and E2 production.

P-631 Alleviating effects of EGCG on ovarian hyperstimulation syndrome and the potential modulating mechanism through the VEGF pathway

Abstract Study question What are the underlying mechanisms of EGCG to alleviate Ovary hyperstimulation syndrome (OHSS)? Summary answer EGCG inhibited VEGF via TGF-β1 classical-SMAD pathway and 67LR-mediated CREB pathway and could reduce the ovarian inflammatory effect and attenuated OHSS progress in rat model. What is known already Ovarian hyperstimulation syndrome (OHSS) is one of the most severe complications of COH during IVF treatment. The pathophysiology of OHSS is characterized by increased capillary permeability, VEGF is an important mediator in OHSS, and serum VEGF levels have been shown to correlate with OHSS severity. (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active polyphenolic catechin in green tea and has been reported to have multiple effects in humans. Many research indicated that in many pathological processes, EGCG could inhibit VEGF and its receptor expression and have an angiogenesis effect. Herein, EGCG might have a therapeutic effect on OHSS. Study design, size, duration We investigated the role of EGCG in OHSS in vitro and in vivo. The primary human granulosa-lutein(hGL) cells and human granulosa-like tumor (KGN) cell line were cultured and treated with different concentrations of EGCG for 24 hours. Animal OHSS model was established in SD rats by injection of pregnant mare serum gonadotropin (PMSG), and randomly assigned to receive vehicle only or EGCG for 3 days. All experiments were performed 3-8 times for comparisons. Participants/materials, setting, methods The effect of EGCG on KGN and hGL cells was determined by MTT assay. The body weight of rats was measured every day, and the ovary size was measured after removal, the permeability was determined by Evans-blue. The serum estrogen and VEGF level in rats were detected by ELISA. RNA and protein expressions of VEGF, VEGFR-2, TGF-β, and TβR II were detected by qPCR and Western-blotting and immunostaining in vitro and in vivo experiments. Main results and the role of chance Our study demonstrated that administration of EGCG attenuated the development of OHSS in rats, as shown by histological examination and ovarian weight and morphology. The ovary weight was significantly decreased in the EGCG treatment group compared with the OHSS group (147.9 vs 206.5 mg). Additionally, compared to OHSS rats, EGCG treated rats exhibit downregulated ovarian VEGF expression determined by IHC and RT-qPCR. VEGF and E2 protein levels significantly decrease in the serum of the EGCG treatment group. EGCG exerted inhibitory effects on cell growth only in high dose (50uM) and longtime (48h) treatment in KGN and hGL cells. In KGN cells and hGL cells, EGCG significantly reduces the expression of VEGF and TGF-β at the RNA and protein levels. Furthermore, EGCG inhibits TGF-β1-induced VEGF production and secretion in KGN cells by suppressing TGF-β expression and its traditional Smad signaling pathway. EGCG also downregulates VEGF expression through the 67-kDa laminin receptor-mediated PKA-CREB pathway. Limitations, reasons for caution EGCG has been reported to employ a wide range of biological effects. Therefore, its suppressive effects on VEGF and TGF-β signaling pathway might be part of the pharmacological mechanisms to alleviate Ovary hyperstimulation syndrome. Further studies are also needed to explore the therapeutic mechanisms of melatonin from other perspectives. Wider implications of the findings Our findings add mechanistic insight in support of using EGCG as an adjuvant therapy in the management of OHSS. EGCG shows the potential to act as a novel alternative therapeutic drug to treat OHSS. Daily intake of green tea might be beneficial for women under COH to prevent OHSS. Trial registration number Not applicable

Short-Term Exposure to Bisphenol A Does Not Impact Gonadal Cell Steroidogenesis In Vitro.

Bisphenol A (BPA) is a ubiquitous, synthetic chemical proven to induce reproductive disorders in both men and women. The available studies investigated the effects of BPA on male and female steroidogenesis following long-term exposure to the compound at relatively high environmental concentrations. However, the impact of short-term exposure to BPA on reproduction is poorly studied. We evaluated if 8 and 24 h exposure to 1 nM and 1 µM BPA perturbs luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e., the mouse tumour Leydig cell line mLTC1, and human primary granulosa lutein cells (hGLC). Cell signalling studies were performed using a homogeneous time-resolved fluorescence (HTRF) assay and Western blotting, while gene expression analysis was carried out using real-time PCR. Immunostainings and an immunoassay were used for intracellular protein expression and steroidogenesis analyses, respectively. The presence of BPA leads to no significant changes in gonadotropin-induced cAMP accumulation, alongside phosphorylation of downstream molecules, such as ERK1/2, CREB and p38 MAPK, in both the cell models. BPA did not impact STARD1, CYP11A1 and CYP19A1 gene expression in hGLC, nor Stard1 and Cyp17a1 expression in mLTC1 treated with LH/hCG. Additionally, the StAR protein expression was unchanged upon exposure to BPA. Progesterone and oestradiol levels in the culture medium, measured by hGLC, as well as the testosterone and progesterone levels in the culture medium, measured by mLTC1, did not change in the presence of BPA combined with LH/hCG. These data suggest that short-term exposure to environmental concentrations of BPA does not compromise the LH/hCG-induced steroidogenic potential of either human granulosa or mouse Leydig cells.

Betacellulin regulates gap junction intercellular communication by inducing the phosphorylation of connexin 43 in human granulosa-lutein cells

BackgroundThe gap junction protein, connexin 43 (Cx43) is highly expressed in human granulosa-lutein (hGL) cells. The phosphorylation of certain amino acid residues in the Cx43 protein has been shown to be related to a decline in gap junction intercellular communication (GJIC), which subsequently affects oocyte meiotic resumption. As a member of the epidermal growth factor (EGF) family, betacellulin (BTC) mediates luteinizing hormone (LH)-induced oocyte maturation and cumulus cell expansion in mammalian follicles. Whether BTC can regulate Cx43 phosphorylation, which further reduces Cx43-coupled GJIC activity in hGL cells remains to be determined.MethodsImmortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing in vitro fertilization in an academic research center were used as the study models. The expression levels of Cx43 and phosphorylated Cx43 were examined following cell incubation with BTC at different time points. Several kinase inhibitors (sotrastaurin, AG1478, and U0126) and small interfering RNAs targeting EGF receptor (EGFR) and receptor tyrosine-protein kinase 4 (ErbB4) were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.ResultsThe results showed that BTC induced the rapid phosphorylation of Cx43 at serine368 without altering the expression of Cx43 in primary and immortalized hGL cells. Additionally, using a dual inhibition approach (kinase inhibitors and siRNA-based expression knockdown), we demonstrated that this effect was mainly mediated by the EGFR but not the ErbB4 receptor. Furthermore, using a protein kinase C (PKC) kinase assay and a scrape-loading and dye transfer assay, we revealed that PKC signaling is the downstream signaling pathway that mediates the increase in Cx43 phosphorylation and subsequent decrease in GJIC activity in response to BTC treatment in hGL cells.ConclusionsBTC promptly induced the phosphorylation of connexin 43 at Ser368, leading to decreased GJIC activity in hGL cells. The BTC-induced cellular activities were most likely driven by the EGFR-mediated PKC-dependent signaling pathway. Our findings shed light on the detailed molecular mechanisms by which BTC regulates the process of oocyte meiotic resumption.

TGF-β1 upregulates secreted protein acidic and rich in cysteine expression in human granulosa-lutein cells: a potential mechanism for the pathogenesis of ovarian hyperstimulation syndrome

BackgroundOvarian hyperstimulation syndrome (OHSS) is a serious complication during in vitro fertilization (IVF) treatment. The upregulation of ovarian transforming growth factor-beta 1 (TGF-β1) is involved in the development of OHSS. The secreted protein acidic and rich in cysteine (SPARC) is a secreted multifunctional matricellular glycoprotein. Although the regulatory effects of TGF-β1 on SPARC expression have been reported, whether TGF-β1 regulates SPARC expression in the human ovary remains unknown. In addition, the role of SPARC in the pathogenesis of OHSS is unclear.MethodsA steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing IVF treatment were used as experimental models. OHSS was induced in rats, and ovaries were collected. Follicular fluid samples were collected from 39 OHSS and 35 non-OHSS patients during oocyte retrieval. The underlying molecular mechanisms mediating the effect of TGF-β1 on SPARC expression were explored by a series of in vitro experiments.ResultsTGF-β1 upregulated SPARC expression in both KGN and hGL cells. The stimulatory effect of TGF-β1 on SPARC expression was mediated by SMAD3 but not SMAD2. The transcription factors, Snail and Slug, were induced in response to the TGF-β1 treatment. However, only Slug was required for the TGF-β1-induced SPARC expression. Conversely, we found that the knockdown of SPARC decreased Slug expression. Our results also revealed that SPARC was upregulated in the OHSS rat ovaries and in the follicular fluid of OHSS patients. Knockdown of SPARC attenuated the TGF-β1-stimulated expression of vascular endothelial growth factor (VEGF) and aromatase, two markers of OHSS. Moreover, the knockdown of SPARC reduced TGF-β1 signaling by downregulating SMAD4 expression.ConclusionsBy illustrating the potential physiological and pathological roles of TGF-β1 in the regulation of SPARC in hGL cells, our results may serve to improve current strategies used to treat clinical infertility and OHSS.66JbE8YWJdrsTB-WmCj_-wVideo

Interleukin-1 increases cyclooxygenase-2 expression and prostaglandin E2 production in human granulosa-lutein cell via nuclear factor kappa B/P65 and extracellular signal-regulated kinase 1/2 signaling pathways

A multitude of cytokines have been reported to participate in the folliculogenesis process in female. Interleukin-1 (IL-1), belonging to interleukin family, is originally identified as an important immune factor involved in inflammation response. Besides the immunity system, IL-1 is also expressed in reproductive system. However, the role of IL-1 in regulating ovarian follicle function remains to be elucidated. In the current study, using the primary human granulosa-lutein (hGL) and immortalized human granulosa-like tumor cell line (KGN) models, we demonstrated that both IL-1α and IL-1β increased prostaglandin E2 (PGE2) production via upregulating its cyclooxygenase (COX) enzyme COX-2 expression in human granulosa cells. Mechanistically, IL-1α and IL-1β treatment activated nuclear factor kappa B (NF-κB) signaling pathway. Using the specific siRNA to knock down endogenous gene expression, we found that the inhibition of p65 expression abolished IL-1α and IL-1β-induced upregulation of COX-2 expression whereas knockdown of p50 and p52 had no effect. Moreover, our results also showed that IL-1α and IL-1β promoted the nuclear translocation of p65. ChIP assay demonstrated the transcriptional regulation of p65 on COX-2 expression. Additionally, we also found that IL-1α and IL-1β could activate the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The inhibition of ERK1/2 signaling pathway activation reversed IL-1α and IL-1β-induced upregulation of COX-2 expression. Our findings shed light on the cellular and molecular mechanisms by which IL-1 modulates the COX-2 expression through NF-κB/P65 and ERK1/2 signaling pathways in human granulosa cells.

HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells

BackgroundHeparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown.MethodsA steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments.ResultsWestern blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway.ConclusionThis study increases the understanding of the physiological role of HB-EGF in human luteal functions.7G2SU9-j2App9A69cYCW26Video

TGF-β1 suppresses de novo cholesterol biosynthesis in granulosa-lutein cells by down-regulating DHCR24 expression via the GSK-3β/EZH2/H3K27me3 signaling pathway

Cholesterol is a precursor to steroid hormones and can be obtained from serum LDL or de novo synthesis in steroidogenic cells. Before luteinizing hormone (LH) surge-induced ovulation, follicles remain avascular, and cholesterol required for progesterone production in granulosa cells (GCs) is derived from de novo biosynthesis. Previous studies have verified that the intrafollicular TGF-β1 plays inhibitory roles in GCs luteinization, vascularization, and progesterone production. Nevertheless, the regulatory function of TGF-β1 on de novo cholesterol synthesis in granulosa-lutein (GL) cells remains largely unknown. We aim to investigate this aspect in this study using in vivo cultured human GL cells. Our results suggested that TGF-β1 significantly suppresses intracellular cholesterol levels and down-regulates the expression of the final step enzyme, DHCR24, that catalyzes de novo cholesterol synthesis. We used specific inhibitors and siRNA-mediated knockdown approaches demonstrate that TGF-β1 suppression of DHCR24 expression in GL cells is mediated by the GSK-3β/EZH2/H3K27me3 signaling pathway. Further ChIP assays revealed that elevated H3K27me3 levels in the promoter region of DHCR24 play a vital role in TGF-β1-induced DHCR24 down-regulation, and RNA-sequencing results confirmed these findings. Notably, our study provides a novel insight into the molecular mechanisms by which TGF-β1 suppresses de novo cholesterol biosynthesis in GL cells.

Characterization of the roles of amphiregulin and transforming growth factor β1 in microvasculature-like formation in human granulosa-lutein cells.

Vascular endothelial-cadherin (VE-cadherin) is an essential component that regulates angiogenesis during corpus luteum formation. Amphiregulin (AREG) and transforming growth factor β1 (TGF-β1) are two intrafollicular factors that possess opposite functions in directing corpus luteum development and progesterone synthesis in human granulosa-lutein (hGL) cells. However, whether AREG or TGF-β1 regulates the VE-cadherin expression and subsequent angiogenesis in the human corpus luteum remains to be elucidated. Results showed that hGL cells cultured on Matrigel spontaneously formed capillary-like and sprout-like microvascular networks. Results of specific inhibitor treatment and small interfering RNA-mediated knockdown revealed that AREG promoteed microvascular-like formation in hGL cells by upregulating the VE-cadherin expression mediated by the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase1/2 (ERK1/2) signaling pathway. However, TGF-β1 suppressed microvascular-like formation in hGL cells by downregulating VE-cadherin expression mediated by the activin receptor-like kinase (ALK)5-Sma- and Mad-related protein (SMAD)2/3/4 signaling pathway. Collectively, this study provides important insights into the underlying molecular mechanisms by which TGF-β1 and AREG differentially regulate corpus luteum formation in human ovaries.

Notch Signaling Induced by Endoplasmic Reticulum Stress Regulates Cumulus-Oocyte Complex Expansion in Polycystic Ovary Syndrome.

Endoplasmic reticulum (ER) stress activated in granulosa cells contributes to the pathophysiology of polycystic ovary syndrome (PCOS). In addition, recent studies have demonstrated that Notch signaling plays multiple roles in the ovary via cell-to-cell interactions. We hypothesized that ER stress activated in granulosa cells of antral follicles in PCOS induces Notch signaling in these cells, and that activated Notch signaling induces aberrant cumulus-oocyte complex (COC) expansion. Expression of Notch2 and Notch-target transcription factors was increased in granulosa cells of PCOS patients and model mice. ER stress increased expression of Notch2 and Notch-target transcription factors in cultured human granulosa-lutein cells (GLCs). Inhibition of Notch signaling abrogated ER stress-induced expression of genes associated with COC expansion in cultured human GLCs, as well as ER stress-enhanced expansion of cumulus cells in cultured murine COCs. Furthermore, inhibition of Notch signaling reduced the areas of COCs in PCOS model mice with activated ER stress in the ovary, indicating that Notch signaling regulates COC expansion in vivo. Our findings suggest that Notch2 signaling is activated in granulosa cells in PCOS and regulates COC expansion. It remains to be elucidated whether aberrant COC expansion induced by the ER stress-Notch pathway is associated with ovulatory dysfunction in PCOS patients.

Activin A promotes hyaluronan production and upregulates versican expression in human granulosa cells†.

Hyaluronan is a structural component of the expanded cumulus matrix, and hyaluronan synthase 2 is the major enzyme for the synthesis of hyaluronan in humans. Versican cross-links the hyaluronan-rich matrix to cumulus cells and is critical for successful ovulation. Activin A is a critical intrafollicular regulator of ovarian function. Although activin A has been shown to promote cumulus matrix expansion in mice, the functional role of activin A in the regulation of cumulus expansion in the human ovary remains to be elucidated. Using primary and immortalized human granulosa-lutein cells as study models, we provide the first data showing that activin A increased the production of hyaluronan by upregulating the expression of hyaluronan synthase 2 in these cells. Additionally, activin A also promoted the expression of the hyaluronan-binding protein versican. Moreover, using inhibitor- and small interfering RNA-mediated inhibition approaches, we found that these stimulatory effects of activin A are most likely mediated through the type I receptor activin receptor-like kinase (ALK4)-mediated Sma- and Mad-related protein (SMAD2)/SMAD3-SMAD4 signaling pathway. Notably, the chromatin immunoprecipitation analyses demonstrated that SMAD4 could bind to human hyaluronan synthase 2 and VERSICAN promoters. The results obtained from this in vitro study suggest that locally produced activin A plays a functional role in the regulation of hyaluronan production and stabilization in human granulosa-lutein cells.

Connective tissue growth factor mediates bone morphogenetic protein 2-induced increase in hyaluronan production in luteinized human granulosa cells

BackgroundHyaluronan is the main component of the cumulus-oocyte complex (COC) matrix, and it maintains the basic structure of the COC during ovulation. As a member of the transforming growth factor β (TGF-β) superfamily, bone morphogenetic protein 2 (BMP2) has been identified as a critical regulator of mammalian folliculogenesis and ovulation. However, whether BMP2 can regulate the production of hyaluronan in human granulosa cells has never been elucidated.MethodsIn the present study, we investigated the effect of BMP2 on the production of hyaluronan and the underlying molecular mechanism using both immortalized (SVOG) and primary human granulosa-lutein (hGL) cells. The expression of three hyaluronan synthases (including HAS1, HAS2 and HAS3) were examined following cell incubation with BMP2 at different concentrations. The concentrations of the hyaluronan cell culture medium were determined by enzyme-linked immunosorbent assay (ELISA). The TGF-β type I receptor inhibitors (dorsomorphin and DMH-1) and small interfering RNAs targeting ALK2, ALK3, ALK6 and SMAD4 were used to investigate the involvement of TGF-β type I receptor and SMAD-dependent pathway.ResultsOur results showed that BMP2 treatment significantly increased the production of hyaluronan by upregulating the expression of hyaluronan synthase 2 (HAS2). In addition, BMP2 upregulates the expression of connective tissue growth factor (CTGF), which subsequently mediates the BMP2-induced increases in HAS2 expression and hyaluronan production because overexpression of CTGF enhances, whereas knockdown of CTGF reverses, these effects. Notably, using kinase inhibitor- and siRNA-mediated knockdown approaches, we demonstrated that the inductive effect of BMP2 on the upregulation of CTGF is mediated by the ALK2/ALK3-mediated SMAD-dependent signaling pathway.ConclusionsOur findings provide new insight into the molecular mechanism by which BMP2 promotes the production of hyaluronan in human granulosa cells.

Growth differentiation factor-11 downregulates steroidogenic acute regulatory protein expression through ALK5-mediated SMAD3 signaling pathway in human granulosa-lutein cells

BackgroundGrowth differentiation factor-11 (GDF-11) belongs to the transforming growth factor-β (TGF-β) superfamily. To date, the expression of GDF-11 in the ovary and its role in regulating ovarian function are completely unknown. Ovarian granulosa cell-mediated steroidogenesis plays a pivotal role in maintaining normal female reproductive function. GDF-11 and GDF-8 share high sequence similarity and exhibit many similar features and functions. Steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis and its expression can be downregulated by GDF-8. Polycystic ovary syndrome (PCOS) is the most common cause of female infertility. The expression levels of GDF-8 are upregulated in the human follicular fluid and granulosa-lutein (hGL) cells of PCOS patients. However, whether similar results can be observed for the GDF-11 needs to be determined.MethodsThe effect of GDF-11 on StAR expression and the underlying molecular mechanisms were explored by a series of in vitro experiments in a primary culture of hGL cells obtained from patients undergoing in vitro fertilization (IVF) treatment. Human follicular fluid samples were obtained from 36 non-PCOS patients and 36 PCOS patients. GDF-11 levels in follicular fluid were measured by ELISA.ResultsGDF-11 downregulates StAR expression, whereas the expression levels of the P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are not affected by GDF-11 in hGL cells. Using pharmacological inhibitors and a siRNA-mediated approach, we reveal that ALK5 but not ALK4 mediates the suppressive effect of GDF-11 on StAR expression. Although GDF-11 activates both SMAD2 and SMAD3 signaling pathways, only SMAD3 is involved in the GDF-11-induced downregulation of StAR expression. In addition, we show that SMAD1/5/8, ERK1/2, and PI3K/AKT signaling pathways are not activated by GDF-11 in hGL cells. RT-qPCR and ELISA detect GDF-11 mRNA expression in hGL cells and GDF-11 protein expression in human follicular fluid, respectively. Interestingly, unlike GDF-8, the expression levels of GDF-11 are not varied in hGL cells and follicular fluid between non-PCOS and PCOS patients.ConclusionsThis study increases the understanding of the biological function of GDF-11 and provides important insights into the regulation of ovarian steroidogenesis.