Human Luteinized Granulosa Cells Research Articles

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.

Bone morphogenetic protein-9 downregulates StAR expression by inducing snail expression via SMAD1/5/8 signaling in human granulosa-lutein cells

Ovarian steroidogenesis mediated by granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis. Bone morphogenetic protein-9 (BMP-9), also known as growth differentiation factor-2 (GDF-2), is a member of the transforming growth factor-beta (TGF-β) superfamily. BMP-9 induces epithelial-mesenchymal transition (EMT) that contributes to cancer progression. However, the function of BMP-9 in the female reproductive system remains largely unknown. It has been recently shown that BMP-9 is expressed in human follicular fluid and can downregulate StAR expression in human ovarian granulosa cells. However, the underlying molecular mechanisms warrant investigation. Our results show that treatment of primary granulosa-lutein (hGL) cells with BMP-9 downregulates StAR expression. In addition, two EMT-related transcription factors, Snail and Slug, are upregulated by the treatment of BMP-9. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we show that BMP-9 upregulates Snail and Slug expression by activating SMAD1/5/8 signaling. We also examine the effects of BMP-9 on SMAD-independent signaling pathways, including ERK1/2, p38, JNK, AKT, and CREB. However, none of them is affected by the BMP-9. Moreover, we use gain- and loss-of-function approaches to reveal that only Snail, not Slug, is required for the BMP-9-induced downregulation of StAR expression in hGL cells. This study increases the understanding of the physiology function of BMP-9 in hGL cells and provides important insights into the regulation of StAR expression.

BMAL1 positively correlates with genes regulating steroidogenesis in human luteinized granulosa cells.

In this study, we examined the relationship between BMAL1 expression and the genes regulating steroid biosynthesis in human luteinized granulosa cells. BMAL1 function is crucial for steroid production and proper ovarian function, highlighting the importance of circadian clock regulation in female reproductive health. Human luteinized granulosa cells were collected to analyze circadian clock gene expression and its effect on the genes regulating steroid biosynthesis. We used siRNA to knock down the expression of BMAL1 in KGN cells. We measured the expression levels of genes regulating steroid biosynthesis and circadian clock RT-qPCR. We demonstrated that BMAL1 expression positively correlates with genes regulating steroid biosynthesis (CYP11A1, CYP19A1, STAR, and ESR2). The knockdown of BMAL1 in KGN cells revealed a significant decrease in steroid synthase expression. In contrast, when BMAL1 was overexpressed in KGN and HGL5 cells, we observed a significant increase in the expression of steroid synthases, such as CYP11A1 and CYP19A1. These results indicated that BMAL1 positively controls 17β-estradiol (E2) secretion in granulosa cells. We also demonstrated that dexamethasone synchronization in KGN cells enhanced the rhythmic alterations in circadian clock genes. Our study suggests that BMAL1 plays a critical role in steroid biosynthesis in human luteinized granulosa cells, thereby emphasizing the importance of BMAL1 in the regulation of reproductive physiology.

#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.

TGF-β1 and TGF-β3, but not TGF-β2, are upregulated in the ovaries of ovarian hyperstimulation syndrome†.

Ovarian hyperstimulation syndrome (OHSS) is a life-threatening and potentially fatal complication during in vitro fertilization treatment. The levels of transforming growth factor-β1 (TGF-β1) are upregulated in human follicular fluid and granulosa-lutein cells (hGL) of OHSS patients and could contribute to the development of OHSS by downregulating steroidogenic acute regulatory protein (StAR) expression. However, whether the same is true for the other two members of the TGF-β family, TGF-β2 and -β3, remains unknown. We showed that all three TGF-β isoforms were expressed in human follicular fluid. In comparison, TGF-β1 was expressed at the highest level, followed by TGF-β2 and TGF-β3. Compared to non-OHSS patients, follicular fluid levels of TGF-β1 and TGF-β3 were significantly upregulated in OHSS patients. The same results were observed in mRNA levels of TGF-β isoforms in hGL cells and ovaries of OHSS rats. In addition, StAR mRNA levels were upregulated in hGL cells of OHSS patients and the ovaries of OHSS rats. Treatment cells with TGF-β isoforms downregulated the StAR expression with a comparable effect. Moreover, activations of SMAD3 signaling were required for TGF-β isoforms-induced downregulation of StAR expression. This study indicates that follicular fluid TGF-β1 and TGF-β3 levels could be used as biomarkers and therapeutic targets for the OHSS.

Copper exposure induces ovarian granulosa cell apoptosis by activating the caspase-dependent apoptosis signaling pathway and corresponding changes in microRNA patterns

Environmental copper (Cu) contamination is a complex worldwide public health problem. However, information on the effects of Cu pollution on human reproduction is limited. Although our previous studies have indicated that Cu exposure disrupts ovarian folliculogenesis, the underlying mechanism needs to be further explored. In this study, human luteinized ovarian granulosa cells and a rat animal model were used to investigate whether Cu exposure affects ovarian follicle development by inducing apoptosis and to elucidate the possible mechanisms. The results showed that Cu exposure from weaning to sexual maturity significantly decreased the proportion of preantral follicles but increased the proportion of atretic follicles (P < 0.05). In addition, 6 mg/kg Cu increased the proportion of antral follicles, while 12 and 25 mg/kg Cu decreased it (P < 0.05). We also found that 6 mg/kg Cu exposure inhibited apoptosis of ovarian granulosa cells, while 12 and 25 mg/kg Cu promoted apoptosis (P < 0.05). Experiments on primary human luteinized ovarian granulosa cells suggested that higher levels of Cu exposure induced a significant increase in the mRNA levels of Bcl2 Bax , Fas, Caspase8, and Caspase3 (P < 0.05), and the protein levels of BAX, BCL2, CASPASE3, CASPASE8, CLE-CASPASE3, CLE-CASPASE8 and BAX/BCL2 were also increased (P < 0.05). miRNA chip analyses identified a total of 95 upregulated and 10 downregulated miRNAs in human luteinized granulosa cells exposed to Cu. Hsa-miR-19b-3p, hsa-miR-19a-3p, miR-548ar-3p, hsa-miR-652–5p, and hsa-miR-29b-5p were decreased after Cu exposure (P < 0.05). Additionally, the level of hsa-miR-144–5p was increased (P < 0.05). Together, our results reveal that Cu exposure induces abnormal ovarian folliculogenesis by inducing ovarian granulosa cell apoptosis, which is triggered by the caspase-dependent apoptosis signaling pathway, and that miRNAs may be involved in this process.

Steroidogenic activity of liposomal methylated resveratrol analog 3,4,5,4′-tetramethoxystilbene (DMU-212) in human luteinized granulosa cells in a primary three-dimensional in vitro model

PurposeSteroid hormone secretion is one of the key functions of granulosa cells (GCs). Resveratrol is a natural polyphenol, known for its beneficial health effects, such as improving reproductive health. However, its application is limited due to poor bioavailability. The methoxy derivative of resveratrol (DMU-212) was demonstrated to be more lipophilic, and therefore of greater bioavailability. However, since the addition of methoxy groups to the stilbene scaffold was found to make the molecule insoluble in water, DMU-212 was loaded into liposomes. This study aimed to evaluate how the liposomal formulation of DMU-212 (lipDMU-212) alters estradiol and progesterone secretion of human ovarian GCs in a primary three-dimensional cell culture model.MethodsDMU-212-loaded liposomes were prepared by thin film hydration followed by extrusion. Cell viability was measured after exposure of GCs spheroids to the liposomal formulation of DMU-212 using CellTiter-Glo® 3D Cell Viability Assay. The secretion of estradiol and progesterone was determined using commercial ELISA kits. RT-qPCR was conducted to analyze the expression of steroidogenesis-related genes. Finally, the western blot technique was used to analyze the effect of lipDMU-212 and FSH treatments on CYP11A1 and HSD3B1 protein levels.ResultslipDMU-212 was found to significantly increase estradiol and progesterone secretion in a dose-dependent manner by enhancing the expression of CYP11A1, HSD3B1, StAR, CYP17A1, CYP19A1, and HSD17B1 genes. We have also shown that lipDMU-212, used alone and in combination with FSH, significantly increased the expression of the HSD3B1 and CYP11A1 proteins in GCs. Furthermore, our study suggests that lipDMU-212 increases FSH activity.ConclusionsThis is the first study to describe the steroidogenic activity of liposomal formulation of DMU-212, possibly through increasing the StAR and CYP19A1 expression. These findings suggest that lipDMU-212 might have a beneficial effect in the treatment of disorders related to estrogen deficiency and hyperandrogenism, such as PCOS.

Integrated bioinformatics analysis elucidates granulosa cell whole-transcriptome landscape of PCOS in China

BackgroundPolycystic ovary syndrome (PCOS) is a common reproductive, neuroendocrine, and metabolic disorder in women of reproductive age that affects up to 5–10% of women of reproductive age. The aetiology of follicle development arrest and critical issues regarding the abnormal follicular development in PCOS remain unclear. The present study aims to systematically evaluate granulosa cell whole-transcriptome sequencing data to gain more insights into the transcriptomic landscape and molecular mechanism of PCOS in China.MethodsIn the present study, the microarray datasets GSE138518, GSE168404, GSE193123, GSE138572, GSE95728, and GSE145296 were downloaded from the Gene Expression Omnibus (GEO) database. Subsequently, differential expression analysis was performed on the PCOS and control groups, followed by functional interaction prediction analysis to investigate gene-regulatory circuits in PCOS. Finally, hub genes and their associated ncRNAs were validated by qPCR in human-luteinized granulosa (hGL) cells and were correlated with the clinical characteristics of the patients.ResultsA total of 200 differentially expressed mRNAs, 3 differentially expressed miRNAs, 52 differentially expressed lncRNAs, and 66 differentially expressed circRNAs were found in PCOS samples compared with controls. GO and KEGG enrichment analyses indicated that the DEGs were mostly enriched in phospholipid metabolic processes, steroid biosynthesis and inflammation related pathways. In addition, the upregulated miRNA hsa-miR-205-5p was significantly enriched in the ceRNA network, and two hub genes, MVD and PNPLA3, were regulated by hsa-miR-205-5p, which means that hsa-miR-205-5p may play a fundamental role in the pathogenesis of PCOS. We also found that MVD and PNPLA3 were related to metabolic processes and ovarian steroidogenesis, which may be the cause of the follicle development arrest in PCOS patients.ConclusionsIn summary, we systematically constructed a ceRNA network depicting the interactions between the ncRNAs and the hub genes in PCOS and control subjects and correlated the hub genes with the clinical characteristics of the patients, which provides valuable insights into the granulosa cell whole-transcriptome landscape of PCOS in China.

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.

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.

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

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.

Investigating the potential role of swertiamarin on insulin resistant and non-insulin resistant granulosa cells of poly cystic ovarian syndrome patients

Background and aimConventional drugs have limitations due to prevalence of contraindications in PCOS patients. To explore the potential effects of swertiamarin, on abrupted insulin and steroidogenic signaling in human luteinized granulosa cells from PCOS patients with or without insulin resistance.Experimental procedurehLGCs from 8 controls and 16 PCOS patients were classified for insulin resistance based on down regulation of protein expression of insulin receptor-β (INSR- β) as shown in our previous paper. Cells were grouped as control, PCOS-IR and PCOS-NIR, treated with swertiamarin (66 µM) and metformin (1 mM). Expression of key molecules involved in insulin signaling, fat metabolism, IGF system and steroidogenesis were compared between groups.ResultsSwertiamarin significantly (P < 0.05) reversed the expression of INSR-β, PI(3)K, p-Akt, PKC-ζ, PPARγ, (P < 0.01) IRS (Ser 307) and IGF system in PCOS-IR group and was equally potent to metformin. In the same group, candidate genes viz SREBP1c, FAS, ACC-1 and CPT-1 were down regulated by swertiamarin (P < 0.001) and metformin (P < 0.001). Significant upregulation was demonstrated in expression of StAR, CYP19A1, 17β-HSD and 3β-HSD when treated with swertiamarin (P < 0.01) and metformin (P < 0.01) in PCOS-IR followed by increase in 17β-HSD and 3β-HSD enzyme activity along with estradiol and progesterone secretions. However, swertiamarin did not reveal any effect on PCOS-NIR group as compared to metformin that significantly (P < 0.01) reversed all the parameters related to steroidogenesis and down regulated basal expression of insulin signaling genes.ConclusionSwertiamarin, presents itself as a potential fertility drug in hLGCs from PCOS-IR patients.

Divergent roles of Sirtuin 1 in human granulosa-lutein cells: similarities to hCG.

Sirtuin 1 (SIRT1) is an NAD+ dependent deacetylase that modifies gene expression through histone deacetylation. It also deacetylates non-histone substrates, e.g. tumor suppressor p53, NOS3, HIF1A, NFKB, FOXO3a, PGC-1α, and PPARγ. Consequently, it regulates a wide range of physiological functions including cell cycle control, energy expenditure, oxidative stress response, apoptosis, and aging. SIRT1 is expressed in ovarian granulosa cells (GCs) of various species including humans at different stages of the reproductive cycle. The importance of SIRT1 in female reproduction is supported by the findings that SIRT1-knockout mice exhibit defects in reproductive tissue development. These mice were found to have a thin-walled uterus, small ovaries, with follicles present but no corpora lutea. This review aims to provide state-of-the-art information on SIRT1's mode of action and its roles in human granulosa-lutein cells and GCs from other species where data is available. It also discusses the overlapping actions of SIRT1 and hCG on the production of critical GC-borne factors.

Wide expression of stemness factor SOX2 in human luteinized granulosa cells.

Wide expression of stemness factor SOX2 in human luteinized granulosa cells.