High androgen might increase the resistance of granulosa cells to ferroptosis by decreasing ROS and mitoROS, increasing GSH/GSSG ratio, and downregulating DHCR7 in PCOS.

Objective To investigate the role of PDCD4 in apoptosis of human granulosa cells and to investigate whether hyperandrogen can promote the up-regulation of PDCD4 and participate in the apoptosis of human ovarian granulosa cells. Methods The PDCD4 was overexpressed by transfection of PDCD4 expression construct in human ovarian granulosa cell line KGN cells and the transfection efficiency was detected by fluorescence quantitative PCR. The effect of overexpression of PDCD4 on apoptosis of KGN cells was detected by flow cytometry and Annexin V staining. The KGN cells were treated with testosterone of different concentration gradients (0, 10-7 mol/L, 10-6 mol/L, 10-5 mol/L). The expression of PDCD4 was detected by real-time quantitative PCR and Western blotting after 24 h and 48 h of testosterone treatment. The apoptotic effect of testosterone on granulosa cells was detected by flow cytometry after 48 h. PDCD4 small interfering RNA was used to knock down the expression of PDCD4 in KGN cells treated with high concentration of testosterone (10-5 mol/L). Flow cytometry was used to detect the effect of PDCD4 knockdown on apoptosis induced by high androgen. Results After overexpression of PDCD4 in KGN cells, results of Annexin V-PE staining and flow cytometry showed a significant increase in apoptosis of KGN cells (P=0.014). KGN cells were treated with different concentrations of testosterone. Compared with NC group, the expression level of PDCD4 mRNA in 10-5 mol/L group and 10-6 mol/L group was up-regulated (24 h: P=0.035, P=0.038; 48 h: P=0.029, P=0.009); the expression level of PDCD4 protein was also up-regulated. After treatment of KGN cells with high concentration of testosterone (10-6 mol/L and 10-5 mol/L) for 48 h, apoptosis of granulosa cells increased significantly (P=0.036 in 10-6 mol/L group compared with NC group; P=0.028 in 10-5 mol/L group compared with NC group). After knockdown of PDCD4 expression in KGN cells treated with 10-5 mol/L testosterone, the apoptosis rate was significantly lower than that in the non knockdown group (P=0.038). Conclusion PDCD4 can promote the apoptosis of human ovarian granulosa cells. High concentration of testosterone can induce the apoptosis and up-regulate the expression of PDCD4 in granulosa cells. The mechanism of apoptosis of granulosa cells induced by high concentration of testosterone may be related to apoptotic pathway mediated by PDCD4. Key words: Androgen; Ovarian granulosa cells; Apoptosis; PDCD4; Polycystic ovary syndrome

Follicular fluid steroid content and theca and granulosa cell steroidogenesis in pelvic congestion cystic ovaries were compared with steroidogenic function in both normal and polycystic ovaries. Ovaries were obtained at oophorectomy for benign gynaecological conditions, and classified according to gross morphology at dissection. Individual follicles were dissected out, follicular fluid aspirated, and granulosa and theca cells cultured in vitro. Androstenedione, progesterone and oestradiol content of the follicular fluid and overlying culture medium were measured by radioimmunoassay. There was a significant elevation of both basal and LH-stimulated androstenedione production by theca from both polycystic ovaries (n = 10; P < 0.005) and pelvic congestion cystic ovaries (n = 8; P < 0.05 and < 0.01 respectively) as compared with normal ovaries (n = 5). Granulosa cells from pelvic congestion ovaries (n = 7) had a diminished oestradiol response to FSH as compared with those from normal ovaries (n = 8). Follicular fluid from the majority of follicles in the pelvic congestion cystic ovaries had a high androgen:oestrogen ratio consistent with atresia. For the first time, pelvic congestion ovaries characterized by predominantly atretic follicles scattered throughout the stroma in a normal volume ovary are reported. Follicular atresia was reflected by reduced granulosa cell responsiveness to FSH, theca cell hyperplasia and increased basal and LH-stimulated androgen production. These ovaries are functionally distinct from polycystic ovaries, which do not have a higher proportion of atretic follicles than normal ovaries.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP activated anion channel and its important role in reproduction has been highlighted by infertility observed in men and women with cystic fibrosis, a disease caused by mutations of CFTR. It has been known that a considerable proportion of CF women suffer from anovulation and abnormal level of ovarian hormone, manifested by higher androgen and lower estrogen in serum, but the exact underlying mechanism is not well understood. In the present study, we investigated the expression of CFTR in mouse granulosa cells and the effect of CFTR inhibitors on aromatase expression, and estrogen production. We found that CFTR is expressed in ovarian granulsoa cells. CFTR inhibitors (CFTRinh172 and glybenclamide) down-regulated the aromatase expression, with reduced estradiol production. Since cAMP - CREB is one of the signaling pathways that is involved in aromatase expression, we further determined that the intracellular cAMP level was decreased and CREB phosphorylation was inhibited in granulosa cells when treated with CFTR inhibitors. Our results indicate that CFTR is involved in regulation of estrogen production by modulating the cAMP-CREB pathway. This finding provides a possible explanation for the disorders in ovarian hormone production and ovulation in CF women. (poster)

Polycystic ovary syndrome (PCOS) is a multifactorial reproductive endocrine metabolism disorder, which is closely related to granulosa cell dysfunction, high androgen, insulin resistance, oxidative stress and cellular autophagy. Phosphatidylinositol 3-kinase/protein kinase B (phosphatidylinositol3-kinase/proteinkinaseB, PI3K/AKT) is the main pathway of intracellular signaling. In recent years, studies have confirmed that PI3K/AKT pathway participates in the repair of ovarian tissue damage and the regulation of glucose and lipid metabolism by regulating granulosa cell function, insulin resistance state, oxidative stress and autophagy. TCM has obvious advantages in the treatment of PCOS. One of the mechanisms of PCOS is the regulation of PI3K/AKT signaling pathway, but there are few systematic reviews and reviews of this field at home and abroad. Based on this, this paper summarizes the regulation mechanism of PI3K/AKT signaling pathway in PCOS in recent years and the role of TCM monomer and compound intervention in diseases, in order to provide theoretical basis for the basic and clinical research of PCOS.

Virilization persists in a woman with an androgen-secreting granulosa cell tumor

Proliferation and differentiation of granulosa cells are involved in the folliculogenesis. Gap junctions of granulosa cells are thought to modulate numerous physiological processes, including folliculogenesis and oogenesis. Connexins (Cx) are membrane-spanning proteins that assemble to form the intercellular channels of gap junctions. At least 21 human connexins have been identified. Among them, Cx43 is identified in every stage of folliculogenesis and the expression level is decreased as the follicle goes to atresia. Although it is well established that overproduction of androgens in polycystic ovarian syndrome (PCOS) results in anovulation, the underlying mechanism is less well understood. We hypothesize that high level of androgen may influence Cx 43 expression and proliferation of human granulosa cells. Human granulosa cell line (HO-23) was treated by various dosages (0, 1, 10, or 100 ng/ml) of dihydrotestosterone (DHT), a 5αreduced metabolite of testosterone. Western blot for PCNA, p27kip1, Cx 43 protein and quantitative RT-PCR for Cx 43 mRNA were performed. Gap junctional intercellular communication (GJIC) was analyzed by using the scrape-loading dye transfer (SLDT) technique. Androgen receptor (AR) antagonist, flutamide , was used to test the specificity of the observed androgen responses. We found that human granulosa cell proliferation was not influenced by DHT. Western blot analysis of DHT-treated granulosa cell protein samples showed a decreased amount of Cx 43 protein in dose-dependent manner. Cx43 mRNA levels determined by quantitative RT-PCR analysis in HO-23 cells grown in DHT medium indicated significant downregulation compared with control group. Flutamide significantly blocked the inhibitory effects of DHT on Cx 43 protein and mRNA expression. DHT-treated cells demonstrated a decreased enhancement of GJIC as assessed by SLDT technique. In conclusion, while both androgen and gap junctions are reported to have important roles in follicular development, this is the first report describing a potential regulation of Cx 43 expression by DHT in cultured human granulosa cells. High level androgen reduces Cx 43 expression and impairs GJIC between human granulosa cells through androgen receptors .It may induce the follicles atresia with the impairment of the folliculogenesis and explain part of the underlying mechanism of anovulation in PCOS women.

Insulin resistance (IR) of the granulosa cells may account for the ovarian dysfunctions observed in polycystic ovarian syndrome (PCOS). The underlying mechanism remains largely unresolved. The objective of the study was to investigate the relationship of IR of the granulosa cells with cortisol in the follicular fluid and 11β-hydroxysteroid dehydrogenase 1 and 2 (11β-HSD1 and -2) in the granulosa cells in PCOS. Follicular fluid and granulosa cells were collected from non-PCOS and PCOS patients with and without IR to measure cortisol concentration and the amounts of 11β-HSD1 and -2, which were then correlated with IR status. The effects of cortisol on the expression of genes pertinent to IR were studied in cultured human granulosa cells. Cortisol concentration in the follicular fluid, 11β-HSD1 but not 11β-HSD2 mRNA in the granulosa cells were significantly elevated in PCOS with IR. Increased reductase and decreased oxidase activities of 11β-HSD were observed in granulosa cells in PCOS with IR. In cultured granulosa cells, insulin-induced Akt phosphorylation was significantly attenuated by cortisol. Cortisol not only increased phosphatase and tensin homolog deleted on chromosome 10, an inhibitor of Akt phosphorylation, but also 11β-HSD1 in the cells. Increased 11β-HSD1 expression and its reductase activity in granulosa cells are the major causes of increased cortisol concentration in the follicular fluid of PCOS with IR. The consequent excessive cortisol might contribute to IR of the granulosa cells in PCOS patients by attenuating Akt phosphorylation via induction of phosphatase and tensin homolog deleted on chromosome 10 expression, which might be further exacerbated by the induction of 11β-HSD1.

Objective To investigate the biological effects of Forskolin on insulin resistant porcine granulosa cells. Methods Porcine ovarian granulosa cells were cultured in medium added with 50 μmol/L dimethyl sulfoxide (DMSO) or 250 nmol/L PI-3K inhibitor Wortmannin (WT) for 48 hours. Granulosa cells in DMSO medium were added with 20 μmol/L Forskolin or blank agent for another 48-hour incubation, which were defined as the control group induced by Forskolin and the control group without Forskolin. Similarly, granulosa cells in WT medium were also added with 20 μmol/L Forskolin or blank agent for the next 48 hours, which were defined as the insulin-resistant group induced by Forskolin and the insulin-resistant group without Forskolin. The level of progesterone, testosterone and estradiol were detected by chemoluminescence. The mRNA expression of 17 alpha-hydroxylase( CYP17 ) and aromatase cytochrome P450 (P450arom) were assessed by RT-PCR. t test was used for data analysis. Results The secretion of progesterone in both insulin-resistant group and control group induced by Forskolin showed no statistical significance (P>0.05), and the levels of CYP17 and P450arom genes were elevated. Compared the insulin-resistant group with control group, the level of testosterone was obviously elevated (P 0.05). Conclusions The luteinizing hormone effects of Forskolin were amplified, whereas the follicle stimulating hormone effects were inhibited, which may help to increase androgen secretion in insulin resistant granulosa cells. Key words: Insulin; Ovary; Androgen

The ovary is the main regulator of female fertility. Granulosa cell dysfunction may be involved in various reproductive endocrine disorders. Here we investigated the effect of insulin resistance on the metabolism and function of ovarian granulosa cells, and dissected the functional status of the mitogen-activated protein kinase signaling pathway in these cells. Our data showed that dexamethasone-induced insulin resistance in mouse granulosa cells reduced insulin sensitivity, accompanied with an increase in phosphorylation of p44/42 mitogen-activated protein kinase. Furthermore, up-regulation of cytochrome P450 subfamily 17 and testosterone and down-regulation of progesterone were observed in insulin-resistant mouse granulosa cells. Inhibition of p44/42 mitogen-activated protein kinase after induction of insulin resistance in mouse granulosa cells decreased phosphorylation of p44/42 mitogen-activated protein kinase, downregulated cytochrome P450 subfamily 17 and lowered progesterone production. This insulin resistance cell model can successfully demonstrate certain mechanisms such as hyperandrogenism, which may inspire a new strategy for treating reproductive endocrine disorders by regulating cell signaling pathways.

A 31-yr-old hirsute female with oligoamenorrhea since menarche had markedly elevated peripheral plasma testosterone (T) concentrations of 250-255 ng/100 ml (normal 20-60 ng/100 ml), which lacked a diurnal rhythm, were not suppressed by dexamethasone, were decreased by ACTH, and were massively increased to 2,530 ng/100 ml by human chorionic gonadotropin (hCG). The binding capacity of T-binding globulin (TeBG) was 0.2 mug/100 ml (normal = 1.1-3.3 mug/100 ml). Plasma delta 4-androstenedione (A) was elevated at 374-681 ng/100 ml (normal = 90-135 ng/100 ml). Plasma estrone (E1) and estradiol (E2) were normal. The endometrium was proliferative. A T-secreting tumor was suspected because the plasma T levels were higher than those observed in polycystic ovarian disease. Exploratory surgery revealed bilateral polycystic ovaries and a pure thecoma in the right ovary which was not visible on surface examination. The thecoma did not contain granulosa cells. Plasma T in the right ovarian vein, draining the tumor, was 28,200 ng/100 ml and in the left ovarian vein was 2,600 ng/100 ml. Plasma A was elevated in both ovarian veins: 11,170 ng/100 ml on the left and 8,360 ng/100 ml on the right. The thecoma contained 1.35 mug/g of T and only 0.014 mug/g and 0.007 mug/g of E2 and E1, respectively. Plasma A and T after bilateral oophorectomy and removal of the thecoma were normal at 184 ng/100 ml and 40 ng/100 ml, respectively. 1) This pure thecoma produced primarily T rather than E1 OR E2 and was gonadotropin-responsive. 2) A very high plasma androgen level in a female is an important clue to the presence of a tumor. A T-secreting tumor should be ssupected when the peripheral plasma T is over 250 ng/100 ml and when plasma T increases to over 1,000 ng/100 ml following hCG stimulation. 3) Tumors cannot be classified as estrogenic or androgenic on the basis of the character of the endometrium.

P-876: Berberine improves insulin resistance in granulosa cells in a similar way to metformin

BackgroundInsulin resistance (IR) contributes to ovarian dysfunctions in polycystic ovarian syndrome (PCOS) patients. Serum amyloid A1 (SAA1) is an acute phase protein produced primarily by the liver in response to inflammation. In addition to its role in inflammation, SAA1 may participate in IR development in peripheral tissues. Yet, expressional regulation of SAA1 in the ovary and its role in the pathogenesis of ovarian IR in PCOS remain elusive.MethodsFollicular fluid, granulosa cells and peripheral venous blood were collected from PCOS and non-PCOS patients with and without IR to measure SAA1 abundance for analysis of its correlation with IR status. The effects of SAA1 on its own expression and insulin signaling pathway were investigated in cultured primary granulosa cells.ResultsOvarian granulosa cells were capable of producing SAA1, which could be induced by SAA1 per se. Moreover, the abundance of SAA1 significantly increased in granulosa cells and follicular fluid in PCOS patients with IR. SAA1 treatment significantly attenuated insulin-stimulated membrane translocation of glucose transporter 4 and glucose uptake in granulosa cells through induction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression with subsequent inhibition of Akt phosphorylation. These effects of SAA1 could be blocked by inhibitors for toll-like receptors 2/4 (TLR 2/4) and nuclear factor kappa light chain enhancer of activated B (NF-κB).ConclusionsHuman granulosa cells are capable of feedforward production of SAA1, which significantly increased in PCOS patients with IR. Excessive SAA1 reduces insulin sensitivity in granulosa cells via induction of PTEN and subsequent inhibition of Akt phosphorylation upon activation of TLR2/4 and NF-κB pathway. These findings highlight that elevation of SAA1 in the ovary promotes the development of IR in granulosa cells of PCOS patients.

Our previous study have demonstrated the elevated cortisol concentration in the follicular fluid (FF) contributed to the insulin resistance of the granulosa cells (GCs) in PCOS, but the complicated cortisol generation mechanisms are still unknown. 11β-hydroxysteroid type 1(11β-HSD1) mainly functions as reductase in intact cells, converting cortisone to cortisol. Cortisol and IL-1β are known to induce 11β-HSD1 in number of tissues, but few results were obtained in ovarian GCs In this study, FF and GCs from PCOS and non-PCOS patients were collected to study the interaction of cortisol and IL-1β in 11β-HSD1 expression. The ELISA and qRT-PCR revealed that the cortisol and IL-1β concentration in FF and 11β-HSD1 abundance in GCs were elevated in PCOS patients. By using cultured GCs in vitro, we demonstrated that both cortisol and IL-1β could stimulate 11β-HSD1 expression. The induction of 11β-HSD1 by IL-1β was further inducted by cortisol, whereas the induction of IL-1β and IL-6 expression by IL-1β was completely inhibited by cortisol. In conclusion, cortisol and IL-1β preformed a synergistically upregulation of 11β-HSD1 expression in GCs, contributing to the accumulation of cortisol in FF of PCOS patients. This may lead to the metabolic disorders of the ovary.

Background and objective: Polycystic ovary syndrome (PCOS) is a complex metabolic disorder associated with ovulatory dysfunction, hyperandrogenism, obesity, and insulin resistance, which leads to subfertility. PCOS is the most frequent metabolic disorder in women and the major cause of infertility. Susceptibility to developing PCOS is determined by a complex interaction between environmental and genetic factors. Although different mechanisms have been proposed to explain PCOS manifestations, defects in insulin actions or in the insulin signaling pathways are central in the pathogenesis of the syndrome. However, the mechanisms (molecular players and signaling pathways) underlying its primary origin still remain an unsolved issue. Current research is increasingly focusing on the discovery of novel biomarkers to further elucidate the complex pathophysiology of PCOS. Sam68, an RNA-binding protein, is recruited to insulin signaling, mediating different insulin actions. We aimed to investigate the role of Sam68 in insulin signaling and the possible implications of Sam68 in the insulin resistance in PCOS. Materials and methods: Granulosa cells were taken from women with PCOS (n = 25) and healthy donors (n = 25) and, within the age range of 20 to 42 years, from GINEMED, Assisted Reproduction Centre, Seville, Spain. The Sam68 expression level was analyzed both by qPCR and immunoblot. Statistical significance was assessed by one-way ANOVA, followed by a post-hoc test. A p value of < 0.05 was considered statistically significant. Results: We found that insulin stimulation increases the phosphorylation and expression level of Sam68 in granulosa cells from normal donors. The downregulation of Sam68 expression resulted in a lower activation of both the MAPK and the PI3K pathways in response to insulin. Moreover, the granulosa cells from the women with PCOS presented a lower expression of Sam68, as well as insulin receptor and insulin receptor substrate-1 (IRS-1). In these cells, the overexpression of Sam68 resulted in an increased activation of both the MAPK and the PI3K pathways in response to insulin. Conclusions: These results suggest the participation of Sam68 in insulin receptor signaling, mediating the insulin effect in granulosa cells, and they suggest the possible role of Sam68 in the insulin resistance of PCOS.

Insulin resistance (IR) disrupts ovarian functions in polycystic ovary syndrome (PCOS). The contributing factors remains elusive. High mobility group box 1 (HMGB1), a damage-associated molecular pattern molecule, has been shown to be related to IR and autophagy, respectively, in peripheral tissues. Here, we investigated whether increased HMGB1 contributes to IR in granulosa cells of PCOS patients via induction of aberrant autophagy. Results showed that HMGB1 abundance in the follicular fluid was significantly increased with enhanced autophagy in granulosa cells in PCOS patients with IR. HMGB1 exacerbated autophagy in granulosa cells as evinced by increased LC3B II/I ratio and ATG7 as well as decreased p62, the markers for autophagy. Concurrently, HMGB1 impaired insulin sensitivities by attenuating the abundance of insulin receptor substrate-1, Akt phosphorylation, GLUT4 translocation, and glucose uptake in granulosa cells, which were reversed by blocking autophagy pathways with siRNA-mediated knockdown of ATG7 or with chloroquine and bafilomycin A1, the lysosome inhibitors. In conclusion, our results indicate that increased HMGB1 contributes to IR development in granulosa cells of PCOS patients, which is associated with exacerbation of autophagy by HMGB1. Control of HMGB1 production may be benefical for the improvement of insulin sensitivity in granulosa cells in PCOS.