Analysis of the correlation between 11β-HSDs expression in the ovarian granulosa cells and cortisol metabolism in the ovary of PCOS / 上海交通大学学报（医学版）

Objective: To explore the relationship of 11β-hydroxysteroid dehydrogenases (11β-HSDs) expression in the ovarian granulosa cells and cortisol regeneration in the ovary of polycystic ovary syndrome (PCOS). Methods: In accordance with the revised Rotterdam inclusion/exclusion criteria (2003), 24 patients in the PCOS group were included, meanwhile 21 patients in the control group were included. Follicular fluid and luteinized granulosa cells were collected from patients underwent in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) in the Center for Reproductive Medicine of Renji Hospital. Enzyme linked immunosorbent assay (ELISA) was used to detect the cortisol concentration and the cortisone levels of follicular fluid. Real-time quantitative PCR (RT-qPCR) was used to measure 11β-HSDs abundance in the luteinized granulosa cells. The expression of 11β-HSD1 and 11β-HSD2 in the luteinized granulosa cells were verified by immunofluorescence. The correlation of local cortisol level in the ovary with 11β-HSDs expression in the luteinized granulosa cells was further analyzed. Results: Compared with the control group, the cortisol level in the follicular fluid was elevated in the PCOS group [(477.3±35.3) nmol/L vs (292.0±36.4) nmol/L, P=0.014], while no change was observed in the cortisone levels in two groups. Both 11β-HSD1 and 11β-HSD2 were detected in the luteinized granulosa cells. Compared with the control group, the 11β-HSD1 mRNA abundance (P=0.033) and reductase activity (P=0.006) were increased significantly in the PCOS group, and there was no significant difference in the 11β-HSD2 expression and oxidase activity between two groups. Consistently, Pearson correlation analysis showed that the 11β-HSD1 but not 11β-HSD2 mRNA levels in the luteinized granulosa cells was correlated with the cortisol level in the follicular fluid (r2=0.347 6, P=0.001). Conclusion: Elevation of local cortisol in the ovary of PCOS patients may be related to the increased expression of 11β-HSD1 and reductase activity in the ovarian granulosa cells.

Influence of Tumor Necrosis Factor-alpha on Expression of Insulin-like Growth Factor-II, Insulin-like Growth Factor Binding Protein-1, 2, 3, 5 in Cultured Human Luteinized Granulosa Cells / 대한산부인과학회지

OBJECTIVE: To investigate the influence of tumor necrosis factor (TNF)-alpha on the expression of insulin-like growth factor (IGF)-II, insulin-like growth factor binding protein (IGFBP)-1, 2, 3, and 5 mRNA in cultured human luteinized granulosa cells. METHODS: Human luteinized granulosa cells were obtained from the follicular fluid by transvaginal oocyte aspiration from infertile patients undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF). The cells were cultured for 72 hours with TNF-alpha at concentrations of 1.0, 10.0, and 100.0 ng/mL, respectively. The cells not treated with TNF-alpha were served as control. Riverse transcription-polymerase chain reaction (RT-PCR) had been used to examine the expression of IGF-II, IGFBP-1, 2, 3, and 5 mRNA. Results were analyzed with Kolmogorov-Smirnov test and analysis of variance (ANOVA) and statical significance was defined as p<0.05. RESULTS: The expressions of IGF-II mRNA in 10.0 and 100.0 ng/mL of TNF-alpha groups were significantly lower than the control group (p<0.05, p<0.05, respectively). The expressions of IGFBP-2 mRNA were seemed to be decreased in 10.0 and 100.0 ng/mL of TNF-alpha groups than the control group (p=0.05, p=0.06, respectively). The expression of IGFBP-3 mRNA was seemed to be increased in 100.0 ng/mL of TNF-alpha group than the control group (p=0.08). There were no statistically significant differences in the expressions of IGFBP-1 and 5 in all groups. CONCLUSION: TNF-alpha might play a role as a regulator of human ovarian physiology by modulating the expression of IGF-II in luteinized granulosa cells.

Transforming Growth Factor-alpha Regulation of Insulin-like Growth Factor-II, Insulin-like Growth Factor Binding Protein-1 and 3 secretion and Epidermal Growth Factor receptor expression in human luteinized granulosa cells / 대한산부인과학회잡지

OBJECTIVE: To investigate the effects of transforming growth factor (TGF)-alpha on insulin-like growth factor (IGF)-II, insulin-like growth factor binding protein (IGFBP)-1, and 3 secretion and epidermal growth factor (EGF) receptor expression in cultured human luteinized granulosa cells. MATERIALS AND METHODS: Human luteinized granulosa cells were obtained from follicular fluid by transvaginal oocyte aspiration from infertile patients undergoing in vitro fertilization and cultured for 72 hours with TGF-alpha at concentration of 1.0, 10.0, 100.0 ng/ml. The luteinized granulosa cells not treated with TGF-alpha served as control. The secretion of IGF-II, IGFBP-1 and 3 were determined in conditioned media by immunoradiometric assay (IRMA) and reverse transcription-polymerase chain reaction (RT-PCR) was performed for EGF receptor mRNA expression. RESULTS: The cell numbers of 1.0 and 10.0 ng/ml supplement groups were significantly decreased compared to control (p<0.05, p<0.05, respectively), although the cell viabilities were similar in all groups. IGF-II levels were significantly higher in TGF-alpha treatment group at 1.0 and 10.0 ng/ml (p<0.01, p<0.01, respectively), but lower in 100.0 ng/ml (p<0.01). However, the concentrations of IGFBP-1, and 3 per one granulosa cell in each group were no statistically significant differences among the groups. The mRNA concentration of EGF receptor in cultured human luteinized granulosa cells were not significantly different among the groups. CONCLUSION: This study suggests that TGF-alpha regulate intrafollicular bioavailable IGF-II levels, by which TGF-alpha might involved luteinizations. However, TGF-alpha may not directly regulate EGF receptor mRNA expression in cultured human luteinized granulosa cells.

The Effect of Nitric Oxide on Apoptosis in Human Luteinized Granulosa Cells / 대한산부인과학회잡지

OBJECTIVE: To investigate the effect of nitric oxide on the apoptosis of human luteinized granulosa cells. METHODS : Granulosa cell suspensions were incubated for 48 hours after adding nitric oxide donor (S-nitroso-N-acetyl-penicillamine, SNAP) and nitric oxide synthase inhibitor (nitro-L-arginine methyl ester, L-NAME) at different concentrations. Apoptosis was examined using a terminal deoxynucleotide transferase- mediated dUTP-biotin nick end labeling method, and immunocytochemical staining was performed for six apoptosis-related proteins. RESULTS: Apoptotic rates were significantly lower in cells incubated in SNAP 0.5 mM, but higher in L-NAME 0.5, 1.0, and 5.0 mM. SNAP 0.5 mM lowered the expressions of Fas and p53 in granulosa cells, but Bcl-2 expression was increased, and Fas ligand or Bax remained unchanged. In L-NAME 0.5 and 5.0 mM, the expressions of p53 and Bax were increased, and Bcl-2 was unchanged. Fas/Fas ligands were also activated especially in L-NAME 5.0 mM. CONCLUSION: Nitric oxide may inhibit apoptosis via decreased Fas and p53, and increased Bcl-2 expression in human luteinized granulosa cells.

Influence of Tumor Necrosis Factor-alpha on the Secretion of Estradiol, Progesterone, Insulin-like Growth Factor-II, Insulin-like Growth Factor Binding Protein-1, 2, 3 in Cultured Human Luteinized Granulosa Cells / 대한산부인과학회잡지

OBJECTIVES: To investigate the influence of TNF-alpha on the secretion of estradiol (E2), progesterone (P4), insulin-like growth factor (IGF)-II, insulin-like growth factor binding protein (IGFBP)-1, 2, and 3 in cultured human luteinized granulosa cells MATERIALS AND METHODS: Human luteinized granulosa cells were obtained from the follicular fluid by transvaginal oocyte aspiration from infertile patients undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF). The cells were grouped into the control, 1.0, 10.0, and 100.0 ng/ml of TNF-alpha group according to the concentrations of TNF-alpha. The cells were cultured for 72 hours with the different concentrations of TNF-alpha as descibed above. The cells not treated with TNF-alpha served as control. The concentrations of E2, P4, IGF-I, IGFBP-1, 2, and 3 were determined in conditioned culture media by immunoradiometric assay (IRMA) or radioimmunoassay (RIA). RESULTS: The cell number in 100.0 ng/ml of TNF-alpha group was significantly higher than those in other groups, although the cell viabilities were similar in all groups. There were no statistically significant differences in the concentrations of E2 in all groups. However, the concentrations of P4 were seemed to be decreased as the concentrations of TNF-alpha were increased and the concentration of P4 in 100.0 ng/ml of TNF-alpha group was significantly lower than those in the control and other TNF-alpha groups. The concentrations of IGF-II, IGFBP-1, 2, and 3 were not different among the control and each TNF-alpha group. The secretion of E2 and P4 was not affected by IGF type I receptor antibody pretreatment. CONCLUSION: TNF-alpha might play a role as a regulator of ovarian physiology by modulating luteinized granulosa cellular proliferation and P4 secretion, and this mechanism might not be related to IGF system.

Apoptosis of human luteinized granulosa cell via change of mitochondria / 대한산부인과학회잡지

OBJECTIVE: To determine the effects of extracellular ATP on mitochondrial function and apoptosis during human luteinized granulosa cell cultures. METHODS: The addition of various concentrations of ATP (0, 0.1, 0.25, 0.5, 0.75 mM) to luteinized granulosa cells obtained during In vitro fertilization ovum pickup procedures. After culture for 24 hours, purinoceptor activity and functional changes in mitochondria were measured by patch clamp, flow cytometry, and confocal microscopy. RESULTS: Measurement by patch clamp of the granulosa cell membrane potential after ATP addition to cultured granulosa cells showed that both the inward and outward currents were expressed. After treatment of the granulosa cells with JC-1, measurement of the mitochondrial activity by confocal microscopy showed that the with increasing concentrations of ATP the relative ratio of undamaged mitochondria (red/green ratio) tended to decrease (P=0.027). After double staining of the cultured granulosa cells with Annexin V and Propidium Iodide, quantitative flow cytometry analysis showed that apoptosis increased with increasing concentrations of ATP (7.88%, 8.44%, 11.40%, 13.52%, 18.57%). CONCLUSION: The results of this study shows that apoptosis of granulosa cells increases with increasing extracellular ATP concentrations in cultured human luteinized granulosa cells. This is observed to be a consequence of cell membrane purinoceptor activity and functional changes in the mitochondria. It is therefore thought that remodelling processes of the ovarian tissue is regulated by neuroendocrine factors of the extracellular ATP.