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.