Selective ovary resistance to insulin signaling in women with polycystic ovary syndrome  Xiao Ke Wu, M.D., Ph.D., Shan Ying Zhou, M.D., Jin Xia Liu, M.D., Pasi Pöllänen, M.D., Ph.D., Kirsimarja Sallinen, M.D., Marjaana Mäkinen, M.Sc., Risto Erkkola, M.D., Ph.D.  Fertility and Sterility  Volume 80, Issue 4, Pages 954-965 (October 2003) DOI: 10.1016/S0015-0282(03)01007-0

FIGURE 1 Mitogenic effect of insulin and IGF-1 on granulosa cells from women with PCOS or with NO. Cells were pretreated with troglitazone (Trog) or vehicle (DMSO) for 48 hours then stimulated with insulin (A) or IGF-1 (B), and the incorporation of [methyl-3H]thymidine into DNA was measured. Results are expressed as a percentage of the basal DNA radioactivity values in the absence of hormone stimulation. Each value is the mean ± SD of triplicate determinations. Filled squares = PCOS cells without Trog pretreatment; open squares = PCOS with Trog pretreatment; filled circles = NO cells without Trog pretreatment; and open circles = NO cells with Trog pretreatment. *P<.05; **P<.01 vs. NO cells; †P<.05; ‡P<.01 vs. vehicle-treated cells. Wu. Ovary insulin resistance in PCOS.Fertil Steril 2003. Fertility and Sterility 2003 80, 954-965DOI: (10.1016/S0015-0282(03)01007-0)

FIGURE 2 Metabolic effect of insulin and IGF-1 on granulosa cells from women with PCOS or NO. Cells were stimulated with insulin (A) or IGF-1 (B) and incorporation of D-[U-14C]glucose into glycogen was measured. Results are expressed as a percentage of the basal glycogen radioactivity values in the absence of hormone stimulation. Each value is the mean ± SD of triplicate determinations. Filled squares = PCOS cells without troglitazone pretreatment; open squares = PCOS with troglitazone pretreatment; filled circles = NO cells without troglitazone pretreatment; and open circles = NO cells with troglitazone pretreatment. *P<.05; **P<.01 vs. NO cells; †P<.05; ‡P<.01 vs. vehicle-treated cells. Wu. Ovary insulin resistance in PCOS.Fertil Steril 2003. Fertility and Sterility 2003 80, 954-965DOI: (10.1016/S0015-0282(03)01007-0)

FIGURE 3 Competition curves by insulin (A) or IGF-1 (B) for their individual [125I]-labeled ligands on granulosa cells from women with PCOS or with NO. Cells were incubated for 4 hours at 4°C with 0.1 μCi [125I]-labeled insulin or IGF-1 and an increasing concentration of unlabeled ligands after exposure to vehicle or troglitazone for 48 hours. Results are presented as the percentage of the tracer-labeled hormone specifically bound to cells. Each value is the mean ± SD of triplicate determinations. Filled squares = PCOS cells without troglitazone pretreatment; open squares = PCOS with troglitazone pretreatment; filled circles = NO cells without troglitazone pretreatment; and open circles = NO cells with troglitazone pretreatment. *P<.05; **P<.01 vs. NO cells; †P<.05; ‡P<.01 vs. vehicle-treated cells. Wu. Ovary insulin resistance in PCOS.Fertil Steril 2003. Fertility and Sterility 2003 80, 954-965DOI: (10.1016/S0015-0282(03)01007-0)

FIGURE 4 Expression of the IR, the IGF-1 receptor, IRS-1, and IRS-2 genes after treatment with vehicle (DMSO) or troglitazone (1 μg/mL) in cultured human granulosa cells from infertile women with PCOS or with NO. The RT-PCR products were electrophoresed on 2.5% agarose gel containing 0.5 μg/mL ethidium bromide (A). The relative mRNA expression levels of these signal molecules were determined by measurements of the intensity of the ethidium bromide. The data represent the mean ± SD of three independent experiments, and β-actin as a housekeeping gene was used as the internal standard (B). NO− = NO cells with vehicle only; NO+ = NO cells with troglitazone; PCOS− = PCOS cells with vehicle only; and PCOS+ = PCOS cells with troglitazone; and M = the DNA size marker. *P<.05; **P<.01 vs. NO cells; †P<.05; ‡P<.01 vs. vehicle-treated cells. Wu. Ovary insulin resistance in PCOS.Fertil Steril 2003. Fertility and Sterility 2003 80, 954-965DOI: (10.1016/S0015-0282(03)01007-0)

FIGURE 5 Expression of the IR, the IGF-1 receptor, IRS-1, and IRS-2 proteins after treatment with vehicle (DMSO), or troglitazone (1 μg/mL) in cultured human granulosa cells from infertile women with PCOS or with NO. Equal amounts of protein from cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred, and immunoblotted with appropriate antibodies against the specific proteins. The filters were then incubated with avidin-biotinylated horseradish peroxidase complex and stained with 3,3′-diaminobenzidine and hydrogen peroxide (A). The relative protein expression levels of these insulin/IGF-1 signal proteins were determined by measurements of the intensity of scanned bands. The data represent the mean ± SD of three independent experiments and are presented as the fold of β-actin (B). NO− = NO cells with vehicle only; NO+ = NO cells with troglitazone; PCOS− = PCOS cells with vehicle only; and PCOS+ = PCOS cells with troglitazone. *P<.05; **P<.01 vs. NO cells; †P<.05; ‡P<.01 vs. vehicle-treated cells. Wu. Ovary insulin resistance in PCOS.Fertil Steril 2003. Fertility and Sterility 2003 80, 954-965DOI: (10.1016/S0015-0282(03)01007-0)

FIGURE 6 Optical sections of granulosa cells visualized with a laser confocal microscope by double immunofluorescence under a ×60 objective. Cells were stained with anti-IRS-1 (A) antibody, followed by horse anti-mouse antibodies conjugated to tetramethylrhodamine isothiocyanate (red) and at the same time with anti-IRS-2 (B) antibody, followed by goat anti-rabbit antibodies conjugated to fluorescein isothiocyanate (green). Note the similar distribution (yellow) of IRS-1 and IRS-2 (C, overlay of A and B). Wu. Ovary insulin resistance in PCOS.Fertil Steril 2003. Fertility and Sterility 2003 80, 954-965DOI: (10.1016/S0015-0282(03)01007-0)