Insulin Resistance Directly Contributes to Androgenic Potential Within Ovarian Theca Cells  JunWei Qu, M.D., Yong Wang, Ph.D., Xiaoke Wu, M.D., Ph.D., Lei Gao, M.D., Lihui Hou, M.D., Risto Erkkola, M.D., Ph.D.  Fertility and Sterility  Volume 91, Issue 5, Pages 1990-1997 (May 2009) DOI: 10.1016/j.fertnstert.2008.02.167 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 In vitro cultured theca cells. Combined theca–granulosa membranes were removed from porcine immature follicles (diameter 6–10 mm). After granulose cells were discarded, residual tissue was cut into small pieces and cultured for 2 days (A). Theca cells expanded more fluently from the fringe of the tissue after culture for 4 days (B). Cell suspension was collected and divided in dishes (C) and cultured up to 10 days with well morphology and viability (D). Fertility and Sterility 2009 91, 1990-1997DOI: (10.1016/j.fertnstert.2008.02.167) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Medium glucose levels in theca cells. Porcine theca cells were cultured without (Con) or with dexamethasone (IR) and were further treated by Tro and Met in IR cells or vehicle dimethylsulfoxide (DMSO) only in IR and Con cells. The medium glucose levels were determined for indirect evaluation for cell glucose uptake. The data represent the mean ± SE of three independent experiments. Con = control cells with vehicle DMSO-only treatment; IR = Dex-induced IR in cells with vehicle DMSO-only treatment; Tro = IR cells with Tro treatment; Met = IR cells with Met treatment. aP<.05 vs. Con; bP<.05 vs. IR. Fertility and Sterility 2009 91, 1990-1997DOI: (10.1016/j.fertnstert.2008.02.167) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Messenger RNA expression of IRS-1, GLUT4, and PPARγ in theca cells. Porcine theca cells were induced without (Con) or with Dex (IR) and were further treated by Tro and Met in IR cells or vehicle dimethylsulfoxide (DMSO) only in IR and Con cells. Messenger RNA expression was detected by RT-PCR for β-actin (A), IRS-1 (B), GLUT4 (C), and PPARγ (D) in theca cells. The target genes were determined by the intensity of the ethidium bromide × area/β-actin × area with arbitrary unit (E). The data represent the mean ± SE of three independent experiments. Con = control cells with vehicle DMSO-only treatment; IR = Dex-induced IR cells with vehicle DMSO-only treatment; Tro = IR cells with troglitazone treatment; Met = IR cells with Met treatment. aP<.05 vs. Con; bP<.05 vs. IR. Fertility and Sterility 2009 91, 1990-1997DOI: (10.1016/j.fertnstert.2008.02.167) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Protein expressions of IRS-1 and GLUT4 in theca cells. Porcine theca cells were induced without (Con) or with Dex (IR) and were further treated by Tro and Met in IR cells or vehicle dimethylsulfoxide (DMSO) only in IR and Con cells. Both the IRS-1 and GLUT4 protein levels were significantly lower in Dex-treated IR cells than in Con cells. IRS-1 protein levels increased significantly in Tro/Met vs. IR cells (A), and the GLUT4 protein levels did not change by Tro but were increased significantly by Met (B), as shown by expression quantification (C). Fertility and Sterility 2009 91, 1990-1997DOI: (10.1016/j.fertnstert.2008.02.167) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Medium steroid levels and mRNA expression of CYP17 in theca cells. Porcine theca cells were induced without (Con) or with Dex (IR) and were further treated by Tro and Met in IR cells or by vehicle dimethylsulfoxide (DMSO) only in IR and Con cells. The media of four cell groups were collected for assay of P (A) and T (B) by RIA, and remaining cells were collected for mRNA expression of β-actin (C) and CYP17 (D); β-actin as a housekeeping gene was used as the internal standard. The target gene was determined by the intensity of the ethidium bromide ×area/β-actin × area with arbitrary unit (E). The data represent the mean ± SE of three independent experiments. Con = control cells with vehicle DMSO-only treatment; IR = Dex-induced IR cells with vehicle DMSO-only treatment; Tro = IR cells with Tro treatment; Met = IR cells with Met treatment. aP<.01 vs. Con; bP<.01 vs. IR. Fertility and Sterility 2009 91, 1990-1997DOI: (10.1016/j.fertnstert.2008.02.167) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions