1. Hyperexpression of epidermal growth factor receptors in granulosa cells from women with polycystic ovary syndrome 
Ghanim Almahbobi, Ph.D., Aileen Misajon, Paul Hutchinson, Nicholas Lolatgis, Alan O Trounson, Ph.D. 
Fertility and Sterility 
Volume 70, Issue 4, Pages (October 1998)
DOI: /S (98)

2. FIGURE 1
Epidermal growth factor receptor localization and quantitation in A431 cells. (A), Light scatter histogram of A431 cell line distributed as a homogeneous population focused in a small range of size and granularity. (B), The gated cell population in A shows low fluorescence background when incubated with the mouse isotype–matched negative control antibody. (C), Cells incubated with EGF receptor antibody. The whole cell population is displaced on the fluorescence scale with strong (9,521%) and homogeneous levels of receptor labeling. Inset: Confocal micrograph of a central plane of A431 cell showing intense EGF receptor staining confined to the cell membrane. FALS = forward-angle light scatter. Original magnification, ×100.
Fertility and Sterility  , DOI: ( /S (98) )

3. FIGURE 2
Identification of granulosa cell fraction using flow cytometric analysis. Light scatter analysis shows that the gated viable granulosa cells (GC) are large (A), with noticeable levels of autofluorescence (B). The arrow in A indicates the gated contaminating cells, mainly leukocytes and red blood cells. Flow cytometric histograms showing CD45 labeling of the gated granulosa (C and D) and leukocyte (E and F) fractions. Parts C and E were incubated with negative control antibody; parts D and F were incubated with anti-CD45 antibody. Most of the cells from the leukocyte fraction show positive labeling as displaced to the right side of the fluorescence scale (F). Only a few cells are labeled in the granulosa fraction (D), and they probably are contaminating cells. FALS = forward-angle light scatter. Cell number scales refer to parts C to F.
Fertility and Sterility  , DOI: ( /S (98) )

4. FIGURE 3
Representative histograms of EGF receptor labeling in human granulosa cells of different patients. Flow cytometric analysis of granulosa cells from an untreated patient with normal ovaries (A and B), an ovulatory patient with PCO (D and E), and an anovulatory patient with PCOS (G and H). Parts A, D, and G were incubated with negative control antibody, parts B, E, and H were incubated with EGF receptor antibody. The levels of EGF receptor labeling, expressed as a percentage of the positive labeling values relative to the control background values, are 36% (patient with normal ovaries, B), 100% (ovulatory patient with PCO, E), and 563% (anovulatory patient with PCOS, H). (C, F, and I), Immunofluorescence micrographs of granulosa cells of the corresponding cell populations in B, E, and H, respectively. Epidermal growth factor receptor staining is confined to the cell membrane. Unlike in C and F, the staining intensity in I is strong and seen in all granulosa cells. Original magnification, ×100 (C) and ×40 (F and I).
Fertility and Sterility  , DOI: ( /S (98) )

5. FIGURE 4
Mean (±SD) levels of EGF receptor (EGFR) labeling in granulosa cells of different groups of patients. The levels of EGF receptor labeling are expressed as percentages of the positive labeling values of a given sample relative to the control values of the corresponding sample. (A), In untreated patients, the levels of EGF receptor labeling in granulosa cells from both ovulatory patients with PCO and anovulatory patients with PCOS are significantly (P <0.001) higher than in granulosa cells from patients with normal ovaries. (B), The solid bars indicate that low levels of EGF receptor labeling were observed in granulosa cells from all gonadotropin-treated patients regardless of the health of their ovaries (normal or polycystic) or the type of treatment they received (minimal stimulation with FSH or full gonadotropin stimulation for superovulation). The low levels are comparable to those seen in granulosa cells of normal ovaries obtained from untreated patients (A). The open bars indicate that granulosa cells of patients with normal ovaries who were treated with clomiphene citrate show significantly higher levels of EGF receptor labeling than those of untreated patients with normal ovaries (A) but comparable to those of untreated ovulatory patients with PCO (A). The numbers at the top of the bars are the number of patients in each group.
Fertility and Sterility  , DOI: ( /S (98) )

6. FIGURE 5
Production of E2 in cultured human granulosa cells. The addition of human pituitary FSH (20 ng/mL) to the cultured granulosa cells obtained from superovulated patients significantly increased the production of E2 (n = 15 patients/separate experiments). ∗∗P <0.001, mean ± SD. Both EGF and TGFα abolished the FSH-stimulated E2 production in the same manner when added with FSH.
Fertility and Sterility  , DOI: ( /S (98) )