Analysis of Binding Sites for IGF-I on Membranes from Granulosa Cells of Small, Medium, and Large Porcine Follicles
We examined binding of IGF-I to granulosal membranes from small (<3 mm), medium (3–6 mm) and large (>8 mm) porcine follicles. Granulosa cells were aspirated and membranes prepared by dounce homogenization. Membranes from each group were incubated for 16 h at 4°C (optimal conditions) with I-IGF-I in the absence or presence of unlabeled IGF-I (0.01–1 μg/ml), IGF-II (0.5–5 μg/ml), or insulin (0.1–20 μg/ml). Scatchard analysis of the data demonstrated a curvilinear plot for all three groups. The high-affinity sites had the following Ka values (mean ± SEM; nM): small = 6.50 ± 0.09; medium = 6.45 ± 0.44; large = 6.48 ± 0.44. The binding capacities of the high-affinity sites for each group were (pmol/mg protein): small = 1.51 ± 0.17; medium = 1.15 ± 0.14; large = 1.40 ± 0.15. The low-affinity sites had the following Ka values (nM): small = 0.053 ± 0.008; medium = 0.077 ± 0.019; large = 0.077 ± 0.007. The low-affinity sites had the following binding capacities (pmol/mg protein): small = 20.40 ± 3.10; medium = 13.76 ± 0.78; large = 12.99 ± 0.55. Preferential binding for the high-affinity sites was IGF-I > IGF-II > insulin. These data show that although IGF-I plays a role in granulosal differentiation, the affinity and number of high-affinity binding sites do not change during follicular maturation. However, the number of low-affinity binding sites appears to be greater in granulosa cells from small follicles, suggesting a possible role for IGF-II and/or insulin in early follicular maturation.
KeywordsGranulosa Cell Follicular Fluid Scatchard Analysis Large Follicle Follicular Maturation
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