Statistical analyses

Each experiment was repeated three-times using different animal sources of granulosa cells (8–12 animals per experiment). Each experimental group was represented by four chamber-slide wells. In each well, at least 1000 randomly selected cells were assessed by a Trypan blue test and quantitative immunocytochemical assays. The amount of IGF-I in each experiment was analysed in groups of four wells per measurement. After the IGF-I assay, the values of the blank controls were subtracted from the specific values determined in the cell-conditioned medium to exclude any non-specific background values (which were less than 7% of the total values). Rates of secretion were calculated as a proportion of 1 × 10⁶ viable cells/day. Differences among groups were evaluated using one-way ANOVA followed by Tukey’s test using Sigma Plot 11.0 (Systat Software, GmbH, Erkhart, Germany). Values are presented as means ± SEM. Differences were considered to be statistically significant if p < 0.05.