Figure 2 A-C. Western blot analysis on the effect of metformin on pACC and ACC abundance in granulosa cells.

Granulosa cells from prehierarchical follicles (3–5 and 6–8 mm) and preovulatory follicles (9–12 mm) were dispersed into single-cell suspension and treated with 0, 1, 10, or 20 mM metformin for 60 min in the presence or absence of rhFSH (10 ng/ml) at 41°C. Cell lysates were prepared following treatment, and a Western blot analysis was performed under reducing conditions to quantify the abundance of pACC, ACC, and α-tubulin. Control received no rhFSH and no metformin. α-tubulin is used as a protein loading control.^A,BP < 0.05 by ANOVA; n=3 replicates of 4 broiler breeder hens each. Inset: Photograph of prehierarchical follicles and the most-recently recruited preovulatory follicle to denote the source of granulosa cell (open box).