Fig. 7. Mkp3 is a KRAS^G12D-induced gene involved in normal and abnormal granulosa cell development. (A) Semi-quantitive RT-PCR shows that Mkp3 mRNA levels were elevated in LSL-Kras^G12D;Amhr2- Cre ovaries as compared with those of wild-type mice (n=3 for each genotype). (B) Mkp3 mRNA expression was induced in wild-type granulosa cells by eCG/hCG treatment. (C-H) In situ hybridization for Mkp3 in wild-type and Kras mutant ovaries. Bright-field images show ovarian histology (C,E,G), whereas dark-field images show the signals of Mkp3 antisense probe (D,F,H). Mkp3 mRNA was detected in granulosa/cumulus cells at 4 hours after hCG (D), but not in immature wild-type ovaries (F). By contrast, Mkp3 mRNA was detected in some preantral follicles (H, arrows) in Kras mutant ovaries of the same age. (I,J) Mkp3 siRNA decreased the Mkp3 mRNA levels in unstimulated granulosa cells (I) or those stimulated with AREG (J). (K,L) AREG induced transient phosphorylation of ERK1/2 in control granulosa cells; however, in the granulosa cells treated with Mkp3 siRNA, levels of phospho-ERK1/2 remained elevated for longer (K). (L) Intensity comparison of phospho-ERK2/total ERK2. (M,N) PD98059 blocked AREG-induced Mkp3 expression (M), when the ERK1/2 activation is blocked (N).