PARENT SESSION
OVARY - B

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(454) EVIDENCE THAT DIHYDROTESTOSTERONE INHIBITS INSULIN-MEDIATED MITOGENIC SIGNALING BY REDUCING THE CYCLIN D2 mRNA EXPRESSION IN OVARIAN GRANULOSA CELLS.

Kayampilly, Pradeep¹, Menon, K.M.J. ¹, ¹ Departments of Ob/Gyn and Biological Chemistry, Univeristy of Michigan Medical School, Ann Arbor, MI

ABSTRACT- FSH and insulin/IGF play an important role in granulosa cell proliferation and the sequential and combined actions of these mitogenic factors are critical for ovulation. Polycystic ovary syndrome (PCOS) is one of the major endocrinopathies affecting women of reproductive age. Hyperandrogenism and hyperinsulinemia are two of the characteristic features of PCOS. Studies from our laboratory have shown that under hyperandogenic conditions increased levels of dihydrotestosterone (DHT) inhibit FSH-stimulated granulosa cell proliferation by inhibiting cyclin D2 mRNA expression. In the present study we examined the effects of DHT on insulin-mediated mitogenesis in rat granulosa cells using cyclin D2 as a marker of cell proliferation. Granulosa cells from immature rats were cultured in serum-free DMEM/F12 medium. After attachment, cells were treated with DHT (90 ng/ml) or vehicle for 24 h. Both groups were then treated with insulin (100 nM) for 2 h and northern blot analysis was performed for cyclin D2 mRNA. Insulin treatment produced more than 2-fold increase in cyclin D2 mRNA expression in control cells while DHT treatment reduced (75%) this stimulation. To determine the site of inhibitory action of DHT in insulin-mediated cyclin D2 mRNA expression, we examined the phosphorylation of Akt and MAPK, two key molecules involved in insulin signaling. Granulosa cells were treated with DHT for 24 h followed by treatment with insulin (100nM) for 10 min and western blot analysis was performed for total and phosphorylated Akt and MAPK. Both control and DHT treated cells showed increased phosphorylation of Akt in response to insulin indicating that DHT exerted no inhibitory effect on this pathway. By contrast, although insulin treatment resulted in greater than 75% increase in MAPK phosphorylation, DHT treatment abolished this response. These results suggest that DHT selectively inhibits the mitogenic actions of insulin through the MAPK signaling pathway while exerting no effect on its signaling through Akt pathway. Thus, while insulin signals through multiple pathways in granulosa cells of developing follicles, in the hyperandrogenic state, DHT selectively blocks insulin-stimulated cell proliferation by inhibiting MAPK phosphorylation (supported by NIH grant HD 38424).

KEY WORDS: Cyclin D2, Akt, Insulin, MAPK