HORMONE-INDUCED PROLIFERATION AND DIFFERENTIATION OF GRANULOSA-CELLS - A COORDINATED BALANCE OF THE CELL-CYCLE REGULATORS CYCLIN D2 AND P27(KIP1)

The proliferation and terminal differentiation of granulosa cells are critical for normal follicular growth, ovulation, and luteinization. T herefore, the in situ localization and hormonal regulation of cell cyc le activators (cyclin D1, D2, and D3) and cell cycle inhibitors (p27(K ip1) and p21(Cip1)) were analyzed in ovaries of mice and rats at defin ed stages of follicular growth and differentiation. Cyclin D2 mRNA was specifically localized to granulosa cells of growing follicles, while cyclin D1 and cyclin D3 were restricted to theca cells. In hypophysec tomized (H) rats, cyclin D2 mRNA and protein were increased in granulo sa cells by treatment with estradiol or FSH and were increased maximal ly by treatment with both hormones. In serum-free cultures of rat gran ulosa cells, cyclin D2 mRNA was rapidly elevated in response to FSH, f orskolin, and estradiol, indicating that estradiol as well as cAMP can act directly and independently to increase cyclin D2 expression. The levels of p27(Kip1) protein were not increased in response to estradio l or FSH. In contrast, when ovulatory doses of human CG (LH) were admi nistered to hormonally primed H rats to stimulate luteinization, cycli n D2 mRNA and protein were rapidly decreased and undetectable within 4 h, specifically in granulosa cells of large follicles. Also in respon se to LH, the expression of the cell cycle inhibitor p27(Kip1) was ind uced between 12 and 24 h (p21(Cip1) was induced within 4 h) and remain ed elevated specifically in luteal tissue. A critical role for cyclin D2 in the hormone-dependent phase of follicular growth is illustrated by the ovarian follicles of cyclin D2(-/-) mice, which do not undergo rapid growth in response to hormones, but do express markers of FSH/LH action, cell cycle exit, and terminal differentiation. Collectively, these data indicate that FSH and estradiol regulate granulosa cell pro liferation during the development of preovulatory follicles by increas ing levels of cyclin D2 relative to p27(Kip1) and that LH terminates f ollicular growth by down-regulating cyclin D2 concurrent with up-regul ation of p27(Kip1) and p21(Cip1).