The Cyclin-dependent Kinase Inhibitors p27 and p21 Cooperate to Restrict Proliferative Life Span in Differentiating Ovarian Cells*

The timing of cellular exit from the cell cycle during differentiation is specific for each cell type or lineage. Granulosa cells in the ovary establish quiescence within several hours after the ovulation-inducing luteinizing hormone surge, whereas they undergo differentiation into corpora lutea. The expression of Cdk inhibitors p21 and p27 is up-regulated during this process, suggesting that these cell cycle inhibitors are involved in restricting proliferative capacity of differentiating granulosa cells. Here we demonstrate that the lack of p27 and p21 synergistically renders granulosa cells extended an proliferative life span. Immunohistochemical analyses demonstrated that corpora lutea of p27, p21 double-null mice showed large numbers of cells with bromodeoxyuridine incorporation and high proliferative cell nuclear antigen expression, which were more remarkable than those in p27 single-deficient mice showing modest hyperproliferation. In contrast, differentiating granulosa cells in p21-deficient mice ceased proliferation similarly to those in wild-type mice. Interestingly, granulosa cells isolated from p27, p21 double-null mice exhibited markedly prolonged proliferative life span in culture, unlike cells with other genotypes. Cultured p27, p21 doublenull granulosa cells maintained expression of steroidogenic enzymes and gonadotropin receptors through 8–10 passages and could undergo further differentiation in responses to cAMP accumulation. Thus, the cooperation of p27 and p21 is critical for withdrawal of granulosa cells from the cell cycle, in concert with luteal differentiation and possibly culture-induced senescence.