Function Of P27～(kip1) In Mice Ovarian Development

The molecular mechanisms underlying mammalian ovarian follicle formation and activation are poorly understood. In this thesis, we provide several lines of genetic evidence showing that the cyclin dependent kinase (Cdk) inhibitior p27^kip1 (p27) controls ovrian development in mice, by suppressing follicle formation and activation, and by promoting follicle death.In order to study the function of p27, we used p27-deficient mice (p27^-/-). We extracted the genomic DNA from mice tail tips to determine the genotypes. To quantify the total numbers of ovarian follicles at various development stages, we counted all sections of an ovary that stained with hematoxylin for morphological observation and immunohistochemistry for detection of oocytes. Western blot were performed to measure protein levels in mice ovary and the expression /phosphorylation levels of components of phosphatidylinositol-3 kinase (PI3K) pathway in oocytes. Kinase assays using histone H1 as a substrate were performed to determine kinase activities.We first quantified the oocyte numbers at PD1 (postnatal day) and follicle numbers at PD8 by counting oocytes in serial sections of whole ovaries from p27(-/-) and p27^+/+ mice. In p27-deficient mice (p27^-/-), postnatal follicle assembly was accelerated, and the number of endowed follicles was doubled as compared to wild type mice (p27(+/+)). It indicates that p27 can suppress primordial follicle endowment. In p27-deficient mice, the activities of Cdk2 were elevated and may enhance follicle formation. The formation of ovarian follicles in p27-deficient mice was completely eliminated by simultaneous loss of Cdk2, indicating that Cdk2 is an essential molecule for oocyte survival and follicular formation in wild type and p27-deficient mice. p27 suppresses follicle formation in mice through supression the activition of Cdk2.By staining with hematoxylin for morphological analysis and staining with GCNA1 or VASA for immunohistochemistry analysis, we observed p27^+/+ and p27^-/- mice ovaries of PD2, PD4, PD8, PD13 and PD35 and quantified the number of primordial follicles and activated follicles of PD8, PD18, PD23 and PD35. Our data suggest that primordial follicles were premature activated in p27^-/- mice ovaries. Protein p27 supresses primordial follicle activation in mice. We also detected protein expression levels of component of PI3K pathway in p27^-/- mice ovaries by Western blot, the expression levels of Akt, p-Akt, Foxo3a and p-Foxo3a in p27^-/- oocytes were similar compared to those in p27^+/+ oocytes. That indicated that PI3K pathways in p27-deficient mice oocyte can control primordial follicle activation.However, at early adulthood (i.e.in 12-week-old mice), the overactivated follicles were depleted, causing premature ovarian failure, which is a symptom similar to that of premature ovarian failure in humans. Thus, the p27^-/- mice may be a useful model for the study of ovarian failure, where follicles are depleted due to overactivation.In mice, a large proportion of follicles disappear from non-growing follicle pool during the first wave of folicle development. However, in p27^-/- ovaries this rapid follicle death is largely prevented, indicating that p27 is a key molecule in the promotion of follicle atresia prior to sexual maturity. By performing kinase assays, we showed that Cdk2/Cdc2-cyclinA/Bl complexes-associated kinase activities are elevated in p27^-/- ovaries and suppressed follicle atresia before sexual maturity. Protein p27 prevents follicle atresia through suppression the activation of Cdk2/Cdc2 - cyclinA/B1 complexes.In conclusion, the p27 gene is the critical molecule in determining mammalian ovarian development. We propose that deregulation of p27 may lead to defect in folliclar development, which may cause disturbed ovarian function and pathological changes in ovary. The findings in this study may provide some useful knowledge in the search for genetic aberrations of the ovary that lead to defects in follicle development in human diseases, such as premature ovarian failure.