Recently, we reported that female mice possess germline stem cells (GSC) that replenish the ovaries with new oocytes during postnatal life. Elimination of GSC with busulfan, a chemical known to impair male GSC and hematopoietic stem cell (HSC) function, results in loss of the primordial follicle pool within 3 weeks. Although busulfan is thought to cause spermatogenic failure by eliminating GSC through cytotoxic actions, no studies have been published that have directly proven this. Indeed, very recently busulfan has been shown to suppress HSC function by inducing premature senescence rather than by activating apoptosis. Further, busulfan-induced 'aging' in HSC was accompanied by increased levels of two proteins linked with replicative senescence, both of which are encoded by the Ink4a locus. Expression of Ink4a is actively repressed in non-senescent cells by the polycomb gene product, Bmi-I. In fibroblasts, Bmi-1 expression is downregulated coincident with senescence, and overexpression of Bmi-I extend replicative lifespan by suppressing Ink4a expression. Studies of Bmi-1 mutant mice have shown that neural and hematopoietic stem cells exhibit a reduced rate of self-renewal. These defects are partially rescued by loss of Ink4a, confirming that the Ink4a locus is a critical target for Bmi-I to maintain replicative potential of stem cells in vivo. We hypothesize that GSC are present and required for regenerating the ovarian follicle pool during prepubertal and reproductive life in the mouse. However, with advancing chronological age female GSC activate a program of replicative senescence involving increased expression of Ink4a, due to a loss of Bmi-I . As a consequence, depletion of follicles through atresia is no longer offset by primordial follicle renewal, ultimately resulting in exhaustion of the follicle reserve and ovarian senescence. To test these hypotheses, the following Specific Aims are proposed: l) to characterize age-related changes in the number and proliferative potential of GSC, as well as in Bmi-I and lnk4a expression, in the mouse ovary from neonatal through reproductive life; 2) to evaluate if mutant mice lacking Bmi-1 possess a reduced follicle reserve due to failed primordial follicle renewal resulting from premature replicative senescence of Bmi-I deficient GSC; and 3) to determine if mutant mice lacking expression of Ink4a gene products exhibit extended ovarian function due to impaired activation of GSC senescence with advancing age.
