Mechanisms of Female Germline Stem Cell Senescence

描述（由申请人提供）：最近，我们报道了雌性小鼠具有生殖干细胞（GSC），其在出生后的生命中用新的卵母细胞补充卵巢。用白消安（一种已知会损害男性GSC和造血干细胞（HSC）功能的化学物质）消除GSC会导致3周内原始卵泡池的丧失。虽然白消安被认为通过细胞毒性作用消除GSC而导致生精障碍，但尚未发表直接证明这一点的研究。事实上，最近白消安已被证明是通过诱导过早衰老而不是通过激活细胞凋亡来抑制HSC功能。此外，白消安诱导的HSC“衰老”伴随着与复制性衰老相关的两种蛋白质水平的增加，这两种蛋白质都由Ink 4a基因座编码。Ink 4a的表达在非衰老细胞中被polycomb基因产物Bmi-I主动抑制。在成纤维细胞中，Bmi-I表达下调与衰老一致，并且Bmi-I的过表达通过抑制Ink 4a表达来延长复制寿命。对Bmi-1突变小鼠的研究表明，神经干细胞和造血干细胞的自我更新速率降低。这些缺陷通过Ink 4a的缺失而部分得到挽救，证实Ink 4a基因座是Bmi-I维持干细胞体内复制潜力的关键靶标。我们假设，GSC的存在和所需的再生卵泡池在青春期前和生殖生活中的小鼠。然而，随着年龄的增长，女性GSC激活一个复制衰老的程序，涉及增加表达的Ink 4a，由于Bmi-I的损失。因此，通过闭锁导致的卵泡耗竭不再被原始卵泡更新所抵消，最终导致卵泡储备耗尽和卵巢衰老。为了验证这些假设，提出了以下具体目的：1）表征从新生到生殖生命的小鼠卵巢中GSC的数量和增殖潜力以及Bmi-1和lnk 4a表达的年龄相关变化; 2）评估缺乏Bmi-1的突变小鼠是否由于Bmi-1的过早复制性衰老导致原始卵泡更新失败而具有减少的卵泡储备。I缺乏GSC;和3）确定缺乏Ink 4a基因产物表达的突变小鼠是否由于GSC衰老的激活随年龄增长而受损而表现出延长的卵巢功能。

DESCRIPTION (provided by applicant): 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.