The Role and Regulation of TRP53 Activity in Oocytes and Granulosa Cells After Radiation-induced Damage

项目摘要 电离辐射（IR）是一种用于治疗癌症和消融患者的骨髓之前， 造血干细胞移植IR诱导损伤（双链断裂或活性氧） 但也可以杀死健康的旁观者细胞。原始卵泡（PF）是卵巢中数量有限的卵泡。 含有未成熟卵母细胞的卵泡，极易受到损伤。IR或其他引起的损害 遗传毒性化疗可耗尽卵巢的PF，导致卵巢功能不全（POI）， 不孕因此，有必要了解损伤如何影响卵巢细胞并导致POI， 可以开发毒性较小的治疗性或预测性治疗。该项目的目标是确定 哺乳动物中辐射诱导卵母细胞消除的机制。PF对损害的反应在很大程度上是 除了几个主要玩家之外，检查点激酶2（CHEK 2）主要激活促凋亡TRP 63 TA同种型（TAp 63）对损伤的反应。相比之下，体细胞主要使用另一个CHEK 2靶标， TRP53.我们的实验室显示，接受IR的Chek 2-/-雌性小鼠保留了其PF储备并产生了健康的幼崽。 接受低剂量IR的TAp 63-/-雌性维持PF，而Trp 53-/-小鼠失去其PF储备。表明 TRP 53在卵母细胞中与凋亡有关。在较高IR剂量或化疗的治疗中，TAp 63-/- 雌性失去PF储备，而Chek 2-/-雌性保留PF储备。我们预测了更高剂量的IR， 化疗引起更多的损伤，这激活了TAp 63非依赖性机制。事实上，TAp 63-/- 暴露于较高剂量IR的Trp 53-/-双突变雌性保留PF储备，表明TRP 53触发 当达到一定的损伤阈值时，卵母细胞被清除。TRP 53蛋白表达分析 高剂量IR在纯化的卵母细胞中鉴定了TRP 53的独特形式，并且不存在典型的~ 53 kDa蛋白， 在体细胞中检测到。基于这些观察，我们假设卵母细胞中的TRP 53活性受到调节， 通过卵母细胞特异性机制激活TRP 53或限制其作用，直到达到特定阈值 损害发生。该提案将利用遗传方法（嵌合重构卵巢）和蛋白质组学 方法（质谱法），以确定TRP 53调节卵巢损伤的反应。的目的 这项研究的目的是：（1）确定TRP 53依赖性PF损失是否在 或者是由于从体细胞颗粒细胞到卵母细胞的损伤信号，以及2）确定TRP 53 通过鉴定独特的翻译后修饰和/或蛋白质相互作用， 与TRP 53依赖性PF损失相关。确定卵母细胞特异性机制调节促凋亡 TRP 53活性对损伤的反应将提高我们对PF对遗传毒性剂反应的理解 并为治疗提供了靶点，以预防接受遗传毒性药物治疗的患者的卵母细胞丢失、不育和POI。 治疗。

Project Summary Ionizing radiation (IR) is a treatment used against cancer and to ablate patient's bone marrow prior to hematopoietic stem cell transplantation. IR induces damage (double strand breaks or reactive oxygen species) in target cells but can also kill healthy bystander cells. Primordial follicles (PFs) are a limited population of ovarian follicles that contain immature oocytes and are highly susceptible to damage. Damage induced by IR or other genotoxic chemotherapies can deplete the ovary of PFs resulting in premature ovarian insufficiency (POI) and infertility. Therefore, there is a need to understand how damage affects ovarian cells and leads to POI before less toxic therapeutic or predictive treatments can be developed. The goal of this project is to determine the mechanism of radiation-induced oocyte elimination in mammals. How PF's response to damage is largely unknown beyond a few major players. Checkpoint kinase 2 (CHEK2) primarily activates the pro-apoptotic TRP63 TA isoform (TAp63) in response to damage. In contrast, somatic cells predominantly use another CHEK2 target TRP53. Our lab showed Chek2-/- female mice receiving IR retained their PF reserve and produced healthy pups. TAp63-/- females receiving low dose IR maintained PFs while Trp53-/- mice lost their PF reserve. Suggesting that TRP53 is dispensable for apoptosis in oocytes. In treatments with higher IR dose or chemotherapies, TAp63-/- females lose PF reserve while Chek2-/- females retain PF reserve. We predicted higher doses of IR and chemotherapies cause more damage, which activates a TAp63-independent mechanism. Indeed, TAp63-/- Trp53-/- double mutant females exposed to higher dose IR retained PF reserve suggesting TRP53 triggers oocyte elimination when a certain threshold of damage is reached. Analysis of TRP53 protein expression after high dose IR identified a unique form of TRP53 in purified oocytes and the absence of the typical ~53kDa protein, detected in somatic cells. Based on these observations we hypothesize TRP53 activity in oocytes is regulated by an oocyte-specific mechanism which either activates TRP53 or restricts its action until a specific threshold of damage occurs. This proposal will utilize genetic approaches (chimeric reconstituted ovaries) and proteomic approaches (mass spectrometry) to determine TRP53 regulation in response to ovarian damage. The aims of this proposed research are to (1) determine whether TRP53-dependent PF loss is triggered intrinsically in the oocyte or is due to damage signals from somatic granulosa cells to the oocyte and 2) determine how TRP53 activity is regulated in oocytes by identifying unique post-translational modifications and/or protein interactions associated with TRP53-dependent PF loss. Defining oocyte-specific mechanisms regulating pro-apoptotic TRP53 activity in response to damage will improve our understanding of the PF response to genotoxic agents and provide targets for therapeutics to prevent oocyte loss, infertility, and POI in patients receiving genotoxic treatments.