Characterization of age-related changes in spermatogonial dedifferentiation

项目摘要 这一应用的广泛的、长期的目标是表征与年龄相关的基因的内在变化 生态位微环境中的表达和与年龄相关的外在变化影响着 去分化。这项提案将使用精原细胞去分化来研究这些问题--其中 在果蝇的睾丸中，分化的生殖细胞恢复为精原干细胞。最先进的技术 这些技术将被用来检测RNA聚合酶II在精原细胞去分化过程中的全基因组结合。 这项提议将利用果蝇强大的遗传学和血统追踪，以及 明确识别精原干细胞、精原细胞和躯体支持的能力 果蝇睾丸中的细胞。这一建议得到了已发表的研究结果的支持，这些结果表明，精原细胞 脱分化是维持一生中强大的精原干细胞库所必需的，而且这一点 这一过程需要Jun N末端激酶(JNK)信号。这项工作也得到了未出版的初步研究的支持 数据显示，随着年龄的增长，精原细胞脱分化率急剧下降。此外，这一点 这一提议得到了未发表的结果的支持，这些结果表明，生态位微环境发生了重大变化 衰老过程中：在老年男性，异位细胞外基质聚集在壁龛周围。第一个目标是有针对性的 DAMID将仅用于在睾丸中恢复精原细胞时获得RNA Pol II的全基因组结合 从年轻的和年老的雄性。对这些数据集的分析将揭示年轻但不是老年人被激活的基因 精原细胞。在第二个目标中，来自目标1的一个或两个经过验证的基因将被用来测试该模型 年轻睾丸中候选基因缺失应降低去分化率，其过度表达应 提高老年男性的返老率。第二个目标中的实验也将检验减少的假设 老年男性的去分化是细胞恢复而不是上调细胞外基质结合蛋白的结果。 这项提案中的研究将增加更多关于衰老如何影响去分化的知识基础 复杂的系统，将为年龄相关男性的机制和治疗研究开辟新的途径 不孕不育。

Project Summary The broad, long-term objectives of this application are to characterize how age-related intrinsic changes in gene expression and age-related extrinsic changes in the niche microenvironment impact the process of dedifferentiation. This proposal will examine these issues using spermatogonial dedifferentiation - in which differentiating germ cells revert to become spermatogonial stem cells - in the Drosophila testis. State of the art techniques will be used to detect genome-wide binding of RNA Polymerase II in dedifferentiating spermatogonia. The proposal will capitalize upon the powerful genetics and lineage tracing available in Drosophila, as well as the ability to unequivocally identify the niche, spermatogonial stem cells, spermatogonia and somatic support cells in the Drosophila testis. This proposal is supported by published results demonstrating that spermatogonial dedifferentiation is required to maintain a robust pool of spermatogonial stem cells over a lifetime and that this process requires Jun N-terminal kinase (JNK) signaling. This work is also supported by unpublished preliminary data showing that the rate of spermatogonial dedifferentiation declines steeply during aging. Additionally, this proposal is supported by unpublished results showing that the niche microenvironment changes substantially during aging: in old males, an ectopic extracellular matrix accumulates around the niche. In the first aim, targeted DamID will be used to obtain the genome-wide binding of RNA Pol II only in reverting spermatogonia in testes from young and old males. Analyses of these data sets will reveal genes that are activated in young but not old spermatogonia. In the second aim, one or two validated genes from Aim 1 will be used to test the model that candidate gene depletion in young testes should decrease dedifferentiation rates and its overexpression should increase reversion rates in old males. Experiments in the second aim will also test the hypothesis that decreased dedifferentiation in old males is a result of reverting cells not upregulating extracellular matrix-binding proteins. The studies in this proposal will increase the knowledge base about how aging affects dedifferentiation in more complex system and will foster new avenues of research into mechanisms and treatments for age-related male infertility.