The role of UCH-L1 in mammalian spermatogonia

UCH-L1 在哺乳动物精原细胞中的作用

• 批准号: RGPIN-2016-03767
• 负责人: Dobrinski, Ina
• 金额: $ 3.93万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=614616
• 关键词: role; UCH; L1; mammalian; spermatogonia

Stem cells in the testis are one of the most important stem cell systems in the adult because they produce sperm that transmit genetic information across generations. Stem cells can divide in two distinct ways: they can self-renew to generate more stem cells or differentiate to a committed cell type that will proliferate and ultimately produce sperm. This process, known as stem cell fate decision, is essential for mammalian reproduction. If this delicate balance is skewed toward self-renewal, sperm cells will eventually disappear. If there is too much differentiation, however, the testis will lose all of its stem cells and consequently, sperm production will cease. In either case infertility will ensue. Given the importance of stem cell fate decision, it is perhaps surprising that the mechanisms that govern it are largely unknown. We previously identified a protein, called UCH-L1 that is specifically expressed in the nervous system and in the undifferentiated germ cells of the testis in many species. As germ cells begin to differentiate toward sperm, UCH-L1 is no longer present. Yet, while the expression of UCH-L1 is restricted to undifferentiated germ cells, its function in these cells is unknown. We have designed a series of experiments that help characterize the molecular and cellular pathways by which UCH-L1 influences germ cell fate decision. First, we will use a mouse model with a specific deletion of UCH-L1 to identify how its absence affects progression of spermatogenesis. We will then identify which proteins UCH-L1 binds to in germ cells and how it is involved in controlling germ cell differentiation. Studies in other cell types such as neurons or cancer cells show that UCH-L1 interacts with the mTORC1 pathway in the control of cell metabolism. We will investigate if this also occurs in the testis. We will also monitor differences in gene expression in the presence or absence of UCH-L1. The definitive test as to whether a cell is a true stem cell is the ability to recreate its entire cell lineage. In case of the testis, a true stem cell can colonize the testis of a recipient animal depleted of germ cells, and produce sperm. We hypothesis that the ability to reconstitute spermatogenesis is impaired in stem cells without UCH-L1 and we will use transplantation assays to test this hypothesis. Finally, we will translate our findings in the mouse model to the pig as an agriculturally important species. We will block the function of UCH-L1 in pig germ cells using a specific inhibitor or suppression of gene expression and will investigate whether the same pathways are affected as in the mouse model. We will then use transplantation assays previously developed by us to further define the role of UCH-L1 in pig germ cell differentiation. Knowing what regulates stem cell maintenance versus differentiation will allow us to develop systems to propagate testis stem cells in vitro and help design strategies for fertility regulation.

睾丸中的干细胞是成年人最重要的干细胞系统之一，因为它们产生精子，将遗传信息传递给后代。干细胞可以以两种不同的方式分裂：它们可以自我更新以产生更多的干细胞，或者分化为一种定向细胞类型，这种细胞类型将增殖并最终产生精子。这个过程被称为干细胞命运决定，对哺乳动物的繁殖至关重要。如果这种微妙的平衡倾向于自我更新，精子细胞最终会消失。然而，如果分化过多，睾丸将失去所有干细胞，从而停止精子产生。在任何一种情况下，不孕症都会随之而来。鉴于干细胞命运决定的重要性，也许令人惊讶的是，管理它的机制在很大程度上是未知的。我们以前发现了一种名为UCH-L1的蛋白质，它在许多物种的神经系统和睾丸未分化的生殖细胞中特异性表达。随着生殖细胞开始向精子分化，UCH-L1不再存在。然而，虽然UCH-L1的表达仅限于未分化的生殖细胞，但其在这些细胞中的功能尚不清楚。我们设计了一系列实验，帮助表征UCH-L1影响生殖细胞命运决定的分子和细胞途径。首先，我们将使用UCH-L1特异性缺失的小鼠模型来确定其缺失如何影响精子发生的进展。然后，我们将确定UCH-L1与生殖细胞中的哪些蛋白质结合，以及它如何参与控制生殖细胞分化。在其他细胞类型（如神经元或癌细胞）中的研究表明，UCH-L1在控制细胞代谢中与mTORC 1通路相互作用。我们将研究这是否也发生在睾丸中。我们还将监测存在或不存在UCH-L1时基因表达的差异。一个细胞是否是真正的干细胞的决定性测试是重建其整个细胞谱系的能力。在睾丸的情况下，真正的干细胞可以定殖于生殖细胞耗尽的受体动物的睾丸，并产生精子。我们假设没有UCH-L1的干细胞重建精子发生的能力受损，我们将使用移植试验来验证这一假设。最后，我们将把我们在小鼠模型中的发现转化为作为农业重要物种的猪。我们将使用特异性抑制剂或抑制基因表达来阻断猪生殖细胞中UCH-L1的功能，并将研究是否与小鼠模型中相同的途径受到影响。然后，我们将使用我们以前开发的移植试验来进一步确定UCH-L1在猪生殖细胞分化中的作用。了解是什么调节干细胞的维持与分化将使我们能够开发体外繁殖睾丸干细胞的系统，并帮助设计生育调节策略。