Cisplatin and Mechanisms of Long-term Male Infertility

描述（由申请人提供）：化疗药物顺铂可高效清除体内的致瘤细胞。然而，它不是选择性的，并导致睾丸正常生殖细胞的损失。历史上一直认为，在化疗治疗过程中干精原细胞的损失是长期不育和/或精子发生恢复延迟的原因。我们最近发现，在小鼠中，很大比例的干精原细胞存活临床反复顺铂暴露，没有随之而来的精子发生恢复。这表明睾丸内的其他体细胞受损。顺铂处理小鼠的睾丸组织学显示支持细胞空泡较大、顶端脱落、细胞物质脱落进入管腔和生殖细胞因凋亡而丢失，这些都是支持细胞损伤的表现。在这项研究计划中，我们将首先测试的主要假设，顺铂直接损伤支持细胞，并创建一个睾丸微环境不适合的发展和分化的常驻生殖细胞在化疗后的恢复期。顺铂处理的W/W-v小鼠将用作正常干细胞移植的受体，以测试睾丸微环境是否确实由于顺铂暴露而改变。这些小鼠由于c-kit受体突变而缺乏内源性生殖细胞，但具有正常的支持细胞和干细胞因子信号传导，这创造了一个独特的系统来研究正常的支持细胞，而没有生殖细胞的干扰存在。 在本建议的第二部分中，我们将重点阐明支持细胞损伤的机制。顺铂已被证明通过利用已经进化用于管理铜稳态的转运蛋白进入和离开靶细胞。我们的初步体外评价顺铂处理的TM 4支持细胞显示下调的主要铜外排转运蛋白ATP 7 B（低于我们的检测限的水平）。未观察到主要内流转运蛋白Ctrl水平的变化。我们提出顺铂破坏支持细胞中ATP 7 B的表达，导致顺铂（和铜）以威尔逊病样方式积累增加，从而导致对该细胞的损伤加重。我们将通过测量暴露于顺铂的小鼠睾丸中铂和铜的细胞内水平来测试这一假设，并将其水平与支持细胞特异性因子分泌的改变相关联。另外的实验将使用由于Ctrl突变而显示铜（顺铂）摄取减少50%的小鼠，以证明支持细胞损伤的敏感性与顺铂从细胞的摄取和排泄相关。本研究的长期目标是了解顺铂诱导的支持细胞损伤和生精损伤的机制。本项目过程中获得的数据将提供见解，将允许开发的战略，预防和/或逆转顺铂诱导的支持细胞损伤和重建功能性精子发生。

DESCRIPTION (provided by applicant): The chemotherapeutic drug, cisplatin, is highly efficient at removing tumorigenic cells from the body. However, it is not selective and results in loss of normal germ cells from the testes. It has been historically believed that loss of stem spermatogonia during chemotherapeutic treatment accounts for long-term infertility and/or delayed recovery of spermatogenesis. We recently showed, in mice, that a large proportion of stem spermatogonia survive clinic-like repeated cisplatin exposure, without an ensuing recovery in spermatogenesis. This indicates damage to other somatic cells within the testis. Histopathology of testes from cisplatin-treated mice revealed large Sertoli cell vacuoles, apical sloughing, shedding of cellular material into the lumen and loss of germ cells by apoptosis, which are all manifestations of Sertoli cell injury. In this research proposal, we will first test the primary hypothesis that cisplatin directly injures Sertoli cells and creates a testicular microenvironment unsuitable for the development and differentiation of resident germ cells during the recovery period after chemotherapeutic treatment. Cisplatin treated W/W-v mice will be used as recipients of normal stem cell transplants to test if testicular microenvironment is indeed altered as a result of cisplatin exposure. These mice lack endogenous germ cells due to a mutation in c-kit receptor, but have normal Sertoli cells and stem cell factor signaling, which creates a unique system to study normal Sertoli cells, without the interfering presence of germ cells. In the second part of this proposal, we will focus on elucidating the mechanism(s) of the injury to Sertoli cells. Cisplatin has been shown to enter and exit target cells by utilizing transporters that have evolved for the management of copper homeostasis. Our preliminary in vitro evaluation of cisplatin-treated TM4 Sertoli cells revealed the downregulation of the main copper efflux transporter ATP7B (to levels below our detection limits). No alteration in the levels of the main influx transporter, Ctrl, was observed. We propose that cisplatin disrupts the expression of ATP7B in Sertoli cells, resulting in increased accumulation of cisplatin (and copper) in a Wilson's disease-like fashion resulting in exacerbation of the injury to this cell. We will test this hypothesis by measuring intracellular levels of platinum and copper in testes of mice exposed to cisplatin and correlate their levels with alterations in secretion of Sertoli cell specific factors. Additional experiments will employ mice that show a 50% reduction in copper (cisplatin) uptake, due to a mutation in Ctrl, to demonstrate that the sensitivity of Sertoli cell injury correlates with cisplatin uptake and excretion from the cell. The long-term goal of this research is to understand the mechanism(s) of cisplatin-induced Sertoli cell injury and thus spermatogenic injury. Data obtained during the course of this project will provide insights that will allow for the development of strategies for prevention and/or reversal of cisplatin-induced Sertoli cell injury and the reestablishment of functional spermatogenesis.