Cisplatin and Mechanisms of Long-term Male Infertility

描述（由申请人提供）：化疗药物顺铂在清除体内致瘤细胞方面非常有效。然而，它不是选择性的，会导致睾丸中正常生殖细胞的丢失。历史上一直认为，化疗期间失去精原细胞是导致长期不育和/或精子发生延迟恢复的原因。我们最近在小鼠实验中发现，很大比例的干精原细胞在临床样的反复顺铂暴露中存活下来，没有随后的精子发生恢复。这表明睾丸内其他体细胞受损。顺铂治疗小鼠睾丸组织病理学显示，大的支持细胞液泡，顶端脱落，细胞物质脱落到腔内，生殖细胞凋亡丢失，这些都是支持细胞损伤的表现。在本研究计划中，我们将首先验证顺铂直接损伤支持细胞并在化疗后恢复期产生不适合驻留生殖细胞发育和分化的睾丸微环境的主要假设。顺铂治疗的W/W-v小鼠将被用作正常干细胞移植的受体，以测试睾丸微环境是否确实因顺铂暴露而改变。由于c-kit受体的突变，这些小鼠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，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。

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.