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. ? ?
