Sertoli cell toxicant injury and mechanisms of testicular germ cell apoptosis

描述(申请人提供)：这个项目的目标是破译暴露于单-(2-乙基己基)后引起白细胞渗入睾丸的细胞信号。 探讨邻苯二甲酸盐(MEHP)在MEHP诱导生殖细胞凋亡中的功能意义。我们先前已经揭示了支持细胞(SCs)和生殖细胞(GC)之间的旁分泌相互作用，它通过FasL-Fas信号启动生殖细胞的凋亡。这一途径的核心是抑制MEHP暴露后SCs产生TIMP2(金属蛋白酶组织抑制因子2)。这允许随后在肺泡间隙激活基质金属蛋白酶2(MMP2)，两者都产生一种可溶性形式的肿瘤坏死因子-？(sTNF？)以及导致组成相邻SC之间的血-睾丸屏障(BTB)的蛋白质的解体。MEHP(1g/kg，P.O.)对Fischer大鼠睾丸影响的初步研究在两个青春期周围年龄(PND 28和35)的治疗显示，CD11b免疫反应细胞在GC凋亡诱导高峰之前在间质中有很强的渗透。CD11b抗体识别巨噬细胞、中性粒细胞、单核细胞、自然杀伤细胞和树突状细胞上表达的抗原。有趣的是，我们观察到，与成熟大鼠相比，这些细胞的最显著渗透发生在青春期周围的大鼠；而C57BL/6J小鼠在这两个年龄段对MEHP暴露没有明显的渗透反应。对MEHP引起的睾丸损伤的不同物种和年龄相关的敏感性已得到很好的认识，尽管解释这些差异的机制仍未解决。此外，初步研究还表明，PND 28大鼠睾丸小管周围肌样细胞(PTMC)单核细胞趋化蛋白-1(MCP-1)表达显著增加，但成熟大鼠未见表达。单核细胞趋化蛋白-1(MCP-1)是一种典型的趋化因子，可在细胞因子如sTNF？、IL-1？、IL-1？和/或干扰素-？综上所述，我们以前的工作和初步数据导致了一种假设的发展，即MEHP诱导的SC损伤会刺激细胞因子的产生，如sTNF？，从而触发PTMCs释放趋化因子，启动白细胞向睾丸的渗透，并作用于 增强GC细胞凋亡的程度。此外，这一建议的一个具有挑衅性的潜在假设是，白细胞在一定程度上解释了观察到的年龄和物种依赖的MEHP敏感性的机制。为了验证这些假设，第一个具体目标将使用急性和重复的MEHP治疗来表征白细胞亚型及其在暴露后流入睾丸的时间。在第二个目标中，将检测所激发的趋化因子和细胞因子，以及它们在睾丸中的特定细胞来源。最后，将直接检验睾丸白细胞浸润在MEHP损伤发病机制中的功能意义。据预测，从这项工作中获得的见解将对预测易感人群和预防人类生殖健康风险这类化学物质有用。

DESCRIPTION (provided by applicant): The goal of this project is to decipher the cellular signals that instigate leukocyte infiltration into the testis after exposure to mono-(2-ethylhexyl) phthalate (MEHP) and to determine the functional significance of these cells in the pathogenesis of MEHP-induced germ cell apoptosis. We have previously revealed a paracrine interaction between Sertoli cells (SCs) and germ cells (GCs) that initiates GCs to undergo apoptosis via FasL- Fas signaling. Central to this pathway is the inhibition of TIMP2 (tissue inhibitor of metalloproteinase 2) production by SCs after MEHP exposure. This allows for the consequent activation of matrix metalloproteinase 2 (MMP2) in the adluminal space that both generates a soluble form of tumor necrosis factor-? (sTNF?) as well as cause a disorganization of proteins that compose the blood-testis barrier (BTB) between adjacent SCs. A preliminary characterization of Fischer rat testis after MEHP (1 g/kg, p.o.) treatment at two peripubertal ages (PND 28 and 35) showed a robust infiltration of CD11b+ immunoreactive cells in the interstitial space prior to the peak induction of GC apoptosis. The CD11b antibody recognizes an antigen expressed on macrophages, neutrophils, monocytes, natural killer and dendritic cells. Intriguingly, we observed that the most pronounced infiltration of these cells occurs in peripubertal aged rats versus mature rats; and that C57BL/6J mice at both ages do not have a significant infiltration in response to MEHP exposure. The differential species and age- dependent sensitivity to MEHP-induced testicular injury is well recognized, although the mechanisms that account for these differences remain unresolved. Further, preliminary studies also show a dramatic increase in the expression of monocyte chemoattractant protein-1 (MCP-1) in peritubular myoid cells (PTMCs) in testis from PND 28 rats, but not in mature rats. MCP-1 is a prototypical chemokine for signaling the influx of leukocytes into tissues and is itself secreted in response to cytokines such as sTNF?, IL-1?, Il-1? and/or interferon-?. Taken together, our previous work and preliminary data, have led to the development of the hypothesis that MEHP-induced SC injury incites the production of cytokines, such as sTNF?, that trigger PTMCs to release chemokines that initiate the infiltration of leukocytes into the testis and act to enhance the extent of GC apoptosis. Moreover, a provocative underlying hypothesis of this proposal is that leukocytes account, in part, for the mechanism of the observed age- and species-dependent sensitivity to MEHP. To test these hypotheses, the first specific aim will use both acute and repeated MEHP treatments to characterize the leukocyte subtypes and timing of their influx into the testis following exposure. In the second aim, the chemokines and cytokines elicited, as well as their specific cellular source in the testis, will be examined. In the last ai, the functional significance of testis leukocyte infiltration in the pathogenesis of MEHP-induced injury will be directly tested. It is predicted that insights gained from this work will be useful or predicting susceptible individuals and preventing human reproductive health risks this class of chemicals.