Giammona C John, Sawhney Pragati, Chandrasekaran Yamini, Richburg John H
College of Pharmacy, Division of Pharmacology and Toxicology, The University of Texas at Austin, Austin, Texas 78712-1074, USA.
Toxicol Appl Pharmacol. 2002 Dec 1;185(2):119-27. doi: 10.1006/taap.2002.9536.
Apoptosis of testicular germ cells is critical for the maintenance of functional spermatogenesis. Previously, we have demonstrated that the Fas (Apo-1, CD95) receptor participates in the regulation of germ cell apoptosis, particularly after toxicant-induced Sertoli cell injury. In this study, we show that germ cells from B6.SMNC3H-Fas(gld,gld) (gld) mice that express a dysfunctional form of FasL still undergo significant apoptosis, albeit at a lower incidence than seen in B6 mice, following mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury. In addition, we show the presence of Fas, TRAIL-R1 (Death Receptor-4, DR4), and TRAIL-R2 (DR5) in the testis of C57BL/6 (B6) and gld mice (4 weeks old), and Sprague-Dawley rats (5 weeks old) and their responsiveness after MEHP treatment. More importantly, Western blot analysis of cellular fractions showed an increase in death receptor localization on the membrane fractions taken from Sprague-Dawley rats. Immunohistochemical analysis indicated localization of Fas and DR5 primarily to the spermatocyte subpopulation of germ cells. Examination of downstream receptor-mediated signals (i.e., cleavage of procaspase-8 and NFkappaB activation) revealed an early increase in NFkappaB-DNA binding and an increase in procaspase-8 processing in mutant gld mice. In summary, germ cell-associated death receptors, as well as downstream signaling elements, appear to be responsive to MEHP-induced Sertoli cell injury. Whether this is directly responsible for the increases in germ cell apoptosis after MEHP exposure is yet to be determined. The observed robust and early increase in Fas in wild-type testis and diminished rates of germ cell apoptosis in mutant testis (gld and lpr(cg)) reiterates the importance of the Fas signaling pathway.
睾丸生殖细胞凋亡对于维持功能性精子发生至关重要。此前,我们已经证明Fas(Apo-1,CD95)受体参与生殖细胞凋亡的调控,特别是在毒物诱导的支持细胞损伤后。在本研究中,我们发现,表达功能失调形式的FasL的B6.SMNC3H-Fas(gld,gld)(gld)小鼠的生殖细胞在邻苯二甲酸单(2-乙基己基)酯(MEHP)诱导的支持细胞损伤后仍会发生显著凋亡,尽管其发生率低于B6小鼠。此外,我们还展示了C57BL/6(B6)和gld小鼠(4周龄)、Sprague-Dawley大鼠(5周龄)睾丸中Fas、TRAIL-R1(死亡受体-4,DR4)和TRAIL-R2(DR5)的存在及其在MEHP处理后的反应性。更重要的是,细胞组分的蛋白质印迹分析显示,来自Sprague-Dawley大鼠的膜组分上死亡受体的定位增加。免疫组织化学分析表明,Fas和DR5主要定位于生殖细胞的精母细胞亚群。对下游受体介导信号(即procaspase-8的切割和NFκB激活)的检测显示,突变gld小鼠中NFκB-DNA结合早期增加,procaspase-8加工增加。总之,生殖细胞相关死亡受体以及下游信号元件似乎对MEHP诱导的支持细胞损伤有反应。这是否直接导致MEHP暴露后生殖细胞凋亡增加尚待确定。在野生型睾丸中观察到的Fas的强烈早期增加以及突变睾丸(gld和lpr(cg))中生殖细胞凋亡率的降低再次强调了Fas信号通路的重要性。