内质网IREl信号介导镉诱发睾丸生殖细胞凋亡的机制

Cadmium (Cd) is one of major environmental endocrine disruptors and reproductive toxicants, has potentially adverse effects on male reproduction and spermatogenesis. Increasing evidence demonstrated that environmental exposure to Cd was associated with male infertility and the poor semen quality in humans and the molecular mechanisms remain poorly understood. Our previous study showed that Cd could cause endoplasmic reticulum stress (ER stress) and induce germ cells apoptosis in mouse testes, crosstalk between ER stress signaling and mitochondrial pathway mediated Cd-induced testicular germ cell apoptosis, however, the molecular mechanisms linking ER stress to apoptosis remained to be elucidated. In the present study, We will further investigate whether chemical molecular chaperones or GRP78 overexpression can attenuate the Cd-induced ER stress in mice and explore the role the known pro-apoptotic signalling pathways emanating from activated PERK, ATF6 and IRE1 and discuss how their signalling switches to a pro-apoptotic response during Cd-induced testicular germ cell apoptosis. Moreover, We will clarify the regulation role of IRE signal pathway on Cd-induced germ cell apoptosis by using the siRNA technology to down-regulation IREla expression in vitro. Finally, we will further observe the effects of controlling the separate regulation of IRE1's RNase and kinase activity on Cd-induced testicular germ cell apoptosis by specific inhibitors or transfection of miR mimics in vitro and elucidate the molecular mechanisms of IRE1 in this process through RIDD or JNK pathway. These studies are significant for researchers to clarify the molecular mechanisms of human male reproductive impairments under Cd exposure and establish preventive methods.

课题组前期研究发现，内质网应激和线粒体信号通路的交互作用介导镉所致睾丸生殖细胞凋亡，然而作用机制尚不清楚。本课题拟开展以下研究：采用内质网化学分子伴侣或GRP78过表达抑制内质网应激，观察镉对睾丸生殖细胞内质网应激IRE1、PERK和ATF6三个信号通路的影响；采用转染siRNA沉默IRE1α基因，观察体外条件下抑制IRE1信号通路对镉诱导睾丸精原细胞和初级精母细胞凋亡的影响，阐明内质网IRE1通路在镉诱导睾丸生殖细胞凋亡中的作用。采用特异性抑制剂或体外转染miR mimics等抑制IRE1核糖核酸酶或激酶活性，观察选择性抑制IRE1核糖核酸酶或激酶活性对镉诱导睾丸精原细胞和初级精母细胞凋亡的影响，重点阐明IRE1通过RIDD或JNK信号分子调控镉诱导睾丸生殖细胞凋亡的分子机制。本课题为最终阐明镉暴露对人类男性生殖过程的损害作用及其分子机理并制定防治对策提供理论依据。

镉（Cadmium，Cd）是一种重要的环境内分泌干扰物和生殖毒物，能够降低精子数量，引起生殖损伤。睾丸生殖细胞凋亡是引起精子数量下降的主要原因之一。课题组之前的研究证实，镉能够诱导睾丸生殖细胞凋亡，内质网应激和线粒体信号通路的交互作用介导镉所致睾丸生殖细胞凋亡，然而作用机制尚不清楚。本课题主要探讨镉对睾丸生殖细胞内质网应激IRE1、PERK和ATF6三个信号通路的影响；探讨内质网IRE1通路在镉诱导睾丸生殖细胞凋亡中的作用。研究结果显示：镉能够激活IRE1、PERK和ATF6信号通路，启动内质网应激与非折叠蛋白反应；在体内外条件下，镉均能够诱导睾丸生殖细胞凋亡，采用内质网小分子伴侣PBA，不仅抑制镉诱导的内质网应激，同时抑制镉诱导的睾丸生殖细胞凋亡，提示内质网应激参与了镉诱导的睾丸生殖细胞凋亡。IRE1在内质网三条信号通路中，处于中心地位，是许多细胞事件的关键调控子。STF-083010是选择性IRE1α核糖核酸酶抑制剂，可特异性抑制IRE1α的核糖核酸酶活性。CdCl2明显增加IRE1以及IRE1下游分子JNK蛋白的磷酸化，升高GC-1spd细胞活化型XBP-1水平，而STF-083010预处理明显缓解镉诱导的IRE1及JNK蛋白的磷酸化，减轻镉诱导的XBP1蛋白的表达，提示STF-083010对镉激活的IRE1信号通路具有拮抗作用。此外，STF-083010能够明显抑制镉诱导的睾丸生殖细胞凋亡。以上结果提示，IRE1信号通路可能介导镉诱发的睾丸生殖细胞细胞凋亡。综上所述，镉可能通过启动睾丸内质网应激，激活IRE1促凋亡信号通路，导致睾丸生殖细胞凋亡。本课题为最终阐明镉暴露对人类男性生殖过程的损害作用及其分子机理并制定防治对策提供理论依据。