GDNF/MAP2K1/2通路调控精原干细胞自我更新在电磁辐射致精子发生障碍中的作用及机制

It was reported that the electromagnetic fields (EMF) could lead to spermatogenesis failure. However, the molecular mechanism was unclear. It was well known that the balance of spermatagonial stem cells (SSCs) self-renewal and differentiation plays an important role in sustaining the normal spermatogenesis. Recently, it was reported that the glial cell-line-derived neurotrophic factor (GDNF) could modulate SSCs self-renewal and differentiation via MAP2K1/2 pathway. Till now, the role of GDNF in SSCs self-renewal regulation that involves in spermatogenesis failure caused by EMF has not yet been reported. Previously, we found that SSCs proliferation was significantly promoted, SSCs differentiation was obviously inhibited, and the level of testis GDNF and MAP2K1 increased after EMF exposure. Taken together, we put forward the following hypothesis: GDNF/MAP2K1/2 pathway may be involved in spermatogenesis failure caused by EMF exposure through modulating SSCs self-renewal. Based on previous studies, we plan to investigate: the SSCs proliferation and differentiation status after exposure to EMF and its relationship with testicular GDNF level; the mechanism of GDNF/MAP2K1/2 pathway involved in the imbalance of SSCs renewal and differentiation induced by EMF; the effects of intervention of GDNF signaling pathway on SSCs renewal and differentiation imbalance as well as spermatogenesis failure induced by EMF exposure by using the methods such as laser confocal microscope, real time RT–PCR, RNA interference and so on. The research would lay a foundation for elucidating the molecular mechanism of spermatogenesis failure induced by EMF and seeking novel targets in prevention of male reproduction injury.

电磁辐射可致精子发生障碍，但机制尚未阐明。已知精原干细胞（SSCs）更新分化平衡是维持精子正常发生的关键。据最新报道，睾丸GDNF可通过MAP2K1/2调控SSCs自我更新和分化。但该通路在电磁辐射致生精障碍中的作用，尚未见报道。我们前期研究发现，电磁辐射可促进SSCs增殖并抑制其分化，同时上调GDNF和MAP2K1的表达。为此，提出假设：电磁辐射可能通过GDNF/MAP2K1/2通路调控SSCs引起更新分化失衡，最终导致精子发生障碍。本项目拟在既往研究基础上，应用激光共聚焦、RT-PCR和RNA干涉技术等，研究：电磁辐射暴露后SSCs增殖分化与睾丸GDNF水平之间的关系；GDNF/MAP2K1/2调控SSCs更新分化失衡的分子机制；GDNF通路干预对电磁辐射后SSCs更新分化失衡和生精障碍的影响等。该研究将为阐明电磁辐射致精子发生障碍的分子机制，探寻生殖危害防治新靶点奠定基础。

我们前期研究发现，射频辐射（RFR）可促进精原干细胞（SSCs）增殖并抑制其分化，同时上调GDNF和MAP2K1的表达。为此，我们假设RFR可能通过GDNF/MAP2K1/2通路调控SSCs引起更新分化失衡，最终导致精子发生障碍。本课题通过病理学、分子生物学、细胞培养等方法研究了生活和职业环境射频辐射暴露后SSCs增殖分化与睾丸GDNF水平之间的关系及GDNF/MAP2K1/2调控SSCs更新分化失衡的分子机制。首先，研究了职业环境射频辐射（220 MHz RFR）致大鼠精子发生障碍的损伤特点。结果显示：220 MHz RFR连续暴露30 d，大鼠精子数量减少，畸形率升高、存活率降低，提示该条件下RFR可致精子质量下降。然后，研究了生活环境手机RFR（1.8 GHz、1950 MHz和4.85 GHz RFR）连续暴露对成年男性或小鼠精子质量的影响。结果显示：手机使用年限是成年男性精液质量降低的危险因素；1.84 GHz（3G和4G通讯工作频段）RFR连续暴露2 W可显著降低成年雄性小鼠精子质量，且辐照后早期睾丸组织GDNF和MAP2K1水平明显升高。提示生活环境RFR可能作用于精子发生过程的早期阶段，最终导致精子发生障碍；生活环境RFR（4.85 GHz RFR，5G通讯工作频段）连续暴露7 d也可致雄性小鼠精子发生障碍，且RFR诱导的GDNF/MAP2K1水平改变参与了精子发生障碍过程。最后，研究了1950 MHz（3G和4G通讯工作频段）RFR对离体培养的SSCs和支持细胞的影响，结果显示：1950 MHz RFR可促进支持细胞分泌GDNF的水平，但对精原干细胞的增殖和分化无明显影响，经条件培养液置换后，精原干细胞增殖标志物PCNA和OCT4蛋白表达明显增加，分化标志物C-kit蛋白表达明显降低，MAP2K1蛋白表达水平显著升高。提示，GDNF/MAP2K1/2通路调控SSCs自我更新参与了RFR致精子发生障碍的过程。上述研究结果为探寻生殖危害防治新靶点奠定了基础。