重金属镉暴露影响子代睾丸Leydig细胞发育的线粒体相关机制研究

Heavy metals are environmental toxigen to male reproductive health. Although there are many studies disclosed the toxicology effects on Leydig cells by decreasing testosterone secretion directly, the toxicology effects on Leydig cells' development and its mechanism is still poorly understood. Our previous study found that cadmium (Cd) significantly reduced the production of cAMP and mitochondrial membrane potential (ΔΨm) in a dose dependent manner with altered expression of proteins related to mitochondria, which suggests that mitochondria is the target to damage by Cd. Based on these results, the mechanism of toxicity caused by Cd in the development of Leydig cells will be explored in this study. Rats will be exposed to Cd in Gestation and (or) lactation, then several experiments will be conducted as below: (1) morphous and functions of Leydig cells in different developmental stages will be investigated; (2) the altered expression of signaling molecules and the phosphorylation levels in cAMP-PKA-ERK1/2 pathway will be detected by Western bolts and qPCR; (3) mitochondrial functions related to energy metabolism and the production of Reactive Oxygen Species (ROS) will be detected too; (4) mitochondrial protein will be analyzed by Isobaric tags for relative and absolute quantification (iTRAQ) to find out the proteins with altered expression, and the proteins will be validated by in vitro an in vivo experiments. This study will help to disclose the origin of male sexual dysfunction in the developmental stages of Leydig cells, and provide new targets in clinical therapy.

重金属污染严重影响男性生殖健康，在毒性毒理学方面已经取得了相应的研究进展；但在与精子产生相关睾丸间质细胞（Leydig cell，LC）的分化发育方面的研究尚未见报道。本课题组前期研究发现，重金属镉暴露导致LC线粒体功能相关蛋白出现大量差异表达，损伤机制与线粒体损伤和细胞内cAMP水平的下降密切相关。在此基础上，本研究拟对孕期和（或）哺乳期大鼠进行镉染毒，观察子代雄性个体不同发育阶段LC的形态和功能变化，分离各发育阶段LC，检测雄激素生成相关的cAMP-PKA-ERK1/2通路信号分子的表达及其磷酸化水平，检测能量代谢相关线粒体功能和活性氧的生成，最后定位于线粒体，采用iTRAQ蛋白组学技术筛选出差异表达蛋白，在细胞和动物水平上验证，结合生物信息学手段，总结归纳出不同剂量镉暴露对子代LC发育的不同毒理作用机制。该研究将有助于明确男性性功能低下的早期病因，并为其治疗提供新靶点和实验依据。

重金属污染严重影响男性生殖健康，在毒性毒理学方面已经取得了相应的研究进展；但在与精子产生相关睾丸间质细胞（Leydig cell，LC）的分化发育方面的研究尚未见报道。出生后，LC的发育阶段包括Leydig祖细胞（Progenitor Leydig Cells，PLC）、未成熟型Leydig细胞（Immature Leydig Cells, ILC）和成熟型Leydig细胞（Adult Leydig Cells， ALC）。本研究中，首先采用曲细精管体外培养和提取的原代PLC体系进行镉（Cadmium，Cd）暴露，发现Cd阻碍PLC发育为成熟的ALC，并通过抑制cAMP-PKA-ERK1/2信号通路活化和类固醇合成酶（StAR，CYP11A1，3β-HSD和CYP17A1）减少类固醇激素的生成。接着，我们对孕期和哺乳期母鼠进行Cd染毒，观察雄性子代不同发育阶段LC的形态和功能变化。发现Cd导致LC发育明显滞后，在ALC阶段仍然大量表达PLC和ILC阶段的蛋白标志5-还原酶1（5-Reductase 1，SRD5A1），形态上也呈现ILC的长梭形和卵圆形，同样通过抑制cAMP-PKA-ERK1/2信号通路活化和类固醇合成酶减少类固醇激素的生成。最后，我们提取了母代Cd暴露子代各发育阶段的LC，采用iTRAQ蛋白组学技术筛选出差异表达蛋白。结果发现随着子代大鼠性发育成熟，与正常大鼠比较，不同阶段LC的差异表达蛋白逐渐减少。其中，与脂滴合成相关的Perilipin蛋白在PLC阶段显著下调，Coronin-1A在PLC和ILC阶段都呈现显著上调，Phosphoinositide phospholipase C在ALC阶段显著上调，我们用Western blotting技术分别对其进行验证，并探讨其调控类固醇激素合成的相关信号通路。该研究有助于明确男性性功能低下的早期病因，并为其治疗提供新靶点和实验依据。