MTA1/ERα对附睾主细胞功能的影响及其机制

In the epididymis, spermatozoa acquire their ability to become motile and to fertilize an egg. A proper luminal milieu is crucial to this process. Recent studies have discovered that infertility of ERα knock out mice appear to result from an inability of the epididymis to properly acidify the luminal contents. However, the regulative mechanism is still not clear. Our initial work indicated that MTA1, the corepressor of ERα, was mainly restricted to the nucleus of principle cells in mouse epididymis, and overexpression of MTA1 in the principle cells may obviously reduce the levels of ERα target molecules such as aquaporin 9 and Na+ ?H+ exchanger 3. Thus, certain level of MTA1 is possible to regulate ERα-mediated transcription to maintain a stable milieu in epididymal lumen. This project plans to illuminate the interaction in MTA1, ERα as well as ERα target molecules containing estrogen response element in principle cells with luciferase reporter and CHIP assays; to examine the regulation of MTA1/ERα pathway in the reabsorption and secretion activity of principle cells in both primary culture of epididymal epithelia and in vivo RNA interference rats; and to observe the effect of blocking MTA1 on the role of principle cells and the milieu in epididymal lumen with the use of the model of microperfused tubules. It is very important to study comprehensively the role of MTA1 and ERα in the regulation of epididymal milieu, to further elucidate the mechanism of sperm maturation.

精子在附睾中获得运动和受精能力,其中管腔液适宜的微环境对这一过程至关重要。新近研究发现，ERα敲除鼠因附睾管腔液酸性环境破坏而不育，但调控机制仍不清楚。我们的前期工作表明，ERα的辅阻遏物MTA1主要定位于小鼠附睾上皮主细胞胞核，而且过表达MTA1可显著抑制主细胞表达ERα靶分子水通道蛋白9和Ш型钠氢交换体，提示ERα介导的基因转录可能受一定水平的MTA1调控，从而维持着附睾管腔微环境的稳定。本项目拟用荧光素酶启动子检测和ChIP技术，阐明MTA1、ERα以及具有雌激素反应元件的靶分子在主细胞的相互作用；在附睾上皮细胞培养和在体RNA干扰动物两个层次上，探讨MTA1/ERα通路对主细胞重吸收和分泌活性的调节；以及利用附睾管腔微灌注模型，观察阻断MTA1前后对主细胞功能和附睾管腔微环境的影响。全面认识MTA1和ERα在附睾管腔微环境调节中的功能，对进一步阐明精子成熟的调控机制具有重要的意义。

精子在附睾中获得运动和受精能力,其中管腔液适宜的微环境对这一过程至关重要。新近研究发现，ERα敲除鼠因附睾管腔液酸性环境破坏而不育，但调控机制仍不清楚。我们的前期工作表明，ERα的辅阻遏物MTA1主要定位于小鼠附睾上皮主细胞胞核，提示ERα介导的基因转录可能受一定水平的MTA1调控，从而维持着附睾管腔微环境的稳定。本项目首先观察了MTA1与ERα在大鼠、小鼠附睾上皮的表达规律；然后在小鼠附睾上皮细胞系PC1，给予不同浓度的ERα抑制剂MPP，转染MTA1过表达载体，观察相关靶基因的表达变化。最后在MTA1-KO小鼠和输精管切除小鼠层次上，阐明MTA1、ERα以及具有ERE的靶分子在主细胞的相互作用，探讨MTA1/ERα通路对主细胞重吸收和分泌活性的调节和附睾管腔微环境的影响。结果：MTA1高表达于大鼠附睾组织，定位于大鼠附睾上皮的主细胞，基细胞，亮细胞和狭窄细胞的细胞核；MTA1与ER共定位于大鼠、小鼠附睾主细胞的细胞核；ER的靶基因CA12、AQP9和NHE3在小鼠附睾上皮细胞系PC1均有表达，10 µM的MPP可显著抑制ERα及其靶基因CA12，AQP9和NHE3的表达；PC1细胞中过表达MTA1后，CA12、AQP9和NHE3表达降低；与野生型相比，MTA1-KO小鼠附睾管腔pH值降低，ER表达不变，AQP9，CA12，NHE3等含有ERE的基因表达升高；小鼠双侧输精管切除4周后，附睾管腔pH值显著增高；附睾管腔直径增大、管壁厚度变薄，管腔间隙狭窄，附睾尾部管腔内精子数量增多，CA12、AQP9和NHE3表达降低。结论：附睾上皮主细胞核的MTA1调控ERα介导的CA12、AQP9和NHE3的基因转录，影响主细胞的重吸收和分泌功能，从而维持着附睾管腔微环境的稳定，使精子得以成熟和储存。全面认识MTA1和ERα在附睾管腔微环境调节中的功能，对进一步阐明精子成熟的调控机制具有重要的意义。