小鼠ERɑ309位点磷酸化对输出小管和附睾管腔微环境的影响和作用机制

Spermatozoa acquire their ability to become motile and to fertilize an egg after spermatogenesis. A proper luminal milieu is crucial to this process. Recent studies have discovered that infertility of ERα knockout 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 the S309AERKI homozygous males were subfertile with lower sperm motilities, as well as dilated and thinner efferent ducts. Analysis of the epididymal fluid revealed that the S309AERKI mice maintain a higher luminal pH throughout the epididymis. The expression levels of aquaporin 9 (AQP9) as well as carbonic anhydrase 12 (CA12) were reduced in the efferent duct and different segments of S309AERKI epididymis, respectively. Thus, phosphorylation at S309 site plays an important role in the process of ERα-mediated transcription to maintain a stable fluid/ion transportation in epididymal lumen. This project plans to explore the upstream signal pathway activating phosphorylation at ERα S309 site, as well as the mechanism of ERα interacting with downstream target genes in the mouse efferent ductules and epididymis by integrated use of morphological and molecular biological methods. The correlation between the phosphorylation at ERα S309 site and sperm motility will be also analyzed by screening the genetic mutation of the patients with weak spermatozoa. It is very important to study comprehensively the role of ERα phosphorylation at S309 site in regulation of epididymal milieu, to further elucidate the mechanism of sperm maturation.

精子生成后需获得运动和受精能力，其中输出小管和附睾管腔中适宜的微环境对这一过程至关重要。新近研究发现，ERαKO鼠因输出小管液体吸收障碍和附睾管腔酸性环境破坏而不育，但具体调控机制仍不清楚。我们的前期工作表明，通过基因替代的方式阻断ERα309位点的磷酸化，雄性小鼠的生育力降低，精子动力下降，输出小管肿胀，附睾管腔pH值升高，而且输出小管水通道蛋白9和附睾管碳酸酐酶12的表达降低，提示小鼠ERα309位点的磷酸化对维持输出小管和附睾管腔微环境的稳定起着重要作用。本项目拟综合运用形态学和分子生物学方法，探讨输出小管和附睾上皮细胞中ERα309位点磷酸化的上游激活信号，以及与下游靶基因的作用机制；并通过筛查临床弱精症患者基因突变情况，分析ERα309位点磷酸化与精子动力的相关性。全面认识ERα309位点磷酸化在输出小管和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。

雌激素受体α (ERα)在男性生殖系统中起着重要作用，其活性可由多个氨基酸残基的磷酸化调节。人ERα305位点丝氨酸(S305)的磷酸化(与小鼠丝氨酸309同源)可诱导不依赖配体的ERα活性。在本项目中，我们采用同源重组的方法在小鼠胚胎干细胞(ES)中构建ERα309 (p.S309A, ERαS309A)敲入(KI)小鼠模型，研究ERαS309位点磷酸化对雄性小鼠生殖功能的影响。研究发现ERαS309A KI雄性小鼠表现为生育能力下降，精子活力下降，输出小管管腔扩张，上皮变薄，附睾液pH升高。进一步检测与附睾管腔微环境相关且含有雌激素反应元件的基因在ERαS309A KI雄性小鼠输出小管和附睾各段的表达情况，发现水通道蛋白9(AQP9)在输出小管的表达水平降低，碳酸酐酶XII (CA12)在附睾中的表达水平降低。在体外研究中，我们分别用模拟ERα309组成性磷酸化和未磷酸化状态的质粒转染人胚胎肾293T (293T)细胞。与野生型ERα质粒转染的细胞相比，磷酸化ERα309质粒转染293T细胞中AQP9和CA12的表达增加，而非磷酸化ERα309质粒转染细胞中AQP9和CA12的表达减少。附睾转录组测序发现在纯合型小鼠附睾起始部、头部、体部和尾部存在多个差异基因，对这些差异基因进行功能富集分析，发现在起始部和头部，差异基因功能主要集中在膜转运和离子转运相关方面，在体部和尾部，差异基因功能主要集中在抗原结合和受体信号结合相关方面，表明ERα309磷酸化位点在附睾的不同节段影响不同基因的表达和功能。本项目研究结果表明，ERαS309位点的磷酸化分别通过调节输出小管和附睾管水和离子转运蛋白的表达，对雄性小鼠的生殖发挥重要作用。