磷酸酶和内源性大麻系统对昆明小鼠精子运动性的调控作用及机制研究

Spermatozoa with normal motility guarantee successful fertilization. Our study finds that phosphatases PP1γ2 and PP2A play an important role in the regulation of epididymal sperm motility initiation in Kunming mice. However, the regulatory mechanism of the enzyme activity and the interaction between these phosphatases and other regulatory molecules of sperm motility are largely unknown. Endocannabinoid system is a newly discovered lipid regulator involved in the regulation of sperm function. One study finds that the levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) in caput and cadal epididymis spermatozoa are significantly different, suggesting that it may be involved in epididymal sperm maturation, but the mechanism is unknown. A recent study in Science (2016) finds that 2-AG inhibits sperm motility by blocking human spermatozoa CatSper cation channel. When progesterone is present, the enzyme ABHD2 is activated, breaking down 2-arachidonoylglycerol (2-AG) on the sperm membrane, and then triggers sperm motility. This Science paper also confirms that 2-AG can inhibit mouse CatSper cation channel, but the enzyme ABHD2 is absent in mouse sperm fragella. We hypothesize that the enzyme MAGL in mice or ABHD6/12 is responsible for degrading 2-arachidonoylglycerol (2-AG) on the sperm membrane, and that the enzyme MAGL or ABHD6/12 may be interacted with PP1γ2/PP2A, and the enzyme activity of PP1γ2/PP2A may be regulated by the protein sds22.These molecules and their interactions collectively regulate sperm motility in mice. This proposal is of great significance for elucidating the regulation and mechanism of sperm motility in mice.

正常运动的精子是受精的保证，我们研究发现磷酸酶PP1γ2和PP2A对昆明小鼠附睾精子运动性获得起重要调控作用，但此酶活性调节机制、与精子运动性调控其他分子的关系都远未清楚。内源性大麻系统是新近发现参与精子功能调控的因子，小鼠附睾头、尾精子中内源性大麻素2-AG含量有明显差别，提示它可能参与附睾精子成熟，但机制不明。Science（2016）上的研究表明，2-AG通过阻断人精子CatSper而抑制精子运动，当有孕酮时，酶ABHD2被激活，将精子膜上的2-AG分解，激发精子运动活性。该文证实2-AG能抑制小鼠CatSper，但小鼠精子尾部不存在ABHD2。我们假定：小鼠中的酶MAGL或ABHD6/12负责降解精子膜的2-AG，其可能与PP1γ2/PP2A发生关联，PP1γ2/PP2A酶活性可能受控于sds22蛋白，共同调控着小鼠精子运动性。本项目对阐明小鼠精子运动性调控作用及机制具有重要意义。

研究发现磷酸酶PP1γ2/PP2A对小鼠附睾精子运动性获得起重要调控作用，但此酶活性调节机制、与精子中其他重要分子的关系都远未清楚。内源性大麻系统是新近发现参与精子功能调控的因子，但其对小鼠精子运动的调控作用知之甚少。本项目通过系统研究，阐明了昆明小鼠附睾尾和附睾头精子中PP1γ2和PP2A的磷酸化水平有显著差异且这种磷酸化水平与其酶活性有关， PP1γ2和PP2A对小鼠附睾精子成熟表现出负调控的作用方式，即PP1γ2/PP2A的高活性导致精子运动度较低，PP1γ2/PP2A的低活性导致精子运动度较高；阐明了PP1γ2/PP2A在小鼠精子附睾头、尾的活性变化特性及调节机制，发现sds22通过与PP1γ2/PP2A分子的结合与解离而对PP1γ2/PP2A酶活性作出调控，在附睾尾精子中，当sds22与PP1γ2/PP2A结合时，PP1γ2/PP2A被抑制，导致PP1γ2/PP2A酶活性降低或丧失，当sds22脱离PP1γ2/PP2A时，PP1γ2/PP2A解除抑制，则其酶活性得到恢复；发现p17通过与sds22的结合与解离而实现对PP1γ2/PP2A活性及附睾精子成熟的调控；阐明了内源性大麻系统对小鼠精子运动性调控的作用机制，发现2-AG对小鼠精子运动具有抑制作用；免疫组化的结果显示MAGL位于昆明小鼠精子鞭毛中段，ABHD12位于精子鞭毛中段和主段，ABHD2和ABHD6位于精子顶体部位，提示其在小鼠精子中有不同分工；我们的研究表明小鼠精子中的MAGL、ABHD2、ABHD6和ABHD12四种脂质水解酶通过水解内源性大麻素2-AG，从而调控精子运动，但这四种酶活性大小具有差异，其中MAGL水解作用最强， ABHD12次之，ABHD6水解作用较弱；我们发现 ABHD6和ABHD12磷酸化状态在附睾头、尾精子中明显不同，暗示其具有不同活性并可能与不同的精子功能相关；PP1γ2与MAGL或ABHD6/12 的关联性研究表明，PP1γ2与MAGL、ABHD6或 ABHD12无相互作用；探究了PP1γ2/PP2A 调控小鼠附睾精子成熟的细胞信号转导途径，发现PP1γ2/PP2A受SRC激酶调控，即SＲC通过抑制PP1γ2/PP2A 活性以调控精子运动。.本项目对阐明小鼠精子运动调控作用及机制具有重要意义，对探讨相关人类生殖问题也具有重要的借鉴作用。