陆川猪精子去获能蛋白的分离鉴定及作用机理研究

It is indispensable for sperm to capacitate before undergoing the process to naturally fertilize the oocyte and this process is believed to be initiated following the release of surface-associated decapacitation factors. Moreover,capacitated sperm will reversibly lose their capacitated state when mixed with decapacitation factors. The reversible process between capacitation and decapacitation promprs that decapacitation factors play the role of a fatal bridge in this pair of progresses. In recent years, the apllicant has conducted a series of researches on the function and in vitro preservation of boar sperm, preliminarily understanding the change in morphology, structure and function of in vitro preserved sperm. To deepen the preliminary study, this project will be designed to discover decapacitation proteins and be expected to probe into the potential mechanism of capacitation from a perspective of decapacitation protein functions via separating/identifying decapacitation proteins released from capacitated sperm and on a molecular and protein level, from the standpoint of signal pathway, researching into the mechanism of their inhibition effect on capacitation by PCR, western blot, GST pull-down, and MS, using Luchuan pig (a local breed in Guangxi) spermatozoa as the treated samples and choosing decapacitation proteins as the breakthrough point. Furthermore, the theory of protein interaction will be practised to find sperm menbrane proteins which interacted with decapacitation proteins and provide a reference for the elucidation of the reversible process between capacitation and decapacitation. The results maybe impact the improvement of pig IVF and the commercialization of pig embryo production, thereby providing a theoretic reference for the conservation and improvement of a local pig breed.

获能是精子完成受精过程的一个必需条件，而精子表面去获能因子的释放是触发这一过程的关键。获能精子与去获能因子再次接触,又可呈现非获能状态。获能与去获能的可逆过程，提示去获能因子在这一对生理过程中起着重要的作用。申请人近年来对猪精子的功能及体外保存进行了一些研究，对猪精子体外保存时形态结构及功能的变化有一定的了解。为了深化前期的研究，本课题以广西地方猪种陆川猪精子为材料，以去获能蛋白为切入点，通过分离鉴定获能时精子表面释放的去获能蛋白，并用PCR、western blot、GST pull-down、质谱等多种技术，从分子、蛋白水平和信号通路角度研究其抑制获能的作用机制，试图发现新的去获能蛋白。同时，利用蛋白互作原理鉴定与去获能蛋白互作的精子膜蛋白，为阐明获能与去获能的可逆过程提供依据。研究结果可能对猪体外受精技术的完善和猪胚胎生产的商业化进程产生影响，为地方猪种的保存和改良提供理论基础。

获能是精子完成受精过程的一个必需条件，而精子表面去获能因子的释放是触发这一过程的关键。获能精子与去获能因子再次接触,又可呈现非获能状态。获能与去获能的可逆过程，提示去获能因子在这一对生理过程中起着重要的作用。因此，项目组进行了以下4方面的研究：1.基于iTRAQ技术的猪精子获能前后差异蛋白分析。结果：猪精子在获能前后共检测到475种差异蛋白，其中303种上调，172种下调；这些蛋白是构成细胞膜（13.71 %）、细胞器（10.66 %）、细胞质（9.40 %）等组分的主要成分，主要参与氮化合物代谢、氧化还原、生物合成、有机循环化合物代谢、细胞芳香化合物代谢等生物过程和氧化还原酶活性、转运活性、底物特异性转运体活性、核酸结合、离子跨膜转运活性、辅因子结合等分子功能；氧化磷酸化、胆固醇代谢、酮体合成与降解、氨酰-tRNA生物合成、鞘脂信号通路、赖氨酸降解为精子获能的主要信号通路。2.获能精子中与去获能蛋白相互作用蛋白的筛选与鉴定。结果：去获能蛋白磷酸果糖激酶（PFK）能够与获能精子的Q28920、Q29380、P26776、Q68J42等蛋白相互作用，使精子达到良好的获能状态。3.去获能蛋白对精子获能的作用验证。结果：去获能蛋白PFK基因含有2个N-糖基化修饰位点，14个O-糖基化位点，35个磷酸化位点，可促进精子表面发生修饰；存在PKC、PKA特异性蛋白激酶的结合位点，能够参与己糖代谢和单糖代谢；PFK能显著增强获能精子抗氧化和糖代谢能力。4.去获能蛋白对精子获能相关信号通路的影响。结果：PFK可通过提高cAMP/PKA、cAMP/PKG通路中PKA、PKG活性及ERK1/2-Akt通路中关键蛋白磷酸化程度促进精子获能；氧戊二酸盐能够抑制PFK活性，进而抑制精子的被动获能。总之，研究为猪精子获能机制的阐明奠定了基础，将加快体外受精技术的完善和胚胎生产的商业化，促进猪种的保存和改良。