cAMP信号通路调节线虫精子细胞成熟和运动的分子机制研究

The second messenger cyclic adenosine 3’,5’ monophosphate (cAMP), modulates cell proliferation, differentiation and migration in various tissues of organisms ranging from bacteria to higher eukaryotes. The sperm must undergo a maturation process called sperm activation in nematode or capacitation in mammalian, which consists of morphological, biochemical and physiological processes in sperm in order to acquire their fertilizing potential. It has been demonstrated that bicarbonate stimulates an evolutionarily conserved soluble adenylyl cyclase (sAC) from bacteria to mammals to produce the cAMP. sAC was first identified in rat sperm and is essentially required for the sperm maturation in mammals. The bicarbonate is also essential for sperm maturation in nematode Ascaris suum, however, the roles bicarbonate plays during sperm maturation remain elusive. The previous bioinformatic studies indicate that no sAC homologs exist in fly and worm. We propose to continue our investigation of the mechanism(s) of sperm maturation in nematode Ascaris suum, with an emphasis on the signal transduction initiated by the extracellular bicarbonate. These studies will involve a range of experimental techniques and will require an integration of project objectives from the molecular to cellular levels. We plan to focus our attention on the following aims: (a) examine the roles of bicarbonate on nematode sperm activation and investigate whether a bicarbonate-responsive sAC-like sensor functions during sperm activation; (b) analyze the intracellular cAMP level changes induced by bicarbonate in nematode sperm and study the sAC-cAMP signal pathway functions during sperm maturation; (c) investigate the role of the cAMP-specific phosphodiesterases (PDEs) during sperm maturation; (d) the crosstalk between the cAMP and calcium is also the focus of this proposal. These studies will provide the framework for future studies on the evolution and function of bicarbonate-sensing sACs ranging from bacteria to higher animals. Information from this nature will likely potentiate a wide range of fundamental biological studies and might provide clues for developing new strategies for parasite control and nontoxic male contraceptive.

cAMP作为细胞内关键的信号分子，在多种组织中调节着细胞的生长、分化和运动；精子细胞必须经过一个成熟的过程，在信号刺激下其形态，生化和生理发生一系列变化才能发育为功能性精子。然而在模式动物线虫中，cAMP信号与精子成熟和运动之间的调控关系目前还未见报道。本项目从碳酸氢根在线虫精子成熟过程中发挥的重要功能着手，分离鉴定响应碳酸氢根信号的可溶性腺苷酸环化酶(sAC)，研究sAC活性调节及其合成的cAMP信号分子传递网络在精子成熟过程中的调控作用；分析胞内cAMP水平在精子成熟过程中的动态变化。同时研究磷酸二酯酶(PDEs)对胞内cAMP水平的调控及其对精子成熟的影响，探讨钙信号与cAMP信号之间的调控关系，全面解析cAMP信号通路对线虫精子成熟和运动的调节机理。预期研究结果不仅对于控制动植物寄生虫的传播，治疗不育等具有重要的理论指导意义，更是填补了cAMP及腺苷酸环化酶在线虫研究中的空白。

精子发育是一个极其复杂的事件，包括精子发生（减数分裂阶段）、在胞外信号刺激下的精子变形（精子激活阶段）以及精子运动和受精能力的获得（精子获能）三个主要阶段，其中任一阶段的失调都将导致精子功能丧失，造成雄性不育。本项目围绕线虫精子发育展开研究，鉴定新的参与线虫精子发生的功能调控蛋白，探讨精子细胞极性建立和形态变化的调控机理以及这一过程中涉及到的离子平衡调节，分析精子运动能力获得的调控机制，主要成果包括：1）分离鉴定了Ascaris线虫精子内响应碳酸氢根离子刺激产生cAMP信号分子调节精子激活运动的可溶性腺苷酸环化酶sAC蛋白，相关结果正在投稿之中。2）鉴定了离子转运蛋白Na+/K+ ATPase（NKA）复合物在精子激活运动过程中的调控作用，分析了NKA复合物在精子激活时细胞定位发生极性分布，调节膜电势及胞内离子平衡最后影响精子运动的分子机制，相关结果已投稿至Development Cell杂志，处于修改稿的评审阶段（DEVELOPMENTAL-CELL-D-20-0080，under Revising）。3）鉴定线虫精子细胞特有的膜结构Membranous Organelle（MO）作为胞内钙库调节胞质钙水平，发现MO钙储存对于锌离子诱导的精子激活和已活化精子运动能力的获得是必需的，钙离子在MO中的定位依赖于MO的酸化以及锌离子转运蛋白ZIPT-7.1，进而阐述了ZIPT-7.1蛋白调控精子激活运动的网络机制，相关结果正在整理投稿之中。4）鉴定并解析RNA结合蛋白PRP-38在线虫精子发生中的调控机制，解析了PRP-38通过影响精子发生过程中重要调控蛋白例如SPE-5，SPE-6等的RNA剪切调节精子减数分裂进程，进而调控精子发育，相关结果正在整理投稿之中。5）鉴定线虫生殖腺特异表达蛋白AMG-1（C04E6.11）调控精子发生及激活运动的分子机理，阐明了线粒体定位的AMG-1蛋白通过影响线粒体形态、线粒体基因转录、线粒体能量产生以及线粒体膜电势和未折叠蛋白反应等一系列事件调节生殖腺细胞的分化与发育，影响精子的产生、激活和随后的运动能力，相关结果正在整理投稿之中。6）研究了Formin蛋白AtFH14偶联微管微丝调控细胞分裂的分子机理，相关结果作为共同通讯作者已被Biology of the Cell接受发表。