钠肽受体2在精子趋化中的作用和机制

发布时间：2018-07-29 11:29

【摘要】：钠肽受体2 (natriuretic peptide receptor 2, NPR2)在维持卵母细胞减数分裂阻滞中起着关键的作用。本课题研究了NPR2在精子上的表达特征、功能及作用机制,同时利用卵丘卵母细胞复合体的体外培养模型研究了NPR2失活的调节机制。哺乳动物交配后,射入雌性生殖道的几百万精子快速到达输卵管峡部储存起来,只有当排卵发生时,才有少数获能精子恢复运动能力,向受精部位壶腹部运动,最终与卵母细胞相遇,导致受精。该现象通常被称为精子趋化作用。采用免疫沉淀和蛋白免疫印迹的方法,发现NPR2主要表达在精子尾部,而使用荧光标记的配体C-型钠肽(Natnuretic peptide type C, NPPC),即FAM-NPPC,进行结合实验,发现FAM-NPPC主要结合在小鼠精子尾部中段,表明NPR2表达在小鼠精子尾部中段。NPR2的配体NPPC在青春期小鼠输卵管中表达很低,在排卵后输卵管壶腹部的表达显著增加。免疫组化实验表明NPPC蛋白定位在输卵管壶腹部的黏膜层细胞中。NPPC能够增加精子平均路径速度VAP,直线运动速度VSL及进行性运动的精子比率。采用毛细管趋化模型来研究NPPC对小鼠精子的趋化作用,结果表明NPPC能够诱导精子在毛细管中的积累。进一步研究发现,NPPC与精子上的NPR2结合,能够引起精子胞内cGMP水平的升高。NPPC能诱导精子胞内钙离子水平的升高,并且钙离子升高的应答首先出现在精子尾部中段,随后传播到了精子头部。cGMP类似物8-Br-cGMP能够模拟NPPC的作用,引起精子的积累和胞内钙离子水平的升高。NPPC不能诱导Npr2ca-2J/Npr2cn-2J突变鼠精子积累,而且Npr2cn-2J /Npr2cn-2J突变鼠精子人工输精的受精率非常低(Npr2cn-2J/Npr2n-2J,5.4±2.2%; Npr2wt/Npr2cn-2J,77.7±3.40%)。因此排卵发生时,精子上的NPR2与壶腹部来源的配体NPPC结合,通过cGMP-Ca2+信号通路诱导精子朝向壶腹部的卵母细胞运动,这对于排卵和受精的同步化及正常受精是非常重要的。为了研究NPR2失活的机理,我们利用卵丘卵母细胞复合物的体外培养模型进行实验。NPR2与NPPC结合产生的cGMP维持了卵母细胞减数分裂的阻滞。本研究表明EGF能够引起卵丘细胞中钙离子水平升高,导致NPR2的失活。钙离子激动剂离子霉素(ionomycin)和磷酸鞘氨醇(lipid sphingosine-1-phosphate, SIP)能模拟EGF的作用。说明NPR2与NPPC结合诱导了卵丘细胞中钙离子水平的升高,导致了NPR2的失活。以上结果说明NPR2表达在精子尾部中段,通过结合NPPC,产生cGMP,促进了胞外钙离子内流,增加了精子的活力,诱导精子朝向卵母细胞趋化运动,完成受精。卵丘细胞中的钙离子水平的升高导致了NPR2鸟苷酸环化酶的失活。利用NPR2在精子趋化中的作用,对于未来筛选优质的精子进行体外受精及制备避孕药有着很好的应用前景。
[Abstract]:Natriuretic peptide receptor 2 (NPR2) plays a key role in maintaining meiotic arrest in oocytes. In this study, the expression, function and mechanism of NPR2 in spermatozoa were studied, and the regulation mechanism of NPR2 inactivation was studied by using cumulus oocyte complex culture model in vitro. After mating in mammals, millions of sperm injected into the female reproductive tract reach the isthmus of the fallopian tube for storage. Only when ovulation occurs does a few of the capacitated sperm recover their motility and move toward the ampulla, where they are fertilized. Eventually, it meets oocytes and results in fertilization. This phenomenon is often referred to as sperm chemotaxis. By immunoprecipitation and Western blotting, we found that NPR2 was mainly expressed in the tail of spermatozoa, while FAM-NPPC, a fluorescent labeled ligand of C- type natriuretic peptide (Natnuretic peptide type C, NPPC), was used to carry out binding experiments. It was found that FAM-NPPC was mainly bound to the middle part of mouse sperm tail. The results showed that the expression of NPR2 was very low in the oviduct of puberty mice, and increased significantly after ovulation. Immunohistochemical study showed that the localization of NPPC protein in the mucosal layer cells of the ampulla of fallopian tube could increase the average speed of sperm pathway, the velocity of linear motion, VSL, and the rate of progressive motility of spermatozoa. The chemotactic effect of NPPC on mouse spermatozoa was studied by capillary chemotaxis model. The results showed that NPPC could induce the accumulation of spermatozoa in capillaries. Furthermore, it was found that the combination of NPR2 with NPR2 in spermatozoa could induce the increase of intracellular cGMP level in spermatozoa. NPPC could induce the increase of intracellular calcium level in sperm, and the response of increased calcium ion first appeared in the middle part of sperm tail. Then it was transmitted to the sperm head. 8-Br-cGMP, an analogue of cGMP, could mimic the effect of NPPC and induce the accumulation of spermatozoa and the increase of intracellular calcium level. NPPC could not induce the accumulation of sperm in Npr2ca-2J/Npr2cn-2J mutant mice. Moreover, the fertilization rate of Npr2cn-2J / Npr2cn-2J mutant spermatozoa was very low (Npr2cn-2J / Npr2n-2J = 5.4 卤2.2; Npr2wt / Npr2cn-2JN 77.7 卤3.40%). Therefore, during ovulation, NPR2 on the sperm binds to the ligand NPPC from the ampulla, which induces the oocyte movement towards the ampulla through the cGMP-Ca2 signaling pathway, which is very important for the synchronization of ovulation and fertilization and normal fertilization. In order to study the mechanism of NPR2 inactivation, we used the cumulus oocyte complex culture model in vitro to carry out the experiment. The cGMP produced by the combination of NPR2 and NPPC maintained the block of meiosis of oocytes. This study suggests that EGF can increase calcium level in cumulus cells and lead to NPR2 inactivation. Calcium agonists, ionomycin (ionomycin) and lipid sphingosine-1-phosphate (SIP) can mimic the effects of EGF. These results suggest that the combination of NPR2 and NPPC induces the increase of calcium ion level in cumulus cells and leads to the inactivation of NPR2. These results suggest that NPR2 is expressed in the middle part of the sperm tail. By combining with NPR2, cGMPs are produced, which promote the influx of extracellular calcium ions, increase the motility of sperm, induce sperm to move towards oocytes, and complete fertilization. Increased calcium levels in cumulus cells resulted in inactivation of NPR2 guanosine cyclase. The role of NPR2 in sperm chemotaxis has a good prospect for the future screening of high quality spermatozoa for in vitro fertilization and the preparation of contraceptive pills.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q492

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