中华绒螯蟹（Eriocheir sinensis）精子顶体反应诱导与BK通道功能初探

发布时间：2018-03-30 07:24

本文选题：大电导钙激活钾通道　切入点：Ca~(2+)载体　出处：《河北大学》2017年硕士论文

【摘要】：为了探索中华绒螯蟹(Eriocheir sinensis)(简称河蟹)精子大电导钙激活钾通道(large-conductance calcium-activated potassium channel,简称BK通道)功能和顶体反应的关系,本研究采用副性腺蛋白溶解法获得游离中华绒螯蟹精子,以精子为试验对象,开展了以下研究,首先以Ca~(2+)载体A23187进行精子顶体反应的体外诱导,并通过Fluo-4 AM荧光标记检测精子顶体反应前后胞内游离Ca~(2+)浓度的变化情况。其次,采用全细胞膜片钳技术记录精子的全细胞钾电流并结合阻断剂分析钾通道的电生理特性及其类型,并模拟顶体反应时Ca~(2+)浓度变化,观察对中华绒螯蟹精子钾电流的影响,探讨钾通道在中华绒螯蟹精子顶体反应过程中的作用规律。主要研究结果如下:1.以不同浓度的Ca~(2+)载体A23187,分别采用ASW(artificial sea water)和Ca~(2+)-FASW(Calcium-free artificial sea water)配制,对精子进行顶体反应诱导,发现Ca~(2+)载体A23187可显著提高精子的顶体反应率,且发生顶体反应的精子多处于III~IV期,最佳诱导浓度为80μg/m L,诱导时间为30 min。同时说明,Ca~(2+)在中华绒螯蟹精子顶体反应中具有主导作用。2.以Ca~(2+)载体A23187诱导中华绒螯蟹精子发生顶体反应,同时采用钙荧光染料Fluo-4AM进行标记,以激光共聚焦技术追踪单个精子顶体反应发生前后胞内Ca~(2+)浓度的变化。研究发现,中华绒螯蟹精子未发生顶体反应前,Ca~(2+)主要分布于细胞质和顶体囊的顶体管上,诱发精子发生顶体反应的瞬间,精子内Ca~(2+)浓度明显升高,主要分布于细胞质和延长的顶体管前端。此外,在无钙缓冲液中,精子自身荧光并无显著增强,提示顶体反应的发生主要依赖于精子外Ca~(2+)的内流。3.以全细胞膜片钳技术记录精子全细胞钾电流,发现该电流由快速IA样电流和延迟IK样电流组成。在无钙内液条件下,加入BK通道阻断剂IbTX(Iberiotoxin,10 nmol/L)后,IA样和IK样电流钾电流均被显著抑制,抑制幅度分别为17.74%和21.51%。提示中华绒螯蟹精子存在BK通道,部分通道可以被膜去极化激活。4.在内液中加入Ca~(2+)(50μmol/L)模拟顶体反应时Ca~(2+)内流,发现精子IA样和IK样钾电流显著提高,提高幅度分别为61.49%和44.11%。说明Ca~(2+)可以增强全细胞外向钾电流,加入IbTX后,IA样和IK样电流被显著抑制,抑制率分别为27.17%和22.65%。说明BK通道在精子外向钾电流中具有重要作用,而且对Ca~(2+)具有依赖性。以上研究结果表明,以Ca~(2+)载体A23187体外诱导中华绒螯蟹精子发生顶体反应时,内流的Ca~(2+)可激活BK通道,造成K+大量外流,改变精子膜电位,引发精子发生顶体反应。说明BK通道在中华绒螯蟹精子顶体反应中具有重要作用。
[Abstract]:In order to explore the Chinese Mitten Crab (Eriocheir sinensis) (the crab) of large conductance calcium activated potassium channels in sperm (large-conductance calcium-activated potassium channel, referred to as BK channel) function and acrosome reaction, this study uses accessory gland protein dissolution method to obtain free Eriocheir sinensis sperm, the sperm as test object, carry out the following research, firstly the Ca~ (2+) A23187 vector induced sperm acrosome reaction in vitro, and by detection of Fluo-4 AM fluorescence labeled sperm acrosome reaction and intracellular free Ca~ (2+) of concentration. Secondly, using the whole cell potassium current in the whole cell patch clamp techniques combined with electrophysiological blocking sperm analysis of characteristics of agent potassium channel and its types, and to simulate the acrosome reaction of Ca~ (2+) concentration changes, observe the effect of Eriocheir sinensis sperm potassium current, on potassium channels in Eriocheir sinensis sperm The top sub rule body in the reaction process. The main results are as follows: 1. with different concentrations of Ca~ (2+) vector A23187, respectively using ASW (artificial sea water) and Ca~ (2+) -FASW (Calcium-free artificial sea water) with the sperm acrosome reaction induced by Ca~ (2+) vector A23187 can significantly improve the acrosome reaction rate and acrosome reaction sperm at III~IV phase, the best induced concentration is 80 g/m L, the induction time was 30 min. at the same time, Ca~ (2+) in Eriocheir sinensis sperm acrosome reaction in the leading role of.2. in the Ca~ (2+) Eriocheir sinensis sperm acrosome reaction induced by plasmid A23187 were labeled by using calcium fluorescent dye Fluo-4AM, single sperm acrosome reaction and intracellular Ca~ by confocal laser tracking technology (2+) concentration. The study found that Eriocheir sinensis sperm acrosome reaction did not occur before Ca~ (2+), mainly distributed in the cytoplasm and acrosome acrosomal vesicle tube, induced sperm acrosome reaction of the sperm in the moment, Ca~ (2+) concentration increased significantly, mainly distributed in the cytoplasm and acrosome extended at the front end of the pipe. In addition, in the absence of calcium buffer, sperm autofluorescence was significant enhanced prompt acrosome reaction depends mainly on sperm Ca~ (2+) by whole cell potassium whole cell patch clamp recording current flow in sperm.3., found that the current by fast IA like current and delayed IK like current. In the absence of calcium in the liquid condition, adding BK channel blocker (IbTX Iberiotoxin, 10 nmol/L), IA like and IK like current potassium currents were significantly inhibited and the inhibition rate were 17.74% and 21.51%. showed that the BK channel in the presence of Eriocheir sinensis sperm, part of the channel can be activated with Ca~ membrane depolarization (2+),.4. solution (50 mol/L) to simulate the acrosome reaction at Ca ~ (2+) influx, found in sperm IA like and IK like potassium currents increased significantly, increased by 61.49% and 44.11%. Ca~ (2+) can enhance the whole cell outward potassium current, after joining IbTX, IA like and IK like current was inhibited, the inhibition rate was 27.17% and 22.65%. respectively shows that the BK channel has an important role in sperm outward potassium current, and the Ca~ (2+) has the dependence. The above results indicated that Ca~ (2+) of Eriocheir sinensis sperm acrosome reaction induced by plasmid A23187 in vitro, the flow within the Ca~ (2+) can activate BK channels, resulting in a massive outflow of K+, change the sperm membrane potential induced sperm acrosome reaction. It plays an important role in BK channel acrosome reaction in Eriocheir sinensis sperm.

【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q954.43

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