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题名: 水通道蛋白7、8及精液pH值对大菱鲆(Scophthalmus maximus)精子活力影响研究
作者: 安皓
学位类别: 硕士
答辩日期: 2017-05
授予单位: 中国科学院大学
授予地点: 北京
导师: 李军
关键词: 大菱鲆,AQP,pH,精子,精子活力
学位专业: 水产养殖
中文摘要: 鱼类精子活力的影响因素复杂多样,其中水通道蛋白和pH是重要的影响因素。水通道蛋白是一种位于生物细胞膜上的内在膜蛋白,可以控制水分子及其他小分子物质进出细胞,对生物体的正常生理活动至关重要。目前在哺乳类中共发现13种水通道蛋白,其中有8种在鱼类生殖系统中被发现。有研究表明,AQP7在心肌细胞中能转运甘油提供能量;AQP7对精子转运水的能力并无影响,可能通过运输甘油来提供能量基质来影响精子活力;AQP8在调节精子体积和线粒体内活性氧簇运输方面十分重要。关于pH对鱼类精子活力的影响,以往研究多集中在外界不同pH对精子活力的影响,对精液自身pH与精子活力关系研究的较少。在我国大菱鲆(Scophthalmus maximus)是重要的海水经济鱼类,本论文以大菱鲆为研究对象,阐明AQP7、AQP8在大菱鲆精子发育及成熟过程中的表达特征及对精子活力的影响机制,同时通过对大菱鲆精子活力的检测和评价,探究精液活力与pH的关系。
主要结果如下:
  1. 获得AQP7基因全长共1315bp,获得了AQP8部分片段共584bp;AQP7、AQP8 mRNA在精巢表达情况从2期到5期先升高后降低,其最高峰分别为3期和4期,但二者6期又有所升高;AQP7、AQP8原位杂交情况与mRNA表达水平相一致,二者在精巢表达情况从2期到5期先升高后降低,其最高峰分别为3期和4期,6期表达量又有所升高。AQP7蛋白从精原细胞到精子都有表达,且活力好的精子AQP7蛋白表达量较高,这暗示了AQP7对精子活力有促进作用。
  2. 精子活化率高、寿命长的大菱鲆精液pH偏弱碱性(pH=8.0左右),即大菱鲆精子适宜在弱碱性环境中活动;大菱鲆精子运动特性受pH影响显著,精液pH与平均路径速度(VAP)、平均直线运动速度(VSL)、平均曲线运动速度(VCL)、精子平均鞭打频率(BCF)、精子头侧摆幅度(ALH)、呈显著正相关(p<0.05),而与运动前向性 (STR)、运动直线性(LIN)、运动摆动性(WOB)等参数无显著相关性;大菱鲆精子运动特性与精子活化率相关性显著,其中精子活化率与平均路径速度(VAP) 平均直线运动速度(VSL)、平均曲线运动速度(VCL)、精子平均鞭打频率(BCF)、精子头侧摆幅度(ALH)、呈显著正相关(p<0.05),与运动前向性 (STR)、运动直线性(LIN)、运动摆动性(WOB)等参数无显著相关性。
  3. 向激活海水中加入梯度甘油,发现可以显著提高大菱鲆精子寿命,其中甘油浓度为100 mmol/L时,精子寿命延长至15.17±0.29 min,推测甘油在精子激活后可能被精子吸收并氧化分解提供能量。
英文摘要: Factors influencing sperm motility are various. Aquaporin and pH are important influencing factors. Aquaporin is a kind of integral membrane proteins located in cell membrances . It is essential to the nomal physiological activities of the organism because it modulates hydrone and other  small molecules influx or efflux. Up to now, 13 kinds of aquaporin was found inmammalia and 8 kinds of them was found in fish reproductive system. The study showed that AQP7 can provide energy by glycerin transportation whlie has no function of water transprotation for sperm. AQP8 is important in regulating sperm volume and the transportation of ROS in miochondrial. Factors influencing sperm motility are various, and pH is one of the factors. In regred to the influence of pH on fish sperm vitality, the previous studies focuse on the influence of different pH of extermal environment on sperm motility, but there are seldom research focuse on the influence of different pH of sperm itself on sperm motility. Turbot (Scophthalmus maximus) is one of the most important fish in the china sea. This paper seting turbot as a study focus, aims to clarify the influence of AQP7 and AQP8 on sperm motility and the  expression characteristics in the process of sperm development. At the same time, study the relationship between sperm motility and pH by the detection and evaluation of sperm motility.
The main results were as follows:
  1. Get the full-length gene of AQP7 and part gene fragment of AQP8, 1513bp and 584bp respectively. The expression of AQP7 and AQP8 mRNA increased at first and then decreased from stage 2 to 5. The highest peak is stage 3 and stage4 respectively but the expression of both of them increased in stage 6. The results of situ hybridization of AQP7 and AQP8 are consistent with the expression of AQP7 and AQP8 mRNA. AQP7 was expressed from spermatogonia to  spermatozoa. It was highly expressed in high quality sperm, which suggest AQP7 has a promotion to sperm viability.
  2. The sperm with higher motility and longer life pH has a weak alkaline pH (pH ≈ 8.0) which means turbot sperm are active in the weak alkaline environment. We found pH of turbot sperm were in significantly positive correlation with motility, VCL, VSL, VAP, ALH, BCF(p < 0.05), while there was no significant correlation with LIN, WOB, STR. The motility of turbot sperm were in significantly positive correlation with VCL, VSL, VAP, ALH, BCF(p < 0.05), while there was no significant correlation with LIN, WOB, STR.
  3. In addition, we found appropriate amount of glycerol in sea water was useful for maintaining longevity, and the optimal concentration of glycerol was 100mmol/L.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.qdio.ac.cn/handle/337002/136683
Appears in Collections:海洋生物技术研发中心_学位论文

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作者单位: 中国科学院海洋研究所

Recommended Citation:
安皓. 水通道蛋白7、8及精液pH值对大菱鲆(Scophthalmus maximus)精子活力影响研究[D]. 北京. 中国科学院大学. 2017.
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