中国科学院海洋研究所机构知识库

精子储存是一种广泛存在于体内受精物种中十分普遍的繁殖策略。卵胎生硬骨鱼许氏平鲉从交尾到受精, 精子储存时间长达6个月。目前, 对于卵胎生鱼类精子在雌鱼体内的储存位置、活力状态、能量供应相关研究较少。本研究以许氏平鲉为研究对象, 观察许氏平鲉精子超微结构, 并与卵生硬骨鱼大菱鲆比较分析体内受精许氏平鲉精子特殊结构特征; 通过组织学与精子生理状态检测，分析了许氏平鲉精子从交尾到受精期间在卵巢的储存位置的动态变化；通过精子、血清和卵巢液成分组成、药物实验以及卵巢液组分解析初步明确精子能量底物与供应途径。主要研究结果显示:

1. 分析了许氏平鲉的超微结构: 在10月份交尾时采集精子进行结构分析, 许氏平鲉精子由头部, 中段和尾部构成, 且头部无顶体。许氏平鲉精子头部细长, 短棒状, 长径: 3.15 ± 0.17 µm, 短径: 1.44 ± 0.10 µm; 中段长1.23 ± 0.48 µm, 两侧不对称, 呈囊袋状, 线粒体呈垛叠状紧密排列在中段, 约有3-4层, 约30-40个线粒体; 尾部侧鳍较发达, 尾部鞭毛轴丝为“9 + 2”微管结构。交尾时, 许氏平鲉精子结构发育完整, 且可被卵巢液激活迅速运动。与体外受精硬骨鱼相比许氏平鲉精子拥有细长的头部, 发达的中段, 线粒体数量较多，这些结构的差异也是对其体内受精和长期储存特性的适应性进化。

2. 描述了精子在卵巢内长期储存的动态变化: 10月份许氏平鲉交尾完成, 至次年3-4月份受精, 精子储存于卵巢长达半年之久。10月至12月, 可以直接从卵巢液中观察到快速游动的精子(VCL = 67.04 ± 2.51 µm/s), 大多数精子随机散布在卵巢腔内或集中在滤泡复合体外侧上皮细胞形成的隐窝中, 卵巢处于Ⅲ期前卵黄积累期, 卵巢中以皮质小泡期卵母细胞为主, 细胞膜边缘开始出现卵黄颗粒; 12月下旬2月，卵巢处于Ⅳ期卵黄积累期, 由于间质细胞的增生, 部分精子散布在间质细胞之间, 大多数精子储存在滤泡层外的小腔中, 此时不能从卵巢液中观察到活动的精子; 3月份卵巢逐渐发育成熟，精子储存于滤泡层外的小腔中。由此推测: 交尾后, 精子在卵巢中游动, 随机分布, 于次年12月, 精子停止运动, 由于卵子周边间质细胞发育, 精子主动或被动裹入卵母细胞外滤泡层与间质细胞外小腔, 直至受精前，被卵巢液激活完成受精过程。

3. 初步查明精子长期储存主要能量供应物质与能量代谢方式: 借助相关试剂盒，对许氏平鲉卵巢液与精子葡萄糖、果糖、甘油、丙酮酸、乳酸、柠檬酸进行检测，发现许氏平鲉卵巢液底物组分含量与精子相似, 能量物质组分中果糖含量、甘油、乳酸含量较高, 卵巢液物质含量在1月达到峰值, 3月达到最低值。许氏平鲉精子可以利用外界底物支持自身生命活动, 体外实验证明外源能量底物葡萄糖、果糖等添加可以有延长精子寿命, 最长可达85 ± 5 h，增长了40 %，但未显著影响精子运动学参数, 同样添加碘醋酸钠抑制糖酵解途径, 运动相关参数变化不显著。与之相反添加FCCP与鱼藤酮抑制氧化磷酸化途径, 可以使精子的运动率指标(运动率、前向运动率), 速率指标(VCL、VAP、VSL), 运动特征参数(BCF、STR、LIN、WOB)显著降低。由此推测: 体外实验糖酵解底物的添加可以有效延长精子搜狐名，氧化磷酸化可能在交尾和受精前的快速激活运动的能量供应中发挥了重要作用。

4. 通过非靶向代谢组初步探索了许氏平鲉精子储存期微环境: 在正离子模式下, 许氏平鲉卵巢液相较于大菱鲆卵巢液共筛选出9077种物质, 其中122种物质含量显著上调, 20种物质含量显著下调, 差异倍数大于40倍的物质共20种, 17种为二肽; 负离子模式下, 筛选出7125种物质, 其中44种物质显著上调, 23种物质显著下调。Protein digestion and absorption(蛋白质消化与吸收), ABC transporters(ABC转运超家族)等通路发生了显著变化。其中protein digestion and absorption通路中共有15种物质富集到该通路。在ABC transporters通路种21种差异代谢物富集到本通路, 富集到差异代谢物最多。

The phenomenon of female sperm storage is ubiquitous reproductive strategy for species with internal fertilization. In viviparity teleost Sebastes schlegelii, sperm were stored in ovary up to 6 months and the sperm storage location and motility in the ovary was not elaborated. The purpose of this study was to compare the sperm structure and physiological characteristics of S. schlegelii and Scophthalmus maximus, to explore the adaptive structure of sperm storage and internal fertilization; histological method was utilized for examining the sperm storage position and the development of ovaries during process of sperm storage; The main energy substance of sperm, serum and ovarian fluid was examined, inhibition experiments executed for elaborating the energy metabolism of sperm in S. schlegelii. The main results were as follows:

1. The ultrastructure of S. schlegelii was elucidated: Sperm were collected in October for structural analysis. The Spermatozoa of S. schlegelii was composed of head which devoid of acrosome, midpiece and the tail. In black rockfish: the head was rod-shaped with 3.15 ± 0.17 µm in major axis and 1.44 ± 0.10 µm in minor axis. The asymmetrical midpiece was 1.23 ± 0.48 µm long, consisted of 30 to 40 mitochondria closely arranged in stacks, the tail was exhibited “9 + 2” microtubules. At the time of copulation, the sperm of S. schlegelii was mature and can be activated by ovarian fluid. The sperm of S. schlegelii had longer head, longer midpiece contained with a large number of mitochondria which compared with external fertilized teleost. These differences of structure were adaptive evolution of internal fertilization and long-term storage.

2. The dynamic changes of long-term sperm storage in the ovary were revealed: Sperm storage of S. schlegelii started in November and ended in April of the following year. The sperm was stored in the ovary for half a year. From November to December, the sperm of S. schlegelii swam in the ovarian fluid at high speed (VCL = 67.04 ± 2.51 µm/s) and most of the sperm were randomly scattered in the ovarian cavity or concentrated in the crypt formed by the follicle combined with the outer epithelial cells. The ovary was in vitellogenesis onset period (stage Ⅲ), the oocytes in oil droplet stage accounted for the main part of the ovary, and yolk granules began to appear at the edge of membrane; In the last days of December to February, the ovary was in the stage IV(yolk accumulation stage). Due to the proliferation of interstitial cells, some sperm were scattered between the interstitial cells, and most sperm were stored in the cavity outside the follicular layer. At this time, the ovary was in the stage V(maturation) and no active sperm could be observed from the ovarian fluid. From March to April, the sperm were stored in the crypt outside the follicle layer until fertilization. It could be inferred that after mating, the sperm moved into the ovary and distributed randomly. From late December to February of the following year, the sperm stopped moving. Due to the development of interstitial cells around oocytes, the sperm actively or passively wrapped in the outer follicle layer and the cavity of the interstitial cells, until the fertilization process was completed.

3. The sperm energy supply substances and energy metabolism during the process long-term storage were preliminarily identified: The composition of ovarian fluid substrate was similar to sperm, the contents of fructose, glycerin and lactate in the energy components were high, and the metabolism was mainly completed by glycolysis. The concentration reached the peak in January and the lowest in March. The spermatozoa of S. schlegelii could use the external metabolic substrates to support vital movement. In vitro experiments showed that the addition of glucose, fructose, glycerin and pyruvic acid could prolong the life span of sperm, the longest life span as 85 ± 5 h and 40 % longer than that of culture without substrate. The addition of exogenous energy substrate could not increase sperm kinetic parameters.

In the exploration of sperm energy metabolism pathway, by adding FCCP and rotenone to inhibit oxidative phosphorylation pathway, sperm motility index (motility rate, forward motility rate), velocity index (VCL, VAP, VSL), motility characteristic parameters (BCF, STR, LIN, WOB) were significantly reduced, while sodium iodoacetate to inhibit glycolysis pathway, while related parameters were not changed significantly. It suggested that glycolysis played an important role in the long-term storage process, and oxidative phosphorylation may play an important role in the energy supply of rapid activation movement before mating and fertilization.

4. The microenvironment of the ovary during the spermatozoa storage period of S. schlegelii was studied by metabolome: In the analysis of metabolism of ovarian fluid of S. schlegelii and S. maximus, 9077 substances were screened out in the positive ion mode compared with that of S. maximus, of which 122 substances were significantly up-regulated, 20 substances were significantly down regulated, 17 were dipeptides. In the negative ion mode, 7125 substances were screened out of which 44 substances were significantly up-regulated, 23 substances were down-regulated significantly. Significant changes have taken place in the important pathways such as protein digestion and absorption and ABC transporters pathway, etc. There are 15 kinds of differential metabolites in the protein digestion and absorption pathway, and 21 kinds of differential metabolites in the ABC transporters pathway are enriched in this pathway, which was the most enriched pathway.

第一章 文献综述... 1

1.1 鱼类的生殖策略... 1

1.2 精子储存... 2

1.2.1 精子储存时间... 2

1.2.2 精子储存器官... 2

1.2.3 精子储存的分子机制... 3

1.3 精子的代谢底物与途径... 4

1.4 精子结构与精子储存的适应性关系... 6

1.4.1 脊椎动物精子结构... 6

1.4.2 鱼类精子结构... 8

1.5 许氏平鲉的生物学特征... 9

1.6许氏平鲉精子储存及能量代谢的研究现状... 9

1.7 本研究的目的及意义... 10

第二章 体内受精许氏平鲉与体外受精大菱鲆精子结构及生理特性比较... 10

2.1 前言... 10

2.2 材料与方法... 11

2.2.1 亲鱼来源及精液采集... 11

2.2.2 实验仪器与试剂... 11

2.2.3 扫描电镜... 12

2.2.4 透射电镜... 12

2.2.4 精子运动指标... 12

2.2.5 数据分析... 12

2.3 结果... 12

2.3.1 许氏平鲉精子结构... 12

2.3.2 大菱鲆精子结构... 14

2.3.3 许氏平鲉与大菱鲆精子生理特性及运动特征比较... 15

2.4 讨论... 16

第三章 许氏平鲉精子长期储存及其周围卵巢发育变化特征... 20

3.1 前言... 20

3.2 材料与方法... 20

3.2.1 实验仪器与试剂... 20

3.2.2 实验材料... 21

3.2.3 生物学指标的测量与计算... 21

3.2.4 组织学切片... 21

3.2.5 精子储存期间精子运动状态观察... 22

3.2.6 数据分析与处理... 22

3.3 结果... 22

3.3.1 许氏平鲉精子发生过程... 22

3.3.2 许氏平鲉繁殖周期生理指标... 24

3.3.3 许氏平鲉卵巢的组织学观察... 26

3.3.4 精子存储期间滤泡层细胞发育规律... 31

3.3.5 许氏平鲉精子存储状态... 33

3.4 讨论... 36

第四章 许氏平鲉精子长期储存的代谢底物与途径分析... 38

4.1 前言... 38

4.2 材料方法... 39

4.2.1 实验仪器与试剂... 39

4.2.2 实验材料... 39

4.2.3 非靶向代谢组学分析许氏平鲉卵巢液微环境... 40

4.2.4 精子及卵巢液中能量底物的检测... 41

4.2.5 精子的体外培养及代谢底物检测... 41

4.2.6 许氏平鲉精子药物阻断与代谢途径检测... 41

4.2.7 数据处理与分析... 42

4.3 结果... 42

4.3.1许氏平鲉精子储存卵巢内微环境分析... 42

4.3.1 许氏平鲉卵巢液代谢底物含量... 47

4.3.2 体外培养精子寿命观测... 50

4.3.3 许氏平鲉的能量代谢途径探索... 51

4.4 讨论... 59

4.4.1 许氏平鲉卵巢微环境分析... 59

4.4.2 许氏平鲉精子的能量底物... 60

4.4.3 许氏平鲉精子的能量代谢... 62

第五章 结论与展望... 64

5.1 主要结论... 64

5.2 展望... 65

参考文献... 66

致谢... 73

作者简历... 74

攻读学位期间发表的学术论文... 75