【作者】 邹远超；

【导师】 危起伟；

【作者基本信息】 华中农业大学 ， 渔业资源， 2011， 博士

【摘要】 匙吻鲟(Polyodon spathula)隶属鲟形目(Acipenseriformes)、白鲟科(Polyodontidae)、匙吻鲟属(Polyodon),是美国特有的大型淡水经济鱼类,也是世界上现有的白鲟科鱼类之一,与我国白鲟是同科异属的珍稀动物。匙吻鲟具有生长快、品质优、适应性广、抗逆能力强等特点,且肉质细腻、营养丰富,是公认的优良养殖鱼类。我国于20世纪90年代初从美国引进匙吻鲟受精卵进行人工孵化并试养成功,目前匙吻鲟已成为我国水产养殖业广受欢迎的一个名优新品种。由于匙吻鲟与白鲟(psephurus gladius)同属于白鲟科,因此开展匙吻鲟雌核发育的人工诱导实验可为长江白鲟的种群恢复奠定技术基础。此外,近几年随着匙吻鲟养殖产业的形成,国内每年可人工繁殖的匙吻鲟苗种达数百万尾,获得抗病强,生长快等优良性状的匙吻鲟品系是育种工作的首要任务。人工诱导雌核发育技术是快速建立其纯系,选育优良品种的有效途径,为此本研究以我国人工养殖的匙吻鲟为研究对象,采用施氏鲟(Acipenser schrenckii)精子诱导匙吻鲟雌核发育二倍体,通过形态学、细胞遗传学方法与微卫星分子标记对雌核发育群体进行了鉴定,同时,采用扫描电镜、透射电镜和慧星实验对紫外线照射的精子的超微结构和核DNA损伤程度进行了研究,最后,对雌核发育匙吻鲟受精细胞学及其性别决定机制进行初步的探讨,主要研究结果如下：1.在以匙吻鲟为母本,施氏鲟为父本的杂交试验中发现,杂交胚胎能发育到原肠期,但无孵出存活,胚胎在出膜前因发育畸形而死亡,经胚胎染色体计数分析表明其染色体为n=60,从而确定,施氏鲟精子与匙吻鲟卵子杂交所得后代即为匙吻鲟的单倍体。基于这个研究结果,本研究首次采用施氏鲟鲜精作为诱导匙吻鲟雌核发育的激活源,通过对雌核发育热休克起始时间,热休克温度和热休克持续时间的筛选和优化,确定了利用施氏鲟鲜精诱导匙吻鲟卵雌核发育的最佳条件是：在18℃水温中授精18 min后,用37℃的淡水对卵进行热休克处理2 min。该组受精率为76.4±9.2%,胚胎孵化率为7.8±1.4%。本研究同时筛选出了施氏鲟精子遗传失活的条件,即采用波长为254 nm的紫外线照射(照射强度为863μw/cm2) 5 min能使施氏鲟精子遗传失活。采用遗传失活的施氏鲟精子与匙吻鲟卵授精后热休克的方法诱导匙吻鲟雌核发育,该组受精率为56.4±1.2%,胚胎孵化率为28.7±1.8%。在雌核发育后代鉴定的实验中,分别采用了形态学、染色体计数和核型分析、流式细胞仪检测DNA含量和微卫星标记技术对雌核发育鱼苗进行了鉴定。结果发现,对照组匙吻鲟和雌核发育匙吻鲟在形态上没有差异,对照组和雌核发育组匙吻鲟的染色体组均由80条基本染色体和40条微型染色体组成。根据Levan, etal的分类标准,对匙吻鲟10个中期分裂相进行放大测定,按染色体臂比及相对长度把匙吻鲟染色体分为3组：中部着丝点粒染色体(m)44条、亚中部着丝粒染色体(sm)32条、亚端部着丝粒染色体(st)和端部着丝粒染色体(t)44条。染色体臂数(NF)为196,其核型公式为44m+32sm+44st,t。对对照组二倍体血液样品和雌核发育匙吻鲟血液样品进行DNA相对含量的检测,雌核发育匙吻鲟红细胞的DNA含量与二倍体匙吻鲟红细胞DNA含量比为1：1,其相对含量在3.50-3.59 pg。2.采用扫描电镜和透射电镜观察了经紫外线辐射前后施氏鲟精子形态结构和超微结构的变化。结果表明,成熟的施氏鲟精子由顶体、细胞核、中段和尾部三部分构成,其平均长度(顶体+细胞核+中段)为8.95μm,加上尾长(36.80μm)共约45.75μm。鲜精具有明显的顶体及后外侧延伸物,顶体中含有亚顶体、顶体泡和顶体腔,顶体腔中有少量颗粒状物质。经紫外线照射后,精子表现出顶体不明显或顶体与核发生糅合,后外侧延伸物消失或变短,顶体膜受损,顶体腔增大,颗粒物质丢失,有的精子甚至顶体脱落；顶体泡、亚顶体与核之间的排列松弛,线粒体内嵴弥散或线粒体脱落。可以推断,紫外线照射对精子膜、顶体、线粒体和鞭毛的损伤可能是造成精子活力和受精率降低的原因。为了更好地了解紫外线辐射与精核DNA相互作用的机理,本文首次采用慧星实验探明了紫外线照射对精子DNA完整性的影响。实验结果表明：随着UV照射剂量的增加,施氏鲟精核DNA断片增多,慧星状细胞数和慧尾长度也增加。紫外线照射时间大于5 min时,精子的彗星率、损伤系数与鲜精差异显著(P<0.05)。紫外线照射剂量与出现彗尾的频率及长度呈明显剂量-效应关系。3.运用组织学方法研究了人工诱导匙吻鲟卵子雌核发育的细胞学过程。在光镜下对第二极体抑制型雌核发育匙吻鲟二倍体卵在受精过程中的核相变化进行了详细观察。结果表明,精子入卵后精核始终不解凝,也不与雌性原核融合,是典型的雌核发育鱼类的受精细胞学特征。同时,对培育1年后的3个雌核发育群体和2个对照组群体经解剖取性腺,Bouin氏液固定,常规组织切片,性别鉴定结果显示2个对照组家系子代的性别比例都接近于理论值1：1,没有显著性差异(P>0.05)；而3个雌核发育群体中,雌性所占百分比为100%,可初步推测匙吻鲟的性别决定机制为XX/XY型。更多还原

【Abstract】 The American paddlefish Polyodon spathula (Walbaum) is a large freshwater fish belonging to the family Polyodontidae of order Acipenseriformes. The paddlefish of the Mississippi River basin and its tributaries and adjacent Gulf Slope drainagesand and the Chinese paddlefish Psephurus gladius (Martens) of the Yangtze River are the only two species in the family Polyodontidae. The American paddlefish has characteristics such as fast growth, high quality, wide adaptation, etc. which has become increasingly important as a high-value species in aquaculture. Chinese sturgeon aquaculture companies introduced the fertilized eggs of paddlefish and successfully culture in the 1990s. At present, the species has become more and more popular and famous.Paddlefish is an important cultured freshwater fish as well as a promising model fish for the study of conservation of endangered sturgeons and paddlefishes species. In the present study, a protocol for artificial gynogenesis of paddlefish was developed in order to produce a pure line and to obtain a technique routing of conservation of endangered the Chinese paddlefish. On the other hand, the paddlefish aquaculture industry has appeared in China. Therefore, it is necessary to breed new stains that have a good economic characteristic which is grow fast, strong disease tolerance and so on. Artificial induction of gynogenesis is an effective method of constructing homozygote quickly, which is used to select choiceness breed.Artificial induction of gynogenesis is one of the main methods of genome engineering, which can help to solve problems of fish genetics and selection. In this study, we report the methods of inducting meiotic gynogenesis in paddlefish using Amur sturgeon (Acipenser schrenckii) sperm. And apply four methods (the morphometric and chromosome counting analysis, flow cytometry and microsatellite analysis) to confirm the success of the offspring of the gynogenesis. At the same time, electron microscopies (Scanning electron micrograph and Transmission electron micrograph) were used to investigate the changes in morphology and ultrastructure of UV-irradiated Amur sturgeon sperm. And comet assay was also used to detect DNA integrity of Amur sturgeon sperm following UV irradiation. Cytological studies on artificially gynogenetic paddlefish and the sex determinination mechanism in paddlefish were discussed.The following results were received:1. Paddlefish eggs were fertilized with normal Amur sturgeon sperm, the embryo were haploid (n=60) which were identified by the chromosome counting analysis. Based on this result, apply hybridization-heatshock to induce gynogenesis in paddlefish was made. The optimal iniation time for heat shock of gynogenetic eggs was determined, the optimal temperature and treatment time were determined. The gynogenetic diploid were obtained by eggs fertilized with normal Amur sturgeon sperm then heatshocked at 37℃for 2 min, at 18 min post-fertilization. With this treatment the fertilization rate and hatching rate was 76.4±9.2%,7.8±1.4%, respectively. On the other hand, we also obtained gynogenesis diploid in paddlefish using ultraviolet (UV)-irradiated Amur sturgeon sperm for 5-min and consequent heat shock treatment, and UV irradiation of sperm for 5 min at a UV intensity of 863μw/cm2 was the optimum dose to achieve diploid gynogenesis on the basis of observations on hatching rate of eggs. Morphologically examination, chromosome testing, flow cytometry, andmicrosatellite DNA analysis were involved in the identification of offspring of gynogenes and confirmed that all offspring were gynogenetic diploid paddlefish. Thus both distant hybridization-heatshock and irradiatation-heatshock were effective methods at producing gynogenetic paddlefish.2. In the present study, the effect of UV irradiation on sperm morphology and ultrastructure was examined by scanning electron microscopy (SEM) and transmission electron micrograph (TEM). The result shows that the mature spermatozoa of Amur sturgeon consistes of an elongated head with a distinct acrosome and nudeus region, a midpiece and a flagellum. The mean length of the head and midpiece, the flagellum and total length of spermatozoon were 8.95,36.80 and 45.75μm, respectively. The acrosome contains posterolateral projection, subacrosome and granular material and actin filament found within the anterior acrosome and etc. The flagellum was thin and long with a typical "9+2" microtubular structure. Scanning electron microscopy(SEM) observation showed that after iradiated by UV, the acrosome membranes of some of sperm were cracked,and acrosome substances were released to form acrosome filament. Transmission electron micrograph (TEM) observation showed that after iradiated by UV, the plasma membrane swelled and broke off from the nuclear membrane; the acrosome disrupted and its contents lost; some mitochondria lost external membrane, accompanied by the disaggregation of mitochondria or the deformation of mitochondria cristae; swelling of flagellum membrane or loss of flagellum. UV irradiation damage on plasma membrane, acrosome, mitochondria and flagellum probably caused the decrease in motility and fertility of the UV-irradiated sperm. It was estimated that UV irradiation on plasma membrane, acrosome, mitochondria and flagellum probably caused the decrease in motility and fertility of the UV-irradiated sperm.In this paper, detection of DNA damage in response to a UV irradiation process in Amur sturgeon spermatozoa was carried out. The results demonstrated that there were no significant differences The results demonstrated that there were no significant differences(P>0.05) between sperm by UV-irradiated 1,3min and fresh sperm. However, a significant drop in sperm motility and fertilization rate was observed in sperm by UV irradiated 5,7,9 min. The comet rate and damage coefficient of UV irradiated sperm with 1,3 min was similar to fresh sperm, but UV irradiated 5,7,9min were significantly differed to fresh sperm. In fact, there was a positive correlation between comet rate of UV irradiated sperm and intensity and time of UV irradiation in protocol. The majority of sperm with DNA damage within the nucleus were slightly and mildly damaged, while minorities were heavily damaged. Few were totally damaged, and only occurred under the conditions of 7min and 9min UV irradiaton. Our analysis suggests that high intensity of UV irradiation is the main factor that causes the DNA damage in UV irradiated sperm nucleus.3. This paper reports the cytological process on Amur sturgeon sperm induced gynogenesis in paddlefish. It was observed that after being inseminated, Amur sturgeon sperm can enter the egg, spermatozoon nucleus beside female pronucleus was condensed and cannot fuse with female pronucleus.At the same time, whe fry growth over 1 year, the sex of the fry from gynogenesis diploid group and normal paddlefish group were identified. We successfully checked the sex ratios in 3 families from gynogenesis diploid group and 2 families from normal fish. The results showed the male to female ratios in the two normal fish group were not significantly different from theoretical 1:1 ratio (P>0.05), while there were all female in gynogenesis diploid group. Based on this result, We suggest these sex determination mechanism in paddlefish is XX/XY type.更多还原