【作者】 刘文彬；

【导师】 张轩杰；

【作者基本信息】 湖南师范大学 ， 动物学， 2004， 硕士

【摘要】 本文以洞庭湖区的黄颡鱼(Pelteobagrus fulvidraco)为研究对象，综合运用组织切片和超薄切片技术、染色体制片技术、同工酶和PCR(Ploymerase Chain Reaction)技术等对黄颡鱼的生殖生理及一些生物学特性进行了研究和分析，所得结果与结论如下： 1．采用石蜡切片技术对黄颡鱼卵巢进行了观察和研究，实验结果证明，黄颡鱼卵子发生按其组织学特点，可分为6个时相，卵巢依其形态结构特征，可分为6期。30-60日龄的鱼处于Ⅰ期卵巢，且终身只出现一次。60-270日龄的鱼发育至第Ⅱ期，Ⅱ时相卵母细胞出现明显的卵黄核。270-300日龄的鱼发育至Ⅲ期，Ⅲ时相卵母细胞未出现皮质液泡。310日龄鱼进入Ⅳ期卵巢，Ⅳ时相卵母细胞的受精孔和精孔细胞明显。330日龄后发育至性成熟，即Ⅴ期，产后卵巢处于Ⅵ期。成熟卵巢的成熟系数达10.9％-26.5％。最小性成熟年龄为1龄，繁殖季节为每年的5月至7月，属一次产卵类型。 2．用光镜和电镜技术观察研究了黄颡鱼的精巢发育和周年变化及精子的发生和形成，结果显示，黄颡鱼精巢发育按其组织学特点和形态学特征，可分为6期。30-60日龄的鱼精巢发育处于Ⅰ期，60-90日龄的鱼精巢发育处于Ⅱ期，90日龄的鱼精巢发育至Ⅲ期，150-200日龄左右的鱼精巢发育至Ⅳ期，300日龄以上的鱼精巢发育至Ⅴ期，产后精巢处于Ⅵ期。成熟系数可达0.3％-0.65％，最小性成熟年龄为1龄，繁殖季节为每年的5月至7月，且其繁殖周期中出现两次波峰。精子的发生经历精原细胞、精母细胞、精子细胞及成熟的精子等阶段，精子无顶体，核凹窝和线粒体发达，精子尾部细长，两侧有明显的侧鳍，轴丝横切面为典型的“9+2”结构。 3．利用组织切片和超薄切片技术对性成熟雌黄颡鱼的脑垂体进行了组织学和超微结构的研究。黄颡鱼的脑垂体由垂体神经部和垂体腺部组成，垂体腺部又由前叶、间叶和后叶三部分构成。含有嗜酸性和嗜碱性细胞，促肾上腺皮质激素细胞和催乳激素细胞组成腺垂体前叶，促性腺激素分泌细胞、促生长激素分泌细胞和促甲状腺激素分泌细胞组成腺垂体间叶，腺垂体后叶由促黑色素激素细胞构成。 4．采用PCR技术，参照人SRY(Sex Determining Region Y gene)基因及其它动物HMG一box(High Mobility Group，H鹏)保守区的序列，设计l对兼并引物，扩增和克隆了黄颗鱼的Sox基因(PFSox)。结果在雌雄个体中筛选出了5个不同的Sox基因，长度为220bp左右，无性别特异性。经DNA序列分析显示，Sox基因在系统进化上十分保守，其侧A序列与人、鼠、鸡和斑马鱼相应的Sox基因序列的相似性均在85%以上，其编码的氨基酸序列与人、鼠、鸡和斑马鱼相应的氨基酸序列的相似性均在94%以上，而且呈现这样一种现象，与之发育相近的鱼类的斑马鱼、两栖类的爪蟾及爬行类的鳄鱼的同源性要低些，与之发育较远的鸡、鼠及人的同源性反而高些，这在进化上是一个比较特殊的现象，这种异常的现象，与其雌雄外观上两性的差异是否有直接的联系，还需进一步的研究，当然，它与其它动物序列上的高度相似性可能说明了它们在功能上的保守性。 5.采取活体肾细胞染色体直接制片法，获得黄颗鱼体细胞染色体标本，并对其染色体众数值及组型进行了初步的研究，确定其染色体数目为ZN二52，染色体组型公式为:ZN二24m+1 4Sm+1 OSt十4t，NF二90。与黄颗鱼〔P.fu了vz’d1刁c口(Rz’ch。厂击on)〕为同一种属。 6.采用聚丙烯酞胺凝胶电泳(PAGE)及特异性组织化学染色技术，研究了成体黄颗鱼的眼、心脏、肾脏、肝脏、肌肉及尾鳍等6种组织中的4种同工酶(LDH、MDH、SOD、EST)分化表达模式。结果表明，黄颧鱼的同工酶EST、SOD和MDH具有明显的组织特异性，LDH无明显的组织差异，并对同工酶的组织表达特异性的生理意义进行分析和讨论。更多还原

【Abstract】 In this paper, Reproduction Physiology and some Biological Characteristics of Pelteobagrus fulvidraco of Dongting Lake in Hunan Province were studied by the method of histological section and ultramicrotomy, chromosomes preparation, PAGE ( polyacrylamide gradient gel electrophoresis ) and PCR (Ploymerase Chain Reaction). The main results and conclusions were as follows:1 . With the method of the histological section, the morphology, histology and annual cyclical changes of ovary in Pelteobagrus fulvidraco were observed. According to the morphological and histological features, the development of ovary can be devided into six stages and the change of the oocytes of the female can be devided into six phases. The ovaries are in stage I when the fishes are 30 to 60 days old and only appears once in their life. When they are 60-270 days old, the ovaries began to grow to stage II and the yolk nucleus are very distinct. When they are 270-300 days old, the ovaries enter into stage III, the unilayer vacuoles can not be seen in the stage III. The ovaries grow to stage IV when they are 310 days old, the micropyle and the micropylar cell are very clear. After 330 days old, the fishes reach to sexual maturation. The mature coefficent of ovaries is 10. 9%-26. 5%. The maturation age of female is 1 year old. The breeding season of Pelteobagrus fulvidraco is from May to July. The results of this paper indicate that this fish spawns only once a year.2 . The development and annual change of testis, spermatogenesis and spermatoleosis in Pelteobagrus fulvidraco were studied with the method of histological section and ultramicrotomy. According to the morphological and histological features, the development of testis can be devided into six stages. The testis is stage I in 30 days to 60 days oldand stage II in 60 days to 90 days old and stage III in 90 days old and it grows to stage IV in 150 days to 200 days old. After 300 days old, the fishes reach to sexual maturation. The mature coefficient of testis is 0. 3%-0. 65%. The maturation age of male is 1 year old. The breeding season of the fishes is from May to July. The results of this paper indicate that the male appears for twice. The spermatogenesis and spermatoleosis are from spermatogonia, through the following stages including spermatogonium , spermatocytes, spermatids and spermatozoa. The spermatozoa have no acrosome. The tail is long, mitochondria and lateral fins are very developed. The f lagellum shows a typical microtube organization "9+2" in the view of cross-section.3. The histological and ultrasstructure of pituity gland from sexually matured female Pelteobagrus fulvidraco were studied. The pituitary gland consisted of neurohypophysis and adenohypophysis. The adenohypophysis was devided into anterior lobe, intermediate lobe and posterior lobe. There aretwo types of cells including the acidophile and the basophile. Prolactin cell (PROL) and adrenocorticotopic hormone cell (ACTH) exist in the anterior lobe, the gonadotropic hormone cell (GTH), growth hormone cell (GH) and thyrotropic hormone cell (TSH) exist in the intermediate lobe, the posterior lobe cosists of melanotropic stimulating hormone cell (MSH).4. The Sox genes of Pelteobagrus fulvidraco were amplified and cloned using Ploymerase Chain Reaction(PCR) with primers designed according to the known SRY or Sox sequence from the Human genomic DNA and other animal genomic DNAs. We selected five different fragment of Sox gene HMG-box DNA and sequenced, the length of sequences is 220bp or so. The results indicated that Sox gene was conservative in evolution, in which the nucleotide sequences have about 85% or above 85% identical toother animals ( Human, Mus, Chicken and Zebrafish), the amino acid sequences encoded by PFSox have about 94% or above 94% identical to those encoded by other animals (Human, Mus , Chicken and Zebrafish). The nucleotide and amino acid sequences identities of Sox gene fragments of Zebrafish or Alligator from Genebank were compared with the Sox gene fragments of Pelteobagrus更多还原