【作者】 吴莹莹；

【导师】 王清印； 柳学周； 包振民；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2008， 博士

【摘要】 运用组织切片及电子显微镜技术对半滑舌鳎(Cynoglossus semilaevis Günther,1873)性腺分化过程、精巢结构、精子发生过程和成熟精子的超微结构及半滑舌鳎和大菱鲆(Scophthalmus maximus)的受精过程进行了研究。在卵黄囊期,中肾管附近的肠管边缘处,即可发现半滑舌鳎的原始生殖细胞(PGCs);5日龄,PGCs沿腹腔后端向生殖嵴方向移动;10日龄,在肾管下方和肠管之间的体腔膜基部可观察到刚发育形成的生殖嵴;15～26日龄,可以清晰的看到左右两个生殖嵴伸入腹腔;36日龄生殖腺原基中PGCs数目明显增多,已经从体壁游离出来,进入腹腔后部。此时的生殖嵴称为原始性腺。经历一个较长的原始性腺期侯,进入性腺分化阶段。62日龄,半滑舌鳎原始性腺开始快速有丝分裂形成成簇卵原细胞;100日龄,开始形成卵巢腔;190日龄,卵巢腔完全形成,卵巢分化完成。半滑舌鳎精巢分化晚于卵巢分化。80日龄,精原细胞开始快速有丝分裂,性腺开始增大;100日龄,开始出现精小管原基,精巢分化开始;150日龄,观察到精小叶,精小管等结构,同时出现精母细胞,精巢完全分化;190日龄,精巢中精小叶,精小叶腔,精小管,精原细胞,精母细胞等结构明显,进入成熟发育期。精巢属于小叶型,由精小叶、小叶间质及输出管构成。每个小叶由数个精小囊组成。精小叶由精原细胞、初级精母细胞、次级精母细胞、精细胞及成熟精子等生殖细胞和支持细胞构成。对各级生精细胞的结构和形态特征做了详细的描述。半滑舌鳎精子形成过程大致经历了鞭毛发生、核质凝缩、中心粒结构及位置变化、线粒体迁移及多余细胞质外排等过程。经历精子形成过程,精细胞最终发育为成熟的精子。半滑舌鳎精子由头部、中片和尾部三部分组成,为无顶体类精子。细胞核马蹄状,核隐窝深深地陷入其中。核隐窝最里端排列着由近端中心粒和基体组成的中心粒复合体。紧贴细胞核下端排列大约有5～6个线粒体。线粒体和鞭毛之间为狭短的袖套腔。尾部细长,从袖套腔中伸出。尾部的主要结构为轴丝,其起始端与基体后端相连接。鞭毛长约(43.2±1.67)μm,有侧鳍,中心轴丝结构为典型的“9+2”结构,轴丝外侧有些部位还有囊泡状结构分布。半滑舌鳎成熟卵为圆球形,卵表布满纵横交错的网纹和分布均匀的微小孔。动物极有“火山口”状的受精孔区。授精后,半滑舌鳎精子入卵速度非常快,授精后2s即可在少数受精孔处发现有精子进入。授精后1min,没有精子进入的受精孔仍然敞开着,未观察到结构有明显变化。精子入卵后,精孔管管壁边缘由锯齿状变为平滑的环状等。通过组织切片,观察到半滑舌鳎成熟卵处于第二次成熟分裂中期。精子入卵后,卵子被激动,第二次成熟分裂继续进行。授精后10min,第二次减数分裂发育到末期,准备向外排出第二极体;与此同时,胚盘内出现精子星光;授精后20min、30min,雄原核和雌原核分别形成。然后两性原核逐渐靠拢,至授精后40min,两原核成为一个合子;授精后50min,合子核处于第一次有丝分裂中期;授精后60min,第一次卵裂完成。对大菱鲆成熟精卵的形态做了详细描述。卵子动物极有受精孔区。受精孔是精子入卵的唯一通道。授精后0-5s,即可以在受精孔处发现精子。精子入卵后,受精孔处会有受精锥和受精塞形成,以防止多精入卵。大菱鲆成熟卵处于第二次成熟分裂的中期。精子入卵后,卵子被激动,第二次成熟分裂继续进行,同时发生皮层反应;授精后15min,出现精子星光;授精后20min,雄原核早于雌原核形成,然后两性原核逐渐靠拢;授精后30min,两原核相互靠拢,结合线清晰;授精后40min,两原核结合线逐渐消失联合成合子核,之后合子核核膜消失;授精后50min,合子核处于第一次有丝分裂中期;授精后60min,第一次卵裂完成。更多还原

【Abstract】 In this paper, gonadal sex differentiation, structure of testis, spermatogenesis, and spermatozoa of Cynoglossus Semilaevis Günther, and fertilization process of C. Semilaevis Günther and Scophthalmus maximus was studied by means of histological staining mehods and electron microscopy.In yolk sac stage, primordial germ cells (PGCs) was located between gut and mesonephric ducts; PGCs was migrating towards gentital ridge in larva 5 days after hatching(DAH); in larva 10 DAH, PGCs arrived at germinal ridge and began to form primordial gonad together; in larva 15~26 DAH, primordial gonad begin to prolongate; in larva 36 DAH, with mitotic multiplication, primordial gonad elongated ventrally, stretching from the ventral end of kidney to the posterior end of abdominal cavity, and had an abundance of connective tissue. After a long period of undifferentiation stage, some of the primordial gonad began to mitotic multiplication and appeared clusters of oogonia in 62 DAH which indicated the ovarian differentiation of semi-tongue sole. Presumptive ovarian cavity began to form at 100 DAH. Ovarian cavity was formed entirely in 190 DAH. Testicular differentiation of semi-tongue sole is posterior to ovarian differentiation. At 80DAH, presumptive testis began to enlarge through mitotic multiplication. Formation of efferent duct and blood vessels occured in 100 DAH, which was anatomical features of testicular differentiation. At 150 DAH, testis began to develop spermatogonial clusters of cysts and form seminal lobule, which was cytological features of testicular differentiation. By 190 DAH, testis developed into maturation stage.Testes of Cynoglossus semilaevis was lobular type, consisted of seminiferous lobular, interlobular septum and spermaduct. Seminiferous lobular consisted of various stages of spermatogenic cells and Sertoli cells. Spermatogenic cells consisted of primary spermatogonia, secondary spermatogonia, primary spermatocytes, secondary spermatocytes and spermatids. Within a spermatogenic cyst, all the spermatogenic cells were at the same developmental stage. Characters of spermatogenic cell in each stage were discribed in detail. Spermiogenesis included formation of a flagellum, condensation of nucleus that will form sperm head, development of middle piece, discarding of residual cytoplasm, formation of sleeve which contained some mitochondria, etc. Mature sperms were released from cysts and transferred into lobular lumen.Spermatozoon of C. semilaevis consisted of three parts: head, midpiece, and tail. Spermatozoon had no acrosome, nucleus of sperm was horse-hoof shaped. Nuclear fossa was deep, and at bottom of it lacated centriolar complex which consisted of proximal centriole and basal body. Five to six mitochondria localized behind nucleus in a circle, forming midpiece of the sperm. Length of tail was about 43±2.4μm (n=5). Structure of axoneme was typical“9+2”pattern. Lateral fins and sacciform structures were found at some parts of tail. Structural characteristics revealed that the sperm of C. semilaevis belonged to primitive type in teleostean fish.Morphology of surface structure and chorion of eggs, site and structure of micropyle, and early process of sperm penetration were studied by electron microscopy. Speed of sperm penetration was vary fast, 2 seconds post insemination, there had been sperm entried egg. Changes of some structure occured during sperm penetration, for the jagged circle in inner wall of the micropyle tunnel turned into slippery circle. Tissue section of fertilized eggs showed that after sperm entering into egg, male pronucleus and female pronucleus was formed one after another. They contacted with each other and formed nucleus of zygote. It could be sure that the pattern of fertiliztion of C. semilaevis is monospermy.Egg and sperm shapes, process of sperm penetration, and structure changes of fertilized egg of Scophthalmus maximus L. were examined by eletron microscopy. And observation results of tissure section showed that mature eggs of S. maximus L. remained at metaphase of second maturation division. The egg was activated as sperm penetrating into the egg, with development of second maturation division and cortex reaction. At 15 minutes after insemination, sperm-aster appeared. At 20minutes after insemination, male pronucleus was formed prior the formation of female pronucleus, and later they got closer to each other. At 30 minutes after insemination, male and female pronuclei combined together and a clear junction line was observed. At 40 minutes after insemination, zygote nucleus formed and later karyotheca became faint. At 50 minutes after insemination, zygote nucleus developed into metaphase of the fist mitosis, and then at 60 minutes, first karyokinetic division was finished. From the results, it was concluded that fertilization type of turbot was monospermism and combine type of male and female pronuclei was conjugation.更多还原