【作者】 李勇；

【导师】 张耀光；

【作者基本信息】 西南大学 ， 动物学， 2007， 硕士

【摘要】 2005年3月—2007年3月，从万州水产研究所获取经人工催产，干法受精，池塘水激活后发育的胭脂鱼胚胎，采用组织学、组织化学、扫描电镜和透射电镜技术、细胞化学等方法，对受精卵的组织学结构、超微结构、精子入卵过程、雌雄原核迁移与结合、受精卵变化、受精过程中Ca²⁺和Ca²⁺-ATPase的变化等进行了详细研究。结果表明：卵子表面除精孔器周围直径约100μm的范围以外均分布有丰富的绒毛状结构。精孔器由前庭和精孔管组成。前庭漏斗状，最大直径64.94μm，深78.64μm。精孔管位于精孔器底部，长2.55μm，直径2.40μm。从扫描电镜看精孔器属于深陷型，其前庭区呈现螺旋形结构。精子在受精后3s开始穿过精孔管，受精后10s形成精子星光。受精后35s精孔管底部形成受精锥。受精后40s精孔器内聚集大量精子，精孔管堵塞，精孔器内的精子开始凝集而失去精子的完整形态。受精后80s，精孔管阻塞，呈蜂窝状，存在于精孔器内的精子进一步凝集成团。受精后180s，精孔器内的精子开始解体，之间的界限模糊，仅见团状物聚集在一起。成熟卵子的染色体一直停滞于第二次减数分裂中期，随着精子的进入，动物极开始逐渐形成胚盘。受精后15min，卵子处于第二次减数分裂后期，开始形成第二极体。受精后20min，卵子处于第二次减数分裂末期，第二极体形成。受精后90min，雌雄原核融合。受精后140min，处于第一次有丝分裂中期。受精后180min，第一次有丝分裂结束，二细胞形成。卵子的皮层小泡是卵子在生长发育过程中所积累的代谢废物。根据形态、内含物生化特性及超微结构特点将胭脂鱼皮层小泡分为Ⅰ—Ⅳ种类型；皮层区内皮层小泡源于卵母细胞生长过程中的生理变化；第Ⅳ类皮层小泡由某种特定的嗜碱性的卵黄颗粒降解转化而来，其分别出现在皮层反应后期和卵裂期。皮层反应过程中皮层小泡的释放次序表现出一定的规律性，Ⅰ—Ⅳ类皮层小泡依次释放。释放过程中，有外推、融合的一个过程，在此过程中皮层小泡内含物电子致密度也有一个减弱的过程。根据皮层小泡释放强度，可将皮层反应分为潜伏期、发展期、高潮期、衰退期四个时期。受精后10s在离精孔器352.85μm的区域开始微弱的皮层反应，为皮层反应的起始点；受精后80s-120s是皮层反应的高潮期；受精后10min，第一次皮层反应结束。受精后50min-60min，动物极皮层区内出现皮层小泡在，且发生胞吐作用，进行第二次皮层反应。推测皮层小泡除参与皮层反应外，还为后期的质膜修复和卵裂提供充足的膜系统。受精过程中胭脂鱼卵子的钙离子含量发生波动性变化，这一变化与卵子的皮层反应过程密切相关，皮层小泡的释放是基于卵子内钙离子增加的结果。Ca²⁺-ATPase在精卵结合过程中也有两次活性较高的时期。受精后80s左右，是皮层反应的高潮时期，钙离子浓度此时最高，并且Ca²⁺-ATPase活性也是最高。因此钙离子和Ca²⁺-ATPase在卵子受精过程的一系列动态变化中起相关作用，且与皮层反应相关联。为了对卵子激活机制做出更为合理的解释，我们综合前人研究，同时根据胭脂鱼卵子受精过程中整个卵子钙离子含量的变化，提出了钙离子通道理论假说：外源信号物质（各种离子甚至水）激活卵膜上的受体，通过多种信号转导途径诱导细胞内钙离子释放，引发皮层反应。皮层反应中皮层小泡内含物的释放及吸水致使卵膜膨胀，卵膜的亚结构发生改变，使卵膜上的钙离子通道打开。离子通道的打开使钙离子进入卵内，而表现出含量的增加。钙离子的大量流入，使膜电位发生改变，为维持膜电位的平衡，Ca²⁺-ATPase将钙离子泵出卵外，因而表现出活性的波动，钙离子含量降低，因此Ca²⁺-ATPase活性波动与钙离子含量变化相关；Ca²⁺和Ca²⁺-ATPase的作用最终导致卵子激活。更多还原

【Abstract】 From March 2005 to March 2007, we got the Chinese sucker （Myxocyprinus asiaticus） embryos which were developed by artificial oxytocin, dry fertilization and pond water activation from the Wanzhou Fisheries Institute. Disscussed the histological structure and ultrastructure of the fertilized eggs, the process of sperm going into eggs, the male and female nuclear migration and integration, the change of zygotes, and the variety of the Ca²⁺ and Ca²⁺ during the process of fertilization by the means of histology, histochemistry, scanning electron microscopy, transmission electron microscopy, cell chemistry and so on. The results indicates that: There is rich in villiform structures in addition to the area which is of a diameter about 100μm arounds the micropylar apparatus in the egg surface. The micropylar apparatus is constituted by the atria and the micropylar tube. The atria is funnelform, and the largest diameter is 64.94 urn and in the depth of 78.64μm. The micropylar tube with 2.55μm long and a diameter of 2.40μm is on the bottom of the micropylar apparatus. From the scanning electron microscope the micropylar apparatus is of a type which is up to the chin, and its atria erea is formed in spiral structure. 3s after fertilizition, sperm starts going through the micropylar tube, and it forms sperm stars in 10s after fertilizition. 35s after fertilization, there forms fertilization cone in the bottom of the micropylar apparatus. 40s after fertilization, there gatheres alarge number of sperms in the micropylar apparatus, then the hole is pluged, and the sperms inside the micropylar apparatus begins to get together and lose their integrated morphology. 80s after fertilization, the micropylar tube were blocked up and were in the form of fertilization cone. The sperms inside the micropylar apparatus get more collective to form a agglomerate. 180s after fertilization, the sperms inside the micropylar apparatus start to break up, and the boundery between two sperms gets illegible which only can see Mission membranes together. The chromosomes of mature egg’s have stagnated in the second meiotic metaphase, with the entry of sperm, the Animal pole begins forming the blastoderm. 15min after fertilization, the egg is in the second anaphase, which starts to form the second polar body. 20 minutes after fertilization, the egg is at the end of the second meiosis and the second polar body is formed completely. 90 minutes after fertilization, the male and female nuclears integrates. 140min After fertilization, the egg is in the M phase of the first mitosis. 180 minutes after fertilization, the first mitosis ends, and two cells formed.The egg cortex vesicles is the metabolic waste which is accumulated during the process of growth and deveploment. According to the characteristics of the morphologic, contents, biochemical characteristics and ultrastructural of Chinese sucker, the cortex is devided intoⅠ-Ⅳtypes; cortical area cortex vesicles are derived from the physiological changes of oocytes during their growth process; classⅣcortex vesiclesis transformed by a kind of particular degradated yolk particles which are basophilic. They appears in the late phase of cortex reaction and the cleavage period respectively. The release order of the cortex vesicles in the cortical reaction shows some regularity, which isⅠ-Ⅳcortex vesicles are released in order. There is process of extrapolation and integration in the relaesement, in the process, the electronic density of the containing objects is a weakening process too. According to the release intensity of the cortex vesicles, cortical reaction can be divided into the latent period, development period, climactic period, declining period. 10s after fertilization, there starts weak cortical reaction in the region which is 352.85μm away from the micropylar apparatus, and it’s the starting point of the cortical reaction; 80s-120s after fertilization is the climax of cortex reaction; 10 minutes after fertilization, the cortical reaction ends. 50min-60min after fertilization, there appears cortical vesicle in the animal pole cortex, and which occurred exocytosis, the second cortical reaction begin. Cortical vesicles are speculated not only join in the cortical reaction, but also provide adequate membrane systems for the latter part of the plasma membrane repairation and the egg cleavage.During the process of fertilization, the content of calcium fluctuate. This change is closely relates to the cortical reaction of the egg. The release of the cortical vesicles is based on the increase of the calcium in the egg. There are two phases which Ca²⁺-ATPase is in higher activity in the sperm-egg binding process. 80s after fertilization, it’s the climax of the cortical reaction, the calcium ion concentration maximizes at this time, and the Ca²⁺-ATPase activity is the highest too. So, during the fertilization, the calcium and Ca²⁺-ATPase play a relevant role in the process of a series of dynamic changes, and relates to the cortical reaction. To make more reasonable explanation of the egg activation mechanism, we integrated predecessors’ study, and according to the change of the calcium in the egg during the fertilization process, we propose a calcium channel hypothesis: Exogenous signals （various ions and even water） activate receptor which reside in the egg membrane, and induce intracellular calcium release through various signal transduction pathways, then trigger cortex reaction. During the cortical reaction, the release of the inclusion from vesicles and sop up the water causes swelling and the sub-structure of the egg membrane change, so that the calcium ion channels in the egg membrane openes. The opening of the ion channels causes the calcium ions enter into the egg, and shows the increases of the content. The calcium influx causes the change of the membrane potential. In order to maintain the balance of the membrane potential, Ca²⁺-ATPase pumps the calcium out of eggs, which shows active fluctuations, and then the calcium content reduces. Therefore the Ca²⁺-ATPase active fluctuation is relate to the calcium content related; Ca²⁺ and Ca²⁺-ATPase’s role eventually leads to the egg activation.更多还原