【作者】 何建平；

【导师】 奚耕思；

【作者基本信息】 陕西师范大学 ， 动物学， 2002， 博士

【摘要】 黄胫小车蝗(Oedaleus infernalis Saussure)隶属直翅目(Orthoptera)、蝗总科(Acridoidae)、斑翅蝗科(Oedipodidae)，是我国草地的主要害虫。近几年蝗灾在我国许多地区又不断爆发，为避免化学防治造成的环境污染，寻求防治蝗虫的新途径，以黄胫小车蝗为材料，应用细胞学、形态解剖学、组织化学、免疫组织化学等方法，对其卵子发生及受精囊的显微、超微结构，卵子发生的分期，卵子发生过程中核酸、糖复合物、蛋白质、脂肪等物质的动态变化及卵子发生的调控机制等进行研究，阐明黄胫小车蝗卵子发生、发育机制，以充实蝗虫生殖生物学理论，并为其生物防治提供理论依据。研究主要结果如下： 1．通过对黄胫小车蝗卵子发生的显微和超微结构的研究，将黄胫小车蝗卵子发生过程划分为3个时期10个阶段。卵黄发生前期、卵黄发生期和卵壳形成期。 黄胫小车蝗卵黄形成是自体合成与异体合成同时存在。首次在昆虫中发现线粒体参与卵黄形成。首次在直翅目昆虫卵子发生过程发现卵母细胞凋亡，这种凋亡是对其生活环境的适应对策。 在卵子发生的过程中，滤泡细胞包围在卵母细胞周围，为其发育提供一个小环境，不仅为卵母细胞分泌卵黄膜及卵壳，并参与卵母细胞的发育调控。 2．黄胫小车蝗卵子发生过程中，核酸呈现动态变化。卵原区卵母细胞核呈现DNA阳性反应，进入生长区后，阳性反应立即减弱至阴性，直到卵子成熟。胞质中RAN在卵原区为弱阳性，进入生长区后，RNA为强阳性。卵黄发生期，RNA阳性反应减弱。滤泡细胞在卵子发生的整个过程中呈现DNA和RNA的阳性反应，并在卵黄发生期，胞质中的RNA阳性反应增强。核酸的这种动态变化说明，卵母细胞在减数第一次分裂前期的双线期停滞，进入静止期，而RNA的活动非常活跃，使发育初期卵母细胞积累大量的RNA和蛋白质，这些RNA和蛋白可能形成母体mRNA和母体蛋白质，成为生殖质的成分，参与产生未来的原始生殖细胞。滤泡细胞在卵子发生过程中的核酸的活动，一方面形成蛋白质提供给发育中卵母细胞，更重要的是调节卵母细胞的发育。 由蛋白质、脂类和糖的变化说明，黄胫小车蝗卵母细胞沉积的卵黄蛋白是糖脂复合蛋白质，蛋白质来源有两种，即内源性的蛋白质和外源性的蛋白质。这些蛋白质，一部分作为胚胎发育的储备，一部分参与形成生殖质。滤泡细胞不仅在卵子发生后期形成卵黄膜和卵壳，而且在卵子发生过程中也向卵母细胞提供蛋白质。 3．用4种不同凝集素，对黄胶小车蝗卵子发生过程中糖复合物的分布和变化 进行研究表明，在卵黄发生前期，卵母细胞的胞质中，有少量的糖复合物，其糖 基有甘露糖、葡萄糖和N－乙铣胺葡萄糖；卵母细胞膜上为甘露糖、葡萄糖及N－乙 酞胺葡萄糖；滤泡细胞膜上有甘露糖和葡萄糖．在卵黄发生期，卵黄蛋白表面上 有甘露糖、少量葡萄糖和半乳糖糖基；卵母细胞后极有甘露糖及N－乙酞胺葡萄糖 复合物，此时，卵母细胞表面上N－乙酞胺葡萄糖消失，出现半乳糖糖基复合物． 滤泡细胞中糖复合物糖基有甘露糖、葡萄糖和半乳糖颗粒。成熟卵子卵黄膜上有 甘露糖、半乳糖及葡萄糖．黄胜小车黄卵子发生过程中，卵母细胞膜表面上的糖 复合物经过修饰，修饰与滤泡细胞的相互作用有关。而滤泡细胞中糖复合物成分 与卵母细胞的成熟有关。 4．首次用免疫组化方法对黄胜小车蝗卵子发生中原癌基因 c－myc和。－k i t蛋 白表达进行研究，结果表明，这两种原癌基因蛋白在黄胜小车蝗卵子发生中的表 达方式不同。卜k i t蛋白在卵黄发生后期的卵母细胞膜表面开始表达；c－myc蛋白o 在卵母细胞胞质中和滤泡细胞核中均表达。c寸it蛋白的表达与卵母细胞的受精 及减数分裂的再启动相关；而c－myc蛋白在卵母细胞中可能作为母体蛋白参与原 始生殖细胞的产生，而滤沧细胞中的表达则是调节滤泡细胞的生长，并通过调节 滤泡细胞进而调节卵母细胞的发育。 5．通过组织学、组织化学、亲和组织化学及免疫组化等方法研究表明，黄胜 小车蝗受精囊的组织结构由内而外依次为：表皮层、上皮层、基膜、结缔组织、 肌肉层和围脏层等．在上皮细胞边缘与表皮层接触处有大量的微缄毛，而腺细胞 基部亦有大量的指状突起，充分满足大量物质通过的需求，再加上上皮细胞中的 导管，使蛋白质、各种信号分子和因子等物质能够顺利地从血淋巴、上皮细胞或 分泌细胞及受精囊腔中通过，来调节受精囊的活动。各层细胞中含有大量碳水化 合物、蛋白质和少量的脂肪，为精子的存活、运动及改变提供营养和能量，并保 持精子活性。受精囊中糖复合物的糖链主要有半乳糖、甘露糖、a－葡萄糖及N－ 乙酞胺葡萄糖，这些糖复合物对精子的获能和顶体反应起作用。 首次对受精囊中原癌基因的表达做了研究，发现受精囊在受精后，出现原癌更多还原

【Abstract】 Oedaleus infernalis Saussure (Orthoptera: Acridoidae) is one of many pest insects in our country. In recent years, plagues of grasshoppers have broken out on many regions. In order to avoid environment pollution from insecticide. Some effective methods about control the pest expect to be produced. At present, Oogenesis and spermatheca of O. infernalis have been investigated by histology, histochemistry, lectin histochemistry and immunohistochemistry methods on the level of microstructure, ultrastructure, which may offer useful materials for Biology of Reproduction and Biology Harness. The main results describe as follows:1. Three stages can be covered by the oogenesis of O. infernalis in morphology aspect, i.e. previtellogenic stage one, vitellogenic stage two and Chorionation stage three. Vitellogenesis of O. infernalis is autosynthetic and heterosynthetic process. Mitochondrias adding the procedure of vitellogenesis, Germ-cell apoptosis occurred in the oogenesis of O. infernalis, this programmed cell death (PCD) is an adaptation strategy to its habitat.During the oogenesis of O. infernalis, the follicle cells form a monolayer epithelial cells surround the oocyte and provide a microenvironment for the oocyte growth. Not only the follicle cells secrete the vitelline membrane and the chorion, but also regulate the growth of the oocyte.2. In the procedure of oogenesis of O. infernalis, the quantity of nucleic acid changed actively. In germarium, the nucleus of the oocyte shows a Feulgen positive mark and in the vitellarium, the Feulgen positive mark begins to decrease with the oocyte growth and finaly appeared to negative. The cytoplasm of the oocyte exhibits weak UNA positive in germarium and the RNA show strong RNA positive in vitellarium. During the vitellogenesis RNA positive decreases. The follicle cells possess DNA and RNA positive during the oogenesis of O. infernalis, RNA positive increase in the cytoplasm during the vitellogenesis. This active change shows that the oocyte halts in prophase of meiosis I and becomes silent. But the transcription of RNA is active, and a phenomenon-concerned accumulation of RNA and protein may be observed during theininitial stage of the oocyte development. These RNA and the protein involved in the germ plasm and form primordial germ cells (PGCs). The active changes of the nucleic acid jn the follicle cell show that the follicle cells produce the protein to developing the oocyte, meanwhile adjust development of the oocyte.The variations of the protein, lipid and carbohydrate indicated that yolk granules is gly-lipid-protein complex. There are two kinds of proteins, including endogenous and exogenous proteins. A part of these proteins to store up for the embryogenesis, the others may take part in the germplasm component. The carbohydrate provides energy for embryogenesis.3. 4 lectins binding glycoconjugates on the oocyte of O. infernalis were detected with lectin histochemical for better understanding of distribution of glycoconjugates during the oogenesis. In previtellogenic stage, the ooplasm contains litter glycoconjugates, which is GlcNAc, Man, and Glc; the oocyte surface possesses Man, Glc and GlcNAc. The follicle cells membrane own Man and Glc. In vitellogenesis, the surface of the yolk granules hold Man, Glc and Gal, there are Man and GlcNAc in posterior pole, meanwhile GlcNAc is replaced by Gal. On the mature oocyte surface, there are Man, Gal and Glc. The change of the distribution shows that the glycoconjugate on the oocyte surface was modificated and this modification is related with the follicle cells interaction. The glycoconjugate in follicle cell is involved in the oocyte mature.4. Protooncogene c-kit and c-myc protein expression during the oogenesis of O. infernalis were firstly studied by means of immunohistochemistry in our experiment. The patterns of those two-protooncogene expressions are different. At the late vitellogenesis, the c-kit protein initiates expression on the o更多还原