泰山螭霖鱼（Varicorhinus macrolepis）卵巢发育与性激素水平在年周期中变化规律的研究
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摘要
本课题对泰山螭霖鱼(Varicorhinus macrolepis)卵巢的解剖学、组织学、组织化学以及性激素水平在年周期中的变化规律进行了研究，另外对泰山螭霖鱼成熟卵子的超微结构进行了电镜观察。实验一对泰山螭霖鱼的组织学研究结果表明：泰山螭霖鱼卵母细胞在年周期的发育过程中分为6个时相。卵巢发育分期依卵巢切面中所占面积超过50%或居最高比例的卵母细胞的时相决定，分期情况如下：2～4月份，卵巢由Ⅳ期发育至第Ⅴ期，4月份进入繁殖期；4～6月份，为第Ⅴ期；6月下旬～7月份为第Ⅵ期，是排卵后退化阶段；8月份，为Ⅱ期卵巢（休整期）；9月份～翌年的1月份为第Ⅲ期，以Ⅲ期卵巢过冬。根据卵巢发育的解剖学和组织学观察可知，泰山螭霖鱼为短期分批产卵类型，一年产卵一次。实验二利用组织化学的方法就糖原（高碘酸Schiff反应法，PAS）、DNA（Feulgen-Rossenbeek改良反应）、碱性磷酸酶（Gomori改良钙钴法）和酸性磷酸酶（Berry硝酸铅法）四种化学组分在不同时相卵母细胞中的变化情况进行了研究。结果如下：①PAS反应：在各个时相的卵母细胞中，卵膜均呈阳性反应。在Ⅲ、Ⅳ时相中，核周围细胞质、液泡中物质、液泡之间的颗粒呈PAS阳性反应，其它部分呈阴性。②AKP反应：在各期卵巢中的各时相卵母细胞滤泡膜呈强阳性，其它部分均为阴性。③ACP反应：不同时期卵巢中的卵母细胞间隙中的组织呈弱阳性、阳性、强阳性反应。④DNA反应：在所有时相卵母细胞中，滤泡膜上皮细胞胞核均对Feulgen-Rossenbeek改
良法呈阳性反应，被染为紫色；在第Ⅱ时相期的卵母细胞核仁和核质中见到一些呈阳性反应的丝状物，其它部分均呈阴性反应。实验三对泰山螭霖鱼性激素（雌二醇、17α，20β-双羟孕酮）的年周期变化规律进行了研究。E2的变化规律：血清中E2自1月份开始升高，排卵前3月份达到高峰，53.6pg/ml左右，4月份排卵时降为20.22 pg/ml，一直持续到9月份，降至最低点，1.6667pg/ml左右，之后维持在低水平，直到1月份开始上升至3月份；E2自Ⅲ期开始升高，Ⅳ期后期（排卵前）升至高峰，排卵后E2骤然下降。孕酮的变化规律：孕酮在4月份（接近排卵时）达到高峰，56.333pg/ml，然后维持高水平（56.333～50.667 pg/ml）至排卵结束（6月份），11月份～翌年3月份均维持低水平（35～15pg/ml）；孕酮在Ⅳ期后期升高，Ⅴ期升至高峰，高水平维持至Ⅵ期，排卵结束。泰山螭霖鱼的成熟诱导激素：17α，20β-DHP作为泰山螭霖鱼的MIH对卵母细胞的成熟起着重要的作用，17β-E2对泰山螭霖鱼卵母细胞最后成熟没有作用，不作为MIH发挥作用。实验四对泰山螭霖鱼成熟卵子的超微结构进行了研究，说明卵子的大小与鱼的个体无关，而与鱼的发育方式有关；成熟卵子内已有基本完整的细胞器结构。
Abstract： Anatomy, histology, histochemistry, and the electron microscope observation of the matural oocytes of Varicorhinus macrolepis were studied, as well as the changes of sexual steroid in annual reproductive cycle. EXP.1:Ovary histology of Varicorhinus macrolepis was studied. The results showed as follows: Oocytes were divided into 6 phases in ovary development, and Different annual period of ovary was classified based on different phase oocytes existed different proportion in ovary(the ovary was corresponding period of the phase oocyte which the 50% that should exceed the section of field of vision appearance), Ovary of Varicorhinus macrolepis was classified into 5 stages in annual reproductive cycle, which was as follows: (Ⅰ) February - April, it was Ⅳ period. (Ⅱ) April - June : it was Ⅴ period, but it would change with different temperature. (Ⅲ) June -July: entering Ⅵ period after ovlation finishing. (Ⅳ) August: it was Ⅱ period, then resumed promptly into stage Ⅲ, storing energy in order to live through the winter. (Ⅴ) September-January of next year, ovary was in Ⅲ period and spent cold weather in winter. EXP.2 : changes of Gly, DNA 、AKP and ACP in different phase oocytes were studied by histochemistry. Changes of Gly were as follows: membrane was positive to PAS in every Phase.Ⅲ and Ⅳ Phase, The material around nucleus in cytoplasm and in liquid bubble were positive to PAS,as well as granules among liquid bubbles, While other part was negative.
The results of DNA showed that there were negative except membrane to Feulgen- Rossenbeek reaction in every phase oocytes. The amount of AKP changed that it was positive of the epithelium cell in each phase oocyte, while other part was negative. ACP changed that there were shallow black, black and deep black granules in oocyte cell gap, and the granules form of gathering among the oocytes in different stages ovaries. EXP 3: Changes of Sexual steroid (E2 and 17α，20β-DHP) of Varicorhinus macrolepis were studied in annual reproductive cycle. The result showed:E2 went up to peak in March before ovulating (53.6 pg/ml around)，and began to drop to minimum point from April to September and maintained at low level to January then began to rise to March. 17α，20β-DHP rose to the highest point in April , and maintained at high level to the end of ovulating. Then it decreased from July to November and maintained at low level to March of next year. EXP 4: The electron microscope observation of the matural oocytes of Varicorhinus macrolepis was studied.The results showed that the size of oocyte had nothing with the individual of the fish, and was concerning with the growth way of oocyte of the fish; There were basically complete organelle structure in matural oocyte.
良法呈阳性反应，被染为紫色；在第Ⅱ时相期的卵母细胞核仁和核质中见到一些呈阳性反应的丝状物，其它部分均呈阴性反应。实验三对泰山螭霖鱼性激素（雌二醇、17α，20β-双羟孕酮）的年周期变化规律进行了研究。E2的变化规律：血清中E2自1月份开始升高，排卵前3月份达到高峰，53.6pg/ml左右，4月份排卵时降为20.22 pg/ml，一直持续到9月份，降至最低点，1.6667pg/ml左右，之后维持在低水平，直到1月份开始上升至3月份；E2自Ⅲ期开始升高，Ⅳ期后期（排卵前）升至高峰，排卵后E2骤然下降。孕酮的变化规律：孕酮在4月份（接近排卵时）达到高峰，56.333pg/ml，然后维持高水平（56.333～50.667 pg/ml）至排卵结束（6月份），11月份～翌年3月份均维持低水平（35～15pg/ml）；孕酮在Ⅳ期后期升高，Ⅴ期升至高峰，高水平维持至Ⅵ期，排卵结束。泰山螭霖鱼的成熟诱导激素：17α，20β-DHP作为泰山螭霖鱼的MIH对卵母细胞的成熟起着重要的作用，17β-E2对泰山螭霖鱼卵母细胞最后成熟没有作用，不作为MIH发挥作用。实验四对泰山螭霖鱼成熟卵子的超微结构进行了研究，说明卵子的大小与鱼的个体无关，而与鱼的发育方式有关；成熟卵子内已有基本完整的细胞器结构。
Abstract： Anatomy, histology, histochemistry, and the electron microscope observation of the matural oocytes of Varicorhinus macrolepis were studied, as well as the changes of sexual steroid in annual reproductive cycle. EXP.1:Ovary histology of Varicorhinus macrolepis was studied. The results showed as follows: Oocytes were divided into 6 phases in ovary development, and Different annual period of ovary was classified based on different phase oocytes existed different proportion in ovary(the ovary was corresponding period of the phase oocyte which the 50% that should exceed the section of field of vision appearance), Ovary of Varicorhinus macrolepis was classified into 5 stages in annual reproductive cycle, which was as follows: (Ⅰ) February - April, it was Ⅳ period. (Ⅱ) April - June : it was Ⅴ period, but it would change with different temperature. (Ⅲ) June -July: entering Ⅵ period after ovlation finishing. (Ⅳ) August: it was Ⅱ period, then resumed promptly into stage Ⅲ, storing energy in order to live through the winter. (Ⅴ) September-January of next year, ovary was in Ⅲ period and spent cold weather in winter. EXP.2 : changes of Gly, DNA 、AKP and ACP in different phase oocytes were studied by histochemistry. Changes of Gly were as follows: membrane was positive to PAS in every Phase.Ⅲ and Ⅳ Phase, The material around nucleus in cytoplasm and in liquid bubble were positive to PAS,as well as granules among liquid bubbles, While other part was negative.
The results of DNA showed that there were negative except membrane to Feulgen- Rossenbeek reaction in every phase oocytes. The amount of AKP changed that it was positive of the epithelium cell in each phase oocyte, while other part was negative. ACP changed that there were shallow black, black and deep black granules in oocyte cell gap, and the granules form of gathering among the oocytes in different stages ovaries. EXP 3: Changes of Sexual steroid (E2 and 17α，20β-DHP) of Varicorhinus macrolepis were studied in annual reproductive cycle. The result showed:E2 went up to peak in March before ovulating (53.6 pg/ml around)，and began to drop to minimum point from April to September and maintained at low level to January then began to rise to March. 17α，20β-DHP rose to the highest point in April , and maintained at high level to the end of ovulating. Then it decreased from July to November and maintained at low level to March of next year. EXP 4: The electron microscope observation of the matural oocytes of Varicorhinus macrolepis was studied.The results showed that the size of oocyte had nothing with the individual of the fish, and was concerning with the growth way of oocyte of the fish; There were basically complete organelle structure in matural oocyte.
引文
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