【作者】 颜桂军；

【导师】 左嘉客； 申庆祥； 徐万祥；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 输卵管蛋白(Oviduct-specific glycoprotein，Oviductin)是哺乳动物输卵管粘膜上皮细胞特异性表达分泌的高糖基化蛋白质，普遍存在于各种哺乳动物的输卵管分泌物中，为配子的成熟、转运、受精和早胚的发育营造了一种适宜的微环境，但具体的作用机制尚不清楚。本实验室从兔输卵管粘膜上皮细胞的cNDA文库中筛选出兔的全长Oviductin cDNA(1.9kb)，其演绎分子量为64kDa，命名为早胚发育促进因子-1(Development Promocing Factor-1，DPF-1)。DPF-1与其它物种的输卵管糖蛋白具高度同源性。为了研究其在早胚发育过程中潜在的生物学功能，进行了下列实验： 建立了离体未完全分化颗粒细胞、动情前期垂体前叶和Δ-4雄烯二酮组成的“联合培养系统”。该联合培养系统可模拟正常生理状态下卵泡中各相关细胞间的协同作用，更有效地诱导经己烯雌酚刺激后大量源自大鼠早期三级卵泡中卵母细胞同步成熟，并能正常受精和发育。经DES刺激，每侧幼龄大鼠卵巢中平均可取得189枚同步发育的卵母细胞，经在联合培养系统中培养，77.6％的卵母细胞排出第一极体达到成熟。这些成熟的卵母细胞中87.8％可正常受精，93％卵裂；经体外培养96h，有59％的受精卵可发育到桑椹或囊胚。2-细胞期胚胎移植后可发育成为健康个体。为研究大鼠卵成熟、受精和早胚发育的分子机制所需的大量实验模型提供了物质基础。 为了从分子水平研究DPF-1在早期胚胎发育中的作用机制，建立了大鼠成熟卵母细胞pMyr cDNA文库，文库容量为8×10~5pfu／ug cDNA。pSos／1.9kb DPF-1cDNA诱饵蛋白质粒对大鼠成熟卵母细胞pMyr cDNA文库进行了双杂交筛选，从大鼠成熟卵母细胞pMyr cDNA文库中初筛出132个阳性克隆，终筛出1个阳性克隆。经测序提示插入片段约为400bp核苷酸，核苷酸序列同源性分析比较，发现与activating signal cointegrator 1 complex subunit 2(ASC-1 complex subunit p100)蛋白核苷酸序列有93％一致性。p100蛋白在多种组织中表达，特别在卵巢组织和移植前胚胎中广泛表达，并与p50、p200和ASC-1共同组成了ASC-1复合体，来调节SRF(serum response factor)、AP-1(activating protein 1)和NF-B(nuclear factor B)等转录因子的活性。对p100蛋白与DPF-1相互作用关系进行研究可望为进一步深入分析输卵管蛋白的作用机制提供线索。更多还原

【Abstract】 The nonciliated epithelial cells of the oviduct actively synthesize and secrete a high-molecular-weight family of glycoproteins termed oviductins. Due to oviductins bind to the zona pellucida , cell- membranes of gametes and early embryos , it was suggested that oviductins has a potential biological role during the transport of gametes, fertilization and early embryonic development. A rabbit oviductin, here we termed Development Promoting Factor-1(DPF-1), has cloned from cDNA library derived from rabbit oviduct mucosal epithelial cells. DPF-1 appears to be highly conserved, and shares a high degree of identity and similarity in the N-terminal region of oviductins. The results obtained from "loss of function" experiments, indication that the binding site to recognize its target protein is localized in the c-terminal region of DPF-1. Since we decide to identify its target protein/proteins by using yeast two hybrid system. In the same time, we decided to establish a system that can provide mature oocytes in mass to fulfil the need for the further establishment of the target cDNA library.A "combination culture system" , containing primary differentiated granulosa cells, proestrus anterior pituitary lobe and Δ-4-androstenedione, can act in a synergistic manner to effectively induce the maturation, fertilization and development of oocytes from early tertiary. follicles of diethylstilbestrol (DES)-treated rats. This combination culture system provides an experimental model in which the molecular mechanisms of egg maturation, fertilization, and embryonic development can be studied. After DES stimulation, an average of 189 oocytes were obtained from early tertiary follicles of unilateral ovaries; 77.6% of these oocytes, when cultured in our combination culture system, were shown to extrude a first polar body, a criterion of oocyte maturation. Of the mature oocytes, the rate of normal fertilization and egg cleavage were 87.8% and 93%, respectively. After 96 h of culturing in vitro, 59% of the zygotes developed into morulae or blastocysts. Embryos implanted at the two-cell stage were able to develop into healthy individuals.To identify proteins that interact with DPF-1, 1.9kb DPF-1 cDNA was fused to pSos plasmid. And the target cDNA library, rat oocyte pMyr cDNA plasmid library, was constructed. 1 putative positive clone derived from the yeast two hybrid screening was isolated, and its nucleic acid sequence analysis showed that it shared 93% identity with activating signal cointegrator 1 complex subunit 2 (ASC-1 complex subunit p100). Since p100 was expressed in many tissues, especially in ovary and pre-implantation embryos, and ASC-1 complex, including the interactions of ASC-1 with p100, p200 and p50, which is essential for SRF(serum response factor)、 AP-1 (activating protein 1) and NF-B(nuclear factor B) transactivation. So identification of p100 as a novel target for DPF-1, will bring us some supposes to study the mechanism of DPF-1 in regulation of early embryo development.更多还原