【作者】 慈光新；

【导师】 张才乔；

【作者基本信息】 浙江大学 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 家禽卵巢以其卵泡等级发育的特点而成为研究卵巢生物学和卵泡发育的理想模型,而卵泡颗粒细胞因其独特的结构特点及其在卵泡发育中的重要作用,常被看作卵泡发育的一个重要标志。本实验通过小黄卵泡颗粒细胞以及全卵泡体外培养模型,探讨了家禽生产中部分重要的生殖激素、生长因子及与卵泡发育相关的抗氧化性生物活性物质对卵泡粒细胞生长发育和功能的影响及其作用机理,以期为家禽优势卵泡选择及繁殖性能的提高提供理论依据。1.鸡等级前卵泡细胞的形态学变化-卵泡刺激素和前列腺素对等级前卵泡发育的影响本文首先利用形态学方法研究了鸡等级前卵泡中颗粒细胞和膜细胞在发育过程中形态学的变化。从有规则产蛋周期的三黄母鸡中取出等级前卵泡,按照其直径分为小白卵泡(SWF,1～3mm)、大白卵泡(LWF,3～5mm)、小黄卵泡(SYF,5～6 mm)和大黄卵泡(LYF,6-9 mm)。各级卵泡用4%多聚甲醛固定24 h,然后包埋并切片,厚度5μm。切片用苏木精-伊红染色或用增殖细胞核抗原(PCNA)免疫组织化学染色。等级前卵泡组织学研究结果表明：SWF只有一层颗粒细胞,LWF中出现两层颗粒细胞,而SYF和LYF中则有多层颗粒细胞。数据统计显示,卵泡膜层和颗粒细胞层厚度随着卵泡直径的变化而显著增加。SYF和LYF的膜细胞层的厚度是最大的(P<0.05)。颗粒细胞的密度度随着卵泡直径的变化而显著增加(P<0.05)。与LWF、SYF和LYF相比,SWF的颗粒细胞的面积较小(P<0.05)。PCNA免疫组化染色表明：和LYF相比,PCNA标记指数(PCNA-LI)在SWF、LWF和SYF中较高。因此,在卵泡的发育过程中,膜细胞和颗粒细胞在形态和增殖特性上具有阶段特异性。卵泡体外悬浮培养实验表明,卵泡刺激素(FSH)和前列腺素PGE1刺激SYF和LYF中颗粒细胞的增殖,但对膜细胞无显著促增殖作用。由此推测,FSH和PGE1主要通过刺激颗粒细胞的增殖促使等级前卵泡进入等级发育。2.人参皂甙对鸡等级前卵泡发育和小黄卵泡颗粒细胞增殖的影响及其信号转导机制的研究人参属五加科多年生草本植物,其中含有的人参皂甙(GS)因其抗氧化特性而作为保健品,具有调节中枢神经系统、抗疲劳和抗衰老等功效。本文研究了GS对鸡等级前卵泡发育和小黄卵泡颗粒细胞增殖的影响及其信号转导机制。等级前卵泡在0.5%胎牛血清(FCS)中预培养16h后换成无血清的培养液,其中含10 gg／ml胰岛素,5μg／ml转铁蛋白和3×10-8mol／L亚硒酸钠(ITS培养液),同时单独添加10μg／ml GS处理24 h。结果表明：GS显著刺激SWF、LWF、SYF和LYF的膜层和颗粒层细胞的增殖,提示GS可通过刺激等级前卵泡细胞的增殖而促使其进入等级发育。从SYF中分离的颗粒细胞在含有0.5% FCS的M199培养液中培养,16h后换成无血清培养液并继续进行培养,同时用PKC抑制剂H7、PKC激活剂PMA单独或与GS联合处理24 h。随后进行细胞形态学和GS作用机理的研究。结果显示：当GS浓度在0.1～10μg／ml范围时,颗粒细胞的数量呈显著上升趋势,并呈一定的剂量-效应关系。同时,这一效应可以被H7所抑制,但可被PMA所加强。颗粒细胞的PCNA-LI统计结果显示细胞的增殖活性与颗粒细胞的数目变化呈现类似的变化。Western印迹法表明10μg／ml GS可促进颗粒细胞中PKC从胞质到胞膜的转移,而H7则抑制此效应。同时,GS上调颗粒细胞中细胞周期蛋白D1／周期蛋白依赖性激酶6(CDK6)和周期蛋白E／CDK2mRNA的表达。然而,用PKC抑制剂H7则减弱了这一刺激效应。由此推测,GS通过上调颗粒细胞中细胞周期蛋白D1/CDK6和E/CDK2的表达来刺激颗粒细胞的增殖,从而促使等级前卵泡进入等级发育。3.大豆黄酮对鸡等级前卵泡发育和小黄卵泡颗粒细胞增殖的影响本实验利用全卵泡培养和颗粒细胞单独培养模型探讨了植物性抗氧化剂大豆黄酮(DAI)对产蛋鸡等级前小黄卵泡及颗粒细胞增殖的影响。等级前卵泡在0.5%FCS中预培养16 h后换成无血清的ITS培养液,同时单独添加10-1000 ng／ml的DAI处理24 h。结果表明：DAI显著刺激SWF、LWF、SYF和LYF的膜层和颗粒层细胞的增殖,提示DAI通过刺激等级前卵泡细胞的增殖而促使其进入等级发育。采用机械法分散产蛋鸡等级前小黄卵泡颗粒细胞。培养液为含0.5%FCS的M199培养液。颗粒细胞预培养16h后,撤去血清,换用ITS培养液。细胞用DAI处理24 h后对其增殖活性进行检测。结果显示：10～1000 ng／ml的DAI处理可显著增加颗粒细胞的数目(P<0.05),这一刺激作用可被雌激素受体阻断剂他莫昔芬所阻断,并呈现出一定的剂量-效应关系。同时,颗粒细胞的BrdU-LI结果显示出细胞的增殖活性与其数目的变化呈现出相似的变化。由此推测,DAI可通过雌激素活性刺激颗粒细胞的增殖而促使等级前卵泡进入等级发育。总之,本实验通过在体的形态学和免疫组化方法研究了产蛋鸡等级前卵泡发育过程中颗粒细胞和膜细胞数量的变化,膜细胞和颗粒细胞在形态和增殖特性上呈现出阶段特异性。颗粒细胞的增殖活性随等级前卵泡的发育而降低。全卵泡体外悬浮培养实验表明,FSH和PGE1主要通过刺激颗粒细胞的增殖而促使等级前卵泡进入等级发育；植物性抗氧化剂GS和DAI可显著刺激SWF、LWF、SYF和LYF中颗粒细胞的增殖,促使等级前卵泡进入等级发育。利用等级前卵泡颗粒细胞单独培养模型证明：GS可上调颗粒细胞中细胞周期蛋白D1/CDK6和E/CDK2 mRNA的表达来促进其增殖,PKC信号途径可能参与GS对颗粒细胞增殖的调控；DAI可通过雌激素样作用显著促进颗粒细胞的增殖。这些结果在理论上有助于阐明家禽等级前卵泡发育和优势卵泡选择的调控机理,在生产实际上也可为植物来源的抗氧化性生物活性物质的开发应用及家禽繁殖性能的提高提供了理论基础。更多还原

【Abstract】 The ovary of the domestic chicken constitutes an ideal model for studies of ovarian biology and follicle development. The follicular granulosa cells represent a crucial symbol for follicle development due to their unique morphology and pivotal roles in the process of follicular development. In this study, we established two culture models of chicken whole prehierarchical follicles and separated granulosa cells to investigate the effects and mechanisms of hormones, growth factors and antioxidant bioactive substances involved in follicular development. From these studies, we expected to provide theoretically the mechanisms of development and selection of preovulatory follicles and practically improve the reproductive performance in laying hens.1.Developmental morphological changes in the follicular cells and effects of FSH/prostaglandin on development of chicken prehierarchical follicular cellsTheca cell (TC) and granulosa cell (GC) of the domestic chickens constitute ideal model system for studies of follicular development. At the beginning, we evaluated the morphological changes of chicken TCs and GCs in preovulatory follicles in laying hens. Samples of prehierarchical small and large white follicles (SWF, LWF), small and large yellow follicle (SYF, LYF) were collected in laying hens and fixed in 4% prafoxmaldehyde for 24 h. The paraffin-embedded follicles were serially sectioned at 5μm thickness and placed on coated slides for haematoxylin/eosin staining or immunohistochemical staining of proliferating cell nuclear antigen (PCNA). Morphological changes showed that there is only one stratum of GCs in SWF, double stratum of GCs in LWF, multiple stratum of GCs in SYF and LYF. Moreover, statistic analysis confirmed that the thicknesses of TCs layer and GCs layer were increased in a follicular size-dependent manner. The thickness was the largest in SYF and LYF (P<0.05). Furthermore, the number of GCs per mm2 manifested an increasing trend with the thickness in SWF, LWF, SYF, LYF (P<0.05). During the follicular development, an increase in the area of GCs layer was accompanied by an increase in the size of follicles (P<0.05). PCNA staining showed that PCNA label index (PCNA-LI) of GCs in SWF, LWF and SYF was significantly higher compared with LYF (P<0.05). Therefore, GCs and TCs manifested a stage-relevant pattern in morphology and proliferating activity during the course of follicular development. Meanwhile, effects of follicle-stimulating hormone (FSH) and prostaglandin E1 (PGE1) on theca and granulosa cell proliferation were investigated through whole follicle suspension culture in vitro. Results showed that FSH significantly stimulated the proliferation of GCs, but not the TCs in the prehirarchical follicles; PGE1 significantly stimulated the proliferation of both GCs and TCs, suggesting that FSH and PGE1 promoted hierarchical development mainly through stimulating proliferation of the GCs in these follicles.2. Effects of ginsenosides on follicular development and proliferation of GCs from chicken prehierarchical folliclesGinsenosides (GS) are the main molecular components responsible for the actions of ginseng and exert varying effects on a myriad of cells and tissues, including pharmacological responses to the central nervous systems, cardiovascular, endocrine and immune systems. In this experiment, the effects of GS on GCs proliferation was investigated by the suspension culture model of whole follicles. The follicles were cultured in Medium 199 supplemented with 0.5% fetal calf serum (FCS). After 16 h, the medium was replaced with serum-free medium and challenged with GS alone or in combination with PKC inhibitor H7 for 24 h. Statistical analysis showed that GS significantly increase the number of GCs in SWF, LWF, SYF and LYF, suggesting GS promoted the follicular development via stimulating GCs proliferation. Meanwhile, the separated GCs culture system was adopted to investigate the specific mechanism of the stimulatory effect of GS on chicken GCs. GCs were isolated from SYF and cultured in Medium 199 supplemented with 0.5% FCS.After 16 h, the medium was replaced with serum-free medium and challenged with GS alone or in combination with PKC inhibitor H7 or activator phorbol 12-myristate 13-acetate (PMA) for 24 h. The results showed that GS (0.1-10μg/ml) significantly increased the number of GCs of SYF in a dose-dependent manner, and this stimulatory effect was inhibited by H7, but enhanced by PMA.Meanwhile, the PCNA-LI of GCs displayed similar changes with the number of cells. Western blot analysis showed that 10μg/ml GS promoted the PKC translocation from the cytosolic compartment to the membrane compartment in GCs, which was blocked by combined H7treatment. Furthermore, GS up-regulated the mRNA expression of cyclin D1/CDK6 and cyclin E/CDK2 in GCs. However, inhibition of PKC with H7 attenuated this stimulatory effect of GS. These results indicated that GS could promote proliferation of chicken GCs through PKC-involved up-regulation of cyclin D1/CDK6 and cyclin E/CDK2.3. Effects of daidzein on follicular development and proliferation of GCs from chicken prehierarchical folliclesThe aim of this study was to evaluate the role of a phytoestrogen daidzein (DAI) on proliferation of GCs from prehierarchical follicles of laying hens. The effect of DAI on theca and granulosa cell proliferation was investigated via suspension culture model of whole follicles. The follicles were cultured in Medium 199 supplemented with 0.5% FCS. After 16 h, the medium was replaced with serum-free medium and the cells were challenged with 1μg/ml DAI for 24 h. Results showed that DAI significantly stimulated the proliferation of GCs and TCs in SWF、LWF、SYF and LYF, suggesting that DAI could promote prehierarchical follicles entering hierarchical development through stimulating the proliferation of follicular cells. Meanwhile, the pure GCs were separated by mechanic method and dispersed into single cells. After 16 h pre-incubation in 0.5% FCS-supplemented medium, the medium was replaced with serum-free medium, which was supplemented with 10μg/ml insulin,5μg/ml transferrin and 3×10-8 M selenite. GCs were challenged with 10-1000 ng/ml DAI for 24 h and assessed for proliferation. DAI(10-1000 ng/ml) significantly increased the number of GCs (P<0.05) and this stimulatory effect was inhibited by an estrogen receptor antagonist tamoxifen in a dose-dependent manner. Furthermore, BrdU-LI of GCs displayed similar changes with the number of GCs. These results indicated that DAI promoted proliferation of cultured chicken GCs via estrogenic action.In conclusion, through the methods of morphology and immunohistochemistry, we investigated the developmental changes in number and density of both GCs and TCs during the course of prehierarchical follicular development. GCs and TCs manifested a stage-relevant pattern in morphology and proliferating activity. By the whole follicle suspension culture, it was proved that both FSH and PGE1 promoted hierarchical development through stimulating the proliferation of GCs in these follicles. Moreover, plant-derived antioxidants GS and DAI could stimulate the proliferation of GCs in SWF,LWF, STF and LYF. By using the pure GCs culture model, we found that GS could promote proliferation of chicken GCs through PKC-involved up-regulation of cyclin D1/CDK6 and cyclin E/CDK2; DAI promoted proliferation of GCs from SYF involving estrogenic action. These results could be contributed to elucidate the mechanism of prehierarchical follicular development and selection, as well as to represent a reference for application of dietary-derived antioxidant bioactive substances to improvement of poultry reproductive performance.更多还原