【作者】 金艳梅；

【导师】 张才乔；

【作者基本信息】 浙江大学 ， 动物营养与饲料学， 2007， 博士

【摘要】 家禽卵巢是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体外培养模型,探讨了家禽生产中重要的生殖激素、生长因子及与卵泡发育相关的营养物质对颗粒细胞生长发育和功能的影响及作用机理,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.等级前小黄卵泡颗粒细胞体外培养模型的建立和应用采用机械法和胶原酶法分散成年产蛋鸡等级前小黄卵泡颗粒层,添加不同浓度的血清培养颗粒细胞。细胞培养结束后用四甲偶氮唑蓝比色法测定其活性。结果表明在添加0.5%胎牛血清(FCS)的培养液中预培养16h后换成无血清培养液继续进行培养,同时用药物处理细胞,这一培养模型能较好地维持颗粒细胞的存活和增殖。此外,本实验中选用对卵泡发育起重要调节作用的卵泡刺激素(FSH)验证培养模型的可行性。0.1～10 ng/ml的FSH对颗粒细胞的增殖作用呈显著的剂量-效应关系。FSH对小黄卵泡颗粒细胞促增殖作用的信号转导途径的研究表明：通过添加PKA.PKC信号途径激活剂(forskolin.PMA)及其相应的抑制剂(H89、H7),发现PKA信号转导系统在FSH的促小黄卵泡颗粒细胞增殖过程中起主导作用。细胞核增殖抗原(PCNA)免疫细胞化学测定结果与上述结果呈相同趋势。以上结果表明所建立的鸡小黄卵泡颗粒细胞培养模型是一种评价外源性激素离体生物效应的简易、快速和准确并且易于观察的良好模型。2.前列腺素对颗粒细胞的促增殖作用及其机理研究本实验探讨了前列腺素PGE,对鸡等级前小黄卵泡颗粒细胞增殖的作用及其作用途径。颗粒细胞在低浓度血清(0.5% FCS)中预培养16h后换成无血清培养液继续进行培养,同时单独添加前列腺素PGE1(0.1～100 ng/ml)或FSH(10 ng/ml)处理24h。当PGE,浓度在0.1～10 ng／ml范围时,颗粒细胞的增殖作用呈上升趋势,当浓度达到100ng/ml时,其作用呈下降趋势,其中PGE1 10 ng/ml的作用效果最明显,且其作用效果类似与FSH。PG作用机理研究发现,当前列腺素受体阻断剂SC19220(10-7～10-5mol/l)分别与FSH和PGE1联合处理后,FSH和PGE1的促增值作用均被显著抑制,且呈剂量效应；前列腺素合成酶阻断剂消炎痛(10-7～10-6 mol/l)与FSH联合后,FSH对颗粒细胞的促增殖作用也呈剂量效应被抑制。PGE,对小黄卵泡颗粒细胞促增殖作用的信号转导途径的研究表明：通过添加PKA、PKC信号途径激活剂(forskolin、PMA)及其相应的抑制剂(H89、H,),发现PKA信号转导系统在PGE。的促小黄卵泡颗粒细胞增殖过程中起作用。PCNA测定结果与上述结果呈相同趋势。转录因子CREB1免疫细胞化学实验发现,PGE,可以促进颗粒细胞内CREB1的表达,但其表达强度较forskol in处理组弱。上述结果表明PGE,具有类似于FSH的生理作用,可促进颗粒细胞的增殖。小黄卵泡颗粒细胞上存在PGE受体,FSH可以促进小黄卵泡颗粒细胞合成PGE1,PGE1可通过FSH介导的细胞内PKA信号转导系统促进核内转录因子CREB1的表达,进而促进颗粒细胞的增殖。3.前列腺素和表皮生长因子的互作对颗粒细胞增殖的影响本实验探讨了前列腺素PGE1和表皮生长因子(EGF)间的相互作用对产蛋鸡等级前小黄卵泡颗粒细胞增殖的影响。用12.5μg/ml的胶原酶将颗粒层分散为单个颗粒细胞,在低浓度血清(0.5%FCS)中预培养16h后换成无血清培养液继续进行培养,同时用药物处理细胞。免疫细胞化学染色结果表明小黄卵泡颗粒细胞表达EGF及其受体EGFR,而且这种表达能被PGE1(1～100 ng/ml)和forskolin(10-7～10-5 mol／1)处理所增强。EGFR的表达同样也能被其配体EGF所增强。选择性环氧合酶(COX)抑制剂SC560(10-7～10-5 mol／l,特异性抑制COX-1)和NS398(10-7～10-5 mol／l,特异性抑制COX-2)抑制了EGF诱导的颗粒细胞的增殖,EGF的这种作用被PCNA免疫细胞化学染色结果证实。尽管EGF促进了颗粒细胞内COX-1和COX-2的表达,但是EGF与特异性的PG合成酶抑制剂联合后再添加PGE,的补救实验发现,PGE-对NS398抑制的EGF诱导的颗粒细胞的增殖的补救效果较SC560明显。这表明COX-2在介导EGF作用的过程中占主导地位。上述结果表明细胞内PG和EGF相互促进合成的作用增强了颗粒细胞的增殖作用,进而促进了等级前卵泡的发育。4.花生四烯酸对颗粒细胞增殖、活性及功能的影响花生四烯酸(arachidonic acid,AA)是一类顺式多不饱和脂肪酸,是动物细胞膜磷脂的重要组成部分。除了作为前列腺素合成的前体物调节动物生殖过程外,花生四烯酸在调节细胞功能的过程中也起重要作用。本实验运用低浓度血清颗粒细胞体外培养模型探讨了花生四烯酸对产蛋鸡等级前小黄卵泡颗粒细胞发育及功能的影响。结果发现单独的花生四烯酸、亚油酸和亚麻酸(10-7～10-4 mol／l)对颗粒细胞没有明显的促增殖作用。四甲偶氮唑蓝比色法测定细胞活性发现,花生四烯酸(10-5～10-5 mol／l)可以增强细胞的活性,但当花生四烯酸达10-4mol／l时,细胞活性下降。PKA、PKC免疫细胞化学染色结果发现,花生四烯酸主要通过PKC信号途径发挥作用。此外,花生四烯酸对颗粒细胞功能的研究发现,花生四烯酸(10-7～10-5 mol／l)能够增加细胞外基质成分层粘连蛋白(1aminin,LN)和缝隙连接蛋白Cx43的表达。上述结果表明花生四烯酸通过PKC信号通路增强了细胞活性,并通过促进细胞外基质和细胞间缝隙连接蛋白的合成调控细胞的功能。以上实验结果表明：颗粒细胞在0.5FCS%中预培养16h后换成无血清培养液继续进行培养,同时用药物处理细胞的模型,能够用于研究颗粒细胞的增殖、分化、形态变化以及这种专一性细胞的信号转导途径,并可用于研究与卵泡结构、功能、生长及成熟相关的影响因素的作用及机理。在此模型上发现,PGE1通过FSH介导的细胞内PKA信号转导系统促进EGF、EGFR及核内转录因子CREB1的表达：而EGF主要通过促进COX-2的表达增加了PG的合成,它们形成了一个细胞内的正反馈循环通路,并通过不同信号转导通路的交联,加速了小黄卵泡颗粒细胞的增殖,从而促进了等级前卵泡的发育。而AA通过PKC信号通路,影响颗粒细胞细胞外基质及细胞间缝隙连接蛋白的表达,从而影响颗粒细胞的功能。因此,AA及其代谢物PG可通过改变小黄卵泡颗粒细胞的增殖和分化等功能,进而促进卵泡的发育。这些结果为家禽优势卵泡选择及繁殖性能的提高提供了理论指导和实验平台。更多还原

【Abstract】 The ovary of the domestic chicken constitutes an ideal model system for studies of ovarian biology and follicle development. Chicken granulosa cells are easily isolated and play an important role in follicle development, so it was looked as a crucial symbol in follicle development. In this study, we established the culture model of granulosa cell and discussed the effect and mechanism of hormones, growth factors and nutriment which has relationship with follicle development. From these studies, we expected to provide theoretic guidance for selection of preovulatory follicle and improve the performance of chicken.1.Establishment and application of granulosa cell culture modelThe granulosa layers from prehierarchical small yellow follicles (SYF) were separated by mechanic method and dispersed into single cells by collagenase, and then cells were cultured in different serum media. Cell viability was quantified by measurement of the mitochondrial reduction of MTT to produce a dark-blue formazan product. The results indicated that cultured cell showed the proper proliferation and growth, afterl6h pre-incubation in 0.5% FCS medium. Furthermore, the feasibility of this model was validated by follicle-stimulating hormone (FSH). FSH stimulated proliferation of granulosa cells in a dose-dependent manner from 0.1 to 10 ng/ml. Action of protein kinases A and C system in FSH-stimulated proliferation of granulosa cells was revealed that FSH promoted the proliferation of granulosa cells from SYF was primary mediated by the intracellular PKA signal transduction. The PCNA-LI of granulosa cells displayed similar changes with the number of cells.The above results indicated that this model could be used for studies about regulation of granulosa cell proliferation and differentiation.2. Study in the proliferation effect of prostaglandin on granulosa cells from chicken prehierarchical folliclesThe aim of the present study was to evaluate the role of prostaglandin (PG) on proliferation of granulosa cells from prehierarchical small yellow follicles (SYF) of buff laying hens. The granulosa layers were separated by mechanic method and dispersed into single cells. After 16h pre-incubation in 0.5% FCS medium, the medium was replaced with serum-free medium, which was supplemented with 10μg/ml insulin,5μg/ml transferrin and 3×10μM selenite. Cells were challenged with PGE1 and FSH for 24h and then assessed for proliferation.The results showed that PGE1 (0.1-10 ng/ml) had a similar proliferating effect as FSH on granulosa cells, and these stimulating effects were restrained by the PGE receptor antagonist SC-19220 at 10-7-10-5 M. Prostaglandin synthase antagonist indomethacin (10-7-10-5M) suppressed FSH-induced increase in the number of granulosa cells in a dose-dependent manner. Downstream activation of protein kinase A by forskolin-activated adenylate cyclase resulted in elevated proliferation of granulosa cells, an effect unobserved by phorbol-12-myristrate-13-acetate-activated protein kinase C.In addition, PGE1-stimulated proliferation of granulosa cells was hindered by H89 (PKA inhibitor) but not by H7 (PKC inhibitor). Furthermore, the proliferating cell nuclear antigen labeling index (PCNA-LI) of granulosa cells displayed similar changes with the number of cells. Immunocytochemical staining showed that PGE1 induced the expression of transcription factor CREB1 (cAMP response element binding protein) in granulosa cells, and CREB1 expression stimulated by forskolin was higher than that of PGE1.These results indicated that PGE1 promoted the proliferation of granulosa cells from SYF and was also involved in mediating FSH-stimulated intracellular PKA signal transduction.3.Interactive actions of prostaglandin (PG) and epidermal growth factor (EGF) on proliferation of granulosa cellsThe interactive actions of prostaglandin (PG) and epidermal growth factor (EGF) on proliferation of granulosa cells was investigated in prehierarchical small yellow follicles (SYF) of laying hens. The granulosa layers were dispersed into single cells by 12.5μg/ml collagenase.After 16 h pre-incubation in 0.5% fetal calf serum-supplemented medium, the medium was replaced with serum-free medium.Immunocytochemical staining showed that granulosa cells expressed EGF and its receptor, and their expression was increased by PGE1 (1-100 ng/ml) or forskolin(10-7 to 10-5 M) treatments.EGF receptor was also induced by its ligand EGF. The specific prostaglandin synthase inhibitors SC560 (for COX-1) and NS398 (for COX-2) suppressed EGF-stimulated increase of the granulosa cell number. Furthermore, the effect of EGF was confirmed by the immunocytochemical staining of the proliferating cell nuclear antigen in granulosa cells. Though EGF promoted the expression of both COX-1 and COX-2, the rescue experiment indicated that combined treatment of PGE1 showed better rescuing effect on NS398 inhibition than SC560 at 10-6 M, which implies COX-2 plays the predominant role in mediating EGF action. The above results indicate that reciprocal stimulation of intracellular PG and EGF production may enhance proliferation of granulosa cells, hence to facilitate development of chicken prehierarchical follicles.4. Effect of Arachidonic acid (AA) on proliferation, cell viability and cell function of granulosa cells from prehierarchical small yellow folliclesArachidonic acid is a cis-polyunsaturated fatty acid that is a normal constituent of membrane lipids in animal cells. In addition to its role as a precursor of prostaglandins, AA itself may play an important role in the regulation of cell function. The AA on development and function of granulosa cells was investigated in prehierarchical small yellow follicles of laying hens. The result indicated that there were no significant difference on proliferation of granulosa cell between Arachidonic acid, Linoleic acid and Linolenic acid. Cell viability was measured by MTT showed that the cell viability with increasing of AA concentration(10-6-10-5M), but the cell viability was higher at high concentration of AA (10-4 M). Immunocytochemical staining showed that AA increased the expression of PKA, LN and Cx-43.Therefore, mediated by PKC signal pathway, AA promoted the cell viability and regulated the cell function by increase the production of extracellular matrix (ECM) and connexin (Cx). The above results indicated this cell culture model that cells pre-incubated in 0.5% fetal calf serum-supplemented medium for 16h then the medium was replaced with serum-free medium was feasible. This model system enables studies of differentiation and morphology as well as of signal transduction events specific for this highly specialized follicular cell type.Finally, this system will be instrumental in answering key questions concerning follicular architecture, function, growth, and maturation. PGE1 promoted the expression of EGF, EGFR and CREB1 of granulosa cells from SYF and was also involved in mediating FSH-stimulated intracellular PKA signal transduction. Otherwise, EGF stimulated the production of PG predominant mediatied by COX-2. It forms a positive intracellular feedback cycle and regulated by "cross talk" between different signal pathways. Sequentially, the proliferation of granulosa cell was accelerated and follicle development was facilitated.更多还原