【作者】 孟春花；

【导师】 石放雄；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 卵巢卵泡发育是个十分复杂的过程,经历卵泡发育的启始、募集、选择、优势化、排卵和黄体化等过程。同时,卵泡的生长和发育也是一个高度协调的生理过程,受多种因素（激素、生长因子、芳香化酶、金属离子、物理及化学刺激等）的影响和控制,少数卵泡最终发育为优势卵泡并排卵,99%以上的卵泡都将发生闭锁。香猪是我国特有的小型猪种之一,也医学和繁殖学的理想模式动物。但与其繁殖相关的细胞生物学知识研究很少。哺乳动物中,叉头转录因子家族O亚家族的4个成员:FOXO1、FOXO3a、FOXO4和FOXO6在多种生命活动中起着广泛的作用。近年来的研究表明,FoxO转录因子在小鼠、大鼠、人的卵巢中通过多条通路调节细胞的生长、增殖、分化和凋亡。深入研究香猪卵巢细胞的生物学特性及FoxO蛋白在卵泡发育及发情周期中的作用及其调控机制,在提高家畜繁殖力、人的不孕症治疗、避孕手段的开发以及癌症等疾病的防治等方面均具有重要意义。本实验采用TUNEL法检测了香猪卵巢中的细胞凋亡,利用免疫组织化学的方法就FoxOs及几个相关蛋白在香猪卵巢中的细胞定位进行了研究,利用流式细胞术、RIA、Western blot等方法检测了不同直径猪卵泡中的颗粒细胞和不同处理条件下的体外培养的猪颗粒细胞的存活率、[Ca²⁺]i、E₂的分泌以及FoxO3a、Bcl-2的表达水平,就其可能的作用机制进行了深入探讨。主要结论如下:1.用TUNEL法检测香猪卵巢中的细胞凋亡的结果表明:健康卵泡中颗粒细胞排列整齐,仅在靠近卵泡腔处有少量颗粒细胞发生凋亡,而在闭锁卵泡中,颗粒细胞排列松散,部分进入卵泡腔,且凋亡比例较高。香猪卵巢切片的免疫组织化学检测结果表明:抑制素α在发育各阶段卵泡的颗粒细胞细胞质中有表达;PKB在香猪卵巢原始卵泡的颗粒细胞、次级卵泡和有腔卵泡的近卵泡膜细胞的颗粒细胞内有表达,在闭锁卵泡、黄体和卵巢间质细胞中均不表达;FoxO1在原始卵泡、次级卵泡、有腔卵泡的颗粒细胞的细胞质中有表达,在闭锁卵泡中凋亡颗粒细胞及黄体细胞中没有表达;FoxO3a在发育各阶段卵泡的颗粒细胞、颗粒黄体细胞以及血管内皮细胞的细胞质中均有表达;FoxO1和FoxO3a均在卵母细胞中表达;未检测到FoxO4在香猪卵巢细胞中的表达;Bax和Bcl-2在各发育阶段卵泡的颗粒细胞、黄体细胞和血管内皮细胞中均有表达。表明抑制素α、PKB、FoxOs、Bax和Bcl-2在香猪卵巢中呈卵泡发育阶段特异性和细胞特异性表达,可能在香猪卵泡的发育、闭锁和黄体化中起重要调控作用。2.为了研究FoxO6在猪生殖系统的作用,本实验从4个成年猪卵巢和4只成年小鼠的脑组织中提取RNA,根据已报道的小鼠Foxo6基因引物进行RT-PCR,结果从小鼠脑组织中扩增出一条约2500 bp的片段,而猪卵巢组织中未见此片段。因此,FoxO6基因可能在成年猪卵巢中不表达。3.为了研究猪卵泡发育过程中影响pGC生长和卵泡闭锁的因素,本实验中用机械法从猪卵巢分离有腔卵泡,按直径大小分为3组:小卵泡（1.5-3 mm）、中卵泡（3-5mm）和大卵泡（≧5 mm）,统计健康卵泡和闭锁卵泡分别所占比率,分离pGC并收集pFF。用流式细胞术Annexin V-FITC/PI双染法检测pGC存活率和Fluo 3-AM探针标记pGC检测[Ca²⁺]i,用Western blot检测pGC内FoxO3a表达水平,用化学发光法检测pFF中E₂的浓度。结果表明:随有腔卵泡直径的增大,闭锁卵泡的比率逐渐降低（P<0.05）,健康卵泡的比率逐渐增加（P<0.05）;pGC存活率逐渐升高（P<0.05）;pGC内[Ca²⁺]i逐渐降低（P>0.05）;pGC内FoxO3a表达水平逐渐升高（P<0.05）;pFF中E₂的浓度逐渐升高（P<0.05）。表明[Ca²⁺]i和FoxO3a转录因子可能通过调控不同直径有腔卵泡中pGC的生长和凋亡,从而调节猪有腔卵泡的生长、发育和闭锁。4.为研究细胞外游离Ca²⁺对pGC的影响,在猪颗粒细胞培养液中添加CaCl₂或Ca²⁺螯合剂EGTA,分别于0h、6h、24h和48h收集pGC和培养液。用流式细胞术检测细胞内Ca²⁺浓度([Ca²⁺]i)、用Western blot检测pGC内FoxO3a表达水平、用RIA法检测培养液中E₂的浓度。结果表明:（1）培养液中50 mg/mL CaCl₂对pGC的[Ca²⁺]i没有显著影响;50μg/mL EGTA显著降低了猪颗粒细胞内Ca²⁺浓度（P<0.05）,但500μg/mL EGTA反而对PGC的[Ca²⁺]i无显著影响。（2）培养液中添加CaCl₂ 6h可以显著提高pGC内FoxO3a的表达,而添加EGTA显著抑制了pGC内FoxO3a的表达（P<0.05）,且抑制程度呈剂量依赖性关系。（3）培养液中添加CaCl₂对pGC内E₂的分泌起抑制作用,而添加EGTA对pGC内E₂的分泌起显著的促进作用（P<0.05）,且E₂分泌提高幅度与EGTA的添加量呈剂量依赖性正相关。另外,体外培养的pGC中E₂的分泌随时间的延长逐渐升高的趋势,体外培养的pGC可能存在一个自成熟的过程。所以,细胞外钙离子可能通过FoxO3a信号通路来调控体外培养的pGC的生长、凋亡和成熟。5.为研究pGC中Ca²⁺信号通路是否受PI3K/PKB/FoxO信号通路的调节,用2ng/mL FSH、1mmol/L EGTA(一种Ca²⁺螯合剂)、10μmol/L LY294002（一种PI3K的特异性抑制剂）以及FSH+EGTA、EGTA+LY和FSH+EGTA+LY共处理体外培养的pGC,用流式细胞仪检测pGC存活率、用Western blot检测pGC内FoxO3a等蛋白表达水平、用RIA法检测培养液中E₂的浓度。结果表明:处理后2h,各处理组细胞存活率差异不显著,E₂分泌量差异不显著;处理后6h,EGTA和LY294002共处理组细胞存活率显著降低（P＜0.05）,其他各组细胞存活率差异不显著（P＞0.05）,EGTA处理组FoxO3a的表达水平受抑制,而LY294002处理组pGC内FoxO3a的表达水平升高,FSH对pGC中Bcl-2的表达起促进作用,而EGTA起抑制作用,添加LY促进了Bcl-2的表达;处理后24 h,LY294002+EGTA处理组和LY294002+EGTA+FSH共处理E₂的分泌量显著降低（P＜0.05）。所以,pGC内Ca²⁺信号通路与FoxO蛋白所在的PI3K/PKB信号通路存在交互作用,可能在pGC的成熟过程中起调控作用。更多还原

【Abstract】 Follicular development is a complex procedure,which includes follicular initiation, recruitment,selection,dominance,ovulation and luteinization.This procedure can be influenced by a lot of factors,such as hormones,growth factors,et al.Only very few follicles will develop into dominant follicles and ovulation and more than 99%turn atresic. Xiang pigs are a world famous breed of the miniature pig mainly distributed in Guizhou and Guangxi provinces of China.Miniature pigs have become a perfect animal model for reproductive and biomedical research.However,knowledge of reproductive physiology in this species is extremely limited.Forkhead transcription factors family O subgroup has 4 members:FOXO1,FOXO3a, FOXO4,and FOXO6,which play different roles in vast physical processes.Recent study showed that FoxO transcription factors regulated cell growth,proliferation,differentiation, and apoptosis through many different signal pathways in ovary cells.Follicle development from primordial follicles to dominant follicles was companied by growth,proliferation, differentiation,and maturation of granulosa ceUs（GCs）.Apoptosis of GCs will cause follicular atresia.In this study,apoptosis of follicular cells was firstly detected by TUNEL assay,and cellular localization of FoxOs and several proteins related were studied in porcine ovaries by immunohistochemistry（IHC）,then cellular survival rates,[Ca²⁺]i, secretions of E₂,and the expression of FoxO3a protein et al.were detected by FCM,RIA, Western blot et al.to study the mechanisms underlying follicular development and atresia. The conclusions were as follows:1.The apoptosis of follicular cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling（TUNEL） assay.Results indicated that the apoptosis rate of granulosa cells in healthy follicles was lower than that in atretic follicles. Cellular localization of inhibinα-subunit,PKB/Akt,FoxOs,Bax,and Bcl-2 proteins were studied by immunohistochemistry（IHC） in the ovaries of Xiang pigs.The results indicated that inhibinα-subunits were localized in granulosa cells of follicles at all stages but not in the corpora luteal.PKB was localized in granulosa cells of primordial follicles and in the basal layers of granulosa cells of secondary and antral follicles,but not in atretic follicles and corpora luteal.FoxO1 was localized in the cytoplasm of granulosa cells of follicles at all stages but not in corpra luteal cells and apoptotic cells.FoxO3a was localized in granulosa cells of follicles at all stages,granulosa luteal cells and vascular epithelial cells. Both FoxO1 and FoxO3a were localized in oocytes.Bax and Bcl-2 were localized in the cytoplasm and nuclear of granulosa cells of follicles at all stages and the cytoplasm of vascular epithelial cells and corpra lutcal cells.Altogether,the stage- and cell-specific expression pattern of inhibinα-subunit,PKB,FoxO1,FoxO3a,Bax,and Bcl-2 suggested that these proteins might play potential roles in the follicle development,atresia,and luteinization in Xiang pigs.2.To investigate the functions of FoxO6 on matured porcine reproductive system, RNA were extracted from 4 matured mouse brains and 4 matured porcine ovaries.A fragment about 2500 bp was amplified from the mouse RNA,but not yet from porcine ovary RNA.Therefore,FoxO6 may not be expressed in matured porcine ovaries.3.To investigate the factors that influnt the growth and apoptosis of pGCs,GCs were isolated from small（1.5-3 mm）,middle（3-5 mm）,and large（larger than 5 mm） antral follicles in different diameters.Then,the survival rates of granulosa cells and[Ca²⁺]i were detected by FCM,the expression of FoxO3a was detected by Western blot,and the concentrations of E₂ in pFF were detected.The results showed that the rate of atresic follicles decreased,rate of the healthy follicles increased at the same time;the survival rate of pGC from small follicles was the highest（P＜0.05）;[Ca²⁺]i decreased with the growth of follicles,the expression of FoxO3a increased;concentrations of E₂ in porcine follicular fluid increased with the enlargement of antral follicles.These results indicated that FoxO3a transcription factor and[Ca²⁺]i may be involved in the apoptosis of pGC and hence in the atresia of antral follicles.4.Ca²⁺ plays important roles in cell metabolism,synaptic transmission,gene expression,cell cycle control,and intercellular communication et al.The effects of extracelluar Ca²⁺ on granulosa cells were investigated in this study.CaCl₂ or EGTA was supplied in medium of granulosa cells.[Ca²⁺]i,secretion of E₂,and expression of FoxO3a were detected.The main results were as follows:（1）[Ca²⁺]i was not significantly affected by 50 mg/mL CaCl₂ but decreased after 50μg/mL EGTA;（2） The expression of FoxO3a was significantly promoted by CaCl₂ but inhibited by EGTA（P＜0.05）;（3） The secretion of E₂ was promoted by CaCl₂ but inhibited by EGTA.It suggested that extracelluar Ca²⁺ may influence the growth,apoptosis,and maturation of granulosa cells through FoxO3a-dependent signal pathways.5.Effects of FSH,EGTA,LY294002,FSH+EGTA,EGTA+LY294002 and FSH+EGT A+LY294002 on the expression of FoxO3a and secretion of E₂ in porcine granulosa cells were investigated in this study.Results showed that There were no significant differences among cell survival rates and secretion of E₂ of pGC after 2 h treatments by FSH,EGTA, and LY294002;The secretion of E₂ decreased after EGTA+LY294002 for 6h（P＜0.05）,and expression of FoxO3a is inhibited by EGTA,but promoted by LY294002 for 6h（P＜0.05）; Secretion of E₂ were significantly inhibited by LY294002+EGTA and LY294002+EGTA +FSH treatment for 24h,The expression of Bcl-2 was promoted by FSH and LY294002 but inhibited by EGTA.It was implied that Ca²⁺ signal pathway was related with PI3K/PKB signal pathway in granulosa cells of pigs.更多还原