【作者】 陈阿琴；

【导师】 许梓荣； 俞颂东；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2008， 博士

【摘要】 本研究以湖羊卵泡颗粒细胞为试验对象,利用抑制消减杂交技术筛选和鉴定了与湖羊卵泡成熟相关的差异表达基因,探讨了多胎候选基因骨形态蛋白15(BMP15)和生长分化因子-9(GDF-9)受体基因在湖羊卵泡成熟过程中的表达规律,并以体外培养颗粒细胞为模型研究了激素和茶多酚对差异表达基因的影响。这对充分利用湖羊的多胎性能,揭示绵羊卵泡发育的分子机制和改进绵羊胚胎工程技术具有重要的理论意义和应用价值。1.湖羊卵泡颗粒细胞BMP15和GDF-9受体基因表达的变化及激素对其表达的影响湖羊经促卵泡刺激素(FSH)处理后屠宰,将剥离的有腔卵泡分为成熟卵泡(LF,>5mm)和生长卵泡(SF,3-5mm),分离卵泡颗粒细胞；以体外培养绵羊卵泡(2-5mm)颗粒细胞为模型,添加不同浓度FSH(0、1、5、10 ng/ml)或与E2(1ng／ml)协同作用处理细胞；荧光定量PCR法检测卵泡颗粒细胞中BMPRⅡ、BMPRIB和ALK-5mRNA的表达量。结果显示,与生长卵泡颗粒细胞相比,成熟卵泡颗粒细胞中BMPRⅡ、BMPRIB和ALK-5mRNA的表达量分别提高了1.46、1.86和1.53倍。与对照组相比,添加FSH(1-10ng/ml)显著降低BMPRIB mRNA表达水平(P<0.05),5ng/mlFSH对BMPRⅡ和ALK-5mRNA表达无显著影响。FSH与E2协同作用使BMPRⅡ、BMPRIB和ALK-5mRNA的表达量较对照组分别提高了2.81、2.76和8.39倍(P<0.01)。以上结果提示,BMP15和GDF-9及其受体基因在绵羊卵泡发育、成熟过程中起重要的作用。BMP15和GDF-9受体基因在绵羊卵泡颗粒细胞中差异表达,其机制可能与FSH和E2协同作用调控有关。2.湖羊成熟卵泡颗粒细胞差异表达基因的筛选和鉴定利用抑制消减杂交技术筛选湖羊卵泡发育分化过程中不同阶段之间差异表达的基因。从分离的成熟卵泡和生长卵泡颗粒细胞中提取总RNA,构建了湖羊卵泡颗粒细胞正、反双向cDNA消减文库。在正、反向文库中,分别随机挑选了384个克隆,再经斑点杂交验证。根据斑点杂交信息,挑选了92个克隆进行单向测序,获得90个EST,聚类后通过BLAST比对,共有38个与已知功能的基因相似,15个与已知但功能未知的基因相似,14个为新EST(Genbank登录号为EY202207,FD480266-FD480278)。荧光定量PCR进一步证实了LAPTM4A、SERPINE2、GSTA1和INHBA在成熟卵泡颗粒细胞的表达量均显著高于生长卵泡颗粒细胞(P<0.05)。本研究中这些差异基因的发现为深入分析绵羊卵泡发育机制尤其是卵泡成熟相关的基因,进而用于绵羊多胎的分子育种奠定了基础。3.湖羊GSTA1的克隆及FSH对其表达的影响克隆了湖羊卵泡颗粒细胞谷胱甘肽S-转移酶Al(GSTA1)基因的cDNA序列,并探讨了FSH对GST酶活性及GSTA1mRNA表达的影响。根据湖羊卵泡颗粒细胞SSH文库鉴定出的GSTA1EST序列,用电子克隆及RT-PCR方法获得了840bp的GSTA1基因序列(GenBank登录号：EU399787),其开放阅读框长666bp,编码222个氨基酸。推测的湖羊GSTA1氨基酸序列与牛、猪和人GSTA1序列的同源性分别为98%、88%和82%。RT-PCR结果显示：GSTA1在绵羊黄体、肝、肾、睾丸和肺中都有转录,在黄体、肝和睾丸中表达水平较高,肺和肾中表达次之,在心、子宫和垂体中未见明显表达。分别用0、1、5和10ng／ml的FSH处理体外培养的颗粒细胞。结果显示：与对照组相比,10ng/ml FSH使GST酶活性提高了68.8%(P<0.05),GSTA1mRNA的表达量升高了2.07倍(P<0.05)。上述结果表明,GSTA1基因表达特异性可能与其功能密切相关,且FSH可调控GSTA1mRNA表达,提示GSTA1可在激素调控下参与绵羊卵泡发育,尤其是在卵泡成熟过程中发挥重要作用。4.茶多酚对氧化损伤诱导的绵羊卵泡颗粒细胞凋亡的影响利用次黄嘌呤-黄嘌呤氧化酶(HX/XO)活性氧自由基产生系统建立绵羊卵泡颗粒细胞氧化损伤模型,通过MTT法、荧光显微镜观察、分光光度法、流式细胞仪检测、荧光定量PCR,探讨茶多酚(GTPs)对氧化损伤诱导的绵羊卵泡颗粒细胞凋亡和GSTA1表达的影响。结果显示,HX／XO(1mM／l；1mU／ml)可通过氧化损伤诱导颗粒细胞凋亡,以剂量效应使颗粒细胞GSH水平和GST酶活性显著降低,MDA形成增加(P<0.05),同时可使GSTA1mRNA表达水平显著降低(P<0.05)。低浓度GTPs(<10μg/ml)对颗粒细胞的生长、分化无显著影响,高浓度GTPs(>20μg/ml)对颗粒细胞增殖有抑制作用;与HX/XO组相比,GTPs(5,10μg/ml)能显著缓解HX/XO系统引起的颗粒细胞氧化损伤,使存活的细胞数目显著增加(P<0.05)；GTPs(10μg/ml)处理可提高HX/XO诱导的颗粒细胞GST酶活和GSH水平降低,抑制MDA的产生(P<0.05),同时显著提高GSTA1mRNA表达水平。研究表明,GTPs可抑制氧化应激诱导的绵羊卵泡颗粒细胞凋亡,其机制可能与GTPs调控GSH-GST途径及GSTA1表达有关。更多还原

【Abstract】 The present study was to investigate expression parttens of receptor genes for BMP 15 and GDF-9, identify the differently espression genes of ovine follicular granulosa cells, and examine effects of hormone and green tea polyphenols on these genes mRNA expression in Hu sheep. It is of momentous theoretical significance and practical significance for effective use of Hu Sheep in the breeding, revealing moleculal mechanism of follicle development, and optimizing embryo industry protocol.1. Stage-specific of BMP 15 and GDF-9 receptor genes expression:effects of follicle-stimulating hormone on ovine antral follicleOvaries were collected and granulose cells were harvested from two diameter size follicles (3-5mm,>5mm). For in vitro studies, granulosa cells were obtained from follicles of 2-5mm diameter and cultured in serum-free McCoy’5A medium supplemented with different doses of FSH (0,1,5,10 ng/ml) or combination 5ng/ml FSH with 1ng/ml E2. Total mRNA expression of BMPRII, BMPRIB and ALK-5 was estimated by the quantitative real-time PCR. Our results demonstrated that BMPRII, BMPRIB and ALK-5 expression was significantly higher in the granulosa cells of large follicles than in those of small follicles. Treatment of granulose cells with FSH (1-10ng/ml) alone down-regulated the expression of BMPRIB (P<0.05). BMPRII and ALK-5 mRNA expression were no significantly differentiation by the concentration of FSH (5ng/ml), compared to control. A further increase FSH (10ng/ml) down-regulated the expression of BMPRII and ALK-5 (P<0.05). Combination FSH (5 ng/ml) with E2 (1 ng/ml) up-regulated the expression of the BMPRII, BMPRIB and ALK-5 in the granulosa cells (P<0.05). Therefore, the present study provided the expression levels of receptor genes of BMP15 and GDF-9 and suggested the expression of BMPRII, BMPRIB and ALK-5 might be regulated by FSH and E2 in ovine granulose cells.2. Analysis of gene expression in granulosa cells of ovine antral follicles from Hu sheep using suppression-subtractive hybridizationIn the present study, suppression subtractive hybridization(SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles(LF,>5mm) and small follicles(SF,3-5mm) in Hu sheep during antral follicle phase, and a subtractive cDNA library was constructed. Furthermore, with dot blot analysis and quantitative reverse transcription-polymerase chain reaction, a total of 92 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among of these,38 exhibited high homology to known genes, 14 identified as new EST (Accession No. EY202207, FD480266-FD480278). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR, and the results of confirmed an increase expression of their mRNA in granulosa cells of large follicles compared with that of small follicles. We conclude that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.3. Cloning of GSTA1 and regulation of FSH on expression of GSTA1 of ovine granulosa cellsAccording to the identified ESTs, electic cloning and RT-PCR were used to obtained cDNA of the GSTA1 gene (Genbank Accession No, EU399787). The nucleotide sequence of cDNA of GSTA1 was 840bp and contained open reading frame of 666 nucleotides, encoding a predicted protein of 222 amino acides. The deduced molecular weight was 25.4kD and the theoretical pI was 8.66. Ovine GSTA1 was 82% to Homo sapiens,88% identical to Sus scrofa, and 98% identical to Bos taurus. The mRNA was detected in corpus luteum, testis, liver, lung, and kidney. By using cultured ovine granulosa cells, this study was conducted to analyze the effects of GSTA1 on expression in granulosa cells. Results showed that concomitant to the increase in glutathione S-transferase activity were significantly enhanced when cultured ovine granulosa cells were treated with FSH. Furthermore, relative expression of GSTA1 mRNA in cultured granulosa cells sensitized with lOng/ml FSH has increased significantly (P＜0.05). It was concluded that GSTA1 may have a functional role in ovine ovarian follicular development under endocrine regulation.4. Effects of green tea polyphenols on the granulosa cells against oxidative stress induced apoptosisWe sought to investigate the induction effect of the reactive oxygen species (ROS)-producing system hypoxanthine/xanthenes oxidize (HX/XO) on the expression of GSTA1 in vitro cultured ovine granulosa cells. This study showed that HX/XO (1mM; 1mU/ml) exerted toxic effects on ovine granulosa cells induced oxidative damage and cell death. Granulosa cells GST activity and GSH levels decreased after HX/XO exposure (P<0.05). HX/XO obviously induced the expression of GSTA1 in cultured granulosa cells (P＜0.05). The expression of GSTA1 mRNA was lowest when XO concentration is at 1mU/ml. GTPs obviously attenuated the oxidative stress induced by HX/XO system, increased the cell number, and restored HX/XO-induced decrease in glutathione (GSH) level and HX/XO-induced increase in malondialdehyde (MDA) formation. GTPs also counteracted the HX/XO-induced GST activity decrease and down expression of GSTA1. Taken together; these studies provide insights into the action of GTPs and suggest the GTPs-mediated increased GST may be a mechanism contributing to prevention against reactive oxygen species injury in the follicular development of sheep. Ovine GSTA1 protect against granulosa cells apoptosis caused by HX/XO, suggesting an important role of GSTA1 in protection against oxidative stress in follicular development, selection, and ovulation.更多还原