【作者】 王伟；

【导师】 徐银学；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 繁殖力是影响养猪业生产的最重要因素之一,是决定养猪业效益高低的主要限制因素。繁殖力的高低取决于母体卵巢卵泡的生长发育,因为卵泡发育决定了排卵数,是影响繁殖力的关键因素。掌握母猪卵泡发育规律及其影响因素有利于进一步提高母猪繁殖能力,提高猪生产的经济效益。哺乳动物卵泡发育是十分复杂的过程,主要包括：原始卵泡的募集、腔前卵泡的发育、有腔卵泡的选择和生长及卵泡的成熟或闭锁。随着后基因组信息学的发展,人们发现卵泡的生长发育是一个高度协调的、非独立生理生化过程,受多种相同或者不同的细胞内、外因子及信号通路的影响和控制。卵泡颗粒细胞是卵泡中最重要的体细胞,它对卵母细胞成熟、卵泡发育有至关重要的作用。伴随着哺乳动物卵泡生长和发育,颗粒细胞体积增大、数量增多及逐步分化和成熟。BMPs (bone morphogenetic proteins, BMPs)属于TGF-p超家族成员,近几年,研究人员对BMPs在繁殖上的作用进行了广泛的研究,包括卵泡的生长发育、颗粒细胞和膜细胞的增殖与凋亡、类固醇激素的合成、卵母细胞成熟、排卵和黄体化等。但BMP/Smad信号通路在猪卵泡颗粒细胞中具体作用及机制目前尚未见报道。因此,深入研究BMP/Smad信号通路在卵泡发育过程颗粒细胞中的作用及其调控机制,可以为哺乳动物繁殖性状的选育提供可能的理论基础,在提高家畜繁殖性能方面有重要意义。本研究以猪卵泡颗粒细胞(GCs)为实验材料,采用real time RT-PCR方法检测BMP/Smad信号通路中关键因子(受体和下游信号分子)在猪卵泡不同发育时期的颗粒细胞中的表达水平；利用免疫组织化学方法就BMP/Smad信号通路核心下游信号分子—Smad4,在猪卵巢中的细胞定位进行研究；用RNAi方法敲减Smad4,以阻抑BMP/Smad信号通路,利用MTT、流式细胞术、CLIA、real time RT-PCR等方法检测阻抑该信号通路对猪卵泡颗粒细胞的作用及其分子机制；在阻抑通路的基础上,额外添加FSH,利用MTT、流式细胞术、CLIA、real time RT-PCR等方法检测阻抑该信号通路对FSH影响猪卵泡颗粒细胞的作用的影响；用BMP配体(BMP-6)激活BMP/Smad信号通路,用RNAi方法敲减Smad4,以阻抑该通路,分别利用MTT、流式细胞术、CLIA等方法检测两种条件下体外培养的猪卵泡颗粒细胞的存活率、细胞周期、类固醇激素分泌水平等,并采用高通量基因芯片技术,对阻抑信号通路和阻抑后激活信号通路的猪卵泡颗粒细胞之间的差异表达基因进行比较研究,主要研究结果如下：1用real time RT-PCR法检测不同发育时期猪卵泡颗粒细胞中BMP/Smad信号通路的关键因子(BMP受体和Smads)表达情况,结果表明：BMP受体和Smads在猪卵泡颗粒细胞中均有表达,并且ActR1A和BMPR2 mRNA在大卵泡颗粒细胞中的表达量显著高于小卵泡(P<0.05)。BMPR1A、ActR2、Smad1、Smad5和Smad8在卵泡发育过程中的表达有增加趋势,但差异不显著。相反,BMPR1B、Smad4和Smad7的表达有一定程度的下降。这表明BMP/Smad信号通路关键因子在猪卵泡发育阶段特异性表达,在卵泡颗粒细胞生长与分化过程中起重要的调控作用。2用免疫组织化学方法检测Smad4在猪卵巢组织中的定位与表达,结果显示,Smad4在猪卵泡的多个发育阶段的细胞中均有表达。在原始卵泡中和初级卵泡中,Smad4在卵母细胞的细胞质中表达,而在颗粒细胞中没有表达；在腔前卵泡中,Smad4蛋白在卵母细胞和颗粒细胞中均有表达,在有腔卵泡中,Smad4蛋白主要被定位到颗粒细胞和卵泡内膜细胞中,而卵母细胞中表达较弱。3为了研究BMP/Smad信号通路在猪卵泡颗粒细胞中的作用,本研究从猪卵巢卵泡(3-5mm)内分离颗粒细胞(pGCs)进行体外培养。基于Smad4在该通路中的核心地位,采用RNAi技术“沉默”猪Smad4基因,阻抑BMP/Smad信号通路,以检测阻抑该通路对猪卵泡颗粒细胞生长和类固醇激素分泌的影响。结果表明：采用的RNAi技术成功的“敲减”猪Smad4基因,在mRNA水平该基因被下调89%,蛋白水平被下调56.6%。建立了BMP/Smad信号通路阻抑的细胞模型。阻抑该通路能显著抑制细胞的增殖,增加G1期细胞的百分含量,减少S期的细胞,并能显著下调细胞增殖标志因子Cyclin D2和CDK4的mRNA表达。阻抑该通路能显著增加颗粒细胞的凋亡比率,下调Bcl-2的表达,但不影响Bax的表达。阻抑该通路能显著抑制雌二醇的分泌,并下调P450arom mRNA的表达,表明BMP/Smad信号通路在猪卵泡颗粒细胞中有重要的调控作用。4为研究BMP/Smad信号通路对FSH诱导猪卵泡颗粒细胞的影响,本研究在阻抑BMP/Smad信号通路的细胞中添加FSH。用MTT法检测细胞增殖情况、流式细胞法检测细胞周期变化、CLIA方法检测雌二醇和孕酮的分泌以及real time RT-PCR检测相关基因的mRNA表达。结果表明：阻抑信号通路下调FSH受体的表达,阻抑信号通路使FSH促进细胞增殖的作用减弱,阻抑信号通路能显著增加G1期的细胞比率,即使添加FSH；阻抑通路能降低Cyclin D2的表达,且能抑制FSH促进其表达；阻抑信号通路能抑制CDK4的表达,即使添加FSH；阻抑信号通路能降低FSH诱导细胞分泌雌二醇的能力,但对孕酮没有影响。阻抑信号通路能减少P450arom的表达,并且降低FSH对其表达的影响；阻抑信号通路抑制FSH对P450scc表达的影响。5为研究BMP/Smad信号通路对猪卵泡颗粒细胞的影响,基于Smad4在该通路中的核心地位,采用RNAi技术“沉默”猪Smad4基因,阻抑BMP/Smad信号通路,另一方面,添加BMP配体(BMP-6)激活该通路,分别观察阻抑和/或激活BMP/Smad信号通路对细胞生物学特性的影响。结果表明：阻抑通路显著抑制细胞的增殖,增加G1期的细胞,减少S期细胞；激活通路则促进细胞生长,减少G1期细胞,增加S期细胞,阻抑信号通路能抑制雌二醇的合成,对孕酮没有显著影响,激活该通路抑制两种类固醇激素的分泌。在阻抑通路与阻抑后再激活通路的不同处理组细胞中筛选到37个差异表达探针,上调基因25个,下调基因12个。结合GO分析,37个基因中24个基因可以在生物过程中找到注释,23个基因在分子功能中找到注释,13个基因在细胞组成中找到注释。更多还原

【Abstract】 Prolificacy is one of the most important factors which influence the development of pig industry and it is also the main limiting factor to decide the efficiency of pig industry development. Fertility depends on the level of maternal growth and development of ovarian follicles as ovulation follicle development determines the number of ovum ovulated which is the key factor affecting the fecundity. To master sow follicular development and its impact factors are conducive to further improve sow reproductive capacity and enhance the economic benefits of survival pigs. Mammalian follicular development is a very complex process, including recruitment of the primitive follicle, the development of preantral follicles and antral follicle selection, growth and mature or follicle atresia. It was found that follicular growth and development is a highly coordinated and physiological and biochemical cascade processes affected and regulated by acting downstream of complex signaling pathways that integrate signals from the surrounding microenvironment but not an independent event with the development of post-genomics and bioinformatics.Granulosa cells are key somatic cells in follicules, and play very important roles in oocyte maturation and follicular development. Follicular development is accompanied by growth, proliferation, differentiation, and maturation of granulosa cells. Bone morphogenetic proteins (BMPs) belong to the transforming growth factorβ(TGFβ) superfamily and have implicated in the control and regulation of follicular development and female fertility. Recently, there are lots of research about the role of BMP on reproduction, including to follicular growth and development, granulosa cells proliferation and apotosis, steroidogenesis, oocyte maturation, ovulation and luteinization. However, the roles and the molecular mechanisms of BMP/Smad signaling alters follicular development have not been fully investigated in pigs. To further research the roles and mechanisms of BMP/Smad signaling in follicular granulosa cells provide possible rationale to select of mammalian reproductive trait and have great importance to the improvement of reproductive capacity of domestic animal. In this study, the time-spatial expression of BMP receptors and Smads in porcine follicular granulosa cells (GCs) in different period were investigated, by quantitative real-time PCR and cellular localization of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was studied in porcine ovaries by immunohistochemistry (IHC). In order to repress BMP/Smad signaling, the gene of Smad4 was silenced by RNAi, the effects of repressed BMP/Smad signaling on porcine follicular granulosa cells and underlying molecular mechanism were detected by MTT, FCM, CLIA and real time RT-PCR. Based on the repressed signaling, cells were treated with FSH, and the effects of repressed BMP/Smad signaling on the role FSH to GCs were investigated by MTT, FCM, CLIA and real time RT-PCR. Cells were treated with BMP-6, a activator of BMP/Smad signaling, as well as the Smad4-RNAi which repressed the BMP/Smad signaling, then the survival rate, proliferation and secretions of E2 and P4 of GCs were detected by MTT, FCM and CLIA; Microarray analysis was performed to capture the difference in gene expression in porcine GCs of between repressed the signaling and repressed then activated signaling.The main results achieved were as follows:1 The mRNA expression of BMP receptors and Smads in granulosa cells was detected by real time RT-PCR. The results indicated that endogenous BMP receptors and Smads expressed in porcine GCs, and ActRIA and BMPR2 mRNA levels were significantly higher in DF than that in SF(P<0.05). The mRNA levels of BMPR1A, ActR2, Smadl, Smad5 and Smad8 presented increased tendency, but there’s no significant difference in DFs and SFs. Whereas BMPR1B, Smad4 and Smad7 expression had tended to decrease from SF to DF. Together, the stage-specific expression pattern of BMP receptors and Smads suggested that the BMP/Smad signaling might play potential roles in the follicle GCs growth and differentiation.2 Cellular localization and expression of Smad4 proteins were studied by immunohistochemistry (IHC) in the ovaries of pigs. The results indicated that Smad4 localized in all kinds of cells of follicles at all stages. Smad4 localized in the cytoplasm of oocyte of primordial and primary follicles but not in granulosa cells; in pre-antral follicles, Smad4 localized in the oocytes and granulosa cells; Smad4 mainly localized in granulosa cells and follicular theca cells in antral follicles, and in oocyte, the expression of Smad4 was low. 3 To demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs, granulosa cells were isolated from 3-5mm antral follicles in diameter and cultured in vitro. A strategy of RNAi-mediated ’gene silencing’ of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to repress endogenous BMP/Smad signaling in cells. Results showed that siRNA-Smad4 causes specific inhibition of Smad4 mRNA and protein expression after transfection, with the Smad4 transcript level reduced by approximately 89% and the level of Smad4 protein also markedly decreased 56.6%. Repressed endogenous BMP/Smad signaling significantly inhibited growth and induced apoptosis of porcine GCs, and decreased E2 production. In addition, repressed BMP/Smad signaling significantly changed the mRNA expression of Cyclin D2, CDK4, Bcl-2, and P450 aromatase (P450arom). These results implied that BMP/Smad signaling have key regulation in porcine GCs.4 To demonstrate the effects of BMP/Smad signal on FSH-induced porcine GCs, we repress endogenous BMP/Smad signaling in cells while FSH was supplied in the medium of GCs. The proliferation and cycle of cells, secretions of E2 and P4, and the expression of relating genes were detected by MTT, FCM, CLIA and real time RT-PCR, respectively. The results showed that repressed endogenous BMP/Smad signaling significantly decreased the expression of FSHR, inhibited FSH-induced porcine granulosa cell proliferation, and signaificantly increased the proportion of G1-phase cells even with the existence of FSH; the repressed endogenous BMP/Smad signaling significantly decreased the expression of Cyclin D2, and inhibited FSH-induced augmentation of its expression; the repressed the signaling significantly decreased the expression of CDK4 even with the existence of FSH; besides, the repressed the signaling degraded FSH-induced E2 production, but not P4;the repressed the signaling decreaseed expression of P450 aromatase (P450arom), and inhibited FSH-induced expression of P450arom and cholesterol side-chain cleavage enzyme P450 (P450scc).5 To demonstrate the effects of BMP/Smad signal on porcine GCs, we repressed endogenous BMP/Smad signaling in cells, besides, we activated the signaling by supplyling BMP (BMP-6) in the medium, investigated the effects of repressed and activated BMP/Smad signaling on porcine GCs. The results showed that repressed endogenous BMP/Smad signaling significantly inhibited granulosa cell proliferation, increased the proportion of G1-phase cells, and decreased the cells of S-phase. Activated signaling significantly decreased the proportion of cells in G1-phase, increased the cells of S-phase, and repressed endogenous BMP/Smad signaling significantly inhibited secretion of E2, but not P4;activated signaling significantly inhibited the two steroid hormone production.37 probes showed significant differences in the GCs of between repressed the signaling and repressed then activated signaling, among which 25 were up-regulated, while 12 were down-regulated. By the GO analysis,24 genes were found in biological process, 23 genes in molecular function, and 13 genes in cellular component.更多还原