miR-200家族在鱼类精巢和胚胎发育中的表达和功能研究
发布时间：2018-10-08 12:36
【摘要】：MicroRNAs(mi RNAs)在脊椎动物的性别分化和性腺发育过程中发挥重要调控作用。黄颡鱼作为我国性别异形经济鱼类的代表,其雌、雄性腺的miRNAs表达谱均未见报道,此外黄颡鱼性腺发育的分子机制仍不清楚。本研究首先通过Solexa深度测序技术检测了3种类型的黄颡鱼性腺(XX型雌鱼卵巢,XY型雄鱼精巢以及YY超雄鱼精巢)。测序数据产生了384个保守的miRNAs和113个潜在的新miRNAs,其中分别有23,30和14个miRNAs在XX卵巢,XY精巢以及YY精巢中特异表达。研究发现黄颡鱼性腺中一些性别差异表达的miRNAs,在其他脊椎动物中也呈现出相似的性别相关的表达模式。我们还观察到miR-200家族成员(包括miR-141和miR-429)在XY精巢的表达水平均比YY精巢的低。组织学分析显示YY超雄鱼的精巢比XY雄鱼的精巢成熟度要高,因为YY超雄鱼的精巢有更大的精小囊,且精小囊中有更多的精细胞和更少的精母细胞。此外,当用高浓度的雌激素处理XY型雄性黄颡鱼,损害其精巢发育和细胞增殖时,5个miR-200家族成员在精巢中的表达水平均显著上调。黄颡鱼mi R-141和miR-429的表达水平随着精巢发育的进程而表达下调,该现象与人类中的报道一致。最后,通过荧光定量PCR检测了随机挑选的9个miRNAs,它们在黄颡鱼性腺的表达模式与高通量测序结果一致。我们的研究结果为不同性别类型黄颡鱼性腺miRNAs转录组分析提供了较为全面的信息,并初步证实了miR-200家族参与调控精巢发育和精子生成。脊椎动物中growth hormone/insulin-like growth factor(GH/IGF)轴在躯体生长方面发挥了重要的调控作用。GH/IGF轴基因的表达水平与一些哺乳动物以及水产养殖鱼类的生长速度的快慢呈正相关。然而GH/IGF轴基因在发育早期的直接调控机制仍不清楚。miR-200家族在脊椎动物中非常保守,它在果蝇中的唯一同源成员是miR-8。MiR-8/200s在躯体生长方面的功能最先在果蝇中发现,被认为是促进生长的因子。但miR-200家族在脊椎动物中是否具有调控生长的作用未见报道。我们对斑马鱼的研究揭示了保守的miR-200家族成员能够调控胚胎大小,他们主要通过靶向一些GH/IGF轴基因(GH,GHRa,GHRb和IGF2a)来发挥作用。过表达miR-200s成员导致细胞停滞在G1期,并且在胚胎时期诱导凋亡反应,因而抑制斑马鱼胚胎的躯体生长。值得注意的是,GH能诱导p53和miR-200s的表达,miR-200s又是p53的潜在转录靶基因,而在p53突变的胚胎中,GH过表达不能诱导mi R-200家族上调。结合以上实验结果,我们得出结论,p53/miR-200s和GH/IGF信号途径形成了一个调控胚胎大小的负反馈路线。这为“硬骨鱼发育早期身体生长是如何确定的”这一长期存在的难题提供了重要的见解。
[Abstract]:MicroRNAs (mi RNAs) plays an important role in sex differentiation and gonadal development in vertebrates. The miRNAs expression profiles of female and male gonads of Pelteobagrus fulvidraco are not reported, and the molecular mechanism of gonadal development of Pelteobagrus fulvidraco is still unclear. In this study, three types of sex glands of Pelteobagrus fulvidraco (XX type female ovaries, XY type male spermary and YY supermale testis) were detected by Solexa deep sequencing technique. The sequencing data produced 384 conserved miRNAs and 113 potential new miRNAs, of which 2330 and 14 miRNAs were specifically expressed in XX ovary XY testis and YY testis, respectively. It was found that some sex-differentially expressed miRNAs, in the gonad of Pelteobagrus fulvidraco showed a similar sex-related expression pattern in other vertebrates. We also observed that the expression levels of miR-200 family members (including miR-141 and miR-429) in the XY testis were lower than those in the YY testis. Histological analysis showed that the spermary maturity of YY supermales was higher than that of XY males, because YY supermales had larger spermatozoa and more spermatocytes and fewer spermatocytes. In addition, when high concentration of estrogen was used to treat male XY type male Pelteobagrus fulvidraco, the expression level of five miR-200 family members in spermary was significantly up-regulated when it damaged the development of spermary and cell proliferation of male Pelteobagrus fulvidraco. The expression levels of mi R-141 and miR-429 in Pelteobagrus fulvidraco were down-regulated with the development of spermary, which was consistent with human reports. Finally, the expression patterns of 9 randomly selected miRNAs, in the gonads of Pelteobagrus fulvidraco were detected by fluorescence quantitative PCR, which were consistent with the results of high-throughput sequencing. Our results provide more comprehensive information for miRNAs transcriptome analysis of gonad of different sex types of Pelteobagrus fulvidraco, and preliminarily confirm that miR-200 family is involved in the regulation of spermatogenesis and spermatogenesis. The growth hormone/insulin-like growth factor (GH/IGF axis plays an important role in regulating somatic growth in vertebrates. The expression level of GH / IGF axis gene is positively correlated with the growth rate of some mammals and aquaculture fish. However, the direct regulation mechanism of the GH/IGF axis gene in the early stage of development is still unclear. The .miR-200 family is very conserved in vertebrates. Its only homologous member in Drosophila is the function of miR-8.MiR-8/200s in somatic growth, which was first found in Drosophila. It is thought to be a growth-promoting factor. However, the role of miR-200 family in regulating growth in vertebrates has not been reported. Our study of zebrafish revealed that conserved members of the miR-200 family can regulate embryo size, mainly by targeting a number of GH/IGF axis genes (GH,GHRa,GHRb and IGF2a). Overexpression of miR-200s members resulted in cell arrest in G1 phase and induction of apoptosis at embryonic stage, thus inhibiting somatic growth of zebrafish embryos. It is important to note that GH can induce the expression of p53 and miR-200s, which is a potential transcription target gene of p53, but the overexpression of GH in p53 mutant embryos can not induce the up-regulation of mi R-200 family. Combined with the above experimental results, we conclude that the p53 / miR-200s and GH/IGF signaling pathways form a negative feedback pathway for regulating embryo size. This provides an important insight into the long-standing problem of how early body growth is determined in bone-fish development.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4
本文编号：2256777
[Abstract]:MicroRNAs (mi RNAs) plays an important role in sex differentiation and gonadal development in vertebrates. The miRNAs expression profiles of female and male gonads of Pelteobagrus fulvidraco are not reported, and the molecular mechanism of gonadal development of Pelteobagrus fulvidraco is still unclear. In this study, three types of sex glands of Pelteobagrus fulvidraco (XX type female ovaries, XY type male spermary and YY supermale testis) were detected by Solexa deep sequencing technique. The sequencing data produced 384 conserved miRNAs and 113 potential new miRNAs, of which 2330 and 14 miRNAs were specifically expressed in XX ovary XY testis and YY testis, respectively. It was found that some sex-differentially expressed miRNAs, in the gonad of Pelteobagrus fulvidraco showed a similar sex-related expression pattern in other vertebrates. We also observed that the expression levels of miR-200 family members (including miR-141 and miR-429) in the XY testis were lower than those in the YY testis. Histological analysis showed that the spermary maturity of YY supermales was higher than that of XY males, because YY supermales had larger spermatozoa and more spermatocytes and fewer spermatocytes. In addition, when high concentration of estrogen was used to treat male XY type male Pelteobagrus fulvidraco, the expression level of five miR-200 family members in spermary was significantly up-regulated when it damaged the development of spermary and cell proliferation of male Pelteobagrus fulvidraco. The expression levels of mi R-141 and miR-429 in Pelteobagrus fulvidraco were down-regulated with the development of spermary, which was consistent with human reports. Finally, the expression patterns of 9 randomly selected miRNAs, in the gonads of Pelteobagrus fulvidraco were detected by fluorescence quantitative PCR, which were consistent with the results of high-throughput sequencing. Our results provide more comprehensive information for miRNAs transcriptome analysis of gonad of different sex types of Pelteobagrus fulvidraco, and preliminarily confirm that miR-200 family is involved in the regulation of spermatogenesis and spermatogenesis. The growth hormone/insulin-like growth factor (GH/IGF axis plays an important role in regulating somatic growth in vertebrates. The expression level of GH / IGF axis gene is positively correlated with the growth rate of some mammals and aquaculture fish. However, the direct regulation mechanism of the GH/IGF axis gene in the early stage of development is still unclear. The .miR-200 family is very conserved in vertebrates. Its only homologous member in Drosophila is the function of miR-8.MiR-8/200s in somatic growth, which was first found in Drosophila. It is thought to be a growth-promoting factor. However, the role of miR-200 family in regulating growth in vertebrates has not been reported. Our study of zebrafish revealed that conserved members of the miR-200 family can regulate embryo size, mainly by targeting a number of GH/IGF axis genes (GH,GHRa,GHRb and IGF2a). Overexpression of miR-200s members resulted in cell arrest in G1 phase and induction of apoptosis at embryonic stage, thus inhibiting somatic growth of zebrafish embryos. It is important to note that GH can induce the expression of p53 and miR-200s, which is a potential transcription target gene of p53, but the overexpression of GH in p53 mutant embryos can not induce the up-regulation of mi R-200 family. Combined with the above experimental results, we conclude that the p53 / miR-200s and GH/IGF signaling pathways form a negative feedback pathway for regulating embryo size. This provides an important insight into the long-standing problem of how early body growth is determined in bone-fish development.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4
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