miR-200家族在魚類精巢和胚胎發(fā)育中的表達和功能研究
發(fā)布時間：2018-10-08 12:36
【摘要】：MicroRNAs(mi RNAs)在脊椎動物的性別分化和性腺發(fā)育過程中發(fā)揮重要調(diào)控作用。黃顙魚作為我國性別異形經(jīng)濟魚類的代表,其雌、雄性腺的miRNAs表達譜均未見報道,此外黃顙魚性腺發(fā)育的分子機制仍不清楚。本研究首先通過Solexa深度測序技術(shù)檢測了3種類型的黃顙魚性腺(XX型雌魚卵巢,XY型雄魚精巢以及YY超雄魚精巢)。測序數(shù)據(jù)產(chǎn)生了384個保守的miRNAs和113個潛在的新miRNAs,其中分別有23,30和14個miRNAs在XX卵巢,XY精巢以及YY精巢中特異表達。研究發(fā)現(xiàn)黃顙魚性腺中一些性別差異表達的miRNAs,在其他脊椎動物中也呈現(xiàn)出相似的性別相關(guān)的表達模式。我們還觀察到miR-200家族成員(包括miR-141和miR-429)在XY精巢的表達水平均比YY精巢的低。組織學(xué)分析顯示YY超雄魚的精巢比XY雄魚的精巢成熟度要高,因為YY超雄魚的精巢有更大的精小囊,且精小囊中有更多的精細(xì)胞和更少的精母細(xì)胞。此外,當(dāng)用高濃度的雌激素處理XY型雄性黃顙魚,損害其精巢發(fā)育和細(xì)胞增殖時,5個miR-200家族成員在精巢中的表達水平均顯著上調(diào)。黃顙魚mi R-141和miR-429的表達水平隨著精巢發(fā)育的進程而表達下調(diào),該現(xiàn)象與人類中的報道一致。最后,通過熒光定量PCR檢測了隨機挑選的9個miRNAs,它們在黃顙魚性腺的表達模式與高通量測序結(jié)果一致。我們的研究結(jié)果為不同性別類型黃顙魚性腺miRNAs轉(zhuǎn)錄組分析提供了較為全面的信息,并初步證實了miR-200家族參與調(diào)控精巢發(fā)育和精子生成。脊椎動物中g(shù)rowth hormone/insulin-like growth factor(GH/IGF)軸在軀體生長方面發(fā)揮了重要的調(diào)控作用。GH/IGF軸基因的表達水平與一些哺乳動物以及水產(chǎn)養(yǎng)殖魚類的生長速度的快慢呈正相關(guān)。然而GH/IGF軸基因在發(fā)育早期的直接調(diào)控機制仍不清楚。miR-200家族在脊椎動物中非常保守,它在果蠅中的唯一同源成員是miR-8。MiR-8/200s在軀體生長方面的功能最先在果蠅中發(fā)現(xiàn),被認(rèn)為是促進生長的因子。但miR-200家族在脊椎動物中是否具有調(diào)控生長的作用未見報道。我們對斑馬魚的研究揭示了保守的miR-200家族成員能夠調(diào)控胚胎大小,他們主要通過靶向一些GH/IGF軸基因(GH,GHRa,GHRb和IGF2a)來發(fā)揮作用。過表達miR-200s成員導(dǎo)致細(xì)胞停滯在G1期,并且在胚胎時期誘導(dǎo)凋亡反應(yīng),因而抑制斑馬魚胚胎的軀體生長。值得注意的是,GH能誘導(dǎo)p53和miR-200s的表達,miR-200s又是p53的潛在轉(zhuǎn)錄靶基因,而在p53突變的胚胎中,GH過表達不能誘導(dǎo)mi R-200家族上調(diào)。結(jié)合以上實驗結(jié)果,我們得出結(jié)論,p53/miR-200s和GH/IGF信號途徑形成了一個調(diào)控胚胎大小的負(fù)反饋路線。這為“硬骨魚發(fā)育早期身體生長是如何確定的”這一長期存在的難題提供了重要的見解。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S917.4
本文編號：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S917.4
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