發(fā)布時(shí)間：2019-04-04 14:56

【摘要】：對于雄性動(dòng)物而言,睪丸發(fā)育直接影響其性成熟早晚及精液品質(zhì),從而決定雄性動(dòng)物繁殖性能。精子發(fā)生起始于精原干細(xì)胞(Spermatogonial stem cells,SSCs)的自我更新和分化,睪丸組織中SSCs分布反映了其組織發(fā)育程度,組織學(xué)分析可直接觀察睪丸結(jié)構(gòu)變化規(guī)律及生精細(xì)胞分布情況。lncRNA(long noncoding RNA,lncRNA)是一類轉(zhuǎn)錄本長度在200 nt以上的非編碼RNA,表達(dá)豐度較低,只在細(xì)胞發(fā)育、分化的特定階段表達(dá),具有明顯的組織細(xì)胞表達(dá)特異性。lncRNA參與決定干細(xì)胞命運(yùn),包括維持自我更新和分化,但lncRNA調(diào)控SSCs增殖與分化的研究少有報(bào)道。為探索陜北白絨山羊不同發(fā)育期睪丸組織lncRNA表達(dá)差異,本試驗(yàn)取1月齡、3月齡、6月齡及成年陜北白絨山羊睪丸組織制作切片,進(jìn)行蘇木精-伊紅染色,鏡下觀察曲細(xì)精管形態(tài),統(tǒng)計(jì)各生殖細(xì)胞數(shù),分析不同發(fā)育階段陜北白絨山羊睪丸發(fā)育程度;同時(shí)進(jìn)行免疫組織化學(xué)染色,并用q-PCR檢測了發(fā)育階段睪丸中SSCs標(biāo)記基因(CD49f、PLZF、Thy1、ETV5、VASA、Oct4、Nanog、Nanos2、CyclinA)的相對表達(dá)量,以分析不同發(fā)育階段陜北白絨山羊SSCs相對豐度;對1月齡、3月齡及成年羊睪丸組織進(jìn)行l(wèi)ncRNA測序并分析lncRNA差異表達(dá)。本研究獲得的主要研究結(jié)果如下:1.隨著年齡的增長,絨山羊曲細(xì)精管直徑顯著增大(P0.05),6月齡達(dá)到150.72μm,略低于成年羊(161.94μm)(P0.05)。絨山羊細(xì)胞層數(shù)隨年齡的增長而增長,6月齡達(dá)到7.70層,顯著高于1月齡和3月齡(P0.05)。3月齡陜北白絨山羊曲細(xì)精管中精原細(xì)胞最為豐富,占總細(xì)胞數(shù)的46.40%,明顯高于1月齡(33.10%)、6月齡(9.10%)和成年羊(7.60%)(P0.05)。曲細(xì)精管橫截面細(xì)胞總數(shù)隨年齡增長而增長,6月齡和成年絨山羊曲細(xì)精管橫截面細(xì)胞總數(shù)顯著高于1月齡和3月齡絨山羊(P0.05)。結(jié)果表明,陜北白絨山羊在6月齡達(dá)到性成熟,3月齡睪丸中精原細(xì)胞最為豐富。2.精原干細(xì)胞標(biāo)記因子CD49f、CD90、GFRa1及ETV5在3月齡絨山羊睪丸中陽性率較高,分別為41.67%、40.20%、21.92%、25.36%,且顯著高于6月齡和成年羊(P0.05);6月齡絨山羊睪丸中CD49f、CD90、GFRa1及ETV5陽性率比成年羊略高,但差異不顯著(P0.05)。PLZF在睪丸中陽性率隨年齡增長而降低,差異顯著(P0.05)。精原干細(xì)胞標(biāo)記基因(CD49f、PLZF、Thy1、ETV5、VASA、Oct4、Nanog、Nanos2)均在3月齡睪丸組織中高表達(dá),其中PLZF、Thy1、ETV5、VASA、Nanog及Nanos2顯著高于1月齡、6月齡及成年羊(P0.05)。另外,CyclinA在6月齡羊睪丸中表達(dá)量最高,顯著高于其他組(P0.05)。結(jié)果表明,與1月齡、6月齡和成年羊相比,3月齡羊睪丸中SSCs濃度最高,是SSCs體外分離富集的理想材料。3.在絨山羊睪丸組織中,1月齡與3月齡差異表達(dá)lncRNA共17個(gè),其中13個(gè)表達(dá)上調(diào),4個(gè)表達(dá)下調(diào)。1月齡與成年羊差異表達(dá)lncRNA共247個(gè),其中239個(gè)表達(dá)上調(diào),8個(gè)表達(dá)下調(diào)。3月齡與成年羊差異表達(dá)lncRNA共201個(gè),其中174個(gè)表達(dá)上調(diào),27個(gè)表達(dá)下調(diào)。結(jié)果表明這些差異表達(dá)的lncRNA可能參與調(diào)控生精細(xì)胞發(fā)育。對這些差異表達(dá)的lncRNA進(jìn)行分析,發(fā)現(xiàn)這些lncRNA通過各種途徑發(fā)揮作用,為lncRNA對生精過程作用的研究奠定了基礎(chǔ)。本研究表明,與1月齡、6月齡、成年陜北白絨山羊相比,3月齡羊睪丸中SSCs濃度最高,適合作為SSCs富集材料。在6月齡陜北白絨山羊睪丸中有大量精子細(xì)胞,已經(jīng)達(dá)到性成熟。對1月齡、3月齡及成年羊睪丸組織進(jìn)行l(wèi)ncRNA測序,發(fā)現(xiàn)有l(wèi)ncRNA差異表達(dá),說明這些lncRNA可能參與生精細(xì)胞發(fā)育調(diào)控。
[Abstract]:For male animals, the development of the testis directly influences the sexual maturity and the quality of the semen, thus determining the reproductive performance of the male animals. Spermatogenesis begins with the self-renewal and differentiation of Spermatogonial stem cells (SSCs). The SSCs distribution in the testis of the testis reflects the degree of tissue development, and the histological analysis can directly observe the changes of the structure of the testis and the distribution of the spermatogenic cells. LncRNA (lncRNA) is a kind of non-coding RNA with the length of more than 200 nt, and the expression abundance is low, which is expressed only in the specific stage of cell development and differentiation, and has obvious tissue cell expression specificity. The participation of lncRNA in the determination of the fate of stem cells, including the maintenance of self-renewal and differentiation, was rare in the study of the regulation of the proliferation and differentiation of SSCs by lncRNA. In ord to explore that difference of the expression of lncRNA in the testis of the white cashmere goat in northern Shaanxi, the testis tissue of the white cashmere goat of 1 month,3 months,6 months and the adult of northern Shaanxi were cut, and the hematoxylin-eosin staining was carried out, and the morphology of the fine fine tube was observed under the microscope, and the number of the germ cells was counted. The expression of SSCs marker gene (CD49f, PLZF, Thy1, ETV5, VASA, Oct4, Nanog, Nano2, CyclinA) in the testis of the development stage was detected by using q-PCR. In order to analyze the relative abundance of SSCs of the white cashmere goat in the northern Shaanxi, the ncRNA was sequenced and the expression of ncRNA was analyzed for 1 month,3 months and adult sheep. The main results obtained in this study are as follows:1. With the increase of age, the diameter of the fine-fine tube of the cashmere goat was significantly increased (P0.05), and the 6-month-old reached 150.72. m u.m, slightly lower than that of the adult sheep (161.94. mu.m) (P0.05). The number of the cell layer of the cashmere goat increased with the increase of age, reached 7.70 in 6 months, and was significantly higher than that of 1 month and 3 months (P0.05). The spermatogonial cells in the white cashmere goat in the 3-month-old were the most abundant, accounting for 46.40% of the total number of cells. It was significantly higher than that of 1-month-old (33.10%),6-month-old (9.10%) and adult (7.60%) (P0.05). The total number of the cross-section cells of the curved fine-fine tube increased with age, and the total number of the cross-section cells of the 6-month-old and adult-cashmere goats was significantly higher than that of the 1-month-old and 3-month-old cashmere goats (P0.05). The results showed that the white cashmere goat reached sexual maturity at 6 months, and the spermatogonial cells in the 3-month-old testis were the most abundant. The positive rates of CD49f, CD90, GFRa1 and ETV5 in the testis of the 3-month-old cashmere goat were 41.67%, 40.20%, 21.92%, 25.36%, respectively. The positive rate of CD49f, CD90, GFRa1 and ETV5 in the testis of the 6-month-old cashmere goat was higher than that of the adult sheep. But the difference was not significant (P0.05). The positive rate of PLZF in the testis decreased with age and the difference was significant (P0.05). Spermatogonial stem cell marker genes (CD49f, PLZF, Thy1, ETV5, VASA, Oct4, Nanog, Nano2) were highly expressed in the 3-month-old testis tissue, of which PLZF, Thy1, ETV5, VASA, Nanog and Nano2 were significantly higher than those of 1 month,6 months and adult (P0.05). In addition, the expression of CyclinA in the testis of 6-month-old sheep was the highest, which was significantly higher than that of other groups (P0.05). The results showed that the concentration of SSCs in the testis of the 3-month-old sheep was the highest compared with that of the 1-month-old and 6-month-old sheep, and it was the ideal material for the in vitro separation and enrichment of SSCs. In that testis of the cashmere goat,17 of the lncRNA were express in the 1-month-and 3-month-old,13 of which were up-regulated and 4 were down-regulated. The difference between 1-month-and adult-year-old sheep was 247, of which 239 were up-regulated and 8 were down-regulated. The difference between 3-month-old and adult-sheep was 201. Of these,174 were up-regulated and 27 down-regulated. The results show that these differentially expressed lncRNA may be involved in the regulation of the development of spermatogenic cells. The expression of lncRNA in these differentially expressed lncRNA was found to play a role in the study of the effect of lncRNA on the process of spermatogenesis. The results showed that the concentration of SSCs in the testis of the 3-month-old sheep was the highest in the 3-month-old sheep's testis, and it was suitable as the SSCs-enriched material. In the 6-month-old white cashmere goat's testis, there are a large number of sperm cells, and the sexual maturity has been achieved. LncRNA was sequenced on 1-month-old,3-month-old and adult sheep's testis, and it was found that the lncRNA could be involved in the development and regulation of spermatogenic cells.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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本文編號(hào)：2453922