山羊生精细胞发育及睾丸组织lncRNA差异表达分析
发布时间：2019-04-04 14:56
【摘要】：对于雄性动物而言,睾丸发育直接影响其性成熟早晚及精液品质,从而决定雄性动物繁殖性能。精子发生起始于精原干细胞(Spermatogonial stem cells,SSCs)的自我更新和分化,睾丸组织中SSCs分布反映了其组织发育程度,组织学分析可直接观察睾丸结构变化规律及生精细胞分布情况。lncRNA(long noncoding RNA,lncRNA)是一类转录本长度在200 nt以上的非编码RNA,表达丰度较低,只在细胞发育、分化的特定阶段表达,具有明显的组织细胞表达特异性。lncRNA参与决定干细胞命运,包括维持自我更新和分化,但lncRNA调控SSCs增殖与分化的研究少有报道。为探索陕北白绒山羊不同发育期睾丸组织lncRNA表达差异,本试验取1月龄、3月龄、6月龄及成年陕北白绒山羊睾丸组织制作切片,进行苏木精-伊红染色,镜下观察曲细精管形态,统计各生殖细胞数,分析不同发育阶段陕北白绒山羊睾丸发育程度;同时进行免疫组织化学染色,并用q-PCR检测了发育阶段睾丸中SSCs标记基因(CD49f、PLZF、Thy1、ETV5、VASA、Oct4、Nanog、Nanos2、CyclinA)的相对表达量,以分析不同发育阶段陕北白绒山羊SSCs相对丰度;对1月龄、3月龄及成年羊睾丸组织进行lncRNA测序并分析lncRNA差异表达。本研究获得的主要研究结果如下:1.随着年龄的增长,绒山羊曲细精管直径显著增大(P0.05),6月龄达到150.72μm,略低于成年羊(161.94μm)(P0.05)。绒山羊细胞层数随年龄的增长而增长,6月龄达到7.70层,显著高于1月龄和3月龄(P0.05)。3月龄陕北白绒山羊曲细精管中精原细胞最为丰富,占总细胞数的46.40%,明显高于1月龄(33.10%)、6月龄(9.10%)和成年羊(7.60%)(P0.05)。曲细精管横截面细胞总数随年龄增长而增长,6月龄和成年绒山羊曲细精管横截面细胞总数显著高于1月龄和3月龄绒山羊(P0.05)。结果表明,陕北白绒山羊在6月龄达到性成熟,3月龄睾丸中精原细胞最为丰富。2.精原干细胞标记因子CD49f、CD90、GFRa1及ETV5在3月龄绒山羊睾丸中阳性率较高,分别为41.67%、40.20%、21.92%、25.36%,且显著高于6月龄和成年羊(P0.05);6月龄绒山羊睾丸中CD49f、CD90、GFRa1及ETV5阳性率比成年羊略高,但差异不显著(P0.05)。PLZF在睾丸中阳性率随年龄增长而降低,差异显著(P0.05)。精原干细胞标记基因(CD49f、PLZF、Thy1、ETV5、VASA、Oct4、Nanog、Nanos2)均在3月龄睾丸组织中高表达,其中PLZF、Thy1、ETV5、VASA、Nanog及Nanos2显著高于1月龄、6月龄及成年羊(P0.05)。另外,CyclinA在6月龄羊睾丸中表达量最高,显著高于其他组(P0.05)。结果表明,与1月龄、6月龄和成年羊相比,3月龄羊睾丸中SSCs浓度最高,是SSCs体外分离富集的理想材料。3.在绒山羊睾丸组织中,1月龄与3月龄差异表达lncRNA共17个,其中13个表达上调,4个表达下调。1月龄与成年羊差异表达lncRNA共247个,其中239个表达上调,8个表达下调。3月龄与成年羊差异表达lncRNA共201个,其中174个表达上调,27个表达下调。结果表明这些差异表达的lncRNA可能参与调控生精细胞发育。对这些差异表达的lncRNA进行分析,发现这些lncRNA通过各种途径发挥作用,为lncRNA对生精过程作用的研究奠定了基础。本研究表明,与1月龄、6月龄、成年陕北白绒山羊相比,3月龄羊睾丸中SSCs浓度最高,适合作为SSCs富集材料。在6月龄陕北白绒山羊睾丸中有大量精子细胞,已经达到性成熟。对1月龄、3月龄及成年羊睾丸组织进行lncRNA测序,发现有lncRNA差异表达,说明这些lncRNA可能参与生精细胞发育调控。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827
本文编号：2453922
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827
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