种公鸡弱（无）精症发生的生理与分子机制初步研究

发布时间：2018-05-24 00:35

本文选题：鸡弱(无)精症 + 精液品质　；参考：《扬州大学》2015年硕士论文

【摘要】：弱(无)精种公鸡因繁殖能力低下而被迫淘汰,据报道,在群体中无精症种公鸡约占10%-20%。而目前对于鸡无精症发生机制研究鲜有报道,致使此类问题未得到有效、根本的解决,这给我国正处在发展关键时刻的养禽业带来一定损失。因此,对于影响种公鸡弱(无)精症的作用机制探究已是势在必行。本研究是以雪山鸡为研究对象,分别从生理、基因表达、转录调控等3个方面对鸡无精子症的发生机制进行较为系统的探究过程。首先利用生长指标测定、精液品质测定及组织学观察等方法将鸡群分为弱(无)精子鸡群体和正常生精鸡群体；Piwi基因作为睾丸特异性表达基因,在精子生成过程中具有重要作用,缺失导致不能形成成熟精子,因此通过实时荧光定量PCR(Real-Time qPCR, RT-qPCR)法,检测Piwi-element induced wimpy testis)基因在正常、弱(无)精症鸡中不同类型生精细胞中mRNA表达差异,探讨Piwill基因表达水平与产精性能的关系；同时本研究利用PCR-SSCP的方法对Piwill基因的核心启动子区和第一外显子区序列共531bp区域进行遗变异位点检测,并分析无精症鸡群体中变异位点对基因转录水平和产精性能的影响,以期寻找有价值的分子遗传标记。最后采用BSP-克隆测序法对该区域CG二核苷酸位点分布情况进行检测,进一步确定其甲基化位点,以期发现在精子发生过程中各级生精细胞中Piwill基因的表达与甲基化调控间的关系,进而阐明Piwill基因在鸡精子发生过程中的转录调控规律。实验结果如下：1.根据鸡群对按摩法的反应、产精情况及精液品质将鸡群分为正常、反应强烈且有精子、反应强烈且无精子、无反应且无精液等四个组别,分别标记为组1、组2、组3和组4。相比之下,组1的精子活力、精子活率显著高于组2、组3(P0.05)；且组间精子畸形率差异显著(P0.05)。在不同时间点不同组间的FSH、LH、To、TSHR血浆激素水平存在差异。2.在组1中鸡睾丸中的曲细精管上皮结构完整,在组2中鸡的曲细精管结构中,未见精子细胞；在组3中未见次级精母细胞及精子细胞；在组4中未见初级、次级精母细胞及精子细胞,仅在靠近基膜存在精原细胞,且深染的精原细胞极少。由此可见,曲细精管组织结构损害较严重,仅有少量精原细胞,精子细胞和精母细胞缺失、稀少,致使睾丸组织生精功能丧失。3.检测Piwill基因在正常、无精症鸡中不同类型生精细胞中mRNA表达差异,发现Piwill在正常鸡无精症鸡群中的表达存在显著差异,且从胚胎中分离的干细胞的Piwill表达水平较精原细胞低,且在成熟精子中几乎不表达,进一步表明Piwill很可能参与精子发生的减数分裂过程。4.本实验扩增出鸡群Piwill基因5’调控区及第1外显子部分序列总长531bp区域,筛选出2个SNPs;同时检测出鸡Piwill基因启动子区-233～+298bp区域存在一个CpG岛,该岛有56个CG二核昔酸位点。第1～10个CG二核苷酸位点(-233～-129bp区域),精子细胞、四倍体细胞、PGCs、SSCs中处于未甲基化状态,而精原细胞中甲基化比例高达0.600；第39～55个CG二核苷酸位点(+105～+252bp区域),PGCs和SSCs中甲基化比例高于精子细胞、精原细胞、四倍体细胞。PGCs、SSCs中DNA高甲基化在一定程度上抑制了Piwill基因的表达。综上,本研究从生理、基因表达、转录调控等3个方面进行弱(无)精症公鸡与正常鸡差异研究,发现鸡曲细精管上皮结构受损程度与其精子发生呈负相关；Piwill基因在精子中表达量最低,在精原细胞的表达量显着高于PGCs、SSCs和四倍体；同时在启动子区筛选出2个SNPs,且-151bp处的SNPs产生了1个新的转录因子结合位点,但其偏离核心启动子区,检测目标序列发现CpG岛各位点的甲基化情况,预测存在该SNP位点通过影响DNA甲基化水平而调控Piwill基因表达的可能性。研究结果为进一步分析鸡弱(无)精症发生的遗传机制奠定前期基础。
[Abstract]:The weak (non) spermatosperm cock is forced to be eliminated because of the low reproductive capacity. It is reported that there are few reports on the mechanism of the azoospermia cocks in the population, and the mechanism of the chicken azoospermia is rarely reported. The problem is not effective, and the fundamental solution is to bring some loss to the poultry industry at the critical time of development in China. Therefore, the 10%-20%. It is imperative to explore the mechanism of the influence of the weak (non) spermatospermia of the cock. This study is based on the 3 aspects of the physiology, gene expression and transcriptional regulation of chicks. First, the growth index, the quality of semen and the histology are used to determine the mechanism of the chicken azoospermia. The chicken group is divided into weak (no) sperm chicken population and normal spermatogenic chicken population. Piwi gene, as a testicular specific expression gene, plays an important role in the process of spermatogenesis, and the deletion leads to the failure to form mature sperm. Therefore, Piwi-element induced wimpy tes is detected by real-time quantitative PCR (Real-Time qPCR, RT-qPCR) method. TIS) mRNA expression in different types of spermatogenic cells in normal and weak (non) spermatospermia chicken, the relationship between the expression level of Piwill gene and the performance of spermatogenesis is discussed. At the same time, this study uses the method of PCR-SSCP to detect the ectopic sites in the core promoter region of the Piwill gene and the sequence of the first exon region, and to analyze the inseminosinosis. The effect of variable ectopic spots on gene transcription and spermatogenesis in the group of chickens in order to find valuable molecular genetic markers. Finally, BSP- cloned sequencing was used to detect the distribution of CG dinucleotide loci in this region, and the methylation site was further determined in order to develop the Piwi in spermatogenesis of the present spermatogenesis. The relationship between the expression of ll gene and the regulation of methylation, and then clarifying the regulation of Piwill gene in the process of chicken spermatogenesis. The experimental results are as follows: 1. according to the reaction of the chicken group to the massage method, the production of spermatogenesis and the quality of the semen are divided into normal, strong reaction and spermatoson, strong reaction and no sperm, no reaction and no semen. Four groups were marked as group 1, group 2, group 3 and group 4., compared with group 1, sperm viability was significantly higher than group 2, and group 3 (P0.05), and the difference of sperm malformation rates between groups was significant (P0.05). The plasma levels of FSH, LH, To and TSHR in different groups of time points were different in.2. in the seminiferous tubule epithelium of chicken testis in group 1. In group 2, there was no spermatocyte and spermatocyte in group 3. No primary spermatocyte and spermatocyte were not found in group 4. There was no primary, secondary spermatocyte and spermatocyte in group 4. There were only spermatogonial cells near the basement membrane, and the deep stained spermatogonial cells were very few. Only a small amount of spermatogonial cells, spermatocytes and spermatocytes are missing and rare, resulting in the loss of spermatogenic function of the testis by.3. detection of the Piwill gene in normal, different types of spermatogenic cells in the azoospermia chicken and the difference in the expression of mRNA in the normal chicken azoospermia chicken group, and the P of the stem cells isolated from the embryo. The expression level of Iwill is lower than that of spermatogonial cells and is almost not expressed in mature spermatozoa. It further indicates that Piwill is very likely to participate in the meiosis process of spermatogenesis..4. experiment has amplified the 531bp region of the chicken group Piwill gene 5 'regulation region and the 1 exon part sequence, and screened 2 SNPs; meanwhile, the chicken Piwill gene promoter was detected. There is a CpG island in the region from -233 to +298bp, which has 56 CG two nucleotides. First to 10 CG dinucleotide loci (-233 ~ -129bp region), spermatids, tetraploid cells, PGCs, SSCs are in the non methylation state, and the methylation ratio in spermatogonial cells is up to 0.600; thirty-ninth to 55 CG dinucleotide loci (+105 ~ +252bp region) The proportion of methylation in GCs and SSCs was higher than that of sperm cells, spermatogonial cells, tetraploid cells.PGCs, and DNA hypermethylation in SSCs inhibited the expression of Piwill gene to a certain extent. In this study, the differences of the weak (non) spermatogonial cocks and normal chickens were studied from 3 aspects of physiology, gene expression and transcription regulation, and the epithelia of chicken fininal tubules was found. The degree of damage to the spermatogenesis was negatively correlated with the spermatogenesis, and the expression of Piwill gene was lowest in spermatozoa. The expression of spermatogonial cells was significantly higher than that of PGCs, SSCs and tetraploid. At the same time, 2 SNPs were screened in the promoter region, and the SNPs at -151bp produced 1 NEW transcription factor binding sites, but it deviated from the core promoter region and detected the target sequence. The methylation of CpG island sites was found to predict the possibility that the SNP locus could regulate the expression of Piwill gene by affecting the level of DNA methylation. The results provided a preliminary basis for further analysis of the genetic mechanism of the occurrence of chicken weak (no) spermatogenesis.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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