本文選題：雞弱(無)精癥 + 精液品質(zhì)��； 參考：《揚州大學》2015年碩士論文

【摘要】：弱(無)精種公雞因繁殖能力低下而被迫淘汰,據(jù)報道,在群體中無精癥種公雞約占10%-20%。而目前對于雞無精癥發(fā)生機制研究鮮有報道,致使此類問題未得到有效、根本的解決,這給我國正處在發(fā)展關鍵時刻的養(yǎng)禽業(yè)帶來一定損失。因此,對于影響種公雞弱(無)精癥的作用機制探究已是勢在必行。本研究是以雪山雞為研究對象,分別從生理、基因表達、轉(zhuǎn)錄調(diào)控等3個方面對雞無精子癥的發(fā)生機制進行較為系統(tǒng)的探究過程。首先利用生長指標測定、精液品質(zhì)測定及組織學觀察等方法將雞群分為弱(無)精子雞群體和正常生精雞群體；Piwi基因作為睪丸特異性表達基因,在精子生成過程中具有重要作用,缺失導致不能形成成熟精子,因此通過實時熒光定量PCR(Real-Time qPCR, RT-qPCR)法,檢測Piwi-element induced wimpy testis)基因在正常、弱(無)精癥雞中不同類型生精細胞中mRNA表達差異,探討Piwill基因表達水平與產(chǎn)精性能的關系；同時本研究利用PCR-SSCP的方法對Piwill基因的核心啟動子區(qū)和第一外顯子區(qū)序列共531bp區(qū)域進行遺變異位點檢測,并分析無精癥雞群體中變異位點對基因轉(zhuǎn)錄水平和產(chǎn)精性能的影響,以期尋找有價值的分子遺傳標記。最后采用BSP-克隆測序法對該區(qū)域CG二核苷酸位點分布情況進行檢測,進一步確定其甲基化位點,以期發(fā)現(xiàn)在精子發(fā)生過程中各級生精細胞中Piwill基因的表達與甲基化調(diào)控間的關系,進而闡明Piwill基因在雞精子發(fā)生過程中的轉(zhuǎn)錄調(diào)控規(guī)律。實驗結(jié)果如下：1.根據(jù)雞群對按摩法的反應、產(chǎn)精情況及精液品質(zhì)將雞群分為正常、反應強烈且有精子、反應強烈且無精子、無反應且無精液等四個組別,分別標記為組1、組2、組3和組4。相比之下,組1的精子活力、精子活率顯著高于組2、組3(P0.05)；且組間精子畸形率差異顯著(P0.05)。在不同時間點不同組間的FSH、LH、To、TSHR血漿激素水平存在差異。2.在組1中雞睪丸中的曲細精管上皮結(jié)構(gòu)完整,在組2中雞的曲細精管結(jié)構(gòu)中,未見精子細胞；在組3中未見次級精母細胞及精子細胞；在組4中未見初級、次級精母細胞及精子細胞,僅在靠近基膜存在精原細胞,且深染的精原細胞極少。由此可見,曲細精管組織結(jié)構(gòu)損害較嚴重,僅有少量精原細胞,精子細胞和精母細胞缺失、稀少,致使睪丸組織生精功能喪失。3.檢測Piwill基因在正常、無精癥雞中不同類型生精細胞中mRNA表達差異,發(fā)現(xiàn)Piwill在正常雞無精癥雞群中的表達存在顯著差異,且從胚胎中分離的干細胞的Piwill表達水平較精原細胞低,且在成熟精子中幾乎不表達,進一步表明Piwill很可能參與精子發(fā)生的減數(shù)分裂過程。4.本實驗擴增出雞群Piwill基因5’調(diào)控區(qū)及第1外顯子部分序列總長531bp區(qū)域,篩選出2個SNPs;同時檢測出雞Piwill基因啟動子區(qū)-233～+298bp區(qū)域存在一個CpG島,該島有56個CG二核昔酸位點。第1～10個CG二核苷酸位點(-233～-129bp區(qū)域),精子細胞、四倍體細胞、PGCs、SSCs中處于未甲基化狀態(tài),而精原細胞中甲基化比例高達0.600；第39～55個CG二核苷酸位點(+105～+252bp區(qū)域),PGCs和SSCs中甲基化比例高于精子細胞、精原細胞、四倍體細胞。PGCs、SSCs中DNA高甲基化在一定程度上抑制了Piwill基因的表達。綜上,本研究從生理、基因表達、轉(zhuǎn)錄調(diào)控等3個方面進行弱(無)精癥公雞與正常雞差異研究,發(fā)現(xiàn)雞曲細精管上皮結(jié)構(gòu)受損程度與其精子發(fā)生呈負相關；Piwill基因在精子中表達量最低,在精原細胞的表達量顯著高于PGCs、SSCs和四倍體；同時在啟動子區(qū)篩選出2個SNPs,且-151bp處的SNPs產(chǎn)生了1個新的轉(zhuǎn)錄因子結(jié)合位點,但其偏離核心啟動子區(qū),檢測目標序列發(fā)現(xiàn)CpG島各位點的甲基化情況,預測存在該SNP位點通過影響DNA甲基化水平而調(diào)控Piwill基因表達的可能性。研究結(jié)果為進一步分析雞弱(無)精癥發(fā)生的遺傳機制奠定前期基礎。
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31

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