本文選題：男性不育 + 減數(shù)分裂�。� 參考：《安徽醫(yī)科大學》2016年博士論文

【摘要】：研究背景:人類精子的發(fā)生是由包括精原細胞的分裂和分化、精母細胞減數(shù)分裂、精子成熟和變形這一系列受到精確調(diào)控的連續(xù)過程組成的。其中任意一個過程出現(xiàn)問題,均會引起精子發(fā)生異常,進而引起少精子癥或無精子癥等生殖障礙的產(chǎn)生,誘發(fā)男性不育。影響精子發(fā)生的因素包括遺傳因素,激素水平調(diào)節(jié)和外界環(huán)境刺激等。精母細胞減數(shù)分裂Ⅰ前期同源染色體的配對、聯(lián)會、重組交換和交叉形成是精子發(fā)生的重要環(huán)節(jié)。減數(shù)分裂過程中,同源染色體不能正常配對、聯(lián)會、遺傳重組或交叉異常都會導致異常配子的發(fā)生。非梗阻性無精子癥(NOA)是男性不育中病因復雜、治療最為困難的一種病癥。目前很多NOA患者病因及機制仍然不明,導致對相關(guān)疾病的診斷、治療依然存在很大障礙。因此深入了解NOA患者遺傳學因素、發(fā)病原因和形成機制的研究對臨床工作很有必要。研究目的和意義:通過對NOA患者精母細胞免疫熒光染色,探討NOA患者精母細胞減數(shù)分裂Ⅰ前期同源染色體聯(lián)會與遺傳重組的進程,對特殊表型患者發(fā)病機制進行深入研究,以探索精子發(fā)生障礙的機制,為臨床診斷、治療和遺傳學咨詢提供理論依據(jù)。方法:對6例NOA患者和5例可育男性睪丸組織進行精母細胞鋪展和免疫熒光染色。用聯(lián)會復合體組分SCP3抗體顯示聯(lián)會復合體的形態(tài),用重組酶MLH1抗體來顯示重組位點,用著絲粒蛋白CREST抗體顯示著絲粒,分析NOA不育患者減數(shù)分裂Ⅰ前期同源染色體配對、聯(lián)會和重組情況。通過SCP3形態(tài)的分析,將減數(shù)分裂Ⅰ前期分為細線期、偶線期、粗線期及雙線期,并分析減數(shù)分裂的進程,同時通過對每條染色體MLH1位點數(shù)統(tǒng)計,確定減數(shù)分裂Ⅰ前期遺傳重組情況;此外分析易位染色體患者γ-H2AX和BRCA1的表達,并檢測易位所在染色體基因的表達;最后,應用外顯子測序技術(shù)檢測雄激素不敏感綜合征患者AR基因突變,并采用HE和免疫組化染色分析睪丸組織病理學特點。結(jié)果:對6例NOA患者精母細胞減數(shù)分裂Ⅰ各時期進行分析發(fā)現(xiàn),NOA患者P1細線期比率,患者P1、P2、P3和P4偶線期比率較對照組明顯增加,患者P1、P2和P4粗線期比率較對照組明顯減少(p0.05);患者P6未見精母細胞。共分析5例NOA患者326個粗線期精母細胞,其中每例患者細胞MLH1位點平均數(shù)目分別為42.2±12.6、48.1±6.5、49.5±5.0、44.0±4.8和45.1±7.0,可見P1和P4平均每個細胞MLH1位點數(shù)較對照組顯著減少(p0.05)。進一步分析發(fā)現(xiàn),P1患者是一例染色體易位患者,核型為46,X,t(Y;1)(p11.3;p31)。該患者睪丸活檢組織免疫熒光顯示其生精過程停滯于第一次減數(shù)分裂粗線期。易位染色體之間形成異常的四價體結(jié)構(gòu),并對其它未易位的正常染色體雙鏈斷裂(DSB)修復和聯(lián)會造成了一定影響。此外,由于易位染色體的影響,造成Y染色體和1號染色體斷裂點附近部分區(qū)段在減數(shù)分裂粗線期無法正常聯(lián)會,進而導致一些基因在粗線期受到了減數(shù)分裂不聯(lián)會染色體沉默機制(meiotic silencing of unsynapsed chromatin,MSUC)的影響,其中一些對減數(shù)分裂進程或細胞存活具有重要作用的基因無法正常表達,從而引起早粗線期精母細胞在的發(fā)育停滯和凋亡。另外,MLH1染色結(jié)果顯示,患者X和Y染色體PAR區(qū)重組率有明顯下降,這將導致第一次減數(shù)分裂前期染色體無法正常排列到赤道板上,進而激活紡錘體檢驗點,這可能是導致患者生精過程停滯的主要原因。P6病例染色體核型為正常的46,XY,根據(jù)表型特點,我們分析為雄激素完全不敏感綜合征(CAⅠS)。通過DNA測序發(fā)現(xiàn)該患者雄激素受體基因AR2號外顯子位置出現(xiàn)了錯義突變,C.1715AG(p.Y572C),導致AR蛋白572位的酪氨酸被半胱氨酸替代。我們對該患者睪丸進行組織和細胞學檢測,發(fā)現(xiàn)該患者多數(shù)曲細精管中僅見支持細胞(Sertoli cell),只在少數(shù)管腔中能發(fā)現(xiàn)極少量精原細胞。通過SOX9和AMH染色,我們發(fā)現(xiàn)該患者的支持細胞成熟障礙,說明AR基因的突變可導致AMH的高表達,進而影響Sertoli細胞的成熟。結(jié)論:NOA患者減數(shù)分裂過程出現(xiàn)延遲,粗線期精母細胞重組頻率有減少的現(xiàn)象,這些異�？赡軐е律系K。其中,染色體易位會造成減數(shù)分裂進程阻滯,染色體遺傳重組頻率降低和不聯(lián)會區(qū)域基因沉默,導致易位患者的精子發(fā)生障礙和不育;而AR基因突變會引發(fā)睪丸支持細胞成熟障礙,導致精子無法形成。因此,我們的研究證實精子發(fā)生不僅受生精細胞內(nèi)染色體結(jié)構(gòu)和基因表達的影響,也同樣需要支持細胞和外來激素信號的參與,其中任何一方面出現(xiàn)問題,均可能引起精子發(fā)生異常,最終導致男性不育。
[Abstract]:Background: the occurrence of human spermatozoa is composed of the division and differentiation of spermatogonial cells, meiosis of spermatocytes, sperm maturation and deformation, which are made up of a continuous process of precise regulation. In any process, any problem may cause abnormal sperm, resulting in oligospermia or azoospermia and other reproductive disorders. The factors affecting spermatogenesis include genetic factors, hormone level regulation and external environmental stimulation. The pairing of homologous chromosomes in the early stage of meiotic meiosis, association, recombination and cross formation is an important link in spermatogenesis. During the process of subtraction division, homologous chromosomes can not be matched normally. The association, genetic recombination or cross abnormalities all lead to the occurrence of abnormal gametes. Non obstructive azoospermia (NOA) is a complicated disease in male infertility and the most difficult treatment. The etiology and mechanism of many NOA patients are still unknown, leading to the diagnosis of related diseases, and there are still great obstacles in the treatment of NOA. The study of genetic factors, pathogenesis and formation mechanism is necessary for clinical work. The purpose and significance of this study are: the process of homologous chromosomes and genetic recombination in the prophase of the spermatocyte meiosis of NOA patients is studied by immunofluorescence staining of spermatocyte in NOA patients, and the pathogenesis of the patients with special phenotypes is studied in depth. In order to explore the mechanism of spermatogenesis disorder and provide theoretical basis for clinical diagnosis, treatment and genetic counseling. Methods: 6 NOA patients and 5 fertile male testicular tissues were spread out and immunofluorescent staining. The morphology of the association complex was displayed by the SCP3 antibody of the union complex, and the recombinant enzyme MLH1 antibody was used to display the recombination. Site, using centromere protein CREST antibody to display centromere, analysis of the homologous chromosome pairing, association and recombination in the early stage of meiosis of NOA infertile patients. Through the analysis of SCP3 morphology, the meiosis phase I was divided into fine line, dieven, roughing and double lines, and the process of meiosis was analyzed, and each chromosome ML was analyzed. H1 points statistics are used to determine the genetic recombination in the early stage of meiosis. In addition, the expression of gamma -H2AX and BRCA1 in translocation chromosomes and the expression of the chromosomal genes in the translocation are analyzed. Finally, exon sequencing is used to detect the AR gene mutation in the androgen insensitive syndrome patients and to analyze the testis by HE and immunohistochemical staining. Results: the analysis of the meiosis of spermatocyte in 6 patients with NOA showed that the ratio of P1 line phase in NOA patients, the ratio of P1, P2, P3 and P4 in patients was significantly higher than that of the control group, and the ratio of P1, P2 and P4 in the patients was significantly less than that of the control group (P0.05), and the patients P6 did not have spermatocytes. A total of 5 cases were analyzed. The average number of MLH1 loci in each patient was 42.2 + 12.6,48.1 + 6.5,49.5 + 5.0,44.0 + 4.8 and 45.1 + 7, respectively. The average number of MLH1 points per cell of P1 and P4 was significantly lower than that of the control group (P0.05). Further analysis showed that the P1 patient was a case of chromosome translocation, and the karyotype was 46, X, t (Y; 1). 1.3; P31). The immunofluorescence of the biopsy tissue of the testis showed that the process of spermatogenesis stagnated at the first meiotic roughing phase. The abnormal tetravalent structure between the translocation chromosomes was formed and a definite effect on other untranslocated normal chromosome double strand breaks (DSB) repair and association. In addition, the effect of translocation chromosomes caused Y Some of the segments of chromosomes and chromosome 1 in the vicinity of the cleavage point can not be normally associated in the meiotic roughing period, which leads to the effect that some genes are affected by the meiotic silencing of unsynapsed chromatin, MSUC in the roughing period, some of which are heavy for meiosis process or cell survival. In addition, MLH1 staining results showed that the recombination rate of X and Y chromosomes in the PAR region of the patients decreased significantly, which would lead to the failure of the chromosomes to be properly arranged on the equatorial plate at the first meiotic pre meiosis and then activate the spindle examination points. The main cause of stagnation in the process of spermatogenesis in the patient was.P6 chromosome karyotype of normal 46, XY. According to the phenotypic characteristics, we analyzed the androgen completely insensitive syndrome (CA I S). Through DNA sequencing, it was found that the position of the exon AR2 of the androgen receptor gene of the androgen receptor gene appeared missense mutation, C.1715AG (p.Y572C), resulting in AR protein 5. 72 cases of tyrosine were replaced by cysteine. We detected the tissue and cytology of the testis of the patient. Most of the patients were found to have only support cells (Sertoli cell) in the seminiferous tubules. Only a few spermatogonial cells were found in a small number of lumens. SOX9 and AMH staining, we found the patient's support cell maturity barrier, indicating the AR base. Mutation can lead to high expression of AMH and further affect the maturation of Sertoli cells. Conclusion: the meiotic process of NOA patients is delayed and the recombination frequency of roughening spermatocytes is reduced. These abnormalities may lead to spermatogenesis disorder. The gene silencing in and in the non union region causes the spermatogenesis and infertility of the translocation patients, and the mutation of the AR gene causes the maturation obstacle of the testis supporting cells, which leads to the failure of the sperm to form. Therefore, our study confirms that spermatogenesis is not only influenced by the structure of chromosomes and the expression of basic genes in spermatogenic cells, but also needs to support cells and outside the spermatogenesis. The involvement of hormonal signals in any of these problems may lead to abnormal spermatogenesis and eventually lead to male infertility.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R698.2

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