非梗阻性无精子症减数分裂的研究

发布时间：2018-06-29 14:44

本文选题：男性不育 + 减数分裂　；参考：《安徽医科大学》2016年博士论文

【摘要】：研究背景:人类精子的发生是由包括精原细胞的分裂和分化、精母细胞减数分裂、精子成熟和变形这一系列受到精确调控的连续过程组成的。其中任意一个过程出现问题,均会引起精子发生异常,进而引起少精子症或无精子症等生殖障碍的产生,诱发男性不育。影响精子发生的因素包括遗传因素,激素水平调节和外界环境刺激等。精母细胞减数分裂Ⅰ前期同源染色体的配对、联会、重组交换和交叉形成是精子发生的重要环节。减数分裂过程中,同源染色体不能正常配对、联会、遗传重组或交叉异常都会导致异常配子的发生。非梗阻性无精子症(NOA)是男性不育中病因复杂、治疗最为困难的一种病症。目前很多NOA患者病因及机制仍然不明,导致对相关疾病的诊断、治疗依然存在很大障碍。因此深入了解NOA患者遗传学因素、发病原因和形成机制的研究对临床工作很有必要。研究目的和意义:通过对NOA患者精母细胞免疫荧光染色,探讨NOA患者精母细胞减数分裂Ⅰ前期同源染色体联会与遗传重组的进程,对特殊表型患者发病机制进行深入研究,以探索精子发生障碍的机制,为临床诊断、治疗和遗传学咨询提供理论依据。方法:对6例NOA患者和5例可育男性睾丸组织进行精母细胞铺展和免疫荧光染色。用联会复合体组分SCP3抗体显示联会复合体的形态,用重组酶MLH1抗体来显示重组位点,用着丝粒蛋白CREST抗体显示着丝粒,分析NOA不育患者减数分裂Ⅰ前期同源染色体配对、联会和重组情况。通过SCP3形态的分析,将减数分裂Ⅰ前期分为细线期、偶线期、粗线期及双线期,并分析减数分裂的进程,同时通过对每条染色体MLH1位点数统计,确定减数分裂Ⅰ前期遗传重组情况;此外分析易位染色体患者γ-H2AX和BRCA1的表达,并检测易位所在染色体基因的表达;最后,应用外显子测序技术检测雄激素不敏感综合征患者AR基因突变,并采用HE和免疫组化染色分析睾丸组织病理学特点。结果:对6例NOA患者精母细胞减数分裂Ⅰ各时期进行分析发现,NOA患者P1细线期比率,患者P1、P2、P3和P4偶线期比率较对照组明显增加,患者P1、P2和P4粗线期比率较对照组明显减少(p0.05);患者P6未见精母细胞。共分析5例NOA患者326个粗线期精母细胞,其中每例患者细胞MLH1位点平均数目分别为42.2±12.6、48.1±6.5、49.5±5.0、44.0±4.8和45.1±7.0,可见P1和P4平均每个细胞MLH1位点数较对照组显著减少(p0.05)。进一步分析发现,P1患者是一例染色体易位患者,核型为46,X,t(Y;1)(p11.3;p31)。该患者睾丸活检组织免疫荧光显示其生精过程停滞于第一次减数分裂粗线期。易位染色体之间形成异常的四价体结构,并对其它未易位的正常染色体双链断裂(DSB)修复和联会造成了一定影响。此外,由于易位染色体的影响,造成Y染色体和1号染色体断裂点附近部分区段在减数分裂粗线期无法正常联会,进而导致一些基因在粗线期受到了减数分裂不联会染色体沉默机制(meiotic silencing of unsynapsed chromatin,MSUC)的影响,其中一些对减数分裂进程或细胞存活具有重要作用的基因无法正常表达,从而引起早粗线期精母细胞在的发育停滞和凋亡。另外,MLH1染色结果显示,患者X和Y染色体PAR区重组率有明显下降,这将导致第一次减数分裂前期染色体无法正常排列到赤道板上,进而激活纺锤体检验点,这可能是导致患者生精过程停滞的主要原因。P6病例染色体核型为正常的46,XY,根据表型特点,我们分析为雄激素完全不敏感综合征(CAⅠS)。通过DNA测序发现该患者雄激素受体基因AR2号外显子位置出现了错义突变,C.1715AG(p.Y572C),导致AR蛋白572位的酪氨酸被半胱氨酸替代。我们对该患者睾丸进行组织和细胞学检测,发现该患者多数曲细精管中仅见支持细胞(Sertoli cell),只在少数管腔中能发现极少量精原细胞。通过SOX9和AMH染色,我们发现该患者的支持细胞成熟障碍,说明AR基因的突变可导致AMH的高表达,进而影响Sertoli细胞的成熟。结论:NOA患者减数分裂过程出现延迟,粗线期精母细胞重组频率有减少的现象,这些异常可能导致生精障碍。其中,染色体易位会造成减数分裂进程阻滞,染色体遗传重组频率降低和不联会区域基因沉默,导致易位患者的精子发生障碍和不育;而AR基因突变会引发睾丸支持细胞成熟障碍,导致精子无法形成。因此,我们的研究证实精子发生不仅受生精细胞内染色体结构和基因表达的影响,也同样需要支持细胞和外来激素信号的参与,其中任何一方面出现问题,均可能引起精子发生异常,最终导致男性不育。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R698.2

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