精子发生过程中NXF3和Crossover Interference的功能研究

发布时间：2018-05-18 23:01

本文选题：男性不育 + BTB　；参考：《中国科学技术大学》2012年博士论文

【摘要】：不育症是影响人类健康的一种常见重大疾病,其发病率很高,然而大部分的非梗阻性无精症(non-obstructive azoospermia,NOA)患者病因不明,临床治疗时无从下手。多年来研究发现NOA患者发病原因可能与睾丸中血-睾屏障(Blood-testis barrier,BTB)的缺陷和减数分裂过程中同源染色体同源重组异常有关,但对这两个事件发生异常的分子调控机理仍然不太清楚。曲精小管中由相邻支持细胞构成的血-睾屏障(BTB)会在生精上皮周期中的第Ⅷ期、第Ⅸ期经历一个破坏、重建的过程来帮助前细线期或细线期的精母细胞完成由basal compartments向adlummal compartments的迁移。此过程的异常会导致前细线期或细线期的精母细胞不能进入adluminal compartments完成减数分裂或者是引起抗自身精细胞抗体的产生,从而引起不育。鉴于BTB破坏、重建的过程受到细胞因子TGF-β3的调控,本文的第一个研究课题是寻找影响TGF-β3表达、分泌的因素及其分子机制。本文研究发现核输出因子蛋白家族(NXF)成员之一的NXF3特异性表达在睾丸组织中的支持细胞的细胞之中,并且它起始表达的时间和小鼠青春期BTB开始建立时间一致；除睾丸中的支持细胞外,NXF3还特异性表达在附睾头部Ⅱ区的主细胞中,起始表达的时间和第一次生精波完成后睾丸液进入附睾的时间一致,并且它在附睾中的表达是睾丸液依赖。探寻NXF3和TGF-β3在不同情况下的表达情况时发现,无论是生理条件还是病理条件,如热刺激、CdCl2处理,二者的表达均呈现负相关。进一步研究发现在支持细胞中,TGF-β信号通路可以对细胞因子TGF-β3的表达进行转录负调控,并且这种转录负调控是NXF3部分依赖的。具体来讲是,TGF-β信号通路可以通过激活Smad2/3途径来抑制TGF-β3的表达,NXF3的可通过增强细胞内Smad2/3途径的径活性从而进一步抑制细胞内TGF-β3的表达。接着我们对NXF3影响TGF-β信号通路的分子机制进行了研究,发现NXF3可以与TGF-β信号通路的抑制因子STRAP结合,二者的相互作用可以通过破坏STRAP与Smad7的结合来影响TβR1(TD)-STRAP-Smad7复合体的形成,从而增强了细胞内TGF-β-Smad信号通路活性。除参与调节TGF-β3的表达、分泌外,我们发现NXF3还可以通过影响TGFβ-Smad信号通路参与调节支持细胞中其它重要基因的表达,如与BTB相关的基因Claudin1、WT1以及与支持细胞分化发育相关的基因GATA1、p21. 减数分裂过程中同源重组发生在减数分裂前期Ⅰ,是在同源配对的非姐妹染色单体之间进行的,它的发生可以确保减数分裂过程中染色体的正确分离。同源重组是个比较复杂的过程,涉及DSBs形成、单链DNA末端入侵、中间体Holliday交叉形成等一系列过程,最后在同源配对染色体间的形成交换位点(Crossovers, COs,由粗线期联会复合体上的MLH1蛋白标记)。COs形成过程中受到正干涉的调控,即一个COs的形成排斥其它COs在其附近形成,本文的第二个课题中对人类染色体上COs间的干涉特征及其影响因素进行研究。首先,通过综合运用细胞免疫荧光技术和多色荧光原位杂交技术(cenM-FISH)对每条粗线期联会复合体(synaptonemal complexs, SCs)及SCs上的COs进行了标记,测量出相邻Cos之间的距离并拟合到gamma model中计算出用于定量交换干涉强度的参数v,而后对其进行分析发现干涉强度在染色体内、染色体间和个体间均存在异质性,具体来讲就是：染色体上臂间干涉强度大于臂内干涉强度,这可能是由着丝粒引起的；小号染色体的干涉强度大于大号染色体上的干涉强度；个体之间的每条染色体上的干涉强度也存在变异,但没发现规律。进一步分析显示,SCs的结构异常会影响Cos间的干涉强度,比如9号染色体上易出现的不连续区域(gaps)和未联会区域(splits)两种结构异常会cis/trans影响本身和其它染色体上干涉强度。另外,我们的研究也表明Cos间的干涉可通过着丝粒发挥作用。综上所述,本文研究了NXF3调控细胞因子TGF-p3表达、分泌中的分子机制及正常精母细胞中COs间干涉的分布模式,这些结果有助于进一步探讨NOA患者中血睾屏障异常以及同源重组缺陷的原因。
[Abstract]:Infertility is a common and major disease affecting human health. The incidence of the disease is very high. However, most of the patients with non-obstructive azoospermia (NOA) are unknown in etiology, and they are ineffective in clinical treatment. For years, the study found that the causes of NOA patients can be associated with the blood testosterone barrier (Blood-testis barrier, BTB) in the testicles. Defects and meiosis are related to the homologous recombination of homologous chromosomes, but the molecular regulation mechanism of these two events is still not clear.
In the seminiferous tubule, the blood testosterone barrier (BTB), composed of adjacent supporting cells, will be in stage VIII in the spermatogenic epithelium cycle. The period IX experience a destruction and reconstructive process to help the spermatocytes from basal compartments to adlummal compartments in the prior fine line or fine line period. Fine line spermatocytes can not enter adluminal compartments to complete meiosis or cause anti self spermatocyte antibody production and cause infertility. In view of BTB destruction, the process of reconstruction is regulated by cytokine TGF- beta 3. The first research topic in this paper is to find the factors affecting the expression and secretion of TGF- beta 3 and its molecular machine. System.
This paper has found that NXF3, one of the members of the nuclear output factor protein family (NXF), is specifically expressed in the cells of the supporting cells in the testicular tissue, and the time of its initial expression is consistent with the time of the establishment of BTB in the puberty of mice; in addition to the supporting cells in the testis, NXF3 is also specifically expressed in the main cells of the epididymal head II region. In the epididymis, the time of the initial expression is consistent with the time when the first secondary spermatologic wave is completed into the epididymis, and its expression in the epididymis is a testicular fluid dependence. The expression of NXF3 and TGF- beta 3 in different conditions is found to be negative in both physiological and pathological conditions, such as thermal stimulation, CdCl2 treatment, and the expression of the two. Further studies have found that TGF- beta signaling pathway can regulate the expression of cytokine TGF- beta 3 in support cells, and this negative regulation is partly dependent on NXF3. Specifically, the TGF- beta signaling pathway can inhibit the expression of TGF- beta 3 by activating the Smad2/3 pathway, and NXF3 can enhance the intracellular Smad2/ by using the Smad2/3 pathway. The diameter activity of the 3 pathway further inhibits the expression of TGF- beta 3 in the cell. Then we have studied the molecular mechanism of the effect of NXF3 on the TGF- beta signaling pathway, and found that NXF3 can bind to the inhibitory factor STRAP of the TGF- beta signaling pathway, and the interaction of the two can affect the T beta R1 (TD) -STRAP-Smad7 complex by destroying the binding of STRAP to Smad7. In addition to regulating the expression of TGF- beta 3 and secreting, we found that NXF3 can also regulate the expression of other important genes in the supporting cells by affecting the TGF beta -Smad signaling pathway, such as BTB related Claudin1, WT1, and the differentiation and development phase of the supporting cells, in addition to the regulation of the expression of TGF- beta 3. The gene GATA1, p21.
Homologous recombination occurs during meiosis in the prophase of meiosis, which is between the homologous pair of non sister chromatid monomers. The occurrence of homologous recombination can ensure the correct separation of chromosomes during meiosis. Homologous recombination is a complex process involving the formation of DSBs, the single strand DNA terminal invasion, and the interintermediate Holliday cross. In a series of processes, the formation of the exchange site between the homologous chromosomes (Crossovers, COs, MLH1 protein markers on the roughen phase complex) is regulated by positive interference, namely, the formation of a COs formation that excludes other COs from its formation. In the second subjects of this article, the COs between the human chromosomes is on the human chromosome. The characteristics of interference and its influencing factors are studied.
First, by using cellular immunofluorescence technology and polychromatic fluorescence in situ hybridization (cenM-FISH) to mark each synaptonemal complexs (SCs) complex (SCs) and SCs, the distance between adjacent Cos is measured and the parameter V, which is used to calculate the intensity of the quantitative exchange interference, is calculated in gamma model. After analyzing it, it is found that interference intensity is heterogeneous in chromosomes, between chromosomes and between individuals. Specifically, the intensity of interarm interference in the upper arm is greater than that of the interarm interference, which may be caused by centromere; the interference intensity of the trumpet chromosome is greater than the interference intensity on the large chromosome; The intensity of interference on each chromosome is also mutable, but no regularity is found. Further analysis shows that the structural abnormalities of SCs affect the interference intensity between Cos, such as the discontinuous region of the 9 chromosome (gaps) and the two structural abnormalities in the unassociated region (splits), which affect the interference intensity of the other chromosomes and the interference intensity on the other chromosomes. Our research also shows that interference between Cos can play a role through centromere.
In summary, this paper studies the NXF3 regulation of the expression of cytokine TGF-p3, the molecular mechanism of secretion and the distribution pattern of interference between COs in normal spermatocytes. These results are helpful to further explore the abnormal blood testis barrier and the cause of homologous recombination in NOA patients.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R339.2

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