NXF3和CaMKIV在精子发生过程中的功能研究

发布时间：2018-06-29 08:45

本文选题：男性不育 + 血睾屏障　；参考：《中国科学技术大学》2014年博士论文

【摘要】：在世界范围内大约有10%-15%的育龄夫妇正在遭受不育症的折磨。不育症夫妇中40%的男性是不育的,并且随着环境和社会等因素的影响,男性不育的比例正在逐渐增加。男性不育患者大部分为非梗阻性无精症(non-obstructive azoospermia, NOA),其病因不明,对临床治疗极为不利。随着对非梗阻性无精症的深入研究,发现男性不育与血睾屏障(Blood-testis barrier, BTB)的功能异常有关,然而导致血睾屏障功能异常的分子机制仍有待阐明。位于生精上皮底部的支持细胞(Sertoli cell)通过彼此间的紧密连接构建血睾屏障,为精子发生提供一个稳定的微环境和独特的免疫屏障,并通过有序的开放调节精子生成。血睾屏障开放机制的异常使得精子发生的微环境和免疫屏障受损,进而影响精子生成,导致男性不育。血睾屏障的开放机制受到众多因子的调节,而TGF-β3是调节血睾屏障开放的主要因子之一,因此研究影响TGF-β3的表达和分泌的因素具有重要意义,这也是本文的第一个研究课题。 NXF3属于核输出因子蛋白家族(nuclear RNA export factor family, NXF),本文研究发现NXF3在小鼠睾丸的支持细胞中特异性地表达,并且在附睾头部、区的主细胞中也检测到NXF3的表达。在支持细胞中首次检测到NXF3的表达是小鼠出生后10天,而此时正是小鼠血睾屏障形成之时,因此NXF3极有可能与血睾屏障相关,这引起我们的极大兴趣。由于TGF-β3是参与血睾屏障调节的主要因子之一,我们检测了NXF3和TGF-β3在睾丸中的表达,发现两者之间具有负相关的关系。进一步的实验也证实了这一关系：在用热或CdC12处理小鼠睾丸后发现,NXF3的表达下降,而TGF-β3的表达上升了。随着研究的深入,发现NXF3参与调节TGF-β3转录负反馈的调控进而影响了TGF-β3的表达,即TGF-β信号通路激活后,NXF3增强了Smad2/3途径的活性,从而使TGF-P3的转录受到抑制。通过更详细的研究我们发现了NXF3的结合蛋白：STRAP, TGF-β信号通路的抑制因子。因此NXF3调节TGF-p3的机制得以阐明：TGF-β信号通路激活后,NXF3与STRAP结合,抑制了STRAP与Smad7的结合,影响了TpR1-STRAP-Smad7复合体的形成,使得Smad2/3途径激活从而抑制了TGF-β3的转录,并且Smad2/3途径的下游靶基因Claudin11、WT1、GATA1和p21也受到调控. 拟染色小体(chromatoid body)是雄性圆形精子时期出现的一个特异性结构,在电子显微镜下呈纤维状结构,主要由蛋白质和RNA组成,不含DNA。由于其含有许多RNA结合蛋白、mRNA和nicroRNA,拟染色小体被认为是精子发生过程中的RNA存储和加工中心,参与精子发生过程中的基因表达调控,因此非常引人关注。距1891年拟染色小体首次被报道,至今已有一百多年,有关拟染色小体的研究取得了很大的进展,许多拟染色小体的蛋白和RNA组分被发现,但是拟染色小体的功能和机制仍然不清楚,需要进一步的探究。本文发现CaMKIV (Ca2+/Calmodulin-dependent Protein Kinase IV, CaMKIV)定位在拟染色小体上,是拟染色小体的一个新的组分。CaMKIV是钙调蛋白激酶家族之一,据报道CaMKIV表达在睾丸的精原细胞和精子细胞中,并且camkiv基因敲除小鼠由于在精子变形过程中组蛋白替换异常而不育。研究发现CaMKIV能够和拟染色小体中的MVH.MIWI和KIF17b相互作用,并且能够促进MVH、MIWI与KIF17b的结合。这是首次报道拟染色小体中蛋白之间的相互作用的调控模式,对理解拟染色小体的结构和功能具有重要意义。综上所述,本文报道了NXF3调控细胞因子TGF-03在睾丸支持细胞中的表达、分泌的机制,有助于分析非梗阻性无精症患者中血睾屏障异常的原因。同时本文报道了CaMKIV作为拟染色小体的一个新的组分,能够调节拟染色小体中的蛋白质之间的相互作用,有助于拟染色小体的进一步研究。
[Abstract]:Around the world, about 10%-15% of child-bearing age couples are suffering from infertility. 40% of male infertility couples are infertile and the proportion of male infertility is increasing with environmental and social factors. Most male infertility patients are non obstructive azoospermia (non-obstructive azoospermia, NOA). The cause of unknown etiology is extremely adverse to clinical treatment. With the in-depth study of non obstructive azoosinoses, it is found that male infertility is associated with the dysfunction of the Blood-testis barrier (BTB). However, the molecular mechanism that causes the dysfunction of the blood testis barrier remains to be elucidated.
The support cells (Sertoli cell) located at the bottom of the spermatogenic epithelium (Sertoli) construct the blood testis barrier through close connections between each other, providing a stable microenvironment and unique immune barrier for spermatogenesis, and through orderly and open regulation of spermatogenesis. The heterogeneity of the opening mechanism of the blood testis barrier causes the microenvironment and immune barrier of sperm to be damaged. The mechanism of the opening of the blood testis barrier is regulated by many factors, and TGF- beta 3 is one of the main factors regulating the opening of the blood testis barrier. Therefore, it is of great significance to study the factors affecting the expression and secretion of TGF- beta 3, which is the first research topic in this article.
NXF3 belongs to the nuclear RNA export factor family (NXF) family (export factor family, NXF). This study found that NXF3 was expressed specifically in the mouse testis supporting cells, and the expression of NXF3 was detected in the main cells of the epididymis and the main cells of the region. The expression of NXF3 was first detected in the support cells for 10 days after the birth of mice. It is when the mouse blood testis barrier is formed that NXF3 is highly likely to be associated with the blood testis barrier, which is of great interest. Since TGF- beta 3 is one of the major factors involved in the regulation of the blood testis barrier, we detected the expression of NXF3 and TGF- beta 3 in the testis, and found a negative correlation between the two. Further experiments confirmed this One relationship: after the treatment of mouse testicles with heat or CdC12, the expression of NXF3 decreased and the expression of TGF- beta 3 increased. With the further study, it was found that NXF3 was involved in the regulation of the negative feedback of TGF- beta 3 transcription and then influenced the expression of TGF- beta 3, that is, NXF3 enhanced the activity of Smad2/3 pathway, thus enabling TGF-P3. Transcriptional inhibition. Through more detailed studies we found the binding protein of NXF3: STRAP, the inhibitory factor of the TGF- beta signaling pathway. Therefore, the mechanism of NXF3 regulating TGF-p3 is elucidated: the binding of NXF3 to STRAP by the activation of the TGF- beta signaling pathway inhibits the combination of STRAP and Smad7, affecting the formation of the TpR1-STRAP-Smad7 complex and making Sma The activation of d2/3 pathway inhibits transcription of TGF- beta 3, and the downstream target genes Claudin11, WT1, GATA1 and p21 of Smad2/3 pathway are also regulated.
The pseudo coloring body (chromatoid body) is a specific structure in the male round sperm period. It is fibrous structure under the electron microscope, mainly composed of protein and RNA, without DNA. because it contains many RNA binding proteins, mRNA and nicroRNA. The quasi dyed corpuscle is considered to be the RNA storage and processing center in the process of spermatogenesis. It is very concerned about the regulation of gene expression in the process of spermatogenesis. It is first reported in 1891. It has been more than 100 years ago. So far, great progress has been made in the study of quasi dyed corpuscles. Many proteins and RNA components of pseudo dyed bodies have been found, but the function and mechanism of the quasi dyed corpuscles are still unclear. Chu, need further inquiry.
This article found that CaMKIV (Ca2+/Calmodulin-dependent Protein Kinase IV, CaMKIV) is located on the pseudo dyed corpuscle. A new component.CaMKIV of the quasi dyed body is one of the calmodulin kinase family. It is reported that CaMKIV is expressed in spermatogonia and spermatogonial cells of the testis, and the CaMKIV knockout mice are deformed in sperm deformation. It is found that CaMKIV can interact with MVH.MIWI and KIF17b in the pseudo dyed corpuscle and can promote the combination of MVH, MIWI and KIF17b. This is the first report on the interaction between proteins in the pseudo dyed bodies, which is important to understand the structure and function of the quasi dyed corpuscle.
To sum up, this paper reports the expression of NXF3 regulating cytokine TGF-03 in the testis support cells and the mechanism of secretion. It is helpful to analyze the cause of abnormal blood testis barrier in non obstructive azoospermia patients. This article reports that CaMKIV is a new component of the pseudo dyed corpuscle, which can regulate the protein between the pseudo dyed corpuscles. The interaction helps to further study the quasi staining bodies.
【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】：2014
【分类号】：R698.2

【共引文献】