【作者】 杨庆岭；

【导师】 史庆华；

【作者基本信息】 中国科学技术大学 ， 遗传学， 2013， 博士

【摘要】 哺乳动物中,精子发生是一个复杂而且受到严格调控的过程,减数分裂前期Ⅰ,同源染色体的配对联会以及重组的发生十分重要,它可以确保同源染色体相互分离并最终形成正常的配子,重组发生的位置和数目异常时往往会导致异常配子的产生。同时,越来越多的研究表明小RNAs在精子发生过程中发挥重要作用,为探讨哺乳动物中遗传重组发生的机制以及精子发生过程中小RNAs可能的调控机制分别做了以下三部分工作：1.麂科动物(Muntiacus, Cervidaei)-直被广泛用来研究染色体及核型演化,然而,对于它们减数分裂过程中同源染色体的行为,尤其是减数分裂重组的频率和分布模式知之甚少。本部分研究中,以中国麂(Muntiacus reevesi)为研究对象,利用微铺展法将其精母细胞联会复合体铺展开,结合免疫荧光技术,用联会复合体侧轴蛋白SCP3,错配修复蛋白MLH1以及着丝粒蛋白CREST的抗体来显示联会复合体以及重组位点和着丝粒的位置。对于170个粗线期细胞进行重组和分布模式分析,结果显示39.4%的XY二价体上没有重组位点,然而常染色体中只有0.5%没有重组位点,平均每个粗线期细胞重组位点数为29.8,范围为24-34；重组位点MLH1在联会复合体上的分布和其他哺乳动物相比存在差异：当一条联会复合体上有一个重组位点时,在联会复合体上分布相对分散；当联会复合体上有两个重组位点时,在亚端粒部位有一个较大的峰,而且总体上雄性中国麂重组位点间的干扰强度要低,这些都和雄性小鼠和人类男性中MLH1分布模式存在明显不同,提示在雄性中国麂中,同源染色体重组的模式和其他的哺乳动物有些许不同,这也为我们更深刻的理解哺乳动物减数分裂同源染色体重组(重组频率和重组数目)发生的机制提供很好的提供有利线索。2.长期以来,减数分裂重组频率与联会复合体长度之间的关系一直是人们研究的兴趣所在,但二者之间相互关系却未被详尽的阐述过。为了更深入的探讨这一问题,我们测量了10个正常男性889个粗线期精母细胞中的19558条联会复合体长度,同时以MLH1作为重组的标记物,统计了联会复合体上的重组频率。我们看到二者之间有一个非常复杂的关系。在这10个人中有8个二者之间呈现正相关关系,其中有3个为较弱的正相关,其他5个为中等程度的正相关,在另外2个研究对象中,我们首次发现单个细胞中联会复合体长度与重组频率无相关性。而且,在不同人之间,甚至在同一个人的不同细胞之间,大多数联会复合体长度相似的细胞,其重组位点数目也不相同,反之亦然。本研究为阐述联会复合体长度和重组频率之间的关系提供了直接的证据,为以后研究二者之间机制关系提供有力的数据支持。3. miRNAs是一类内源性RNA分子,可以在转录和转录后水平调控基因的表达。一直以来人们都致力于在各个物种中发现新的miRNAs并试图了解他们的功能。然而利用第二代深测序技术对于人类睾丸组织中miRNAs的表达谱至今没有相关报道。本部分研究旨在利用Solexa测序技术构建正常人类男性中miRNAs和piRNAs的表达谱。我们共检测到770条已知的miRNAs和5个条新的miRNAs以及20121条piRNAs,同时也表明人类睾丸组织中有大量的小RNAs存在。我们选择了15条已知的和5条新的miRNAs利用qRT-PCR来验证测序的可靠性。我们分析了表达较丰富的以及新的miRNAs的靶基因,并用GO分析预测这些基因可能参与的生物学过程。结果提示这些miRNA参与了很多重要的生物学过程,其中包括精子发生过程中的减数分裂以及p53中相关的调节通路。更多还原

【Abstract】 Spermotogenesis is a complex and regulated progress in mammals. During meiosis I, meiotic pairing and recombination play a crucial role in holding homologous chromosomes together and guaranteeing their accurate segregation into daughter cells. Abnormalities in the frequency and location of crossovers are associated with non-disjunction of homologous chromosomes and the production of aneuploid gametes. In the study, in order to investigate the mechanisms of meiotic recombination and the possible roles of RNAs in spermatogenesis, we done the work including three parts:1. The muntjacs (Muntiacus, Cervidae) have been extensively studied in terms of chromosomal and karyotypic evolution. However, little is known about their meiotic chromosomes particularly the recombination patterns of homologous chromosomes. Spermatocyte preparations were obtained from Chinese muntjac (Muntiacus reevesi) by a drying down spreading technique and immunostained to show synaptonemal complexes (SCs), recombination foci and kinetochores with antibodies against the SC protein SCP3, mismatch repair protein MLH1and kintochores, respectively. The mean number of MLH1foci on autosome SCs per cell was29.8, ranging from25-34.39.4%of XY bivalents lacked MLH1foci compared to less than0.5%of autosomes. The average number of MLH1foci per pachytene cell in M. reevesi was29.8. The distribution of MLH1foci differed from other mammals. On SCs with one focus, the distribution was more even in M. reevesi than in other mammals; for SCs that have two or more MLH1foci, usually there was a larger peak in the sub-centromere region than other regions on SC in M. reevesi. Additionally, there was a lower level of interference between foci in M. reevesithan in mouse or human. These observations may suggest that the regulation of homologous recombination in M. reevesiis slightly different from other mammals and will improve our understanding of the regulation of meiotic recombination, with respect to recombination frequency and position.2. Although the relationship between meiotic recombination frequency and synaptonemal complex (SC) length has been of interest for a long time, how recombination frequency is related to SC length has not been carefully explored. To address this question, we have measured the meiotic recombination frequency as represented by MLH1foci in889pachytene spermatocytes and measured the length of19,558autosomal SCs from10human males. A complex relationship between the number of MLH1foci and total autosomal SC length per cell was observed. A positive correlation with significant correlation coefficients between the two variables was found in eight of the ten donors examined, with three donors showing weak correlation, and five showing moderate correlation. Two donors who did not show any correlation between the two variables were identified for the first time. Moreover, most cells with similar total autosomal SC length showed very different number s of MLH1foci both between individuals and even within an individual, and vice versa. Our data provide the first evidence for a complex relation-ship between the recombination frequency and total length of autosomal SCs per cell in human males.3. MicroRNAs (miRNAs) are a class of small endogenous RNAs that play a regulatory role in cells by negatively affecting gene expression at transcriptional and post-transcriptional levels. There have been extensive studies aiming to discover miRNAs and to analyze their functions in cells from a variety of species. However, there are no published studies of miRNA profiles in human testis using next generation sequencing (NGS) technology. In this study, we employed Solexa sequencing technology to profile miRNAs in normal human testis. We detected770known and5novel human miRNAs, and20121piRNAs, indicating that the human testis has a complex population of small RNAs. The expression of miRNAs detected by NGS was validated by qRT-PCR of15known and5novel miRNAs. We have predicted potential target genes of the abundant known miRNAs and novel miRNAs and subjected them to GO and pathway analysis, revealing the involvement of miRNAs in many important biological functions including meiosis and p53-related pathways that are implicated in the regulation of spermatogenesis.更多还原