X染色体起源的逆转座基因RPL10L在精子发生中的功能及机制研究

发布时间：2018-02-23 17:37

  本文关键词： RPL10 RPL10L X染色体起源的逆转座基因 减数分裂性染色体沉默 补偿假说　出处：《中国科学技术大学》2017年博士论文　论文类型：学位论文

【摘要】：哺乳动物的性染色体是从祖先的常染色体进化而来,在整个进化过程中经历了复杂的变化。例如,X染色体上的基因数量在真兽亚纲哺乳动物中还有两次爆发性的增长。基因的转移和重新分配赋予了 X染色体的特殊功能。大量研究表明起源于X染色体的逆转座基因在基因组中所占的比例远远高于起源于常染色体的比例,这类基因往往定位于常染色体并且具有睾丸特异表达的特点。科学家们也提出了一些基于进化选择的假说用于解释这种现象,包括性别对抗驱动X染色体失活假说以及减数分裂性染色体失活补偿假说等。然而,这些假说都缺乏直接的实验证据。尽管有几个X染色体起源的逆转座基因已被证实对精子发生至关重要,但这些基因与其前提基因的进化、表达及功能相关性仍未见报道。RPL10L是一个位于常染色体并在睾丸中特异表达的逆转座基因,它的前体是X染色体上一个核糖体蛋白编码基因RPL10,它的进化发生在真兽亚纲和后兽亚纲分支后不久的真兽亚纲哺乳动物中。我们选择RPL10L和RPL10作为模型,详细研究它们的表达模式、分子功能及作用方式,以期深入了解X染色体来源的逆转座基因存在的意义及其进化机制。首先,我们利用CRISPR/Cas9的方法制作了Rpl10l敲除小鼠,并发现雄性个体的精子发生障碍并且完全不育,具体表现为绝大多数精母细胞发育停滞在减数第一次分裂前期向中期的转换,没有减数分裂后的生殖细胞产生。其次,RPL10L蛋白特异地参与睾丸细胞中的核糖体组装,缺失它的粗线期及双线期精母细胞中的核糖体生成减弱并且大量蛋白的含量发生变化。再次,Rpl10在雌雄小鼠不同组织以及精子发生的精原细胞、前细线期精母细胞、细线期及偶线期精母细胞中广泛表达,但在粗线期及双线期精母细胞和圆形精细胞中的表达量急剧下降;有趣的是,Rpl10l在精子发生的粗线期及双线期精母细胞和圆形精细胞中特异地高表达。最后,RPL10敲低的细胞中核糖体生成受阻并伴随细胞周期停滞,RPL10敲除的细胞无法增殖和存活;而外源导入RPL1OL表达载体能够补救RRL10敲除细胞的增殖和存活;更重要的是,Rpl10l启动子驱动的Rpl10转基因能够重建Rpl10l敲除小鼠的精子发生和生育力。综上所述,我们的研究证实Rpl10l能够补偿MSCI导致的Rpl10转录沉默以保证正常的精子发生,为基于MSCI的逆转座基因补偿假说提供了直接的实验证据,也为X染色体起源的逆转座基因的进化机制以及其在雄性生殖过程中的作用提供了新的理论依据和见解。
[Abstract]:The sexual chromosomes of mammals evolved from the autosomes of their ancestors. During the course of evolution, there have been complex changes. For example, the number of genes on the X chromosome has increased twice in mammals of the subclass Encalypta. The transfer and redistribution of genes have given the X chromosome special. Specific function. A large number of studies have shown that the proportion of genes originating in the X chromosome in the genome is much higher than that in the autosomal genome. Such genes are often located on autosomes and have testicle-specific characteristics. Scientists have also come up with hypothesis based on evolutionary selection to explain the phenomenon. These include the X chromosome inactivation hypothesis driven by gender antagonism and the meiotic chromosome inactivation compensation hypothesis. However, These hypotheses lack direct experimental evidence. Although several reverse locus genes from the origin of X chromosomes have been proven to be essential for spermatogenesis, these genes and their underlying genes evolved. Expression and functional correlation have not been reported. RPL10L is a reverse locus gene located on autosomal and specifically expressed in testis. Its precursor is RPL10, a ribosomal protein coding gene on the X chromosome, which evolved in mammals of the order Euphorbia and the suborder of Postzoa. We chose RPL10L and RPL10 as models. Their expression patterns, molecular functions and modes of action were studied in detail in order to understand the significance of the existence of the reverse locus gene from the X chromosome and its evolutionary mechanism. Firstly, Rpl10l knockout mice were made by using CRISPR/Cas9 method. It was also found that the spermatogenesis of male individuals was impaired and completely sterile, which was manifested by the transformation of the majority of spermatocytes from the early to the middle stage of the first meiosis. No meiotic germ cells were produced. Secondly, RPL10L protein was specifically involved in ribosomal assembly in testicular cells. The ribosomal production of spermatocytes in the absence of Rpl10 decreased and the content of a large number of proteins changed in the spermatocytes of the two stages. Rpl10 was found again in spermatogonia and spermatogonia in different tissues of female and male mice, and spermatogonia in the spermatogonium of pretenline stage. The expression of spermatocytes was widely expressed in the fine and even-line stages, but decreased sharply in the spermatocytes and round spermatocytes in the coarse and double-line stages. It is interesting to note that Rpl10l is highly expressed in spermatocytes and round spermatocytes in the coarse-line and double-line stages of spermatogenesis. At last, ribosome production in the cells with low RPL10 knockout and RPL10 knockout with cell cycle arrest can not proliferate and survive. The exogenous RPL1OL expression vector can remedy the proliferation and survival of RRL10 knockout cells, and more importantly, the Rpl10 gene driven by Rpl10l promoter can reconstruct the spermatogenesis and fertility of Rpl10l knockout mice. Our study confirmed that Rpl10l can compensate for Rpl10 transcriptional silencing induced by MSCI to ensure normal spermatogenesis, providing direct experimental evidence for the MSCI gene compensation hypothesis. It also provides a new theoretical basis and understanding for the evolutionary mechanism of the reverse locus gene of the origin of X chromosome and its role in male reproduction.
【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】：2017
【分类号】：Q132.1
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本文编号：1527008