BCAS2在小鼠精子发生中的功能和机制研究

发布时间：2018-08-16 15:37

【摘要】：精子发生是一个非常动态和复杂的过程,主要经历三个阶段:精原细胞有丝分裂、精母细胞减数分裂和精子细胞的变形,最终产生单倍体长形精子。精子发生的每个阶段都需要基因表达的精确调控。可变剪接作为非常重要的基因转录后调控方式,极大的增加了高等真核生物和复杂器官中转录和翻译产物的多样性。大量研究数据显示可变剪接调控了精子发生相关基因的表达和功能;此外,很多剪接因子在睾丸中呈现细胞或发育阶段特异性表达模式,这都说明可变剪接可能在精子发生过程中发挥着重要的调控功能。BCAS2(breast carcinoma amplified sequence 2)是Prp19相关复合体的核心组分,参与了前体RNA的剪接等重要生命活动。我们前期的研究发现BCAS2在雄性生殖细胞中具有特异性表达模式本课题采用精原干细胞特异性敲除BCAS2的小鼠模型,研究BCAS2是否参与精子发生过程中的剪接。通过对BCAS2在睾丸发育过程中的免疫染色观察和生精细胞的富集,发现BCAS2在小鼠睾丸的精原干细胞中相对高表达。Vasa-Cre介导生殖细胞特异性敲除BCAS2的雄性小鼠生长正常,具有正常的交配行为,但不能生育后代;进一步检测发现,BCAS2缺失导致精子发生明显异常,表现为减数第一次分裂前期的生殖细胞数目明显下调,并且基本观察不到减数分裂的关键事件(重组和联会)。通过对精原干细胞的数量、定位、增殖和凋亡进行染色和统计发现,BCAS2缺失的睾丸中精原干细胞发育基本正常,但减数第一次分裂前期的精母细胞大量缺失,减数分裂启动的关键因子STRA8的表达也明显下调,说明BCAS2缺失的睾丸无法正常启动减数分裂。为了进一步解析BCAS2的作用机制,我们对出生后第9天(P9)的正常和BCAS2敲除的小鼠睾丸进行了 RNA深度测序分析。结果发现BCAS2缺失的睾丸转录组水平的基因表达基本正常。但是作为剪接目标基因tubulin的表达明显变化,提示BCAS2可能通过剪接发挥功能。为了研究BCAS2在pre-mRNA剪接方面的功能,我们进一步研究了 BCAS2缺失睾丸中的可变剪接的变化。结果显示,在BCAS2缺失的睾丸中有245个基因的可变剪接发生了异常,其中包括6个在精子发生中具有重要功能的基因(Dazl、Ehmt2、Hmga1、Cit、Hs 和 Bcl2l11)。利用半定量 RT-PCR 和 real-timeRT-PCR,我们成功验证出Dazl、Ehmt2和Hmga1的可变剪接异常。此外,BCAS2的缺失会导致"减数分裂促进因子" DAZL不同转录本的表达异常:全长形式的转录本显著性下调,而缺失8号外显子的转录本表达量明显上升。而且DAZL蛋白总体的表达量明显下调。总之,本研究证明了 BCAS2参与小鼠精原干细胞中的可变剪接,并对减数分裂启动和雄性生育力的维持至关重要。该研究首次揭示了可变剪接在精子发生过程中调控减数分裂启动这一重要事件,为研究哺乳动物减数分裂启动提供了新的思路,并为可变剪接的生理功能研究提供了重要理论依据。
[Abstract]:Spermatogenesis is a very dynamic and complex process, which mainly goes through three stages: spermatogonia mitosis, spermatogonial meiosis and sperm cell deformation, resulting in haploid long spermatozoa. Every stage of spermatogenesis requires precise regulation of gene expression. As a very important gene post-transcriptional regulation, variable splicing greatly increases the diversity of transcription and translation products in higher eukaryotes and complex organs. Numerous studies have shown that variable splicing regulates the expression and function of genes associated with spermatogenesis. In addition, many splicing factors present cellular or developmental specific expression patterns in the testis. These results suggest that variable splicing may play an important role in the regulation of spermatogenesis. BCAS2 (breast carcinoma amplified sequence 2) is the core component of Prp19 related complex and participates in important life activities such as splicing of precursor RNA. Our previous study found that BCAS2 has a specific expression pattern in male germ cells. In this study, the mouse model of spermatogonial stem cell knockout BCAS2 was used to study whether BCAS2 was involved in the splicing of spermatogenesis. Through the immunostaining of BCAS2 during testicular development and the enrichment of spermatogenic cells, it was found that the expression of BCAS2 in spermatogonial stem cells of mouse testis was relatively high. Vasa-Cre mediated germ cell specific knockout of BCAS2 in male mice. It was found that the deletion of BCAS2 resulted in abnormal spermatozoa, which showed that the number of germ cells in the early stage of the first meiosis was significantly down-regulated. The key events of meiosis (recombination and association) were not observed. The number, location, proliferation and apoptosis of spermatogonial stem cells were detected by staining and statistics. It was found that the spermatogonial stem cells in the testis with BCAS2 deletion developed normally, but a large number of spermatocytes were absent in the early stage of the first meiosis. The expression of STRA8, a key factor in meiosis initiation, was also down-regulated, indicating that the testis with BCAS2 deletion could not initiate meiosis normally. In order to further elucidate the mechanism of BCAS2, RNA deep sequencing of normal and BCAS2 knockout testis of mice on the 9th day after birth (P9) was carried out. The results showed that the gene expression of testicular transcriptome without BCAS2 was basically normal. But the expression of tubulin, the target gene of splicing, changed obviously, suggesting that BCAS2 may play a role in splicing. In order to study the function of BCAS2 in pre-mRNA splicing, we further studied the change of variable splicing in BCAS2 missing testis. The results showed that the variable splicing of 245 genes was abnormal in the testis with BCAS2 deletion, including 6 genes (Dazll Ehmt2, Hmga1Hmga1), which played an important role in spermatogenesis. Using semi-quantitative RT-PCR and real-time RT-PCR, we have successfully verified the variable splicing anomalies of Dazln Ehmt2 and Hmga1. In addition, the deletion of BCAS2 resulted in abnormal expression of DAZL transcripts: the full-length transcripts were significantly down-regulated, while the expression of deletion exon 8 was significantly increased. Moreover, the total expression of DAZL protein was significantly down-regulated. In conclusion, this study demonstrated that BCAS2 was involved in the variable splicing of mouse spermatogonial stem cells, and was essential to the initiation of meiosis and the maintenance of male fertility. This study revealed for the first time the important event of variable splicing regulating the initiation of meiosis during spermatogenesis, and provided a new idea for studying the initiation of mammalian meiosis. It also provides an important theoretical basis for the study of the physiological function of variable splicing.
【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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