【作者】 张永；

【导师】 吴际；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 哺乳动物发育过程中生殖系统分泌的多种细胞因子相互作用,形成一个复杂的信号网络,对其发育起着调控作用。近年来,大量与生殖系统发育相关的细胞因子相继被发现。我们在研究与小鼠卵巢发育相关基因的过程中,通过差异显示PCR技术(DDRT-PCR),筛选到一个新的小鼠Kinesin超家族成员,命名为Oocyte-G1。该基因在不同年龄小鼠卵巢中具有差异表达,其开放阅读框为2997bp,编码一个含有997个氨基酸、分子量约110kD的蛋白质,其mRNA长度约3.6kb,广泛存在于小鼠卵巢、肺、肾脏、睾丸和大脑组织中。Oocyte-G1在小鼠卵巢中,主要分布于卵母细胞及其周围的一些颗粒细胞中,在15、40日龄时表达量达到峰值,在5、10、20以及120日龄时低水平表达。为进一步研究Oocyte-G1的功能,我们分别在体外和体内进行了相关实验。TUNEL与流式细胞凋亡检测实验结果均显示:在细胞内过量表达Oocyte-G1,能引起细胞凋亡率显著增高。随后以含有Oocyte-G1外源表达片段的逆转录病毒颗粒为载体,构建了Oocyte-G1过表达小鼠模型,通过PCR与Southern blot技术筛选得到Oocyte-G1过表达小鼠。动物实验表明:雄性Oocyte-G1过表达模型小鼠生精小管内的生殖细胞凋亡率也显著增高。同时还发现Ooyte-G1的过量表达还能延缓小鼠发育,表现为体型及内部器官发育迟缓、生殖器官成熟时期推迟。导致雄性小鼠生殖腺发育迟缓,影响生精过程;雌性小鼠卵泡发育也受到影响,使6周龄小鼠不能形成成熟卵泡。这种发育迟缓现象,在随后的发育过程中能逐渐恢复。为探索Oocyte-G1功能的产生机制,我们使用免疫共沉淀方法对多个参与细胞增殖、凋亡或分化的细胞因子进行了筛选,结果显示Oocyte-G1能与Caspase-3相互结合。免疫荧光定位结果显示,这种相互作用发生在细胞核的周围区域。在体外培养的细胞中过量表达Oocyte-G1,能引起Caspase-3的表达量显著增加,而Oocyte-G1沉默会引起Caspase-3表达量显著降低。另外在Oocyte-G1过表达转基因小鼠中,Caspase-3的表达量也显著增高。综上所述,我们筛选并克隆了一个小鼠Kinesin超家族的新成员基因,Oocyte-G1,并对其功能进行了研究。上述结果均提示Oocyte-G1能通过作用于一些细胞凋亡因子而对凋亡过程具有一定促进作用,并进而影响了动物发育过程。深入研究Oocyte-G1在细胞凋亡以及生殖发育过程中的作用,能加深我们对哺乳动物生殖发育过程机制的理解,同时还为人类不孕不育症的治疗以及避孕药物的研发提供了理论依据。更多还原

【Abstract】 Reproductive system is recognized as a source of regulatory molecules that influence animal development. A complicated signal network is formed by these molecules; through an array of complex interaction, the development process is under control. Recently, more and more hormones and signal molecules were identified; however, function of the majority of members of the network and the detailed mechanism are still unclear. During efforts to clone the follicular developmental related genes, we screened and identified a new kinesin superfamily gene, Oocyte-G1 by DDRT-PCR and cDNA library screening. This gene shows different expression levels in mouse ovaries during development. Oocyte-G1 belongs to Kinesin N-8 subfamily. The open reading frame of Oocyte-G1 is 2,994 bp long, which encodes a 997-residue predicted protein. Northern blot analysis revealed the presence of`~3.6 kb Oocyte-G1 mRNA in ovary, lung, kidney, testis and brain. Oocyte-G1 was weakly expressed on day 5 and was expressed at moderate level on day 10. Thereafter, on day 15 or day 40, there was an increase in expression, followed by a decline on day 20 or day 120. Furthermore, we studied the Ooctye-G1 protein by using the antiserum against a peptide sequence unique to this gene in Western blot analysis and immunolocalization. The antiserum recognized a prominent band of ~110 kD in immunoblots, Oocyte-G1 were dispersed in oocytes and some surrounding granulose cells in mouse ovaries.To study the function of Oocyte-G1, a series of experiments in vitro and in vivo was performed. By using TUNEL and flow cytometry methods, increased apoptosis caused by over-expression of Oocyte-G1 was observed in cultured cells. Oocyte-G1 transgenic mouse model was then constructed to verify this result, an increased apoptosis rate of the germ cells was observed in the Oocyte-G1 transgenic mice. In addition, the majority of the Oocyte-G1 transgenic mice experienced developmental retardation releated aspects, such as the body weight or the organ significantly smaller than wild type ones; in adult mouse testis, seminiferous tubules were underdevelopment hence the spermatogenesis was impaired, in female transgenic mice, most follicles are in early developmental stage of folliculogenesis and no mature follicles were detected in 6-week-old age.Immunoprecipitation were then used to screen potential candidates, which can interact with Oocyte-G1. The results revealed a potential interaction exist between Oocyte-G1 and Caspase-3. The results of co-localization show that this interaction was located at the surrounding area of the nucleus. Increased expression level of Caspase-3 was observed in Oocyte-G1 transgenic mice. By using over-expression and RNAi strategy in vitro, we found that expression level of Caspase-3 was up-regulated by Oocyte-G1.In summary, this dissertation describes the cloning, identification, characterization and functional analysis of a new mouse Kinesin N-8 member gene, Oocyte-G1. All results suggest the potential roles of Oocyte-G1 in apoptosis: it may promote apoptosis through act on some members of the apoptotic signaling pathways. These data may provide several clues to further understanding of the animal reproductive system development and the mechanism or physiological roles of Oocye-G1 in apoptosis.更多还原