SPATA2基因敲除小鼠模型的建立及其睾丸相关表型和机理的研究

发布时间：2018-02-28 16:23

本文关键词： SPATA2 支持细胞精子发生抑制素 CRISPR/Cas9基因编辑技术　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：支持细胞是睾丸曲细精管中的一种体细胞,它的主要功能是为精子发生提供了一个稳定的环境,支持生殖细胞的增殖与分化。支持细胞之间紧密联结,形成血睾屏障,其分泌的各种细胞因子也对精子发生产生重要影响。已有研究表明,精子发生相关蛋白2 (spermatogenesis-associated protein 2,SPATA2)高表达于大鼠及人的睾丸支持细胞,但是这一蛋白在雄性个体的生殖系统中所发挥的功能仍然不明确。本论文证明了 SPATA2在小鼠的睾丸支持细胞中高表达,并且通过CRISPR/Cas9技术建立了 SPATA2基因敲除小鼠模型,之后进一步研究了敲除动物在雄性生殖力上相对于野生型的差异,以及其发挥作用的机制。主要取得了以下研究结果:1. Real-time PCR和Western blot定量方法证明,SPATA2在成年小鼠睾丸中的表达量高于其他组织器官和青春发育期的睾丸。免疫组织化学方法证明,SPATA2蛋白定位于睾丸曲细精管中的支持细胞胞质。2.利用CRISPR/Cas9基因编辑技术,采用Cas9的突变体Cas9n核酸酶对SPATA2基因进行定点突变,产生F0代突变个体,经过分析筛选出最优突变进行建系。杂交两代后得到纯合个体,通过RT-PCR、Western blot和免疫荧光验证了睾丸中基因敲除的效率,用于进一步的研究。3.分析了雄性SPATA2敲除小鼠生殖功能的各项指标,发现敲除小鼠的睾丸缩小、重量较同窝的野生型对照降低了三分之一,4月龄的小鼠睾丸出现曲细精管直径较小、生精上皮变薄的现象。进一步分析后发现,敲除小鼠附睾尾中精子数量下降了 40%。并且精子中出现了一些形态异常的状况,精子活力也有所降低。4.配种实验发现,和同窝的对照相比,SPATA2敲除雄鼠的窝产仔数平均降低了 2.36只,差异显著。接下来又利用体内受精实验,证明了与敲除雄鼠交配的超数排卵雌鼠,其卵母细胞受精率表现出明显下降。5.免疫组织荧光染色证明,敲除SPATA2的小鼠睾丸中,PCNA的阳性细胞数量明显减少,而凋亡的细胞没有增加。进一步检测发现,PLZF标记的精原干细胞的数量也有所降低,但差异不显著;SCP3与γH2A.X所标记的精母细胞的数量均有显著的下降。这初步解释了 SPATA2敲除小鼠精子数量减少的直接原因。6.鉴于SPATA2表达于支持细胞,用Wt1标记支持细胞的方法检测了睾丸发育时期及成年后支持细胞的数量,得到的结论是支持细胞的数量没有变化。之后运用生物素示踪法检测血睾屏障的完整性,发现SPATA2敲除小鼠的血睾屏障受损,这进一步揭示了睾丸中生殖细胞增殖减弱、数量减少的原因。7.对睾丸支持细胞表达的基因进行检测,发现抑制素α亚基在敲除小鼠睾丸中表达升高,提示这可能是产生一系列表型的根本原因。同时,检测到了垂体中卵泡刺激素FSHβ亚基表达的降低。综上所述,本论文在小鼠中证明了,SPATA2表达于睾丸的支持细胞胞质,并且运用CRISPR技术建立了 SPATA2敲除小鼠模型,揭示了 SPATA2的敲除会导致睾丸中FSH的负控因子——抑制素的表达上升,从而使得FSHβ表达降低,雄性生殖系统受损,精子数量减少,生殖能力减弱。
[Abstract]:Support is a kind of somatic cells in seminiferous tube, its main function is to provide a stable environment for spermatogenesis, proliferation and differentiation of germ cells. Support between Sertoli cells are closely connected to form the blood testis barrier, the secretion of various cytokines also have an important effect on spermatogenesis. Studies have shown that spermatogenesis related protein 2 (spermatogenesis-associated protein 2, SPATA2) high expression in rat and human Sertoli cells, but play this protein in the male reproductive system of the individual function is still not clear. This paper proves that SPATA2 in mouse Sertoli cells and the high expression. CRISPR/Cas9 established the SPATA2 gene knockout mouse model, after further study of the knockout animal compared to wild type differences in male reproductive capacity, as well as the role of machine System. The main results are as following: Real-time PCR and Western blot proved that the 1. quantitative methods, the expression of SPATA2 in adult mouse testis were significantly higher than that of other organs and puberty testes. Proof of immunohistochemistry, SPATA2 protein was localized in the seminiferous tubules in the Sertoli cell cytoplasm.2. by CRISPR/Cas9 gene editing technology, using Cas9n nuclease Cas9 mutant of the SPATA2 gene mutation, F0 mutation generation, through analysis and screening out the optimal mutation lines. Hybridization for two generations of homozygous individuals, through RT-PCR, Western blot and immunofluorescence verified testis gene knockout efficiency, for the study of.3. further the analysis of the SPATA2 index in addition to knock on male reproductive function in mice, found that knockout mice testicular size, weight compared with the wild type nest was reduced by 1/3, 4 The month old mice testis seminiferous tubule diameter smaller, seminiferous epithelium thinning phenomenon. Further analysis found that knockout sperm epididymal mouse decreased appeared some morphological abnormalities in 40%. and sperm, sperm motility decreased.4. mating experiment, compared with the control littermates SPATA2, knock out the litter size of male rats was decreased 2.36, the difference was significant. Then the fertilization experiment in vivo, and proved that the knockout of superovulation in female rats mated with male rats, the oocyte fertilization rate showed significantly decreased.5. immunohistofluorescence staining demonstrated that SPATA2 knockout mice testis in PCNA, the number of positive cells decreased, and the apoptotic cells did not increase. Further testing found that the PLZF marker of spermatogonial stem cell number decreased, but the difference was not significant; mark SCP3 and gamma H2A.X spermatocytes fine The number of cells was significantly decreased. The preliminary interpretation of the SPATA2 knockout mice sperm directly causes the decrease in the number of.6. in SPATA2 expression in Sertoli cells, Sertoli cells labeled with Wt1 method to detect the testicular development period and adult Sertoli cell number, the conclusion is the number of Sertoli cells did not change after using complete. The detection of the blood testis barrier biotin tracer method, found that SPATA2 knockout mice blood testis barrier damage, which further reveals the proliferation of germ cells in testis decreased,.7. causes decrease in the number of Sertoli cells expressed genes were detected and found that inhibin A subunit knockout increased expression of mouse testis, suggesting that this may be the reason for a variety of phenotypes. At the same time, to detect the pituitary follicle stimulating hormone beta subunit FSH expression decreased. In summary, this paper proved in mice The support of cytoplasmic SPATA2 expression in testis, and the use of CRISPR technology to establish the SPATA2 knockout mice, revealed that SPATA2 knockout leads to increased expression of negative control factor in the testis - inhibin FSH, so that the expression of FSH decreased and impaired male reproductive system, reduced sperm count, reproductive capacity decreased.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q492.4

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