Regulatory mechanism underlying testicular maintenance in a mouse model of Gata6 gene knockdown

doi:10.3969/j.issn.2095-4344.0831

Abstract

Abstract:

BACKGROUND: The GATA transcription factors have two conserved zinc fingers, which are widely distributed in human and vertebrates. This family plays a significant role in tissue development, differentiation and cell proliferation. Especially, Gata6 exerts important effects on heart, gonad, spleen, and liver in mammals.
OBJECTIVE: To study the mechanism of Gata6 gene in mouse testes.
METHODS: Gata6 was knocked down in male mice aged 2 mouths by CRISPR/Cas9 technique, and normal mice were as controls. Histological changes of the mouse testis were observed. Androgen-related synthase levels were detected by ELISA and real-time PCR.
RESULTS AND CONCLUSION: Gata6 was knocked down successfully in mice by CRISPR/Cas9 technique. Testicular developmental retardation, size-reduced testicles and related genitalia, and decreased spermatogonia and spermatogenesis were detected in the knockdown group compared with the control group. The levels of testosterone and androgen-related gene were significantly lower in the knockdown group than the control group (P < 0.05). To conclude, Gata6 plays an important role in mammal testicular development.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Genes, Mice, Knockout, Reproduction, Tissue Engineering

CLC Number:

R318

Wang Shun-de, Li Jing, Wang Shu-hong. Regulatory mechanism underlying testicular maintenance in a mouse model of Gata6 gene knockdown[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(24): 3857-3862.

Figures/Tables 1

2.1 Gata6 敲降序列选择及突变检测将靶序列选择在Gata6的第一个外显子上，靶序列为：GGC GCC TTC ATG CAC AGC GCG GG，酶切位点为AlwI。提取小鼠尾巴基因组DNA，扩增DNA片段，用AlwI限制性内切酶进行酶切。在对照组相比较，AlwI能切开成2明显的DNA条带，一条322 bp，另一条140 bp，而敲降组大部分条带未被切开。将敲降组未切开条带回收、测序，测序得到的序列与对照组相比较，结果显示在22个测序样品共有10个中出现2-103个碱基不同程度的缺失或者插入(图1)。 2.2 小鼠Gata6敲降后表达通过实时定量PCR技术和蛋白免疫印迹检测敲降组和对照组小鼠Gata6表达量，在敲降组中发现Gata6的表达量不论基因水平(图2A)还是蛋白水平(图2B)都显著性降低。 2.3 Gata6基因敲降影响睾丸的发育结果与对照组睾丸相比，2月龄Gata6基因敲降阳性小鼠睾丸与肛门的距离更短(图3A，B)；睾丸和相关生殖器官的大小显著性降低(图3C，D)。组织学观察(苏木精-伊红染色)发现，敲降阳性小鼠个体的精巢发育不完整，出现明显的空腔，精原细胞明显减少(图3E，F)。 2.4 Gata6缺失对小鼠相关雄性相关信号通路中基因的影响取2月龄雄性敲降小鼠和正常小鼠睾丸，提取RNA，反转录成cDNA，通过实时荧光定量PCR方法检测生殖细胞和体细胞相关基因变化情况。如图4所示，与正常对照组相比，敲降组小鼠睾丸中与减数分裂和精原细胞相关的标记基因表达量显著性降低(Stra8，Vasa，Tnp1，Spo11)，同时，与体细胞相关的标记基因表达量也显著性降低(Sox9，17HSDb3，Dmrt1)，但是Cx30.2和Dazl基因无显著性变化(图4)。 2.5 Gata6缺失对小鼠激素水平的影响采用酶联免疫法(EIA)检测2月龄Gata6基因敲降阳性小鼠雄激素(11-KT)水平，结果显示，Gata6基因敲降阳性小鼠雄性个体雄激素水平与对照组雄性个体相比显著降低(图5)。"

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