热应激对小鼠睾丸组织和精原干细胞中Sirt1、PLZF和Oct4基因表达的影响

Effects of Heat Stress on Expression of Sirt1,PLZF and Oct4 Genes in Mouse Testis Tissue and Spermatogonial Stem Cells

高温应激会损伤哺乳动物生殖系统,造成生殖器官组织形态和基因表达的改变,导致精子发生异常。成年动物睾丸中精原干细胞(spermatogonial stem cells,SSCs)具有不断增殖和自我更新的能力,是精子发生的基础,而 PLZF (Promyelocytic Leukemia Zinc Finger)和 Oct4 (octamer-binding transcription factor)证实均是维持精原干细胞自我更新和增殖的重要基因。在精原干细胞中过表达 Sirt1 (Sirtuin type 1)基因能上调 PLZF和Oct4 的表达。此试验以雄性小鼠为研究对象,利用组织切片免疫染色、精原干细胞体外培养、细胞免疫荧光染色和荧光定量PCR等技术,检测热应激处理的活体小鼠睾丸组织和体外培养SSCs中的 Sirt1、PLZF和Oct4 基因表达变化。结果发现,所有热应激组样品中 Sirt1、PLZF和Oct4 基因的核酸、蛋白水平表达量均下调,小鼠睾丸组织切片中热应激组的 Sirt1、PLZF和Oct4 基因在曲细精管基底膜附近细胞群(含精原干细胞)中的特异性染色点减少,定量PCR检测证实该三个基因的相对表达量在热应激组中均显著下调( P <0.01);体外培养SSCs中热应激组的 Sirt1、PLZF和Oct4 基因染色阳性细胞数目减少,定量PCR检测证实该三个基因的相对表达量在热应激组细胞中均显著下调( P <0.01)。以上结果为进一步研究 Sirt1 在精原干细胞热应激过程中的功能奠定基础。

High temperature stress can damage the reproductive system of mammals, causing changes in the morphology and gene expression of reproductive organs, leading to abnormal spermatogenesis. The spermatogonial stem cells (SSCs) in adult animal testes have the ability to proliferate and self-renew and are the basis of spermatogenesis, while PLZF (Promyelocytic Leukemia Zinc Finger) and Oct 4 (octamer binding transcription factor) are confirmed to be an important gene for the self-renewal and proliferation of primary stem cells. Overexpression of Sirt1 (Sirtuin type 1) gene in spermatogonial stem cells up-regulates the expression of PLZF and Oct4 . In this study, male mice were used as the research object, and tissue culture immunostaining, in vitro culture of spermatogonial stem cells, immunofluorescence staining and real-time PCR were used to detect the testicular tissues of heat-treated live mouse and changes of Sirt1, PLZF and Oct4 gene expression in SSCs cultured in vitro. The results showed that the expression levels of Sirt1, PLZF and Oct4 genes in all heat stress groups were down-regulated, and the specific staining points in the cell population (including spermatogonial stem cells) near the basement membrane of seminiferous tubules for Sirtl, PLZF and Oct4 genes in the mouse testicular tissue sections of heat stress group were reduced. Quantitative PCR assay confirmed that the relative expression levels of the three genes were significantly down-regulated in the heat stress group ( P <0.01);for SSCs in vitro group, the number of cells staining positive for Sirt1, PLZF and Oct4 was decreased. The relative expression of these three genes was significantly down-regulated in heat-stressed cells ( P <0.01). These results lay the foundation for further study of the function of Sirt1 in the process of heat stress in spermatogonial stem cells.