Zuo Qisheng, Jin Kai, Zhang Yani, Song Jiuzhou, Li Bichun
Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, P.R. China.
Animal and Avian Sciences, University of Maryland, Baltimore, MD 20741, U.S.A.
Biosci Rep. 2017 Jul 7;37(4). doi: 10.1042/BSR20170179. Print 2017 Aug 31.
The present study investigated the dynamic expression and regulatory mechanism of transforming growth factor β (TGF-β) signaling involved in embryonic stem cells (ESCs) differentiation into male germ cells. Candidate genes involved in TGF-β signaling pathway were screened from RNA-sequencing (RNA-seq), which were further validated by quantitative real-time PCR (qRT-PCR). Bone morphogenetic protein 4 (BMP4) was used to induce differentiation of ESCs Inhibition of TGF-β signaling pathway was reflected by Western blot of SMAD2 and SMAD5 expression. Differentiating efficiency of germ cells was evaluated by immunofluorescence and fluorescence-activated cell sorting (FACS). Germ cell marker genes were assessed by qRT-PCR in the differentiation process, with activation or inhibition of TGF-β signaling pathway. In the process of induction, SMAD2 and SMAD5 were found to significantly up-regulated in BMP4 group versus the control and inhibition groups after 4 and 14 days. Expression of , and were significantly increased in the BMP4 group compared with the control group, while down-regulated in the inhibition groups. The proportion of germ cell-like cells was decreased from 17.9% to 2.2% after 4 days induction, and further decreased from 14.1% to 2.1% after 14 days induction. Correspondingly, expression of marker genes in germ cells was significantly lower. inhibition of TGF-β signaling pathway reduced germ cells formation from 5.5% to 1.6%, and down-regulated the expression of , and In conclusion, our study reveals the mechanism regulating spermatogonial stem cells (SSCs) and lays the basis for further understanding of the regulatory network.
本研究调查了参与胚胎干细胞（ESC）分化为雄性生殖细胞的转化生长因子β（TGF-β）信号通路的动态表达及调控机制。从RNA测序（RNA-seq）中筛选出参与TGF-β信号通路的候选基因，并通过定量实时PCR（qRT-PCR）进一步验证。使用骨形态发生蛋白4（BMP4）诱导ESC分化，通过SMAD2和SMAD5表达的蛋白质免疫印迹法反映TGF-β信号通路的抑制情况。通过免疫荧光和荧光激活细胞分选（FACS）评估生殖细胞的分化效率。在分化过程中，通过qRT-PCR评估生殖细胞标记基因，同时激活或抑制TGF-β信号通路。在诱导过程中，发现与对照组和抑制组相比，BMP4组在4天和14天后SMAD2和SMAD5显著上调。与对照组相比，BMP4组中 、 和 的表达显著增加，而在抑制组中下调。诱导4天后，生殖细胞样细胞的比例从17.9%降至2.2%，诱导14天后进一步从14.1%降至2.1%。相应地，生殖细胞中标记基因的表达显著降低。抑制TGF-β信号通路使生殖细胞形成从5.5%降至1.6%，并下调 、 和 的表达。总之，我们的研究揭示了调节精原干细胞（SSC）的机制，为进一步了解调控网络奠定了基础。
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