GDNF/GFRα1信号通路调控中华鲟精原干细胞体外增殖的作用及机制

The Chinese sturgeon (Acipenser sinensis) is a critically endangered species. Although complete artificial propagation of Chinese sturgeon was achieved, only a few brood fish is capable of spawning, and Chinese sturgeon is an extremely late maturing species. Thus, it is urgent to apply new technology in preserving germplasm resources and species conservation of Chinese sturgeon. Spermatogonial stem cells (SSCs) have the ability to differentiate into sperm and eggs, and establishment of its long-term culture system in vitro will be highly significant for the conservation of endangered species. The GDNF/GFRα1 signal pathway plays a critical role in the SSCs self-renewal and proliferation in mammals. In the present study, we will report the identification of gdnf and gfrα1 genes, and their expression pattern at both RNA and protein levels in the Chinese sturgeon. In order to determine the function of GDNF and GFRα1 recombinant proteins in the proliferation of Chinese sturgeon SSCs in vitro, the GDNF and GFRα1 will be added into the SSCs culture system established previously, and then the proliferation, apoptosis and differentiation of cultured Chinese sturgeon SSCs will be analyzed. Furthermore, by means of transcriptome sequencing of cultured Chinese sturgeon SSCs before and after the GDNF and GFRα1 added, the signal pathways related to the SSCs proliferation will be filtered and confirmed in vitro, which will be beneficial to elucidate the mechanism of GDNF/GFRα1 in regulating the proliferation of Chinese sturgeon SSCs in vitro. In a word, this study will provide a possibly effective means for establishing a long-term culture system for the Chinese sturgeon SSCs, which will provide new technological support for the germplasm preservation and species conservation of Chinese sturgeon.

中华鲟为极度濒危鱼类，其全人工繁殖虽已突破，但养殖亲鱼数量有限且初次性成熟时间过长，亟需新途径来加强其种质资源保存及物种保护。精原干细胞具有分化为两性配子的潜能，建立其体外长期培养体系是构建濒危鱼类保护新途径的关键。GDNF/GFRα1在哺乳动物精原干细胞的自我更新和增殖调控中起关键作用。本研究拟鉴定中华鲟gdnf和gfrα1基因并揭示其表达模式；通过在前期建立的中华鲟精原干细胞培养体系中添加GDNF和GFRα1重组蛋白，研究其对精原干细胞的增殖、凋亡及分化等特性的影响，阐明重组蛋白在中华鲟精原干细胞体外增殖的作用；进一步对重组蛋白添加前后培养的精原干细胞进行转录组测序，比较分析相关信号通路并进行功能验证，探究GDNF/GFRα1调控中华鲟精原干细胞体外增殖的作用机制。本研究的结果将为中华鲟精原干细胞体外长期培养体系的建立提供有效途径，进而为中华鲟的种质资源保存及物种保护提供新的技术支撑。

中华鲟为极度濒危鱼类，连续6年未监测到自然繁殖活动，其养殖亲本数量有限且性成熟周期较长，迫切需要创建中华鲟种质保存与物种恢复的新途径。精原干细胞是雄性动物体内唯一能向子代传递遗传信息的成体干细胞，且具有分化为两性配子的潜能。GDNF/GFRα1在精原干细胞的增殖与分化调控中起着重要作用。本研究克隆得到中华鲟gdnf和gfrα1基因cDNA序列，分别编码223和473个氨基酸。荧光定量PCR结果表明，gdnf基因在性腺和垂体中的表达量最高，而gfrα1基因在下丘脑、垂体和性腺中大量表达；进一步发现，gdnf基因在减数分裂时期精巢的表达量最高，而gfrα1基因在有丝分裂时期精巢的表达量最高。制备得到了GFRα1蛋白特异性抗体。免疫组化结果表明，GFRα1蛋白主要在精原干细胞和B型精原细胞中表达。通过优化培养基组分和条件，建立了中华鲟精原干细胞的体外短期培养体系。经差速贴壁能够显著的将精原干细胞富集到上清中，进一步通过免疫组化、碱性磷酸酶染色等，证明上清中精原干细胞的特性。通过在培养体系中添加GFRα1重组蛋白，发现当浓度为150ng/ml时，能够有效的促进中华鲟精原干细胞增殖。转录组测序比较分析，发现精原干细胞体外增殖时，钙离子信号通路、PI3K-Akt信号通路、细胞粘附分子、神经活动配体-受体相互作用等起着重要的调控作用。此外，通过将冻存1年的中华鲟精原干细胞移植到出膜7-8天的长江鲟仔鱼，移植后2个月检查发现，供体嵌合率达81.25%，且平均嵌合的供体精原干细胞的数目是受体内源生殖细胞的2倍；在随后的2个月，嵌合的供体精原干细胞在受体性腺增殖了2-3次。更为重要的是，移植后18个月，仍有75.00%的受体长江鲟含有供体中华鲟的DNA。本研究的结果为中华鲟精原干细胞体外长期培养体系的建立提供有效途径，通过结合生殖干细胞移植技术，为中华鲟的物种保护提供新途径。