Santos Nassif Lacerda Samyra Maria, Costa Guilherme Mattos Jardim, da Silva Mariana de Araújo, Campos-Junior Paulo Henrique Almeida, Segatelli Tânia Mara, Peixoto Marco Túlio Diniz, Resende Rodrigo Ribeiro, de França Luiz Renato
Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais 31270-901, Brazil.
Gen Comp Endocrinol. 2013 Oct 1;192:95-106. doi: 10.1016/j.ygcen.2013.06.013. Epub 2013 Jun 20.
In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish.
与体外培养和移植相结合，精原干细胞（SSCs）的分离是研究脊椎动物精原细胞生理学的一种极佳方法。然而，在鱼类中，缺乏SSC分子标记是鉴定/纯化这些细胞的一大限制，这使得在养鱼业中应用几种有价值的生物技术变得困难。在此，我们描述了潜在的分子标记，这些标记用于对尼罗罗非鱼的SSCs进行表型特征分析、培养和移植。免疫定位显示，Gfra1仅在单一类型的未分化A型精原细胞（Aund，推测为SSCs）中表达。同样，在Aund细胞中观察到了Nanos2蛋白的表达。然而，在包含A型分化精原细胞（Adiff）的含有2至8个生殖细胞的囊肿中也鉴定出了Nanos2阳性精原细胞。此外，我们还建立了有效的原代培养条件，使尼罗罗非鱼精原细胞能够扩增其数量至少一个月，同时保持其原始的未分化（干性）特征。早期生殖细胞特异性标记物的表达证明了Aund精原细胞表型的维持，更有说服力的是，通过它们在生殖细胞移植后在经白消安处理的成年尼罗罗非鱼受体睾丸中定殖和发育的能力得以证明。除了增进我们对鱼类SSCs的身份和生理学的了解之外，这些发现为建立一个能够使鱼类SSCs在体外扩增的系统迈出了第一步，这代表着水产养殖新生物技术发展的重要进展，包括生产转基因鱼的可能性。
