Transcriptional profiling of flatfish (Solea senegalensis) spermatogenesis: early versus late spermatogenesis. Solea senegalensis

The Senegalese sole (Solea senegalensis) is a marine flatfish of high economic value and a target species for aquaculture. Here, we used a transcriptomic approach to investigate changes in genes expressed in the Senegalese sole testis throughout spermatogenesis in wild-caught fish adapted to captivity. We identified approximately 400 genes that are differentially expressed during the progression of spermatogenesis and that participate in processes such as activation of the ubiquitin-proteasome system, sperm maturation and motility, cell adhesion or cytoskeletal remodeling. The results from this study contribute to our understanding of the molecular changes ocurring during spermatogenesis in the Senegalese sole. Overall design: This study represents spermatogenesis in Solea senegalensis: early versus late spermatogenesis. Total RNA from testes at different stages in spermatogenesis (early, mid, late and functional maturation) from F0 wild Senegalese sole (3-4 animals at each stage) was extracted using the RNeasy extraction kit (Qiagen) and treated with DNAse following the manufacturer’s instructions. Quantitative and qualitative analysis of total RNA was performed using the Agilent 2100 bioanalyzer. RNA samples from each stage were pooled and amplified, labelled and hybridized to a custom-made oligonucleotide microarray containing 5,087 Senegalese sole Unigene sequences. In brief, pooled testicular RNAs from each stage were amplified and the resulting cRNAs labelled with Cy3 and Cy5, respectively, mixed in equal amounts and hybridized to the microarray for 17 h at 60 ºC. Each hybridization was performed at least in duplicate.塞内加尔鳎（Solea senegalensis）是一种经济价值极高的海水比目鱼，亦是水产养殖的目标物种。本研究采用转录组学方法，探究适应圈养环境的野生塞内加尔鳎精巢中，精子发生全过程的表达基因变化情况。本研究共鉴定出约400个在精子发生进程中差异表达的基因，这些基因参与泛素-蛋白酶体系统激活、精子成熟与运动、细胞黏附以及细胞骨架重塑等生物学过程。本研究结果有助于加深我们对塞内加尔鳎精子发生过程中分子变化的认知。 实验整体设计：本研究聚焦塞内加尔鳎的精子发生过程，比较早期与晚期精子发生的差异。本研究从不同精子发生阶段（早期、中期、晚期及功能成熟期）的F0代野生塞内加尔鳎精巢中提取总RNA（每个阶段取3-4尾个体），采用RNeasy提取试剂盒（Qiagen）完成提取，并按照制造商说明书使用DNA酶进行处理。利用Agilent 2100生物分析仪对总RNA进行定量与定性分析。将每个阶段的RNA样本混合后进行扩增、标记，随后与包含5087条塞内加尔鳎Unigene序列的定制寡核苷酸微阵列进行杂交。简言之，将每个阶段的混合精巢RNA进行扩增，所得的互补RNA（cRNA）分别用Cy3和Cy5标记，等比例混合后于60℃下与微阵列杂交17小时，每个杂交实验至少重复两次。