Pacchiarini Tiziana, Sarasquete Carmen, Cabrita Elsa
Institute of Marine Science of Andalusia- ICMAN.CSIC, Av Republica Saharaui 2, 11510 Puerto Real, Cádiz, Spain.
Reprod Fertil Dev. 2014 Jun;26(5):690-702. doi: 10.1071/RD13103.
Interspecific testicular germ cell (TGC) transplantation was investigated in two commercial flatfish species. Testes from donor species (Senegalese sole) were evaluated using classical histological techniques (haematoxylin-eosin staining and haematoxylin-light green-orange G-acid fuchsine staining), in situ hybridisation and immunohistochemical analysis. Both Ssvasa1-2 mRNAs and SsVasa protein allowed the characterisation of TGCs, confirming the usefulness of the vasa gene in the detection of Senegalese sole TGCs. Xenogenic transplants were carried out using TGCs from one-year-old Senegalese sole into turbot larvae. Propidium iodide-SYBR-14 and 4',6'-diamidino-2-phenylindole (DAPI) staining showed that 87.98% of the extracted testicular cells were viable for microinjection and that 15.63% of the total recovered cells were spermatogonia. The vasa gene was characterised in turbot recipients using cDNA cloning. Smvasa mRNA was confirmed as a germ cell-specific molecular marker in this species. Smvasa expression analysis during turbot ontogeny was carried out before Senegalese sole TGC transplants into turbot larvae. Turbot larvae at 18 days after hatching (DAH) proved to be susceptible to manipulation procedures. High survival rates (83.75±15.90-100%) were obtained for turbot larvae at 27, 34 and 42 DAH. These data highlight the huge potential of this species for transplantation studies. Quantitative PCR was employed to detect Senegalese sole vasa mRNAs (Ssvasa1-2) in the recipient turbot larvae. The Ssvasa mRNAs showed a significant increase in relative expression in 42-DAH microinjected larvae three weeks after treatment, showing the proliferation of Senegalese sole spermatogonia in transplanted turbot larvae.
在两种商业养殖的比目鱼物种中研究了种间睾丸生殖细胞（TGC）移植。使用经典组织学技术（苏木精-伊红染色和苏木精-淡绿-橙G-酸性品红染色）、原位杂交和免疫组织化学分析对供体物种（塞内加尔鳎）的睾丸进行评估。Ssvasa1-2 mRNA和SsVasa蛋白均有助于鉴定TGC，证实了vasa基因在检测塞内加尔鳎TGC中的有用性。使用一岁塞内加尔鳎的TGC对大菱鲆幼体进行了异种移植。碘化丙啶-SYBR-14和4',6'-二脒基-2-苯基吲哚（DAPI）染色显示，87.98%的提取睾丸细胞可用于显微注射，且回收的细胞总数中有15.63%是精原细胞。通过cDNA克隆对大菱鲆受体中的vasa基因进行了鉴定。Smvasa mRNA被确认为该物种中生殖细胞特异性分子标记。在将塞内加尔鳎TGC移植到大菱鲆幼体之前，对大菱鲆个体发育过程中的Smvasa表达进行了分析。孵化后18天（DAH）的大菱鲆幼体被证明易于进行操作程序。27、34和42 DAH的大菱鲆幼体获得了较高的存活率（83.75±15.90-100%）。这些数据突出了该物种在移植研究中的巨大潜力。采用定量PCR检测受体大菱鲆幼体中塞内加尔鳎vasa mRNA（Ssvasa1-2）。处理三周后，Ssvasa mRNA在42 DAH显微注射幼体中的相对表达显著增加，表明移植的大菱鲆幼体中塞内加尔鳎精原细胞发生了增殖。
