University of Guilan Aquatic Physiology and Biotechnology 2345-3966 7 1 1970 01 01 Localization of Vasa mRNA in testicular tissue of Caspian trout (Salmo caspius) using in situ hybridization Localization of Vasa mRNA in testicular tissue of Caspian trout (Salmo caspius) using in situ hybridization 23 39 3446 10.22124/japb.2019.8176.1184 FA Samaneh Poursaeed PhD in Fisheries Science, Department of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran Mohammad Reza Kalbassi Professor in Department of Fisheries, Faculty of Marine Sciences, Tarbiat Modares University, Noor, Iran Seyedeh Nafiseh Hassani Assistant Professor in Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran Goro Yushizaki Professor in Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan Hossein Baharvand Professor in Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran Journal Article 1970 01 01 In situ hybridization is a powerful tool to localize a specific sequence of nucleic acids in heterogeneous cell population. Therefore, the aim of this study was to apply this technique for localization of Vasa mRNA expression in testicular tissue of Caspian trout (Salmo caspius). In this study, a digoxigenin-labeled RNA probe was synthesized using the plasmid pGEM-T carrying a Caspian trout Vasa cDNA fragment as a template. The RNA labeling was performed through in vitro transcription reaction with DIG labeling mix. For in situ hybridization of tissue sections, Caspian trout testes were fixed and dehydrated. After rehydration and treatment with proteinase K, sections were incubated with hybridization buffer containing Vasa antisense riboprobe for 16h. After washing with SSC solution and incubation with anti-Dig-digoxigenin Antibody, the sections were treated with NBT/BCIP. The results of in situ hybridization using a Vasa antisense riboprobe showed that all developmental stages of male germ cells (spermatogonial stem cells to spermatozoa) expressed this gene, but the expression level was different among germ cells. Therefore, Vasa gene is introduced as a specific marker to identify germ cells and in situ hybridization can be used as a technique to evaluate the localization and expression level of gene mRNA in testicular tissue. These findings may provide important information to help understand the formation and development of germ cells in this valuable species of Caspian Sea. In situ hybridization is a powerful tool to localize a specific sequence of nucleic acids in heterogeneous cell population. Therefore, the aim of this study was to apply this technique for localization of Vasa mRNA expression in testicular tissue of Caspian trout (Salmo caspius). In this study, a digoxigenin-labeled RNA probe was synthesized using the plasmid pGEM-T carrying a Caspian trout Vasa cDNA fragment as a template. The RNA labeling was performed through in vitro transcription reaction with DIG labeling mix. For in situ hybridization of tissue sections, Caspian trout testes were fixed and dehydrated. After rehydration and treatment with proteinase K, sections were incubated with hybridization buffer containing Vasa antisense riboprobe for 16h. After washing with SSC solution and incubation with anti-Dig-digoxigenin Antibody, the sections were treated with NBT/BCIP. The results of in situ hybridization using a Vasa antisense riboprobe showed that all developmental stages of male germ cells (spermatogonial stem cells to spermatozoa) expressed this gene, but the expression level was different among germ cells. Therefore, Vasa gene is introduced as a specific marker to identify germ cells and in situ hybridization can be used as a technique to evaluate the localization and expression level of gene mRNA in testicular tissue. These findings may provide important information to help understand the formation and development of germ cells in this valuable species of Caspian Sea.

https://japb.guilan.ac.ir/article_3446_e4e49714f099cf1e87eb91f5d9639732.pdf