title: Isolation, Characterization, and In Vitro Culturing of Spermatogonial Stem Cells in Japanese Quail (Coturnix japonica).
abstract: We reported previously testis-mediated germline chimera production and characterization of germline stem cell-like cells from chicken testes. The present study aimed to establish an in vitro system for culture of quail spermatogonial stem cells (SSCs) for practical applications in germline preservation and transgenesis. Testicular cells (TCs) from juvenile (4 weeks old) or adult (8 weeks old) quail testis were isolated using sequential enzymatic digestion. The percentages of viability of isolated TCs were 91.00% ± 2.12% and 88.00% ± 1.87% in juvenile and adult testes, respectively, and immunohistochemical evaluation indicated the expression of integrin alpha-6 (ITGA6), GDNF family receptor alpha-1 (GFRA1), and Deleted in azoospermia-like (DAZL) in specific TCs. SSCs were purified by differential plating of TCs and then subjected to quantitative reverse transcription-polymerase chain reaction, which showed differential expression of SSC-specific, and germness and stemness-related genes. Coculture of quail SSCs with mouse embryonic fibroblasts and Sertoli cells as a feeder layer resulted in the generation of stable SSC colonies and short-term cultivation, and the expression of SSC and germ cell markers was maintained during several passages of culture. Collectively, these results demonstrate the efficient isolation and characterization of quail SSCs and the suitability of Sertoli cells as a feeder layer for in vitro culture of quail SSCs. Quail SSCs will facilitate the production of germline chimeras and transgenesis.
authors.affiliation: 1 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University , Seoul, Korea.
authors.forename: Ravindran Kumar
authors.initials: RK
authors.lastname: Pramod
authors.affiliation: 1 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University , Seoul, Korea.
authors.forename: Bo Ram
authors.initials: BR
authors.lastname: Lee
authors.affiliation: 1 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University , Seoul, Korea.
authors.forename: Young Min
authors.initials: YM
authors.lastname: Kim
authors.affiliation: 1 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University , Seoul, Korea.
authors.forename: Hong Jo
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: 1 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University , Seoul, Korea.
authors.forename: Young Hyun
authors.initials: YH
authors.lastname: Park
authors.affiliation: 2 Division of Animal Science, Faculty of Agriculture, Shinshu University , Nagano-ken, Japan .
authors.forename: Tamao
authors.initials: T
authors.lastname: Ono
authors.affiliation: 1 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University , Seoul, Korea.
authors.forename: Jeong Mook
authors.initials: JM
authors.lastname: Lim
authors.affiliation: 1 Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University , Seoul, Korea.|3 Institute for Biomedical Sciences, Shinshu University , Nagano-ken, Japan .
authors.forename: Jae Yong
authors.initials: JY
authors.lastname: Han
mesh_terms: D000818:Animals; D002465:Cell Movement; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D003370:Coturnix / Q000378:metabolism*; D061252:Feeder Cells / Q000166:cytology; D008297:Male; D012708:Sertoli Cells / Q000166:cytology; D013093:Spermatogonia / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D013737:Testis / Q000166:cytology
keywords: Sertoli cells; in vitro culture; quail; spermatogonial stem cells; testicular cells