title: Spermatogonial culture medium: an effective and efficient nutrient mixture for culturing rat spermatogonial stem cells.
abstract: An economical and simplified procedure to derive and propagate fully functional lines of undifferentiated rat spermatogonia in vitro is presented. The procedure is based on the formulation of a new spermatogonial culture medium termed SG medium. The SG medium is composed of a 1:1 mixture of Dulbecco modified Eagle medium:Ham F12 nutrient, 20 ng/ml of GDNF, 25 ng/ml of FGF2, 100 microM 2-mercaptoethanol, 6 mM l-glutamine, and a 1x concentration of B27 Supplement Minus Vitamin A solution. Using SG medium, six individual spermatogonial lines were derived from the testes of six separate Sprague-Dawley rats. After proliferating over a 120-day period in SG medium, stem cells within the spermatogonial cultures effectively regenerated spermatogenesis in testes of busulfan-treated recipient rats, which transmitted the donor cell haplotype to more than 75% of progeny by natural breeding. Subculturing in SG medium did not require protease treatment and was achieved by passaging the loosely bound spermatogonial cultures at 1:3 dilutions onto fresh monolayers of irradiated DR4 mouse fibroblasts every 12 days. Spermatogonial lines derived and propagated using SG medium were characterized as homogeneous populations of ZBTB16(+) DAZL(+) cells endowed with spermatogonial stem cell potential.
authors.affiliation: Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center in Dallas, Dallas, Texas 75390, USA.
authors.forename: Zhuoru
authors.initials: Z
authors.lastname: Wu
authors.forename: Ilaria
authors.initials: I
authors.lastname: Falciatori
authors.forename: Laura A
authors.initials: LA
authors.lastname: Molyneux
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Richardson
authors.forename: Karen M
authors.initials: KM
authors.lastname: Chapman
authors.forename: F Kent
authors.initials: FK
authors.lastname: Hamra
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D048708:Cell Growth Processes / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D003470:Culture Media / Q000737:chemistry* / Q000494:pharmacology*; D004526:Efficiency; D005260:Female; D005502:Food; D008297:Male; D051379:Mice; D008954:Models, Biological; D010375:Pedigree; D051381:Rats; D017207:Rats, Sprague-Dawley; D055647:Rats, Transgenic; D013093:Spermatogonia / Q000166:cytology* / Q000187:drug effects; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D013737:Testis / Q000637:transplantation
chemical_list: D003470:Culture Media