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 μM 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.
Original language | English (US) |
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Pages (from-to) | 77-86 |
Number of pages | 10 |
Journal | Biology of reproduction |
Volume | 81 |
Issue number | 1 |
DOIs | |
State | Published - Jul 2009 |
Keywords
- Fertility
- Germ cell
- Germline
- Regenerative medicine
- Spermatogenesis
- Spermatogonia
- Spermatogonial
- Spermatozoa
- Stem cell
- Stem cells
- Transgenic rats
ASJC Scopus subject areas
- Reproductive Medicine
- Cell Biology