An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation.

Background: Spermatogonial stem cell (SSC) transplantation (SSCT) has become important for conservation of endangered species, transgenesis and for rejuvenating testes which have lost germ cells (Gc) due to gonadotoxic chemotherapy or radiotherapy during the prepubertal phase of life. Creating a germ cell-depleted animal model for transplantation of normal or gene-transfected SSC is a prerequisite for such experimental studies. Traditionally used intraperitoneal injections of busulfan to achieve this are associated with painful hematopoietic toxicity and affects the wellbeing of the animals. Use of testicular busulfan has been reported recently to avoid this but with a very low success rate of SSCT. Therefore, it is necessary to establish a more efficient method to achieve higher SSCT without any suffering or mortality of the animals.

Methods: A solution of busulfan, ranging from 25 μg/20 μl to 100 μg/20 μl in 50 % DMSO was used for this study. Each testis received two diagonally opposite injections of 10 μl each. Only DMSO was used as control. Germ cell depletion was checked every 15 days. GFP-expressing SSC from transgenic donor mice C57BL/6-Tg (UBC-GFP) 30Scha/J were transplanted into busulfan-treated testis. Two months after SSCT, mice were analyzed for presence of colonies of donor-derived SSC and their ability to generate offspring.

Results: A dose of 75 μg of busulfan resulted in reduction of testis size and depletion of the majority of Gc of testis in all mice within 15 days post injection without causing mortality or a cytotoxic effect in other organs. Two months after SSCT, colonies of donor-derived Gc-expressing GFP were observed in recipient testes. When cohabitated with females, donor-derived offspring were obtained. By our method, 71 % of transplanted males sired transgenic progeny as opposed to 5.5 % by previously described procedures. About 56 % of progeny born were transgenic by our method as opposed to 1.2 % by the previously reported methods.

Conclusions: We have established an efficient method of generating a germ cell-depleted animal model by using a lower dose of busulfan, injected through two diagonally opposite sites in the testis, which allows efficient colonization of transplanted SSC resulting in a remarkably higher proportion of donor-derived offspring generation.