Abstract
BackgroundSpermatogonial 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.MethodsA 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.ResultsA 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0405-1) contains supplementary material, which is available to authorized users.
Highlights
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
After 60 and 90 days of Busulfan treatment (BST), the body weights were significantly (p < 0.0001) reduced in intraperitoneal busulfan treatment group (IP-BST) group mice in comparison to testicular busulfan treatment groups (T-BST) and control group mice where the body weight increased with increase in age after busulfan injection (Fig. 1a)
Effect of BST on testicular parameters Macroscopic observation of testis at day15 post injection revealed a remarkable reduction in testes size of T-BST75 and T-BST-100 injected animals as compared to that of control animals (Fig. 1b; Additional file 1: Figure S2)
Summary
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. In 1994, Brinster and Zimmerman first demonstrated the possibilities of isolation and transplantation of SSC into an evacuated testis followed by successful spermatogenesis from the transplanted SSC [2] Since this approach has been explored to address various issues like transgenesis [3, 4], infertility [5], conservation of endangered species [6], and for understanding the biology of male germinal stem cells and its niche [3], etc. There are several drawbacks of this existing technique of creating GCD by i.p. injection of busulfan It takes almost 6 weeks for successful depletion of Gc. Busulfan doses, less than 40 mg/kg given to adult mice did not result in prolonged depletion of endogenous spermatogenesis in most tubules [14]; higher doses often caused severe hematopoietic suppression requiring bone marrow transplantation and resulted in death [18]. It was hypothesized that after i.p. injection, busulfan may be absorbed and transferred to other organs via blood circulation and exert a nonspecific cytotoxic effect on the body by destroying other cellular niches of the body resulting in bone marrow depression and anemia
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