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Germ Cell Transplantation and Testis Tissue Xenografting in Mice
Published on: February 6, 2012
An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell
Nirmalya Ganguli1, Neerja Wadhwa1,2, Abul Usmani1
1Embryo Biotechnology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
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.
Insights
This study introduces an improved method for creating germ cell-depleted animal models using targeted testicular busulfan injections. This technique enhances spermatogonial stem cell transplantation (SSCT) efficiency and increases the production of donor-derived offspring.
Area of Science:
- Reproductive Biology
- Stem Cell Research
- Animal Models
Background:
- Spermatogonial stem cell transplantation (SSCT) is vital for species conservation and therapeutic applications.
- Current methods for creating germ cell-depleted models involve toxic systemic busulfan, causing adverse effects.
- A need exists for a more efficient and humane method for germ cell depletion.
Purpose of the Study:
- To develop an efficient and less toxic method for creating germ cell-depleted animal models.
- To improve the success rate of spermatogonial stem cell transplantation (SSCT).
- To increase the yield of donor-derived offspring.
Main Methods:
- Utilized targeted testicular injections of busulfan (75 μg) in a 50% DMSO solution.
- Administered two 10 μl injections diagonally within each testis.
- Transplanted GFP-expressing spermatogonial stem cells (SSCs) into treated testes and assessed outcomes.
Main Results:
- A 75 μg dose of busulfan effectively depleted germ cells within 15 days without mortality or systemic toxicity.
- Successful colonization by donor-derived GFP-expressing germ cells was observed.
- 71% of transplanted males sired transgenic offspring, a significant improvement over previous methods (5.5%).
Conclusions:
- An efficient method for generating germ cell-depleted animal models was established using targeted testicular busulfan.
- This technique significantly enhances spermatogonial stem cell transplantation (SSCT) success.
- The improved method results in a remarkably higher proportion of donor-derived offspring generation.
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