Testicular injection of busulfan for recipient preparation in transplantation of spermatogonial stem cells in mice
Yusheng Qin A B , Ling Liu A , Yanan He A , Wenzhi Ma C , Huabin Zhu A , Mingyuan Liang B , Haisheng Hao A , Tong Qin A , Xueming Zhao A and Dong Wang A D
+ Author Affiliations
- Author Affiliations
A The Key Laboratory for Farm Animal Genetic Resources and Utilisation of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193, China.
B College of Animal Science and Technology, Jilin Agriculture University, Changchun, 130118, China.
C Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education and Key Laboratory of Reproduction and Heredity of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan, 750004, China.
D Corresponding author. Email: dwangcn2002@vip.sina.com.cn
Reproduction, Fertility and Development 28(12) 1916-1925 https://doi.org/10.1071/RD14290
Submitted: 29 April 2014 Accepted: 22 May 2015 Published: 26 June 2015
Abstract
Intraperitoneal busulfan injections are used to prepare recipients for spermatogonial stem cell (SSC) transplantation but they are associated with haematopoietic toxicity. Testicular injections of busulfan have been proposed to overcome this limitation. To date, testicular injections have not been studied in the mouse model. Therefore, in the present study we used ICR mice as recipients for SSC transplantation and prepared these mice by testicular injection of busulfan on both sides (2, 3, 4 or 6 mg kg–1 per side). Following this, donor germ cells expressing red fluorescent protein (RFP) from transgenic C57BL/6J male mice were transplanted into recipients via the efferent duct on Days 16–17 after busulfan treatment. Positive control mice were prepared by intraperitoneal injection of 40 mg kg–1 busulfan and negative control mice were treated with bilateral testicular injection of 50% dimethyl sulfoxide. On Day 49 after transplantation, recipient mice that were RFP-positive by in vivo imaging were mated with ICR female mice. Donor-derived germ cell colonies with red fluorescence were observed on Day 60 after transplantation, and donor-derived offspring were obtained. The results demonstrated that endogenous germ cells were successfully eliminated in the seminiferous tubules via testicular busulfan administration, and that exogenous SSCs successfully undergo spermatogenesis in the testes of recipient mice prepared by testicular injections of busulfan. In addition to its effects on recipient preparation, this method was safe in rodents and could possibly be adapted for use in other species.
Additional keywords: donor offspring, haematopoietic toxicity, intraperitoneal injections, vivo imaging.
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