Testicular Injection Of Busulfan For Recipient Preparation In Transplantation Of Spermatogonial Stem Cells In Mice

Transplantation of spermatogonial stem cells (SSCs) has theoretical and practicalsignificance in many fields such as clinical medicine, studying on spermatogenesis, transgeneticanimal production, and some recipient preparation methods are developed such asintraperitoneal injection busulfan, irradiation method and heat shock. However, the shortcomingof toxicity and tediousness can not be avoided. To obtain an ideal SSCs transplantation result,ICR mice treated by bilateral testicular injection of busulfan were used as recipients at the doseof4, or6mg/kg/side, the positive and negative control group mice were respectively treatedwith40mg/kg body weight busulfan and50%DMSO by bilateral testicular injection in ourstudy in the current study. The mortality rate, routine blood components and cavity diameter ofthe seminiferous tubule were analyzed at day14,21,28,35and70after busulfan treatment. Thedetection results of RBC, WBC, HGB, and PLT showed the toxicity of busulfan in testisinjection groups is greatly lowered than in i.p. injection group. The donor germ cells expressingred fluorescent protein (RFP) from transgenic C57BL/6J male mice were transplanted intorecipients by efferent duct at d16after treatment with busulfan. The positive control was treatedwith40mg/kg busulfan by intraperitoneal injection, and the negative control was treated withbilateral testicular of the50%dimethyl sulfoxide (DMSO). The recipient mice were detected byIn Vivo Imaging Systerm at day42after transplantation, and some recipients with RFP-positivewere natural mated with ICR female mice at day49. Donor-derived germ cell colonies with redfluorescence were observed at day42after transplantation, and donor offspring were obtained.The results indicated the endogenous germ cells have been eliminated in the seminiferoustubules, and exogenous SSCs undergo spermatogenesis successfully in the testes of recipientmice prepared by bilaterally testicular injection busulfan, the fertility was restored anddonor-derived offspring were obtained after SSC transplantation, and no recipient died duringthe whole experimental, and the efficiency and safty were demonstrted in this recipientpreparation method using testes-injection of busulfan in rodents.