Testicular Busulfan Injection in Mice to Prepare Recipients for Spermatogonial Stem Cell Transplantation Is Safe and Non-Toxic

Abstract

Current methods of administering busulfan to remove the endogenous germ cells cause hematopoietic toxicity, require special instruments and a narrow transplantation time. We use a direct testicular injection of busulfan method for preparing recipients for SSC transplantation. Male ICR mice (recipients) were divided into four groups, and two experimental groups were treated with a bilateral testicular injection of 4 or 6 mg/kg/side busulfan (n = 60 per concentration group). Mice received an intraperitoneal injection (i.p.) of 40 mg/kg busulfan (n = 60, positive control) and bilateral testicular injections of 50% DMSO (n = 60, negative control). Donor SSCs from RFP-transgenic C57BL/6J mice were introduced into the seminiferous tubules of each recipient testis via efferent duct injection on day 16-17 after busulfan treatment. Recipient mice mated with mature female ICR mice and the number of progeny was recorded. The index detected at day 14, 21, 28, 35 and 70 after busulfan treatment. Blood analysis shows that the toxicity of busulfan treated groups was much lower than i.p. injection groups. Fertility was restored in mice treated with busulfan and donor-derived offspring were obtained after SSC transplantation. Our study indicated that intratesticular injection busulfan for the preparation of recipients in mice is safe and feasible.

Fig 1. The change of testis weight after busulfan treatment over time.

Fig 2. The change of body weight after busulfan treatment over time.

Fig 3. Histological sections of testicular tissue taken from mice in the four groups at different time points.

Hematoxylin & Eosin staining is shown. A-D Numbers 1–5 refer to the day after treatment on which the samples were taken, where 1 = day 14, 2 = day 21, 3 = day 28, 4 = day 35 and 5 = day 70. A1–A5) The negative control group mice were treated with 50% DMSO at corresponding time points. B1–B5) Testis tissue sections from mice in the 4 mg/kg/side testis-treated group at corresponding time points. C1–C5) Testis tissue sections from mice in the 6 mg/kg/side testis-treated group at corresponding time points. D1–D5) Testis tissue sections from mice in the 40 mg/kg I.P. injection group. Scale bars(A = 100μm, B,C,D = 50 μm).

Fig 4. The cavity diameter of seminiferous tubules from mice administered with busulfan at the indicated routes and doses.

Fig 5. Changes in levels of WBC, RBC, HGB and PLT after busulfan treatment among groups.

WBC, white blood count; RBC, red blood count; HGB, hemoglobin; PLT, platelets.

Fig 6. The number of offspring of the recipients treated with different doses of busulfan and different methods over time.