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.
Highlights
The self-renewal and differentiation of spermatogonial stem cells (SSCs) is the foundation of spermatogenesis [1]
The average diameter of the seminiferous tubule cavities of mice in the testis-injection groups gradually increased (Figs 3 and 4, Table 2) and was significantly higher than that of those in the i.p. injection group after busulfan treatment (P < 0.05); it peaked on day 28 at 267.78 μm in the 6 mg/kg/side and at 218.01 μm in the 4 mg/kg/side, before gradually decreasing
The Sertoli cell only was observed clearly on day 14 after busulfan treatment and the largest hollow space appeared on day 21 and remained until day 28 (Figs 3C2 and 4), which was earlier than observed in the other groups
Summary
The self-renewal and differentiation of spermatogonial stem cells (SSCs) is the foundation of spermatogenesis [1]. The mechanisms of spermatogenesis, sperm deformation and the maturation of sperm in humans have not been fully elucidated [2, 3]. Male infertility that resulted from spermatic deformities and abnormal spermatogenesis, amongst other causes, are becoming increasingly common [4,5,6]. In the background of a high incidence of hereditary diseases worldwide [7], it is necessary to intensively research the mechanisms of male spermatogenesis to explore male contraception techniques and other birth control. Testis Injection of Busulfan Is Safe for Mice Recipient of SSCs technologies, which have become the focus of male reproductive research field. The lack of adequate experimental animal models and experimental approaches has delayed progress in this field
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