ABSTRACT
An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg-1) on day 36 or a dose of 40 mg kg-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.
Subject(s)
Animals , Male , Mice , Adult Germline Stem Cells/transplantation , Azoospermia/chemically induced , Busulfan/toxicity , Disease Models, Animal , Infertility, Male/chemically induced , Injections, Intraperitoneal , Spermatogenesis/drug effects , Spermatogonia/drug effects , Stem Cell Transplantation/methodsABSTRACT
An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg-1) on day 36 or a dose of 40 mg kg-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.
ABSTRACT
Transplantation of spermatogonial stem cells (SSCs) in experimental animal models has been used to study germ line stem cell biology and to produce transgenic animals. The species-specific recipient model preparation is important for the characterization of SSCs and the production of offspring. Here, we investigated the effects of surgically induced cryptorchidism in dog as a new recipient model for spermatogonial stem cell transplantation. Artificially unilateral or bilateral cryptorchidism was induced in ten mature male dogs by surgically returning the testis and epididymis to the abdominal cavity. The testes and epididymides were collected every week after the induction of artificial cryptorchidism (surgery) for one month. To determine the effect of surgical cryptorchidism, the seminiferous tubule diameter was measured and immunohistochemistry using PGP9.5 and GATA4 antibodies was analyzed. The diameters of the seminiferous tubules of abdominal testes were significantly reduced compared to those of the scrotal testes. Immunohistochemistry results showed that PGP9.5 positive undifferentiated spermatogonia were significantly reduced after surgical cryptorchidism induction, but there were no significant changes in GATA-4 positive sertoli cells. To evaluate the testis function recovery rate, orchiopexy was performed on two dogs after 30 days of bilateral cryptorchidism. In the orchiopexy group, SCP3 positive spermatocytes were detected, and spermatogenesis was recovered 8 weeks after orchiopexy. In this study, we provided optimum experimental conditions and time for surgical preparation of a recipient canine model for SSC transplantation. Additionally, our data will contribute to recipient preparation by using surgically induced cryptorchidism in non-rodent species.