Two allogeneic descendents derived from the high-dose busulfan-treated infertile mouse model after freeze-thawed spermatogonial stem cell transplantation  Xiang Wang, M.D., Ph.D., Qiang Ding, M.D., Ph.D., Yuanfang Zhang, M.D., Huilin Wang, Ph.D., Lianghong Ma, M.D., Ph.D., Xiayang Xie, M.Sc.  Fertility and Sterility  Volume 90, Issue 4, Pages 1538-1549 (October 2008) DOI: 10.1016/j.fertnstert.2007.08.049 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Western blotting to compare the α6-integrin, c-kit, and SCF expressions at the first, second, and third month checkpoints. I, bilateral testes of allogeneic non-busulfan-treated mice served as the positive control. II, a lateral autogeneic testis injected with an equal amount of cell suspension medium to serve as the internal control. III, the other lateral testis underwent transplantation as in the fresh group. Fertility and Sterility 2008 90, 1538-1549DOI: (10.1016/j.fertnstert.2007.08.049) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Comparison of α6-integrin, c-kit, and SCF mRNA expressions at the first, second, and third month checkpoints by real-time fluorescence quantitative-PCR analysis. The yellow line represents the positive control, the red line represents the fresh group, and the blue line represents the internal control. (a) Comparison of the mean α6-integrin mRNA expressions; (b) comparison of the mean c-kit mRNA expressions; (c) comparison of the mean SCF mRNA expressions. Fertility and Sterility 2008 90, 1538-1549DOI: (10.1016/j.fertnstert.2007.08.049) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (a) Demonstration of α6-integrin expression on cytomembrane and cytoplasm at different checkpoints after transplantation. (b, c, d) Scanning electron micrograph(SEM) demonstration on the spermatogenic cells in lumina of the seminiferous tubules at the third month checkpoint. (b) Internal control, the recipient pretreated with 40 mg of busulfan/kg body weight, injected with transplantation medium and maintained for 3 months. SEM, ×3,000. (c) Fresh group, recipients received the fresh cell suspension immediate after spermatogonial stem cell isolation and purification, and maintained for 3 months. SEM, ×1,125. (d) Freeze-thawed group, the suspension was preserved in liquid nitrogen at −190°C for 3 months before thawing for transplantation, and maintained for 3 months. SEM, ×1,125. Fertility and Sterility 2008 90, 1538-1549DOI: (10.1016/j.fertnstert.2007.08.049) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 IVF result and offspring production of spermatozoa obtained from the third month maintenance in the recipients, who received freeze-thawed allogeneic spermatogonial stem cells that were preserved at −190°C liquid nitrogen for three months. (a) Development of preimplantation, two-cell embryos derived from IVF with recipient spermatozoa and the F1 mice oocytes. Microscope, ×400. (b) Two young pups were born 20 days later: one was black, the other was plum. Fertility and Sterility 2008 90, 1538-1549DOI: (10.1016/j.fertnstert.2007.08.049) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions