Chemokine (C-X-C) Ligand 12 Facilitates Trafficking of Donor Spermatogonial Stem Cells

Figure 3

Inhibition of CXCR4 expression or function reduces stem cell activity of SSC-enriched germ cells. (a) qRT-PCR examination of shRNA-mediated knockdown of CXCR4 gene expression. SSC-enriched germ cell cultures were transduced with lentivirus containing a scrambled control shRNA vector or with one of three CXCR4-targeting shRNA clones (shCXCR4-1, shCXCR4-2, and shCXCR4-3). The transcript levels of CXCR4 were normalized to GAPDH gene expression and compared to untreated controls using . Significance was calculated using a Student’s -test (; ). Data are means ± SEM. (b) Colony forming efficiency of CXCR4-deficient SSC-enriched germ cell cultures. CXCR4 shRNA lentiviral transduced cultures of SSC-enriched germ cells derived from Rosa-lacZ mice were transplanted at 1 × 10⁴ cells/testis into the testes of Busulfan-treated recipient mice. Donor-derived spermatogenesis was quantified by staining recipient testes with X-gal 2 months after transplantation. Significance was calculated using a Student’s -test (; testes per treatment group). Data are means ± SEM. (c) AMD3100 disrupts SSC colony formation. Donor Rosa-lacZ SSC-enriched germ cells were treated with AMD3100 (5 μg/testis) followed by transplantation into recipient testis (data are means ± SEM, , testes per treatment group). (d) Alternatively, recipient mice bearing Rosa-lacZ SSC-enriched transplanted germ cells were repeatedly administered AMD3100 (5 μg/testis) via the intraperitoneal (IP) route at 12 hr intervals for 2 days following transplantation. Donor-derived colonies were detected using X-gal and counted 2 months after transplantation. Data are means ± SEM, where significance was assessed using Student’s -test (, testes per treatment group).

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