Identification of CHD1L as an Important Regulator for Spermatogonial Stem Cell Survival and Self-Renewal

Figure 4

Chd1l modulates SSC gene expression, clonogenicity, proliferation, and apoptosis. (a) Chd1l gene knockdown in mouse SSCs. Mouse SSCs were infected with scrambled shRNA, Chd11 shRNA #1, or Chd11 shRNA #2 lentivirus. After 48 hours, total RNAs were harvested and subjected to quantitative RT-PCR analysis. (b) Oct4, Plzf, Gfrα1, and Nanog gene expressions are modulated by Chd1l knockdown in mouse SSCs. Mouse SSCs were infected with scrambled shRNA or Chd11 shRNA#2 lentivirus. After 48 hours, total RNAs were harvested and subjected to quantitative RT-PCR analysis with indicated primers. Data in (a) and (b) are the mean ± SD of three independent experiments. compared with the control. (c) Clonogenicity of mouse SSCs was significantly inhibited by knockdown of Chd1l. Cell clusters whose diameters were greater than or equal to 50 μm were defined as colonies. Bars = 200 μm. Representative images (bottom) and quantitative data (up, colonies per well) from three independent experiments () were presented here. (versus the scrambled control). (d–f) Chd1l knockdown reduced mouse SSC proliferation. Mouse SSCs were infected with scrambled shRNA or Chd11 shRNA#2 lentivirus. Infected SSCs were cultured for further 48 hours (d and e), or the indicated times (f), followed by BrdU cell proliferation assays (d), quantitative RT-PCR analyses (e), or MTS assays (f), respectively. In panel (d), the representative images (bottom) and quantitative data (up, percentage of BrdU⁺ cells, green) from three independent experiments () were presented. Bars = 50 μm; (versus scrambled control shRNA). Data presented in panels (e) and (f) are mean ± SD of three independent experiments. (g) Chd1l knockdown increased mouse SSC apoptosis. Apoptosis was compared between the scrambled shRNA control and Chd1l shRNA#2 cells using flow cytometry. The cells labeled with Annexin-V but without propidium iodide labeling were counted as apoptotic cells. -axis: intensity of Annexin-V, relative units; -axis: intensity of propidium iodide incorporation, relative units. The percentage of apoptotic cells is the mean ± SD of three independent experiments. compared with the control. shRNA: short hairpin RNA; BrdU: 5-bromo-2′-deoxyuridine; SSCs: spermatogonial stem cells; SD: standard deviation. compared with the control.

(a)

(b)

(c)

(d)

(e)

(f)

(g)