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. 2012 Apr;22(4):773-6.
doi: 10.1038/cr.2012.17. Epub 2012 Jan 31.

Endogenously produced FGF2 is essential for the survival and proliferation of cultured mouse spermatogonial stem cells

Yan Zhang, Si Wang, Xiuxia Wang, Shangying Liao, Yujian Wu, Chunsheng Han

Endogenously produced FGF2 is essential for the survival and proliferation of cultured mouse spermatogonial stem cells

Yan Zhang et al. Cell Res. 2012 Apr.
No abstract available

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Figures

Figure 1
Figure 1
FGF2 is produced by mSPG and is essential for their in vitro propagation (IVP) and stem cell activity. To assess the IVP of mSPG, mSPG were collected by pipetting and trypsinized into single cells and cultured under different conditions for 5 days. The IVP was evaluated in terms of cell recovery fold (RF) values, which were the ratios of the numbers of mSPG at the end of different treatments over those at the beginning. Values with different superscripts were significantly different (P < 0.05). (A) Low-density but not high-density culture of mSPG required the supply of exogenous sFGF2. n = 5. (B) Blocking sFGF2 signaling by FGFR inhibitor SU5402 in exogenous sFGF2-devoid medium downregulated the IVP of high-density mSPG (–F+S) compared with the normal culture (N), while withdrawal of exogenous sFGF2 only (–F) had no significant effect on the high-density culture of mSPG. As a control, withdrawal of GDNF (–G) also reduced the IVP of mSPG significantly. n = 3. (C) mSPG cultured on laminin-coated slide were positively stained for FGF2 both in the cytosol and the nucleus, while stained positively only in the nucleus for the stem cell marker OCT4. (D) sFGF2 and its HMW isoforms were detected in cell lysate of mSPG and hESCs but not of MEFs or mESCs. sFGF2 was detected in conditioned medium (CM) of mSPG but not of MEFs. Recombinant sFGF2 was used as a positive control. (E) FACS analysis revealed that the number of the undifferentiated PLZF+ mSPG was significantly reduced after FGF2 shRNA was introduced into mSPG by lentivirus and that the effect of shRNA could not be rescued by excess amount of sFGF2. n = 5. (F) Colonization of recipient testes by transplanted EGFP-mSSCs that were transiently transfected with FGF2 siRNA or scrambled siRNA. n = 3. (G) Quantitative evaluation of IVP of mSPG cultured on feeder-free laminin-coated dishes in three types of MCM (See text for the preparation of the three types of MEF-conditioned medium). n = 3. (H) Assessment of phosphorylation of AKT and ERK1/2 by FGF2 and GDNF in mSPG in which sFGF2 signaling was blocked by SU5402 or endogenous FGF2 was knocked down by siRNA in advance. mSPG were cultured for 18 h in the absence of both GDNF and sFGF2 but in the presence of SU5402 or siRNA, and GDNF or sFGF2 was then added back. *Represents significant difference from controls with no growth factors (P < 0.05). n = 3. (I) The adverse effect of sFGF2 signaling blockade could not be rescued by GDNF and other factors except for sFGF2 itself in the presence of apoptosis inhibitor Y27632. n = 7. (J) The schematic summary of the source, the signaling pathways and the categories of activated genes of FGF2 as detected by microarray analysis.
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