IGF-1R signaling is essential for the proliferation of cultured mouse spermatogonial stem cells by promoting the G2/M progression of the cell cycle

Abstract

Culture of mouse spermatogonial stem cells (mSSCs) contributes to understanding the mechanisms of mammalian spermatogenesis. Several key growth factors such as GDNF and FGF2 have been known to be essential for the proliferation of cultured mSSCs. However, additional factors regulating SSC proliferation remain to be identified. In this study, we report that IGF-1R signaling is required for the proliferation of cultured mSSCs by promoting the G2/M progression of the cell cycle. IGF-1 and its receptor IGF-1R are expressed in cultured mSSCs as well as in isolated Sertoli cells and interstitial cells. Blockage of IGF-1R signaling either by knockdown of IGF-1R or by the IGF-1R-specific inhibitor picropodophyllin (PPP) significantly reduced the proliferation of mSSCs, increased their apoptosis, and impaired their stem cell activity in an insulin-independent manner. PPP treatment of mSSCs blocked the G2/M progression. In contrast, both GDNF withdrawal and FGF2 signaling blockade decreased the entry of mSSCs into their S phases. Consistently, IGF-1 promoted the G2/M progression of thymidine-treated mSSCs, which were arrested at G1/S boundary synchronously; while GDNF and/or FGF2 stimulated their entry into the S phase. Moreover, IGF-1 activated the phosphorylation of AKT but not that of ERK1/2 in mSSCs. These results indicate that IGF-1R signaling stimulates the proliferation of mSSCs using a distinct mechanism from those by GDNF and FGF2, and will contribute to the establishment of a chemically defined culture system.

FIG. 1.

Expression of IGF-1R signaling genes. (A) The reverse transcription-polymerase chain reaction detection of the expression of Igf-1, its receptor Igf-1r, insulin receptor Insr, and insulin receptor substrate Irs1, Irs2 in the liver, embryonic stem cells (ESC), cultured mouse spermatogonial stem cells (mSSCs), mouse embryonic fibroblasts (MEFs), isolated Sertoli cells (SC), and interstitial cells (IC). (B) IGF-1 was detected in the cytoplasm of PLZF- or MVH-positive mSSCs. (C) IGF-1R protein was localized in the cytoplasm and the nucleus of PLZF-, GFRα1-, or DAZL-positive mSSCs. Color images available online at www.liebertpub.com/scd

FIG. 2.

IGF-1R signaling pathway is required for the proliferation and stem cell activity of mSSCs. (A, B) Blockage of IGF-1R signaling by picropodophyllin (PPP) reduced mSSC proliferation on MEF feeder cells (A), laminin-coated dishes (B). (C, D) The values of cell recovery fold (CRF) of 1 μM PPP-treated cells significantly decreased in comparison with DMSO-treated cells in the presence or absence of insulin. CRFs were evaluated after 3 days of treatment on MEF feeder cells (C) and after 2 days of treatment on a laminin-coated dish (D). (E) Knockdown of Igf-1r with specific siRNAs. After transfection with negative control duplex and specific siRNAs against Igf-1r for 24 h, the protein was collected and western blots were conducted. (F, G) Knockdown of Igf-1r reduced mSSC proliferation of the feeder-free culture at 5 days after transfection as compared with that of the negative control duplex transfected cells; (G) are the quantitative results of (F). (H, I) Blockage of IGF-1R signaling impaired the stem cell activity assessed by transplantation experiments. GFP-labeled mSSCs were treated with DMSO or PPP for 36 h, and then, 1×10⁵ GFP donor cells were transplanted into each testis of busulfan-pretreated recipient mice. One month later, the recipient testes were analyzed (H) and the GFP-mSSC colonies (green fragments≥0.1 mm) were counted and plotted in (I). Controls were set using an equal volume of DMSO, which was the solvent of PPP solution. n=8 recipients for the DMSO group, and n=9 recipients for the PPP group; all the data were from three independent experiments and analyzed by t-test. Asterisks represent statistically significant differences (**P<0.01). Significant differences by multiple comparison are marked with different letters (P<0.05). Color images available online at www.liebertpub.com/scd

FIG. 3.

Blockage of IGF-1R signaling reduced the mitotic division of mSSCs and increased their apoptosis. (A, B) Reduced mSSC proliferation indicated by the decreased percentage of BrdU-labeled cells treated with PPP for 36 h. An aliquot of untreated cells was incubated with mouse isotype IgG, which was used to adjust the flow cytometer. (C, D) Increased mSSC apoptosis detected by TUNEL labeled cells treated with PPP for 48 h. To adjust the flow cytometer, an aliquot of untreated cells was incubated in the negative control buffer that was prepared without rTdT Enzyme. Asterisks represent statistically significant differences (**P<0.01). Color images available online at www.liebertpub.com/scd

FIG. 4.

Blockage of IGF-1R signaling resulted in mSSCs arrested in G₂/M phases. (A, B) mSSCs were treated with DMSO or 1, 2.5 μM PPP for 48 h, and the cell cycle phases were analyzed by propidium iodide staining. The percentages of mSSCs at different cell cycle phases were analyzed by ModFit software (B). (C, D) Mitotic cells marked by pH3 increased when mSSCs were treated with PPP for 24 h. An aliquot of untreated cells was incubated with rabbit isotype IgG, which was used to adjust the flow cytometer. (E) Cell cycle distributions showed that the cell cycle was blocked at G₂ and M phases on the blockage of the IGF-1R signaling by PPP for 24 h. Asterisks represent statistically significant differences (**P<0.01). Color images available online at www.liebertpub.com/scd

FIG. 5.

IGF-1 played a distinct role in maintaining the in vitro proliferation of mSSCs from GDNF, FGF2. (A, B) Cell cycle analysis of mSSCs at 2 days after the withdrawal of GDNF (A) or the addition of the FGFR inhibitor SU5402 (B). (C) The scheme of cell cycle synchronization by thymidine and the re-stimulation by growth factors. (D) The G₁/S-arrested mSSCs were re-stimulated with GDNF, FGF2, and IGF-1 for 2 days, and cell cycles were analyzed. Double asterisks represent statistically significant differences (P<0.01). Values with different lowercase letters were significantly different (P<0.05). Color images available online at www.liebertpub.com/scd

FIG. 6.

IGF-1 activates the phosphorylation of AKT. (A) Both IGF-1 and insulin could activate the phosphorylation of AKT, but not that of ERK1/2 in mSSCs. mSSCs were serum- and growth factor starved for 3 days, and re-stimulated with insulin and/or IGF-1 for 30 min. The starved cells were also restimulated with GDNF and FGF2 that served as a positive control. Cell lysates were prepared, and western blots were conducted. GAPDH was used as an internal control. (B) The p-AKT and p-ERK1/2 protein levels of different treatments were quantified by densitometry. Double asterisks denote statistically significant differences (P<0.01). NS, no significance. Color images available online at www.liebertpub.com/scd

FIG. 7.

Effect of IGF-1R on the proliferation of differentiating spermatogonia. (A) mSSCs were cultured on mitomycin-inactivated Sertoli cells and treated with DMSO or PPP. Two days later, the proliferation of differentiating spermatogonia (c-KIT-positive cells) was significantly decreased by PPP treatment in comparison with that of DMSO-treated cells. (B) Quantification of differentiating spermatogonia with DMSO or PPP treatments. (C) TUNEL assay showed that apoptotic cells were significantly increased after PPP treatment for 2 days. (D) After blockage of IGF-1R signaling by PPP for 2 days, the percentage of G₁ and S phases cells was decreased, while that of G₂/M phase cells was significantly increased. Double asterisks denote statistically significant differences (P<0.01). Color images available online at www.liebertpub.com/scd