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First published online March 6, 2009
doi: 10.1242/10.1242/dev.032243

Development 136, 1191-1199 (2009)
Published by The Company of Biologists 2009
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Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal

Jon M. Oatley1, Melissa J. Oatley1, Mary R. Avarbock2, John W. Tobias3 and Ralph L. Brinster2,*

1 Department of Dairy and Animal Sciences, Center for Reproductive Biology and Health, College of Agricultural Sciences, Pennsylvania State University, University Park, PA 16802, USA.
2 Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
3 Penn Bioinformatics Core, University of Pennsylvania, Philadelphia, PA 19104, USA.


Figure 1
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  		Fig. 1. Isolation of Thy1+ and Thy1-depleted cell populations from 6-dpp mouse testes and quantification of SSC contents by functional germ cell transplantation. (A) Schematic of the experimental strategy using MACS to isolate Thy1+ fractions. Following incubation with anti-Thy1 antibody conjugated to magnetic microbeads, testis cell suspensions were subjected to magnetic column selection. Rosa mice that express lacZ in all germ cells were used as donors to compare SSC content of the Thy1+ and Thy1-depleted populations. Inbred C57BL/6 donors were used as a source of cell fractions for gene expression profiling, which was conducted using Affymetrix 430 2.0 GeneChips. (B) Representative recipient mouse testes transplanted with MACS-isolated Thy1+ or Thy1-depleted cell fractions from 6-dpp Rosa donors. Scale bars: 2 mm. (C) Quantification of SSC content in Thy1+ and Thy1-depleted cell fractions based on donor-derived colonies of spermatogenesis in recipient testes following functional germ cell transplantations. *P≤0.05.

 

Figure 2
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  		Fig. 2. Identification of differential Csf1r gene expression in Thy1+ versus Thy1-depleted testis cell fractions. (A,B) Relative Csf1r transcript expression in cell fractions isolated from 6-dpp mouse pup testes measured by microarray analysis (A) and qRT-PCR (B). (C) Relative Csf1r transcript expression in cell fractions isolated from adult mouse testes measured by qRT-PCR. All data are mean±s.e.m. for three different replicates. *P≤0.05.

 

Figure 3
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  		Fig. 3. Expression of Csf1r protein in mouse pup testes. (A) Immunofluorescence localization of Csf1r expression in testes of 10-dpp mouse pups. Expression was observed in individual spermatogonia within a few seminiferous tubules (arrow), and in macrophages and other blood cells in the interstitial space (arrowheads). (B) FCA of the Csf1r+ cell populations in testes of 8-dpp mouse pups. F4/80 was used as a macrophage-specific marker to distinguish Csf1r+ macrophages (region R2) from other Csf1r+ populations. A population of cells that is F4/80-/Csf1r+ was identified (region R3). (C) Examination of Thy1 expression in pup R3 cell populations using FCA. Red fill, isotype control immunoglobulin; black line (open fill), marker-specific antibody. Data are mean±s.e.m. for three separate testis cell preparations.

 

Figure 4
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  		Fig. 4. Expression of Csf1r in cultured Thy1+ germ cell clumps. Serum-free media with Gdnf and Fgf2 as the only growth factor supplements supports formation of germ cell clumps consisting of both SSCs and non-stem cell spermatogonia. (A) RT-PCR analysis of Csf1r transcript expression. (Top) Csf1r expression analysis of four different Thy1+ germ cell cultures (lanes 1-4). (Bottom) Control expression analysis of identical RNA samples subjected to mock reverse transcription. MW, 100 bp DNA ladder. (B) Immunofluorescence evaluation of Csf1r protein expression in cultured Thy1+ germ cell clumps (arrows). Scale bars: 100 µm. (C) Representative scatter plot for FCA of the percentage of cultured Thy1+ germ cell clumps expressing Csf1r protein. The left-hand plot is cells incubated with isotype control immunoglobulin and the right-hand plot is cells incubated with anti Csf1r primary antibody.

 

Figure 5
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  		Fig. 5. Effects of Csf1 exposure on germ cell proliferation and SSC self-renewal in vitro. (A) Morphology of Thy1+ germ cell clumps (arrows) in control (Gdnf + Fgf2) and Csf1-treated (Gdnf + Fgf2 + Csf1) cultures. Scale bars: 50 µm. (B) Total germ cell expansion in control and Csf1-treated cultures during a 63-day culture period. Graph lines for both conditions are nearly identical and overlap. Regression analysis for homogeneity of slopes revealed no difference (P=0.800) between control (y=112.1 x -1519.7) and Csf1-treated (y=120.8 x -1642.1) cultures. (C) SSC expansion in control and Csf1-treated Thy1+ germ cell cultures as determined by functional germ cell transplantations. Regression analysis for homogeneity of slopes revealed a significant difference (P=0.011) between control (y=9568 x -135,198) and Csf1-treated (y=32,056 x -461,164) cultures. (D) Difference in SSC content within cultured Thy1+ germ cell clumps at specific time-points of analysis throughout a 63-day period in control and Csf1-treated cultures. The concentration of SSCs in the cultured germ cells was significantly higher in cultures treated with Csf1 beginning at day 21 and progressively increased with time in culture. SSC numbers were determined using functional germ transplantation and data are presented as fold difference in SSCs/105 cells injected between Csf1-treated and control cultures. In all graphs, data are mean±s.e.m. for three different independent cultures. Red lines are control cultures (Gdnf + Fgf2) and blue lines are Csf1-treated cultures (Gdnf + Fgf2 + Csf1). *P≤0.05 between control and Csf1-treated cultures.

 

Figure 6
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  		Fig. 6. Expression of Csf1 protein in mouse testes. (A) Immunofluorescent localization of Csf1 expression in 10-dpp pup (top row) and adult (bottom row) mouse testes. Intense expression was observed in clusters of Leydig cells (arrows) located in the interstitial space at both ages. (B) Localization of Csf1 expression in myoid cells (arrow) lining the basement membrane of some seminiferous tubules in pup testes. Scale bars: 50 µm in A; 25 µm in B.

 

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