Journal: Stem Cells (Dayton, Ohio)
Article Title: ETV5 Regulates Sertoli Cell Chemokines Involved in Mouse Stem/Progenitor Spermatogonia Maintenance
Figure Lengend Snippet: Expression of CCL9 in mouse testis. (A): Immunofluorescence of 8-day WT mouse testis showing strong expression of CCL9 in Sertoli cells (green Alexa Fluor 488 staining). Arrows indicate locations of stem/progenitor spermatogonia, which are negative for CCL9. Scale bar = 25 µm. (B): Immunofluorescence of 8-day Etv5 -/- mouse testis showing a sharp decrease of CCL9 expression in Sertoli cells. Scale bar = 15 µm. (C): Immunofluorescence of 8-day WT mouse testis showing expression of CCR1, the receptor for CCL9, in stem/progenitor spermatogonia. Type A spermatogonia show double-staining for CCR1 (green Alexa Fluor 488 staining) and GCNA1 (pink Alexa Fluor 594 staining). The green staining in the lumen is background staining. Scale bar = 25 µm. (D): Immunohistochemistry of 6-day WT mouse testis showing expression of CCR1 in stem/progenitor spermatogonia (arrows). Scale bar = 15 µm. (E): Western blot of protein extracts from isolated WT seminiferous tubules of 4-, 10-, 35- and 90-day-old mice with anti-CCL9 and anti-WT1 antibodies, showing that CCl9 expression is restricted to early steps of spermatogenesis. (F): Electrophoretic mobility shift assays to investigate ETV5 binding to its consensus sequence in the Ccl9 promoter. The left panel shows gel shifts using nuclear extracts of TM4 cells after transfection with the pERMFLAG construct (pERMFLAG TM4). 1: biotinylated probe alone (B EBS3); 2: nuclear extract of pERMFLAG TM4 and B EBS3, with apparent gel shift (arrow); 3: nuclear extract of pERMFLAG TM4 with B EBS3 and nonbiotinylated probe (NB EBS3) showing outcompetition; and 4: nuclear extract of pERMFLAG TM4 with B EBS3 and ETV5 antibody (ETV5 Ab) showing a decrease of DNA/protein binding. The middle panel shows gel shifts using again pERMFLAG TM4 nuclear extracts. 1: B EBS3 alone; 2: pERMFLAG TM4 with B EBS3, showing apparent gel shift (arrow); 3: pERMFLAG TM4 with B EBS3 and ETV5 Ab showing decrease of protein-DNA binding; 4: nuclear extract of TM4 cells after transfection with control vector (Control TM4), and incubated with B EBS3; 5: control TM4 with B EBS3 and NB EBS3, 6: pERMFLAG TM4 with mutant B EBS3, and 7: pERMFLAG TM4 with mutant NB EBS3. Gel shifts with NB EBS3 substantially, but not completely outcompeted the protein-DNA complex suggesting the presence of other factors in the complex. There was no shift of protein-DNA complexes with mutant EBS3 sequences or TM4 cells transfected with control vector. The right panel shows gel shifts of purified ETV5 protein using pET vector (ETV5 protein). 1: ETV5 protein with B EBS3, showing gel shift (arrow) and 2: ETV5 protein with B EBS3 and NB EBS3 showing outcompetition. The data confirms a possible ETV5-EBS3 binding. Abbreviations: CCL9, C-C-motif ligand 9; CCR1, C-C-receptor type 1; GCNA, germ cell nuclear antigen 1; Etv5 , Ets variant gene; ETV5 protein, Ets variant protein; B EBS3, biotinylated Ets binding site 3; NB EBS3, nonbiotinylated Ets binding site 3; WT, wild type.
Article Snippet: Electromobility Shift Assays Electromobility shift assays (EMSAs) were carried out using 3' biotinylated probes (Invitrogen, Carlsbad, CA, http://www.invitrogen.com ) designed to include the three Ets binding sites (EBSs) found in the Ccl9 genomic sequence (Accession number AL596122) and a chemiluminescent kit (Cat. No. 20148, Thermo Scientific, Pittsburgh, PA, http://www.thermo.com ).
Techniques: Expressing, Immunofluorescence, Staining, Double Staining, Immunohistochemistry, Western Blot, Isolation, Mouse Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Transfection, Construct, Protein Binding, Plasmid Preparation, Incubation, Mutagenesis, Purification, Positron Emission Tomography, Variant Assay