Primer sequences for qPCR.

<p>Primer sequences for qPCR.</p

Effect of GILZ deficiency on FOXO1 activity during spermatogenesis.

<p>A: Day 6 old WT and Gilz KO mouse testes were used in immunofluorescence analysis to assess the localization of FOXO1 (green) and GILZ (red) using confocal microscopy (DAPI-stain nuclei shown in blue). High magnification inserts showing localization of FOXO1 and GILZ were also included. Scale bars represent 100 µM and insert scale bars represent 25 µM. B: Quantitative analysis of nuclear FOXO1 positive cells in day 6 old WT and Gilz KO testes. Data represents the mean ± SEM of 6 testes. **, p&lt;0.01. C: Quantitative PCR analysis of <i>Ret</i>, <i>Lhx</i>, <i>Egr4</i>, Sall4, Dppa4 and Bim mRNA expression in day 6, 10 and 14 old WT and Gilz KO testes. Results are expressed as the number of mRNA copies per 10⁶ 18 s rRNA copies. Data represents the mean ± SEM of 4–8 mice per group. **, p&lt;0.01 related to WT controls. D: Protein expression of BIM-EL (extra long) and GILZ was detected in day 20 old WT and Gilz KO testes using Western blotting. Representative images of two individual experiments.</p

Effects of GILZ deficiency on blood-testis barrier and testicular leukocyte.

<p>A: Immunostaining for the blood-testis barrier marker espin was performed on day 20 WT and Gilz KO testes using anti-espin antibody. Scale bars represent 100 µM. Single insert represents the no espin staining negative control. B: Blood-testis-barrier (indicated by arrows) in day 20 old WT and Gilz KO testes samples were examined using transmission electron microscopy. Scale bars represent 5 µM. C: The presence of leukocytes in adult (day 70) WT and Gilz KO testes was examined by immunohistochemistry using CD45 as a marker. Scale bars represent 100 µM.</p

Effect of GILZ deficiency on testicular size.

<p>A: Representative image of WT and Gilz KO testes. B: Testes weights of WT and Gilz KO testes. Data represents the mean ± SEM of 5 mice per group. **P&lt;0.001.</p

Analysis of the SSC population in Gilz KO testis.

<p>A: Immunohistochemistry for PLZF on testis sections from mice of the indicated postnatal ages (days). Higher magnification insets show the presence of PLZF⁺ cells in the periluminal region of day 6 tubules. B: Immunohistochemistry for SALL4 on testis sections from post-natal day 30 mice. Asterisks in A and B indicate day 14 and 30 tubules containing PLZF and SALL4-positive cells respectively. Scale bars represent 50 µM.</p

GILZ deficiency leads to spermatogenic failure.

<p>A: Testes were collected from 6, 10, 14, 20 and 30 day old and adult WT and Gilz KO mice, processed and stained with PAS. Scale bars represent 100 µM. B: The presence of mature sperm was detected in adult WT but not Gilz KO epididymides. Scale bars represent 100 µM. C: Representative images of TUNEL positive cells in 20 day old WT and Gilz KO testes. Scale bars represent 50 µM. D: Chromatin morphology of pachytene cells was examined using PAS staining (scale bars represent 50 µM) and SCP3 immunofluorescence in day 20 WT and Gilz KO testes.</p