Figure 1.
Spatiotemporal expression and testicular localization of JMJD3.
Transcripts of Jmjd3 were examined by semi-quantitative RT-PCR in multiple tissue samples (A) and during post-natal development of the testis (B). Hprt was used as an internal control. Plzf was used as a control of spermatogonial stem cell-specific gene. GS: germline stem cells. C. Relative changes of Jmjd3 and Plzf during postnatal development of the testis. Relative densities of PCR products to the adult are presented after normalization with Hprt. Vertical bars represent the SEM of at least three experiments. D. Localization of H3K27 methylaton and its modifiers in the testis. Representative immunofluorescence images of indicated proteins with PLZF and DAPI in wild type adult testis are shown. Scale bar: 10µm.
Figure 2.
Effect of Jmjd3 knockdown in germline stem cells.
A. Immuno-localization of H3K27me3 and H3K27 modifiers in germline stem (GS) cells. Colocalization of H3K27me3, EZH2, and JMJD3 with PLZF in GS cell are shown. Notably, PLZF strongly localizes in clusters and weakly localized in chains of GS colony. Arrow and solid line indicate chains of GS cells. B. The number of colonies formed by Jmjd3 and mock knockdown. The number of colonies at 1 week after replating of mock (green) and Jmjd3 (red) knockdown GS cells were counted. Bars indicate mean ± se. * indicates that there is significant difference (P<0.05). C. Representative morphology of mock and JMJD3 KD GS cell colony. D. Relative expression of spermatogonial stem cell markers and cell cycle regulators in mock and JMJD3 KD GS cells. Relative expression of indicated genes are quantitatively analyzed after normalization with Gapdh. * indicates that there is significant difference (P<0.05).
Figure 3.
Testis phenotype and fertility analysis of JMJD3 cKO.
A. Gross picture of control (F/-) and JMJD3 cKO (cKO) testes. B. Histology of control (F/-) and JMJD3 cKO (cKO) testis. Scale Bar: 50 µm. C. Testis weights of control (F/-) and JMJD3 cKO (cKO) mouse. * indicates that there is significant difference (P<0.05). D. Average number of pups produced by control (F/-) and JMJD3 cKO (cKO) males over 20 months of breeding (n=7 per genotype). E and F. Average litter sizes (E) and litter period (F) for total period, first 1 year, and after 1 year of breeding produced from control (F/-) and JMJD3 cKO (cKO) males.
Figure 4.
Distribution of methylated H3K27 and H3K27 modifiers in the JMJD3 cKO tsetis.
A. Distribution of methylated H3K27. Immunofluorescence images of di-methylated and tri-methylated H3K27 with PLZF are shown. Insets are higher magnification images of PLZF positive spermatogonia. B. Distribution of H3K27 modifiers. Immunofluorescence images of EZH2 and UTX with PLZF are shown. Insets indicate higher magnification images of PLZF-positive spermatogonia. Scale bar: 25 µm.
Figure 5.
Characteristics of JMJD3 null undifferentiated spermatogonia.
A. Relative expressions of SSC markers in control (F/-) and JMJD3 cKO (cKO) EPCAM-positive testicular cells were quantitatively analyzed. All of the expression levels were normalized to Gapdh expression. * indicates that there is significant difference (P<0.05). B. Flow cytometric analysis of EPCAM and intracellular staining of PLZF in the EPCAM-positive JMJD3 F/- (upper) and JMJD3 cKO (lower) spermatogonia. Ratio of PLZF positive cells is significantly different (P<0.01). C. Flow cytometric analysis of side scatter and CD9 in the JMJD3 F/- and the JMJD3 cKO testicular cell (Left panels). Ratio of side scatter-low and CD9+ cells is statistically different (P<0.001). Right panels: KIT expression in the side scatter-low and CD9+ JMJD3 F/- and JMJD3 cKO cells. Ratio of KIT-negative cells is significantly different (P<0.001).
Figure 6.
Effect of JMJD3 ablation to the spermatogonial chain formation.
A. Whole mount staining of seminiferous tubules for PLZF (green). Arrows indicate As spermatogonia. Scale bar: 100 µm. B. The ratio of PLZF-positive spermatogonial cyst in control (F/-) and JMJD3 cKO (cKO) seminiferous tubules. Total number of colonies counted in the experiment are shown above the graph.
Figure 7.
Disruption of intercellular bridges by loss of JMJD3.
A. Immuno-staining of JMJD3 (red) and TEX14 (green) in mock KD and JMJD3 KD GS cells. Cells were fixed and stained at 5 days after shRNA induction. White arrows indicate TEX14 positive intercellular bridges and red arrowhead indicates connective region of two adjacent cells without intercellular bridge. B and C. Whole mount staining of PLZF (green) and CDH1 (red) in the JMJD3 F/- and the JMJD3 cKO seminiferous tubules. Low magnification images (B) and high magnification images of individual colonies (C) are shown. Scale bar: 100 µm.