Expression profiles of SSC markers and cell cycle regulators in <i>Tex14^+/−</i> and <i>Tex14^−/−</i> SSCs.

<p>Relative expressions of SSC markers (A) and cell cycle regulators (B) in <i>Tex14^+/−</i> (blue) and <i>Tex14^−/−</i> (red) SSCs were quantitatively analyzed. All of the expression levels were normalized to <i>Gapdh</i> expression. * indicates that there is significant difference (P<0.05).</p

Alterations of cell cycle regulators in spermatogonial stem cells by lack of intercellular bridges.

<p>Protein expression of cell cycle regulators in <i>Tex14^+/−</i> and <i>Tex14^−/−</i> SSCs were analyzed by immunoblot (A) and immunostaining (B). Immunofluorescence images (B) of indicated cell cycle regulators (green) and ZBTB16 (red) in <i>Tex14^+/−</i> (+/−) and <i>Tex14^−/−</i> (KO) SSCs are shown.</p

H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts

<div><p>The spermatogonial stem cell (SSC) compartment is maintained by self-renewal of stem cells as well as fragmentation of differentiating spermatogonia through abscission of intercellular bridges in a random and stochastic manner. The molecular mechanisms that regulate this reversible developmental lineage remain to be elucidated. Here, we show that histone H3K27 demethylase, JMJD3 (KDM6B), regulates the fragmentation of spermatogonial cysts. Down-regulation of <i>Jmjd3</i> in SSCs promotes an increase in undifferentiated spermatogonia but does not affect their differentiation. Germ cell-specific <i>Jmjd3</i> null male mice have larger testes and sire offspring for a longer period compared to controls, likely secondary to increased and prolonged maintenance of the spermatogonial compartment. Moreover, JMJD3 deficiency induces frequent fragmentation of spermatogonial cysts by abscission of intercellular bridges. These results suggest that JMJD3 controls the spermatogonial compartment through the regulation of fragmentation of spermatogonial cysts and this mechanism may be involved in maintenance of diverse stem cell niches.</p> </div

Expression profiles of SSC markers in <i>Tex14^−/−</i> testis and spermatogonia.

<p>Relative expression patterns of the indicated genes in <i>Tex14^+/−</i> (blue) and <i>Tex14^−/−</i> (red) testis at 8 weeks of age (A) and in CD9-positive <i>Tex14^+/−</i> (blue) and <i>Tex14^−/−</i> (red) spermatogonia (B) were quantitatively analyzed. All of the expression levels were normalized to <i>Gapdh</i> expression. * indicates that there is significant difference (P<0.05).</p

Spatiotemporal expression and testicular localization of JMJD3.

<p>Transcripts of <i>Jmjd3</i> were examined by semi-quantitative RT-PCR in multiple tissue samples (A) and during post-natal development of the testis (B). <i>Hprt</i> was used as an internal control. <i>Plzf</i> was used as a control of spermatogonial stem cell-specific gene. GS: germline stem cells. C. Relative changes of <i>Jmjd3</i> and <i>Plzf</i> during postnatal development of the testis. Relative densities of PCR products to the adult are presented after normalization with <i>Hprt</i>. 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.</p

Effect of JMJD3 ablation to the spermatogonial chain formation.

<p>A. Whole mount staining of seminiferous tubules for PLZF (green). Arrows indicate A_s 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.</p

Disruption of intercellular bridges by loss of JMJD3.

<p>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.</p

Distribution of methylated H3K27 and H3K27 modifiers in the JMJD3 cKO tsetis.

<p>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.</p

Primer sequences used in the study.

<p>Primer sequences used in the study.</p

Genotyping of the rescued <i>Tex14</i> allele.

<p>A. Strategy of gene replacement of the <i>Tex14</i> mutation. The <i>Pgk-Hprt</i>, which was inserted into exon 10 of the <i>Tex14</i> gene, was replaced with exon 10 of <i>Tex14</i> and a <i>loxP</i> flanked <i>Pgk-Neo</i> cassette. Locations of primers used for genotyping and predicted size of the PCR products are shown. B and C. Genotyping of the <i>Tex14</i> mutation. Mutation of the <i>Tex14</i> gene was confirmed by genomic PCR using primer pairs F1 and R1, F2 and R2 (B), and F1 and F2 and R2 (C). D. Genotyping of the replaced allele of <i>Tex14</i> in <i>Tex14^+/−</i> SSCs. Genomic PCR using indicated primer pairs and conditions are shown. Lane 1: <i>Tex14^+/−</i> SSCs, Lane 2: unreplaced SSC clone, Lanes 3–5: replaced SSC clone. E. Long range genomic PCR to confirm homologous recombination. Genomic PCR using indicated primer pairs are shown.</p