Wnt3a leads to increased cell-cell interactions.

<p>Representative fluorescent photomicrographs (left) and corresponding CellProfiler Image analysis (right) of B6GFP cluster cells following control or Wnt3a treatment after 1 day (<b>A</b>) and 4 days (<b>B</b>). The color gradient labels individual cells according to the number of directly-contacted neighboring cells. (<b>C, D</b>) Percentage of clustering cells after 1 day (solid line) and 4 days (dashed line) following control (C) and Wnt3a treatment (D). Wnt3a stimulates cells to cluster, while most cells remain as singles or doublets in controls. (<b>E</b>) Cluster-formation assay results showing SSC numbers over the course of feeder-free culture with or without Wnt3a. Increased SSC maintenance is observed with Wnt3a.</p

Wnt3a increases SSC numbers under feeder-free conditions.

<p>(<b>A</b>) Germ cell clusters (left) and after 6 day treatment with Wnt3a (right) (<b>B</b>) Quantification of SSCs following Wnt3a treatment measured by spermatogonial transplantation. Results of the cluster-forming (<b>C</b>) and transplantation (<b>D</b>) assays for SSC quantification, after feeder-free culture with Wnt3a. A significant increase in SSC numbers is detected with Wnt3a treatment by both assays. (<b>E</b>) Spermatogonial transplantation results following the addition of the β-catenin signalling inhibitor, DKK1, to Wnt3a-treated cultures. Inhibition of the β-catenin pathway inhibits the effect of Wnt3a on SSC activity. Scale bar: 75 µm.</p

Wnt3a leads to increased “cluster-like” aggregations.

<p>Representative brightfield photomicrographs of cluster cells following control or Wnt3a treatment at each day of culture. At day 1 after seeding, cluster cells are arranged predominantly as singles or doublets under both conditions. At day 2, most cells remain as singles and doublets in both conditions but longer chains are more easily observable (arrowheads; upper panels are higher magnification). In day 3 control cultures, most cells remain as singles but small clumps are occasionally observed (arrowhead). In Wnt3a-treated cultures, larger clumps are observable by day 3 (arrowheads). On day 4, large “cluster-like” cell aggregations are found throughout Wnt3a-treated cultures (arrowheads), while control cultures are mostly singles and infrequently show small cell accumulations or the occasional cell chain (arrowheads) but lack large cell accumulations. Upper panels show higher magnification images. Scale bar: 50 µm, upper panels: 12.5 µm.</p

WNT4 downregulates SSC activity <i>in vitro</i>.

<p>(<i>A</i>, <i>B</i>) Germ cell cluster formation ability of cells treated with the indicated concentrations of FST (<i>A</i>) or WNT4 (<i>B</i>). (<i>C</i>) Colony numbers obtained after transplantation of germ cells treated with WNT4 or WNT4 and FST at the indicated concentrations. (<i>D</i>, <i>E</i>) Representative flow cytometric histograms (<i>D</i>) and analysis (<i>E</i>) showing the cell cycle profiles of cultured germ cells, with or without prior WNT4 treatment (100 ng/ml). (<i>F</i>, <i>G</i>) Representative scatter plots (<i>F</i>) and analysis (<i>G</i>) showing TUNEL assay results of germ cells following WNT4 treatment (100 ng/ml). All data are expressed as mean (columns) ± SEM (error bars). Significant differences from controls (<i>P</i><0.05) are indicated with an asterisk (*) and accompanied by relevant <i>P</i> values.</p

WNT4 acts downstream of CTNNB1 to cause germ cell loss <i>in vivo</i>.

<p>(<i>A</i>–<i>D</i>) Photomicrographs of testes from 8 week-old animals of the indicated genotypes. Insets show sections of epididymides from the corresponding animals. (<i>E</i>) Photomicrograph of a testis of the indicated genotype showing the severe testicular degeneration, coagulation necrosis and intratubular hemorrhage phenotypes described in the text. (<i>F</i>) Testis weights from 8 week-old animals of the indicated genotypes, n = 4 animals/genotype. C;W^f/−: <i>Ctnnb1</i>^tm1Mmt/+;<i>Wnt4</i>^flox/− (control), C;A: <i>Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+}, C;W^+/−;A: <i>Ctnnb1</i>^tm1Mmt/+;<i>Wnt4</i>^+/−;<i>Amhr2</i>^{tm3(cre)Bhr/+}, C;W^f/−;A: <i>Ctnnb1</i>^tm1Mmt/+;<i>Wnt4</i>^flox/−;<i>Amhr2</i>^{tm3(cre)Bhr/+}. (<i>G</i>) <i>Wnt4</i> mRNA levels in the mice described in panel F. Note the logarithmic scale on the Y axis. (<i>H</i>) CTNNB1 immunoblot analyses of testes from 8 week-old animals of the indicate genotypes. The lower band corresponds to the dominant-stable CTNNB1 mutant protein produced by the recombined <i>Ctnnb1</i>^tm1Mmt/+ allele. ACTB was used as a loading control. Animals showing the severe degenerative phenotype described in the text and shown in panel <i>E</i> were excluded from the data analyses shown in panels <i>F</i>–<i>H</i>. All data are expressed as mean (columns) ± SEM (error bars). Groups labeled with different letters (a, b, c) were significantly different (<i>P</i><0.05).</p

Increased germ cell apoptosis in <i>Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+} testis.

<p>(<i>A–F</i>) TUNEL staining (red) in <i>Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+} testes (<i>B</i>, <i>D</i>, <i>F</i>) compared with <i>Ctnnb1^tm1Mmt/+</i> controls (<i>A</i>, <i>C</i>, <i>E</i>) at different ages. Counterstain = DAPI (blue). For clarity, seminiferous tubules are circumscribed with a dotted white line in panel <i>D</i>.</p

<i>Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+} testes are unable to support donor SSCs.

<p>(<i>A</i>, <i>B</i>) Photographs of decapsulated, LacZ-stained recipient <i>Ctnnb1^tm1Mmt/+</i> (<i>A</i>) or <i>Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+} (<i>B</i>) testes 1 week after transplantation of <i>Gt(ROSA)26Sor</i> germ cells. Insets show higher magnification lateral views of seminiferous tubules from the corresponding testes. (<i>C</i>, <i>D</i>) Photographs of decapsulated, LacZ-stained recipient <i>Ctnnb1^tm1Mmt/+</i> (<i>C</i>) or <i>Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+} (<i>D</i>) testes 8 weeks after transplantation of <i>Gt(ROSA)26Sor</i> germ cells. Insets are photomicrographs demonstrating complete regeneration of spermatogenesis in the <i>Ctnnb1^tm1Mmt/+</i> testes (<i>C</i>), whereas no evidence for spermatogenesis was detected in the <i>Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+} testes (<i>D</i>). Original magnification 32× (<i>A</i>, <i>B</i>) or 16× (<i>C</i>, <i>D</i>).</p

<i>Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+} mice lose spermatogonial stem cell activity over time.

<p>(<i>A</i>, <i>E</i>) Photographs of decapsulated, LacZ-stained recipient testes 8 weeks after transplantation of donor cells from 5- (<i>A</i>) or 17- (<i>E</i>) week-old <i>Gt(ROSA)26Sor;Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+} (<i>R</i>;<i>C</i>, control) and <i>Gt(ROSA)26Sor;Ctnnb1</i>^tm1Mmt/+;<i>Amhr2</i>^{tm3(cre)Bhr/+} (<i>R</i>;<i>C</i>;<i>A</i>) mice. Original magnification 12.5×. (<i>B</i>, <i>C</i>, <i>F</i>, <i>G</i>) Photomicrographs demonstrating complete regeneration of spermatogenesis in the testes shown in (<i>A</i>) and (<i>E</i>). (<i>D</i>) Spermatogenic colony numbers obtained after donor cell transplantation, n = 8–9/time/genotype. Results are expressed as colonies per million transplanted cells. (<i>H</i>) Total spermatogonial stem cells present in the donor testes, calculated by multiplying colony numbers (<i>D</i>) by the total number of germ cells harvested from the donor testis. All data are expressed as mean (columns) ± SEM (error bars). Significant differences from controls (<i>P</i><0.05) are indicated with an asterisk (*) and accompanied by relevant <i>P</i> values.</p

WNT4 acts through canonical and noncanonical pathways in different testicular cell types.

<p>(<i>A</i>) Left panel: LacZ-positive cell numbers in cultured germ cells from TCF/Lef-lacZ transgenic mice treated or not beforehand with WNT4 (100 ng/ml). Data is expressed as mean (columns) ± SEM (error bars). Right panel: Timecourse immunoblot analyses of cultured germ cells treated with WNT4 (100 ng/ml). ACTB was used as a loading control. (<i>B</i>) Timecourse immunoblot analyses of cultured Sertoli cells treated with WNT4 (50 ng/ml). ACTB was used as a loading control. (<i>C</i>) Experimental model illustrating WNT4/CTNNB1 signaling mechanisms in Sertoli cells and spermatogonial stem cells.</p