CTNNB1 Signaling in Sertoli Cells Downregulates Spermatogonial Stem Cell Activity via WNT4

Constitutive activation of the WNT signaling effector CTNNB1 (β-catenin) in the Sertoli cells of the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ mouse model results in progressive germ cell loss and sterility. In this study, we sought to determine if this phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ and wild-type mice showed that SSC activity is lost in Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes over time, whereas the mutant testes could not support colonization by wild-type SSCs. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway, which was also found to occur in Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ testes. One CTNNB1 target gene encoded the secreted signaling molecule WNT4. We therefore tested the effects of WNT4 on SSC-enriched germ cell cultures, and found that WNT4 induced cell death and reduced SSC activity without affecting cell cycle. Conversely, conditional inactivation of Wnt4 in the Ctnnb1tm1Mmt/+;Amhr2tm3(cre)Bhr/+ model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Furthermore, WNT4 was found to signal via the CTNNB1 pathway in Sertoli cells, suggesting a self-reinforcing positive feedback loop. Collectively, these data indicate for the first time that ectopic activation of a signaling cascade in the stem cell niche depletes SSC activity through a paracrine factor. These findings may provide insight into the pathogenesis of male infertility, as well as embryonic gonadal development

Genome Sequence of a Hyperthermophilic Archaeon, Thermococcus nautili 30-1, That Produces Viral Vesicles

International audienceThermococcus nautili 30-1 (formerly Thermococcus nautilus), an anaerobic hyperthermophilic marine archaeon, was isolated in 1999 from a deep-sea hydrothermal vent during the Amistad campaign. Here, we present the complete sequence of T. nautili, which is able to produce membrane vesicles containing plasmid DNA. This property makes T. nautili a model organism to study horizontal gene transfer

Functional characterization of Negri Bodies (NBs) in Rabies Virus-Infected cells : evidence that NBs are sites of viral transcription and replication

International audienceRabies virus infection induces the formation of cytoplasmic inclusion bodies that resemble Negri bodies found in the cytoplasm of some infected nerve cells. We have studied the morphogenesis and the role of these Negri body-like structures (NBLs) during viral infection. The results indicate that these spherical structures (one or two per cell in the initial stage of infection), composed of the viral N and P proteins, grow during the virus cycle before appearing as smaller structures at late stages of infection. We have shown that the microtubule network is not necessary for the formation of these inclusion bodies but is involved in their dynamics. In contrast, the actin network does not play any detectable role in these processes. These inclusion bodies contain Hsp70 and ubiquitinylated proteins, but they are not misfolded protein aggregates. NBLs, in fact, appear to be functional structures involved in the viral life cycle. Specifically, using in situ fluorescent hybridization techniques, we show that all viral RNAs (genome, antigenome, and every mRNA) are located inside the inclusion bodies. Significantly, short-term RNA labeling in the presence of BrUTP strongly suggests that the NBLs are the sites where viral transcription and replication take place

Hepatitis C virus (HCV) protein expression enhances hepatic fibrosis in HCV transgenic mice exposed to a fibrogenic agent.

International audienceBACKGROUND & AIMS: During chronic HCV infection, activation of fibrogenesis appears to be principally related to local inflammation. However, the direct role of hepatic HCV protein expression in fibrogenesis remains unknown. METHODS: We used transgenic mice expressing the full length HCV open reading frame exposed to a 'second hit' of the fibrogenic agent carbon tetrachloride (CCl(4)). Both acute and chronic liver injuries were induced in these mice by CCl(4) injections. Liver injury, expression of matrix re-modeling genes, reactive oxygen species (ROS), inflammation, hepatocyte proliferation, ductular reaction and hepatic progenitor cells (HPC) expansion were examined. RESULTS: After CCl(4) treatment, HCV transgenic mice exhibited enhanced liver fibrosis, significant changes in matrix re-modeling genes and increased ROS production compared to wild type littermates despite no differences in the degree of local inflammation. This increase was accompanied by a decrease in hepatocyte proliferation, which appeared to be due to delayed hepatocyte entry into the S phase. A prominent ductular reaction and hepatic progenitor cell compartment expansion were observed in transgenic animals. These observations closely mirror those previously made in HCV-infected individuals. CONCLUSIONS: Together, these results demonstrate that expression of the HCV proteins in hepatocytes contributes to the development of hepatic fibrosis in the presence of other fibrogenic agents. In the presence of CCl(4), HCV transgenic mice display an intra-hepatic re-organization of several key cellular actors in the fibrogenic process

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 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

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