ROS amplification drives mouse spermatogonial stem cell self-renewal
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE106103 | GEO | 2020/10/24
REPOSITORIES: GEO
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Project description:Reactive oxygen species (ROS) play critical roles in self-renewal division for various stem cell types. However, it remains unclear how ROS signals are integrated with self-renewal machinery. Here we report that the MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification. The activation of MAPK14 induced MAPK7 phosphorylation in cultured SSCs, and targeted deletion of Mapk14 or Mapk7 resulted in significant SSC deficiency after spermatogonial transplantation. The activation of this signaling pathway not only induced Nox1 but also increased ROS levels. Chemical screening of MAPK7 targets revealed many ROS-dependent spermatogonial transcription factors, of which BCL6B was found to initiate ROS production by increasing Nox1 expression via ETV5-induced nuclear translocation. Because hydrogen peroxide or Nox1 transfection also induced BCL6B nuclear translocation, our results suggest that BCL6B initiates and amplifies ROS signals to activate ROS-dependent spermatogonial transcription factors by forming a positive feedback loop.
2019-12-01 | GSE118191 | GEO
Project description:The MAPK14/MAPK7/BCL6B pathway creates a positive feedback loop to drive spermatogonial stem cell (SSC) self-renewal via ROS amplification.
| PRJNA484802 | ENA
Project description:Testis immune privilege is thought to be mediated by somatic cells. However, it is not strong enough to protect allogeneic spermatogonial stem cells (SSCs) transplanted into the seminiferous tubules. Here we report successful production of allogeneic offspring by inducing PD-L1 in SSCs. Activation of self-renewal division induced PD-L1 and B7-H3 expression in cultured SSCs, which produced sperm after allogeneic transplantation. While B7-H3 depletion did not influence colonization, PD-L1 depletion prevented donor-derived spermatogenesis. PD-L1 expression was induced by BCL6B via reactive oxygen species (ROS) generation, suggesting that self-renewal stimulation confers immune privilege by ROS. In contrast, reduced ROS or Mapk14 deficiency downregulated PD-L1 expression. Allogeneic offspring were produced by SSC transplantation into congenitally infertile mice and busulfan-treated wild-type mice. Therefore, SSCs can escape rejection when their self-renewal division is stimulated.
2022-04-11 | GSE129658 | GEO
Project description:Testis immune privilege is thought to be mediated by somatic cells. However, it is not strong enough to protect allogeneic spermatogonial stem cells (SSCs) transplanted into the seminiferous tubules. Here we report successful production of allogeneic offspring by inducing PD-L1 in SSCs. Activation of self-renewal division induced PD-L1 and B7-H3 expression in cultured SSCs, which produced sperm after allogeneic transplantation. While B7-H3 depletion did not influence colonization, PD-L1 depletion prevented donor-derived spermatogenesis. PD-L1 expression was induced by BCL6B via reactive oxygen species (ROS) generation, suggesting that self-renewal stimulation confers immune privilege by ROS. In contrast, reduced ROS or Mapk14 deficiency downregulated PD-L1 expression. Allogeneic offspring were produced by SSC transplantation into congenitally infertile mice and busulfan-treated wild-type mice. Therefore, SSCs can escape rejection when their self-renewal division is stimulated.
2022-04-11 | GSE129546 | GEO
Project description:Insight into mechanisms controlling gene expression in the spermatogonial stem cell (SSC) will improve our understanding of the processes regulating spermatogenesis and aid in treating problems associated with male infertility. In this study we explored the global gene expression profiles of glial cell line-derived neurotrophic factor (GDNF) regulated transcription factors, Ets variant gene 5 (Etv5), B-cell CLL/lymphoma 6, member B (Bcl6b) and POU domain, class-3 transcription factor-1 (Pou3f1). We reasoned that these three factors may function as a core-set of transcription factors, regulating genes responsible for maintaining the SSC population. Using transient short-interfering RNA oligonucleotides (siRNA) to individually target Etv5, Bcl6b and Pou3f1 within mouse SSC cultures, we examined changes to the global gene expression profiles associated with these transcription factors. While there were only modest overlaps in the target genes regulated by the three factors, ETV5 was found to be a critical downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes including, Bcl6b and LIM homeobox 1 (Lhx1). Notably, ETV5 was identified as a regulator of Brachyury and CXC chemokine Receptor, type 4 (Cxcr4), and we show that ETV5 binding to the Brachyury gene promoter region is associated with an active state of transcription. Moreover, in vivo transplantation of SSCs following silencing of Brachyury significantly reduced the number of donor cell-derived colonies formed within recipient mouse testes. These results suggest Brachury is of biological importance, and functions as part of GDNF/ETV5 signaling to promote self-renewal of mouse SSCs cultured in vitro. Microarray gene expression analysis was conducted with Affymetrix Mouse 430 2.0 GeneChips (Affymetrix Inc.).Following with gene knockdown, total RNA from spermatogonial stem cells was converted to cDNA. There are total 16 samples (Four groups and four samples per group) Negative control (N), Bcl6b Knockdown (B), Etv5 knockdown (E), and Pou3f1 knockdown (O), respectively.
2011-07-14 | E-GEOD-30683 | biostudies-arrayexpress
Project description:Insight into mechanisms controlling gene expression in the spermatogonial stem cell (SSC) will improve our understanding of the processes regulating spermatogenesis and aid in treating problems associated with male infertility. In this study we explored the global gene expression profiles of glial cell line-derived neurotrophic factor (GDNF) regulated transcription factors, Ets variant gene 5 (Etv5), B-cell CLL/lymphoma 6, member B (Bcl6b) and POU domain, class-3 transcription factor-1 (Pou3f1). We reasoned that these three factors may function as a core-set of transcription factors, regulating genes responsible for maintaining the SSC population. Using transient short-interfering RNA oligonucleotides (siRNA) to individually target Etv5, Bcl6b and Pou3f1 within mouse SSC cultures, we examined changes to the global gene expression profiles associated with these transcription factors. While there were only modest overlaps in the target genes regulated by the three factors, ETV5 was found to be a critical downstream regulator of GDNF signaling that mediated the expression of several known SSC self-renewal related genes including, Bcl6b and LIM homeobox 1 (Lhx1). Notably, ETV5 was identified as a regulator of Brachyury and CXC chemokine Receptor, type 4 (Cxcr4), and we show that ETV5 binding to the Brachyury gene promoter region is associated with an active state of transcription. Moreover, in vivo transplantation of SSCs following silencing of Brachyury significantly reduced the number of donor cell-derived colonies formed within recipient mouse testes. These results suggest Brachury is of biological importance, and functions as part of GDNF/ETV5 signaling to promote self-renewal of mouse SSCs cultured in vitro.
2011-07-15 | GSE30683 | GEO
Project description:Objectives: Fibroblast growth factor 9 (FGF9) is expressed by somatic cells in the seminiferous tubules, yet little information exists about its role in regulating spermatogonial stem cells (SSCs). Materials and Methods: Fgf9 overexpression lentivirus was injected into mouse testes, and PLZF immunostaining was performed to investigate the effect of FGF9 on spermatogonia in vivo. Effect of FGF9 on SSCs was detected by transplanting cultured germ cells into tubules of testes. RNA-seq of bulk RNA and single-cell was performed to explore FGF9 working mechanisms. SB203580 was used to disrupt p38 MAPK pathway. p38 MAPK protein expression was detected by western blot and qPCR was performed to determine different gene expression. Small interfering RNA (siRNA) was used to knock down Etv5 gene expression in germ cells. Results: Overexpression of Fgf9 in vivo resulted in arrested spermatogenesis and accumulation of undifferentiated spermatogonia. Exposure of germ cell cultures to FGF9 resulted in larger numbers of SSCs over time. Inhibition of p38 MAPK phosphorylation negated the SSC growth advantage provided by FGF9. Etv5 and Bcl6b gene expression was enhanced by FGF9 treatment. Gene knockdown of Etv5 disrupted the growth effect of FGF9 in cultured SSCs along with downstream expression of Bcl6b. Conclusions: Taken together, this data indicates that FGF9 is an important regulator of SSC proliferation, operating through p38 MAPK phosphorylation and upregulating Etv5 and Bcl6b in turn.
2020-10-26 | GSE160062 | GEO
Project description:Objectives: Fibroblast growth factor 9 (FGF9) is expressed by somatic cells in the seminiferous tubules, yet little information exists about its role in regulating spermatogonial stem cells (SSCs). Materials and Methods: Fgf9 overexpression lentivirus was injected into mouse testes, and PLZF immunostaining was performed to investigate the effect of FGF9 on spermatogonia in vivo. Effect of FGF9 on SSCs was detected by transplanting cultured germ cells into tubules of testes. RNA-seq of bulk RNA and single-cell was performed to explore FGF9 working mechanisms. SB203580 was used to disrupt p38 MAPK pathway. p38 MAPK protein expression was detected by western blot and qPCR was performed to determine different gene expression. Small interfering RNA (siRNA) was used to knock down Etv5 gene expression in germ cells. Results: Overexpression of Fgf9 in vivo resulted in arrested spermatogenesis and accumulation of undifferentiated spermatogonia. Exposure of germ cell cultures to FGF9 resulted in larger numbers of SSCs over time. Inhibition of p38 MAPK phosphorylation negated the SSC growth advantage provided by FGF9. Etv5 and Bcl6b gene expression was enhanced by FGF9 treatment. Gene knockdown of Etv5 disrupted the growth effect of FGF9 in cultured SSCs along with downstream expression of Bcl6b. Conclusions: Taken together, this data indicates that FGF9 is an important regulator of SSC proliferation, operating through p38 MAPK phosphorylation and upregulating Etv5 and Bcl6b in turn.
2020-10-26 | GSE160061 | GEO
Project description:NOX1 gene is member of the NADPH-dependent superoxide producing enzyme family. It generates reactive oxygen species (ROS) in regulated manner in response to growth factors, cytokines and calcium signal. Low levels of ROS play role in many processes including cell proliferation, inflammatory response and mitogenesis. NOX1 over-expression in colon cancer cell lines as well as surgical samples suggests a role in colon cancer. We identified unique target sequences of siRNAs for post-transcriptional silencing of NOX1. Gene specific and scrambled (control) sequence carrying U6 promoter/shRNA cassettes were cloned into GFP expression vector. Stable clones were selected based on NOX1 expression after transient transfection of HT-29 cells. Clone 6A and 6C showed 70-80% decrease in NOX1 expression. The cells were expanded and analyzed further. We measured decreased ROS production, cell proliferation and G1/S block of the cell cycle. HT-29 parental cells, clone SA (scrambled) and clone 6A (gene specific) were utilized to establish xenografts in athymic mice to investigate the effect of NOX1 silencing on tumor growth and gene expression in vivo. Significant decrease was measured in the volume of forming tumors. The stable clones and corresponding xenografts were analyzed on microarray. The microarray data was validated with real-time PCR and western-blot analysis demonstrating the inhibition of genes important in cell cycle regulation and angiogenesis. The results of this study show silencing NOX1 levels of generated ROS decreased, the tumor formation attenuated in vivo suggesting NOX1 may be a therapeutic target for colon cancer. Experiment Overall Design: We have found that NOX1 over-expression in colon cancer cell lines as well as surgical samples suggests a role in colon cancer. We identified unique target sequences of siRNAs for post-transcriptional silencing of NOX1. Gene specific and scrambled (control) sequences carrying the U6 promoter/shRNA cassettes were cloned into a GFP expression vector. Stable clones were selected based on NOX1 expression after transient transfection of HT-29 cells. HT-29 parental cells, clone SA (scrambled) and clone 6A (gene specific) were utilized to establish xenografts in athymic mice to investigate the effect of NOX1 silencing on tumor growth and gene expression in vivo. A significant decrease was measured in the volume of tumors formed. The stable clones and corresponding xenografts were analyzed on microarray. The microarray data was validated with real-time PCR and western-blot analysis demonstrating the inhibition of genes important in cell cycle regulation and angiogenesis. The results of this study show silencing NOX1 levels of generated ROS decreased, the tumor formation attenuated in vivo suggesting NOX1 may be a therapeutic target for colon cancer.
2008-06-13 | E-GEOD-4561 | biostudies-arrayexpress
Project description:Maintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. In this manuscript we have identified a key role for the nucleosome remodelling protein Chromodomain Helicase DNA binding protein 4 (CHD4) in regulating SSC function. Gene expression analyses revealed that CHD4 expression is largely restricted to spermatogonia in the mouse testis, and is particularly enriched in SSCs. Using spermatogonial transplantation techniques and RNAi mediated knockdown it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, resulting in a ~50% reduction in colonisation of recipient testes. A single cell RNA-seq comparison depicted reduced expression of ‘self-renewal’ genes such as Gfra1 and Pten following Chd4 knockdown, along with increased expression of signature progenitor genes, Neurog3 and Dazl. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only though its traditional association with the remodelling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated fundamental role for CHD4 in controlling fate decisions in the spermatogonial pool.
2021-06-17 | PXD022066 | Pride