Phenotyping spermatogenic defects by single-cell expression profiling
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ABSTRACT:
PROVIDER: PRJNA450677 | ENA |
REPOSITORIES: ENA
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Project description:Using Drop-seq, we generated high-throughput single-cell expression data from wild-type and four mutant models with male infertility phenotype. Our study demonstrates the applicability of single-cell RNA-sequencing in study of male gonadal dysfunction and provides cell atlas resource for testis.
2019-06-30 | GSE113293 | GEO
Project description:RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA – Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo. Comparison of total RNA from post-natal day 15 testis extracted from three wild-type mice and three transgenic mice genetically engineered to express siRNA designed to knock-down Rhox3. RNA hybridised to Affymetrix mouse all exon arrays.
2014-12-19 | E-GEOD-64324 | biostudies-arrayexpress
Project description:RNA interference (RNAi) is widely used to determine the function of genes. We chose this approach to assess the collective function of the highly related reproductive homeobox 3 (Rhox3) gene paralogs. Using a Rhox3 short hairpin (sh) RNA with 100% complementarity to all 8 Rhox3 paralogs, expressed from a CRE-regulated transgene, we successfully knocked down Rhox3 expression in male germ cells in vivo. These Rhox3-shRNA transgenic mice had dramatic defects in spermatogenesis, primarily in spermatocytes and round spermatids. To determine whether this phenotype was caused by reduced Rhox3 expression, we generated mice expressing the Rhox3-shRNA but lacking the intended target of the shRNA – Rhox3. These double-mutant mice had a phenotype indistinguishable from Rhox3-shRNA-expressing mice that was different from mice lacking the Rhox3 paralogs, indicating that the Rhox3 shRNA disrupts spermatogenesis independently of Rhox3. Rhox3-shRNA transgenic mice displayed few alterations in the expression of protein-coding genes, but instead exhibited reduced levels of all endogenous siRNAs we tested. This supported a model in which the Rhox3 shRNA causes spermatogenic defects by sequestering one or more components of the endogenous small RNA biogenesis machinery. Our study serves as a warning for those using shRNA approaches to investigate gene functions in vivo.
2014-12-19 | GSE64324 | GEO
Project description:To reveal distinct transcriptomes associated with various spermatogenic cells, including spermatogonial stem cells and all of their subsequent progeny, single-cell transcriptomes from Adult human spermatogonia, StaPut-enriched spermatocytes and spermatids, or unselected steady-state spermatogenic cells were used for Drop-Seq analysis. We used the 10x Genomics Chromium (Drop-Seq) to perform single-cell RNA-seq
2018-11-06 | GSE109037 | GEO
Project description:Mouse models represent a critical tool to study human diseases, particularly developmental disorders, as well as for advancing our general understanding of mammalian biology. However, it has long been suspected that conventional approaches for phenotyping are insufficiently sensitive to detect subtle defects throughout the developing mouse. Here we set out to establish single cell RNA sequencing (scRNA-seq) of the whole embryo as a scalable platform for the systematic molecular and cellular phenotyping of mouse genetic models. We applied combinatorial indexing-based scRNA-seq to profile 101 embryos of 26 genotypes at embryonic stage E13.5, altogether profiling over 1.6M nuclei. The 26 genotypes included 22 mouse mutants representing a range of anticipated severities, from established multisystem disorders to deletions of individual enhancers, as well as the 4 wildtype backgrounds on which these mutants reside. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibited changes in dozens of trajectories (e.g., the pleiotropic consequences of altering the Sox9 regulatory landscape) whereas the impact of others was considerably more restricted. We further identified differences between widely used wildtype strains, compared phenotyping of gain vs. loss of function mutants, and characterized deletions of topological associating domain (TAD) boundaries. Intriguingly, even among these 22 mutants, some changes are shared by heretofore unrelated models, suggesting that developmental pleiotropy might be “decomposable” through further scaling of this approach. Overall, our findings show how single cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.
2023-05-26 | GSE199308 | GEO
Project description:Purpose: Many young adults are in a state of stress due to social and psychological pressures, which may result in male reproductive dysfunction. To provide new insight into this phenomenon, we investigated the relationship between pathological changes in rat spermatogenic cells and the expression of genes specific to spermatogenic cell types under different stress conditions. Methods: After establishing rat stress models of different time durations, we observed pathological changes in testicular tissues through haematoxylin and eosin staining, and analysed gene expression in spermatogenic cells by RNA-seq, bioinformatic analysis, and reverse transcription qPCR (RT-qPCR).Three testicular samples were taken from each group to construct 12 cDNA libraries. For each sample, 3 μg RNA was used as the starting material. Ribosomal RNA was removed using the Epicentre Ribo-Zero™ Gold kit (Rat) (Epicentre, an Illumina company, Madison, WI, USA). Results:Compared with the control group, there were 1,194 DEGs in the 3-day RS+IS group , including 455 upregulated genes and 739 downregulated genes , 1,774 DEGs in the 14-day RS+IS group including 1,124 upregulated genes and 650 downregulated genes, and 2,267 DEGs in the 21-day RS+IS group including 1,366 upregulated genes and 901 downregulated genes. Mean-while, some same expression patterns were observed in three phenotypes.After comparison with single-cell sequencing data, 349 DEGs of spermatogenic cells at different developmental stages were found. Conclusions: Our study suggest that chronic psychosomatic stress can affect the spermatogenic transcriptome of the testes, leading to a significant change in the expression of the spermatogenic genes. At the same time, histopathological and immunohistochemical results showed that chronic stress may lead to pathological changes in spermatogenic cells and to a significant decrease in key regulatory proteins
2021-05-01 | GSE144485 | GEO
Project description:To reveal distinct transcriptomes associated with various spermatogenic cells, including spermatogonial stem cells and all of their subsequent progeny, single-cell transcriptomes from Adult ID4-EGFP+ spermatogonia (sorted for brightest or dimmest), StaPut-enriched spermatocytes and spermatids, or unselected steady-state spermatogenic cells were used for 10x Genomics analysis. The GFP-bright and dim phenotypes exhibit distinct fates when assayed by transplantation, with ID4-EGFPbright cells highly enriched for SSCs, and ID4-EGFPdim cells enriched for progenitors. We used the 10x Genomics Chromium (Drop-Seq) to perform single-cell RNA-seq
2018-11-06 | GSE109033 | GEO
Project description:These samples are part of the ENCODE consortium’s proposed time-limited Pilot Study for confirmation of the utility of RNA abundance measurements as a standard reference phenotyping tool. Keywords: cell type comparison For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Each batch of H1-ES cells cultured by Cellular Dynamics International for use by the ENCODE labs was processed on Affymetrix Exon 1.0 ST arrays to obtain phenotyping data.
2009-02-16 | E-GEOD-14863 | biostudies-arrayexpress
Project description:These samples are part of the ENCODE consortium’s proposed time-limited Pilot Study for confirmation of the utility of RNA abundance measurements as a standard reference phenotyping tool. Keywords: cell type comparison For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Each of the 7 ENCODE laboratories submitted biological triplicates for at least one of the four Tier2 cell lines. These were processed on Affymetrix Exon 1.0 ST arrays to obtain phenotyping data for each cell ine.
2009-08-24 | E-GEOD-17778 | biostudies-arrayexpress
Project description:We report genome-wide distribution of Linker histone variant H1t in mouse spermatogenic cells. We found that H1t was mainly located at around transcriptional start site of genic area, positively correlated with the gene expression. This study using ChIP-seq analysis provides genomic distribution of H1t in mouse spermatogenic cells.
2023-03-09 | GSE146733 | GEO