The effect of 2HG level on transcriptome at different stages of male mouse meiosis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE169014 | GEO | 2022/05/31
REPOSITORIES: GEO
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Project description:Purpose: Analysing gene expression changes along mose spermatogenesis Method:The novel method of the isolation of 6 populations of cells at sequantial developmental stages -from undifferentiated spermatogonia (Undiff.Spg), differentiating spermatogonia (Diff.Spg), preleptotene (PreL), leptotene/zygotene (LZ), pachytene/diplotene (PD) and round spermatids (RS) was developed. Results: Median sequencing depth was ~18 million reads per sample. Sequencing depth was homogenous across samples in the experiment. We found a profound shift in gene expression between LZ and PD populations.
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Project description:We found that testis of male mice contain high concentrations of L2-hydroxyglutarate (L2HG) specifically in germ cells at pachytene/ diplotene (PD) stage of spermatogenesis. In order to elucidate the function of 2HG we decreased its level by incubating PD cells with 24 mM oxamate and to rescue the putative phenotype by incubation with 24 mM oxamate together with 0.3 mM cell permeable L2HG.
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Project description:To understand the changes in gene expression during spermatogenesis, we did microarray profiling of spermatogonial stem cells, leptotene-zygotene cells and round spermatids. Microarray profiling of pachytene-diplotene cells can be found in E-MTAB-2668.
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Project description:Prophase I of male meiosis involves dynamic chromosome segregation processes during early spermatogenesis, including synapsis, meiotic recombination, and cohesion. Genetic defects in genes participating in these processes consistently cause reproduction failure in mice. To identify candidate genes responsible for infertility in humans, we performed expression profiling of mouse spermatogenic cells undergoing meiotic prophase I. Cell fractions enriched in spermatogonia, leptotene/zygotene spermatocytes, or pachytene spermatocytes were separately isolated from mouse testes for RNA extraction. To minimize the contamination of other cell types, we fractionated the testicular cells undergoing the first round of spermatogenesis using Percoll gradient procedure. The cell fractions were characterized by morphological analysis by phase contrast microscopy and Nomarski interference microscopy, and then expression of cell lineage- and spermatogenesis stage-specific genes were examined by RT-PCR. The most enriched fractions for spermatogonia (fraction2 from day8), leptotene/zygotene spermatocytes (fraction5 from day12), and pachytene spermatocytes (fraction8 from day15) were subjected to hybridization on Affymetrix microarrays.
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Project description:We used Illumina Small RNA and RNA-Seq kits to prepare both small RNA and RNA-Seq libraries from total RNA isolated from either leptotenze/zygotene or pachytene spermatocytes purified from either Dgcr8 or Dicer germline conditional knockout mice. Conditional knockout mice were generated by using a Ddx4 promoter to drive cre excision of either Dgcr8 or Dicer at embryonic day 18. Mixed leptotene/zygotene or pachytene spermatocytes were then isolated from the testis of adult conditional knockout mice, along with paired WT littermates as a control. RNA was isolated from these spermatocytes using Trizol. Small RNA or RNA-Seq libraries were then prepped using Illumina's sequencing library preparation kits.
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Project description:The DSB-machinery, which induces the programmed DNA double-strand breaks (DSBs) in leptotene and zygotene stages during meiosis, needs to be kept in silence after the initiation of pachytene stage to prevent the activation of DSB checkpoint that may lead to meiotic arrest or apoptosis of germ cells. However, the mechanisms underlying this repression remain largely unknown. Here, we report that ZFP541, a germ cell-specific zinc finger protein, is responsible for the suppression of DSBs formation at late pachytene. Lack of Zfp541 in mice leads to generation of DSBs in late pachytene spermatocytes by DSB formation related-proteins and causes male infertility due to meiotic failure. Plated-based scRNA-seq of Zfp541-/- spermatocytes revealed that ZFP541 negatively regulates many meiotic prophase genes, including genes for DSB formation and their upstream transcriptional regulators, in late pachytene spermatocytes. These results were confirmed by 10x single-cell RNA-seq data on spermatogenesis of Zfp541-/- testes, which suggested that Zfp541 is required for repressing the activation of pre-pachytene gene expression programs from early to late pachytene. ZFP541 ChIP-seq on pachytene and diplotene spermatocytes demonstrated that ZFP541 occupies the promoters of meiosis initiators (e.g., Meiosin and Rxra) and a subset of their downstream genes to repress their transcription, and thus prevent the reactivation of pre-pachytene gene expression programs in pachytene spermatocytes. Thus, our results not only revealed the role of ZFP541 in maintaining the repression of pre-pachytene transcriptional programs in pachytene spermatocytes but also provide new insight into the regulation of meiotic progression by timely turning off pre-pachytene genes.
2021-04-16 | GSE172171 | GEO
Project description:The DSB-machinery, which induces the programmed DNA double-strand breaks (DSBs) in leptotene and zygotene stages during meiosis, needs to be kept in silence after the initiation of pachytene stage to prevent the activation of DSB checkpoint that may lead to meiotic arrest or apoptosis of germ cells. However, the mechanisms underlying this repression remain largely unknown. Here, we report that ZFP541, a germ cell-specific zinc finger protein, is responsible for the suppression of DSBs formation at late pachytene. Lack of Zfp541 in mice leads to generation of DSBs in late pachytene spermatocytes by DSB formation related-proteins and causes male infertility due to meiotic failure. Plated-based scRNA-seq of Zfp541-/- spermatocytes revealed that ZFP541 negatively regulates many meiotic prophase genes, including genes for DSB formation and their upstream transcriptional regulators, in late pachytene spermatocytes. These results were confirmed by 10x single-cell RNA-seq data on spermatogenesis of Zfp541-/- testes, which suggested that Zfp541 is required for repressing the activation of pre-pachytene gene expression programs from early to late pachytene. ZFP541 ChIP-seq on pachytene and diplotene spermatocytes demonstrated that ZFP541 occupies the promoters of meiosis initiators (e.g., Meiosin and Rxra) and a subset of their downstream genes to repress their transcription, and thus prevent the reactivation of pre-pachytene gene expression programs in pachytene spermatocytes. Thus, our results not only revealed the role of ZFP541 in maintaining the repression of pre-pachytene transcriptional programs in pachytene spermatocytes but also provide new insight into the regulation of meiotic progression by timely turning off pre-pachytene genes.
2021-04-16 | GSE172160 | GEO
Project description:The DSB-machinery, which induces the programmed DNA double-strand breaks (DSBs) in leptotene and zygotene stages during meiosis, needs to be kept in silence after the initiation of pachytene stage to prevent the activation of DSB checkpoint that may lead to meiotic arrest or apoptosis of germ cells. However, the mechanisms underlying this repression remain largely unknown. Here, we report that ZFP541, a germ cell-specific zinc finger protein, is responsible for the suppression of DSBs formation at late pachytene. Lack of Zfp541 in mice leads to generation of DSBs in late pachytene spermatocytes by DSB formation related-proteins and causes male infertility due to meiotic failure. Plated-based scRNA-seq of Zfp541-/- spermatocytes revealed that ZFP541 negatively regulates many meiotic prophase genes, including genes for DSB formation and their upstream transcriptional regulators, in late pachytene spermatocytes. These results were confirmed by 10x single-cell RNA-seq data on spermatogenesis of Zfp541-/- testes, which suggested that Zfp541 is required for repressing the activation of pre-pachytene gene expression programs from early to late pachytene. ZFP541 ChIP-seq on pachytene and diplotene spermatocytes demonstrated that ZFP541 occupies the promoters of meiosis initiators (e.g., Meiosin and Rxra) and a subset of their downstream genes to repress their transcription, and thus prevent the reactivation of pre-pachytene gene expression programs in pachytene spermatocytes. Thus, our results not only revealed the role of ZFP541 in maintaining the repression of pre-pachytene transcriptional programs in pachytene spermatocytes but also provide new insight into the regulation of meiotic progression by timely turning off pre-pachytene genes.
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