Revealing RNA poly(A) tail dynamics during spermiogenesis and its function to support male fertility [dRNA-seq_busulfan]
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PROVIDER: PRJNA1229588 | ENA |
REPOSITORIES: ENA
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Project description:Revealing RNA poly(A) tail dynamics during spermiogenesis and its function to support male fertility [dRNA-seq_mutants]
| PRJNA1229591 | ENA
Project description:Revealing RNA poly(A) tail dynamics during spermiogenesis and its function to support male fertility [dRNA-seq_wildtype]
| PRJNA1229590 | ENA
Project description:Poly(A) tail processing of mRNAs have been shown to dictate mRNAs metabolism of a few transcripts critical for fertility, but the extent of poly(A) tail-mediated regulation at the transcriptome level remains poorly characterized. Here, we performed whole transcriptome poly(A) tail profiling of purified late spermatocytes and round spermatids using direct RNA sequencing. As the germ cells enter spermiogenesis, we show a global increase in poly(A) tail length up to ~150-nts associated with transcript stabilization.
2025-03-20 | GSE290700 | GEO
Project description:TENT5C is a terminal nucleotidyltransferase with mRNA polyadenylation activity, required for the fastening of the flagella and the head of the sperm. However, TENT5C’s role in shaping the poly(A) tail of the elongating spermatid transcriptome remains limited. Here we show that the poly(A) polymerase activity of TENT5C is required for fertility and spermiogenesis: male mice expressing catalytically inactive TENT5C (Tent5cdCat/dCat) are sterile and produce headless spermatozoa phenocopying TENT5C-deficient animals. We found that the transcript encoding for the structural proteins SMCP and ODF1 are directly targeted by TENT5C. We propose a model in which TENT5C secures the fastening of the flagella to the head of the sperm by targeting ODF1 in elongating spermatids.
2025-03-20 | GSE290699 | GEO
Project description:To investigate the poly(A) polymerase activity of TENT5C in testicular somatic cells, we enriched the testis of wild-type and TENT5cdcat/dcat mice with interstitial cells by depleting germ cells with busulfan. Sequencing of busulfan treated testis revealed Insl3 transcripts with poly(A) tails almost exclusively accumulating at ~180-nts in wild-type samples. In Tent5c mutants, Insl3 transcript show gradual deadenylation down to ~60-nt with a significant depletion of the ~180-nt long tails. A significant decrease in Insl3 transcripts was also observed in TENT5cdcat/dcat busulfan treated testis compared to wild-type. These results indicate that TENT5C poly(A) polymerase is required to maintain or readenylate Insl3 poly(A) tails up to ~180-nts in interstitial cells. The shortening of the poly(A) tail may lead to transcripts decay.
2025-03-20 | GSE290698 | GEO
Project description:Spermiogenesis defines the final phase of male germ cell differentiation. Multiple deubiquitinating enzymes have been linked to spermiogenesis, yet the impacts of deubiquitination on spermiogenesis remain poorly characterized. Here, we investigated the function of UAF1 in mouse spermiogenesis and male fertility. We selectively deleted Uaf1 in premeiotic germ cells using Stra8-Cre knock-in mouse strain (Uaf1 sKO), and found that Uaf1 is essential for spermiogenesis and male fertility. We found that UAF1 interacts and colocalizes with USP1 in testes. Conditional knockout of Uaf1 in testes results in disturbed expression and localization of USP1, suggesting that UAF1 regulates spermiogenesis through the function of the deubiquitinating enzyme USP1. We used tandem mass tag-based proteomics to identify differentially expressed proteins and potential underlying mechanisms, and found that conditional knockout Uaf1 in testes results in reduced levels of proteins essential for spermiogenesis. Thus, the UAF1/USP1 deubiquitinase complex is essential for normal spermiogenesis by regulating the levels of spermiogenesis-related proteins.
2024-02-07 | PXD046120 | Pride
Project description:Taf7l (a paralogue of Taf7) and Trf2 (a TBP-related protein) are components of the core promoter complex required for gene/tissue-specific transcription of protein-coding genes by RNA polymerase II. Previous studies reported that Taf7l knockout mice exhibit structurally abnormal sperm, reduced sperm count, weakened motility and compromised fertility. Here we find that continued backcrossing of Taf7l-/Y mice from N5 to N9 produced KO males that are essentially sterile. Genome-wide expression profiling by mRNA-seq analysis of wild type (WT) and Taf7l-/Y (KO) testes revealed that Taf7l ablation impairs the expression of many post-meiotic spermatogenic specific as well as metabolic genes. Importantly, histological analysis of testes revealed that Taf7l-/Y mice develop post-meiotic arrest at the first stage of spermiogenesis, phenotypically similar to Trf2-/- mice, but distinct from Taf4b-/- mice. Indeed, we find that Taf7l and Trf2 co-regulate post-meiotic genes, but none of Taf4b-regulated germ stem cell genes in testes. Genome-wide ChIP-seq studies indicate that TAF7L binds to promoters of activated post-meiotic genes in testis. Moreover, biochemical studies show that TAF7L associates with TRF2 both in vitro and in testis suggesting that TAF7L likely cooperates directly with TRF2 at promoters of a subset of post-meiotic genes to regulate spermiogenesis. Our findings thus provide a new mechanism for cell-type specific transcriptional control involving an interaction between a ‘non-prototypic’ core promoter recognition factor (Trf2) and an orphan TAF subunit (Taf7l) in mammalian testis-specific gene transcription. Genome-wide mapping of TAF7L and Pol II in testis tissue, and mRNA-seq expression profiling wild type and Taf7l knockout testis.
2013-09-29 | E-GEOD-50807 | biostudies-arrayexpress
Project description:We combined high-resolution tiling microarrays and 5'-end RNA sequencing to obtain a genome-wide map of transcription start sites (TSSs) for Shewanella oneidensis MR-1. To test the reliability of these TSSs, we compared our result to those from differential RNA sequencing (dRNA-seq), which discriminates primary and processed ends of transcripts. We found that our identified TSSs tend to have significantly more mapped reads in the TEX(+) sample than the TEX(-) sample. Overall, the dRNA-seq results support the validity of our predictions for TSS. S. oneidensis MR-1 was grown to mid-log phase in Luria-Bertani broth (LB) or defined lactate minimal medium, and total RNA was isolated and used for differential RNA-sequencing (dRNA-seq) by next-generation sequencing, which is used to verify genome-wide transcription start sites. For dRNA-seq, total RNA was partially treated with Terminator Exonuclease (TEX) to digest processed RNA and thereby enrich for primary transcript ends.
2014-06-12 | E-GEOD-58292 | biostudies-arrayexpress
Project description:Nonobstructive azoospermia (NOA) is one of the most severe forms of male infertility, characterized by the absence of spermatozoa in the ejaculate. The etiology and genetic mechanism(s) of this disease remain largely unclear. Using Sanger sequence, we identified 12 heterozygous missense mutations and 2 heterozygous nonsense mutations in the HIPK4, encoding a serine/threonine kinase. Moreover, K490* mutant cause HIPK4 unstable in 293T cells. In accord with these findings, we observed that homozygous Hipk4-/- male mice were infertility owing to malformed and immotile spermatozoa, which are characterized by a deformed nucleus and a detached acrosome. Furthermore, we performed a phosphoproteomic analysis of testis from Hipk4 wild type and knockout mice, revealing multiple targets regulated by Hipk4 during spermiogenesis. Notably, manchette-associated protein Rimbp3 has multiple phosphorylation sites regulated by Hipk4. Moreover, Hipk4 and Rimbp3 deficiency mice display several common phenotypes, in particular with regard to the ectopic positioning of the manchette within spermatids, a presumed cause of sperm head deformities. Thus, Hipk4 might regulate manchette function through phosphorylation of Rimbp3, thereby playing essential role for sperm head formation and male fertility.
2022-08-02 | PXD019303 | Pride
Project description:During mammalian spermatogenesis, male germ cells undergo dramatic reorganization of chromatin, whereby 90-99% of histones are evicted and replaced by protamines. This reorganization prominently features histone acetylation to loosen chromatin structure. Given the potential role of retained histones in fertility and early embryonic development, the genomic location of retained nucleosomes is of great interest. However, the ultimate position and mechanisms underlying nucleosome eviction/retention are poorly understood, including several studies utilizing MNase-seq methodologies, but reporting remarkably dissimilar locations. Here, we utilized ATAC-seq to determine the location of retained nucleosomes in mouse sperm and found enrichment at promoters, but also retention at inter- and intragenic regions, and repetitive elements. We further investigated nucleosome eviction/retention by generating pre-meiotic, germ cell specific, conditional knockout mice for the histone acetyltransferase Gcn5, a key histone acetyltransferase. Gcn5cKO germ cells exhibited abnormal chromatin dynamics during spermiogenesis, including diminished nucleosome eviction leading to increased histone retention in sperm. These mice exhibited severe reproductive phenotypes: abnormal sperm production, sperm morphology, and ultimately, male infertility. Our findings demonstrate Gcn5 mediated histone acetylation promotes chromatin accessibility and nucleosome eviction in spermiogenesis, and that loss of histone acetylation leads to defects that disrupt male fertility and potentially, early embryogenesis.
2019-11-07 | GSE134560 | GEO