Single-Cell Transcriptomic Profiling of the Mouse Testicular Germ Cells Reveals Important Role of Phosphorylated GRTH/DDX25 in Round Spermatid Differentiation and Acrosome Biogenesis during Spermiogenesis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE221226 | GEO | 2022/12/21
REPOSITORIES: GEO
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Project description:GRTH/DDX25 is a testicular RNA helicase expressed in germ cells that plays a crucial role in completion of spermatogenesis. Previously, we demonstrated a missense mutation (R242H) of GRTH gene in Japanese infertile patients (5.8%) with non-obstructive azoospermia. This mutation upon expression in COS-1 cells revealed absence of the 61 kDa phosphorylated GRTH in cytoplasm and the presence of the 56 kDa non-phosphorylated GRTH in the nucleus. GRTH knock-in (KI) mice carrying the human GRTH (R242H) mutation, lack phosphorylated GRTH, and sperm due to failure of round spermatid elongation during spermiogenesis. To determine the impact of phosphorylated GRTH on molecular events/pathways participating in spermatid development during spermiogenesis, we analyzed transcriptome profiles obtained from RNA-Seq of germ cells from KI and WT mice. RNA-Seq analysis of 2624 differentially expressed genes revealed 1404 down-regulated and 1220 up-regulated genes in KI mice. Genes relevant to spermatogenesis, spermatid development and spermatid differentiation were significantly down-regulated. KEGG enrichment analysis showed genes related to ubiquitin-mediated proteolysis and protein processing in endoplasmic reticulum pathway genes were significantly down-regulated while the up-regulated genes were found to be involved in Focal adhesion and ECM-receptor interaction pathways. Real-Time PCR analysis confirmed considerable reduction in transcripts of ubiquitination related genes Ube2j1, Ube2k, Ube2w, Rnf8, Rnf133, Rnf138, Cul3 and increased expression of Ccnd2, Col1a, Lamb1, Cav1, Igf1, Itga9 mRNA's in KI mice compared to WT. Also, marked reduction in protein expression of UBE2J1, RNF8, RNF138 (ubiquitination network), MOF (histone acetyltransferase), their modified Histone substrates (H2AUb, H2BUb) and H4Ac, H4K16Ac were observed in KI mice. GRTH-IP mRNA binding studies revealed that Rnf8 and Ube2J1 mRNAs from WT mice associated with GRTH protein and the binding is greatly impaired in the KI mice. Immunohistochemistry analysis showed significantly reduced expression of RNF8, MOF, H4Ac and H4K16Ac in round spermatids of KI mice. Absence of phosphorylated GRTH impairs UBE2J1, RNF8 and MOF-dependent histone ubiquitination and acetylation essential for histone replacement, chromatin condensation and spermatid elongation during spermiogenesis.
| S-EPMC7242631 | biostudies-literature
Project description:GRTH/DDX25 is a testis-specific DEAD-box family of RNA helicase, which plays an essential role in spermatogenesis and male fertility. There are two forms of GRTH, a 56 kDa non-phosphorylated form and a 61 kDa phosphorylated form (pGRTH). GRTH-KO and GRTH Knock-In (KI) mice with R242H mutation (lack pGRTH) are sterile with a spermatogenic arrest at step 8 of spermiogenesis due to failure of round spermatids (RS) to elongate. We performed mRNA-seq and miRNA-seq analysis on RS of WT, KI, and KO to identify crucial microRNAs (miRNAs) and mRNAs during RS development by establishing a miRNA-mRNA network. We identified increased levels of miRNAs such as miR146, miR122a, miR26a, miR27a, miR150, miR196a, and miR328 that are relevant to spermatogenesis. mRNA-miRNA target analysis on these DE-miRNAs and DE-mRNAs revealed miRNA target genes involved in ubiquitination process (Ube2k, Rnf138, Spata3), RS differentiation, and chromatin remodeling/compaction (Tnp1/2, Prm1/2/3, Tssk3/6), reversible protein phosphorylation (Pim1, Hipk1, Csnk1g2, Prkcq, Ppp2r5a), and acrosome stability (Pdzd8). Post-transcriptional and translational regulation of some of these germ-cell-specific mRNAs by miRNA-regulated translation arrest and/or decay may lead to spermatogenic arrest in KO and KI mice. Our studies demonstrate the importance of pGRTH in the chromatin compaction and remodeling process, which mediates the differentiation of RS into elongated spermatids through miRNA-mRNA interactions.
| S-EPMC10001410 | biostudies-literature
Project description:Gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25), a member of the DEAD-box protein family, is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells and germ cells (meiotic spermatocytes and spermatids). In this study, we observed that GRTH is present in the nucleus, cytoplasm and chromatoid body of germ cells, and is an integral component of messenger ribonuclear protein particles. Male mice with a null mutation in the GRTH gene displayed normal gonadotropin and androgen profiles. However, they were sterile, with azoospermia caused by a complete arrest of spermiogenesis at step 8 of round spermatids and failure to elongate. Round spermatids of the null mice showed marked diminution in the size of chromatoid bodies. The transcription of relevant messages was not altered, but their translation was abrogated in a selective manner. Protein expression of transition proteins 1 and 2 and angiotensin-converting enzyme was completely absent, whereas that of the transcriptional activator cAMP responsive element modulator was intact. These findings indicate that GRTH participates in translational-associated events during germ cell development. Although significant apoptosis was present at the metaphase of meiosis in the GRTH-null mice, spermatogenesis proceeded to step 8 of spermiogenesis when complete arrest occurred. This progression may relate to compensatory gene function(s) and/or the observed up-regulation of DNA repair proteins Rad51 and Dmc1. This study (i) demonstrates that GRTH is essential for completion of spermatogenesis, (ii) provides insights into intrinsic requirements for spermiogenesis, and (iii) establishes a model for studies of male infertility and contraception.
| S-EPMC404052 | biostudies-literature
Project description:In this study, we examined genome-wide transcriptome profiles of germ cells isolated from GRTH Knock-In and WT mice testis using RNA-Seq. We used homozygous KI mice with R242H mutation in GRTH gene resulting loss of phospho-GRTH to further understand its role in spermatid development during spermiogenesis by analyzing the DEGs from RNA-Seq. The analysis of RNA-Seq data allowed us to find the relevant DEGs related to ubiquitination pathway that are essential for spermatid development. This study using KI mice model demonstrated that loss of phospho-GRTH impairs UBE2J1 and RNF8 dependent histone modifications hampering initial stages of spermatid elongation process.
2020-03-04 | GSE145047 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2023-03-20 | GSE222628 | GEO
Project description:GRTH/DDX 25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. Regulation of spermatogenesis occurs at the levels of transcription, post-transcriptional and translational levels which needs to be explored in detail. miRNAs regulate the expression of important genes involved in spermatid elongation process. Differential miRNA expression in round spermatids (RS) of GRTH KO and GRTH-KI mice in comparison with mRNAs remains unexplored. Our miRNAseq analysis of small RNAs obtained from RS reveal differential expression of important miRNAs that regulate spermatid differentiation, apoptosis, chromatin compaction and ubiquitination. mRNAseq analysis of RS isolated from GRTH-KI and GRTH-KO mice models were also carried-out to study the putative miRNA targets and mRNA-miRNA interaction. mRNAseq analysis revealed specific set of mRNAs that are critical for sperm chromatin compaction, ubiquitination and spermatid elongation etc. miRNA-mRNA interaction prediction highlighted differentially expressed/enriched miRNA transcripts whose predicted mRNA targets were differentially expressed. Based on the interaction information of the miRNAs-mRNAs differential analysis, the miRNA-mRNA network was constructed. These results demonstrate the importance of miRNA in the translational arrest and stability of germ cell specific mRNAs which are critical for later stages of spermiogenesis and fertility.
2023-03-20 | GSE222626 | GEO
Project description:GRTH/DDX 25 is a member of the DEAD-box family of RNA helicases that play an essential role in spermatogenesis. Regulation of spermatogenesis occurs at the levels of transcription, post-transcriptional and translational levels which needs to be explored in detail. miRNAs regulate the expression of important genes involved in spermatid elongation process. Differential miRNA expression in round spermatids (RS) of GRTH KO and GRTH-KI mice in comparison with mRNAs remains unexplored. Our miRNAseq analysis of small RNAs obtained from RS reveal differential expression of important miRNAs that regulate spermatid differentiation, apoptosis, chromatin compaction and ubiquitination. mRNAseq analysis of RS isolated from GRTH-KI and GRTH-KO mice models were also carried-out to study the putative miRNA targets and mRNA-miRNA interaction. mRNAseq analysis revealed specific set of mRNAs that are critical for sperm chromatin compaction, ubiquitination and spermatid elongation etc. miRNA-mRNA interaction prediction highlighted differentially expressed/enriched miRNA transcripts whose predicted mRNA targets were differentially expressed. Based on the interaction information of the miRNAs-mRNAs differential analysis, the miRNA-mRNA network was constructed. These results demonstrate the importance of miRNA in the translational arrest and stability of germ cell specific mRNAs which are critical for later stages of spermiogenesis and fertility.
2023-03-20 | GSE222627 | GEO
Project description:Gonadotropin Regulated Testicular RNA Helicase (GRTH/Ddx25) is a testis-specific multifunctional RNA helicase and an essential post-transcriptional regulator of spermatogenesis. GRTH transports relevant mRNAs from nucleus to cytoplasmic sites of meiotic and haploid germ cells and associates with actively translating polyribosomes. It is also a negative regulator of steroidogenesis in Leydig cells. To obtain a genome-wide perspective of GRTH regulated genes, in particularly those associated with polyribosomes, microarray differential gene expression analysis was performed using polysome-bound RNA isolated from testes of wild type (WT) and GRTH KO mice. 792 genes among the entire mouse genome were found to be polysomal GRTH-linked in WT. Among these 186 were down-regulated and 7 up-regulated genes in GRTH null mice. A similar analysis was performed using total RNA extracted from purified germ cell populations to address GRTH action in individual target cells. The down-regulation of known genes concerned with spermatogenesis at polysomal sites in GRTH KO and their association with GRTH in WT coupled with early findings of minor or unchanged total mRNAs and abolition of their protein expression in KO underscore the relevance of GRTH in translation. Ingenuity pathway analysis predicted association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NF?B/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis. This study has revealed known and unexplored factors in the genome and regulatory pathways underlying GRTH action in male reproduction.
| S-EPMC3316541 | biostudies-literature
Project description:The spermatogenesis process is complex and delicate, and any error in a step may cause spermatogenesis arrest and even male infertility. According to our previous transcriptomic data, CEP70 is highly expressed throughout various stages of human spermatogenesis, especially during the meiosis and deformation stages. CEP70 is present in sperm tails and that it exists in centrosomes as revealed by human centrosome proteomics. However, the specific mechanism of this protein in spermatogenesis is still unknown. In this study, we found a heterozygous site of the same mutation on CEP70 through mutation screening of patients with clinical azoospermia. To further verify, we deleted CEP70 in mice and found that it caused abnormal spermatogenesis, leading to male sterility. We found that the knockout of CEP70 did not affect the prophase of meiosis I, but led to male germ-cell apoptosis and abnormal spermiogenesis. By transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis, we found that the deletion of CEP70 resulted in the abnormal formation of flagella and acrosomes during spermiogenesis. Tandem mass tag (TMT)-labeled quantitative proteomic analysis revealed that the absence of CEP70 led to a significant decrease in the proteins associated with the formation of the flagella, head, and acrosome of sperm, and the microtubule cytoskeleton. Taken together, our results show that CEP70 is essential for acrosome biogenesis and flagella formation during spermiogenesis.
| S-EPMC8116340 | biostudies-literature
Project description:Gonadotropin-regulated testicular RNA helicase (GRTH) (GRTH/DDX25), is a testis-specific protein essential for completion of spermatogenesis. Transgenic mice carrying 5'-flanking regions of the GRTH gene/green fluorescence protein (GFP) reporter revealed a region (-6.4/-3.6 kb) which directs its expression in germ cells (GCs) via androgen action. This study identifies a functional cis-binding element on the GRTH gene for GC nuclear factor (GCNF) (GCNF/RTR) required to regulate GRTH gene expression in postmeiotic testis GCs and explore the action of androgen on GCNF and GRTH transcription/expression. GCNF expression decreased in mice testis upon flutamide (androgen receptor antagonist) treatment, indicating the presence of an androgen/GCNF network to direct GRTH expression in GC. Binding studies and chromatin immunoprecipitation demonstrated specific association of GCNF to a consensus half-site (-5270/-5252) of the GRTH gene in both round spermatids and spermatocytes, which was abolished by flutamide treatment in round spermatids. Moreover, flutamide treatment of wild-type mice caused selective reduction of GCNF and GRTH in round spermatids. GCNF knock-down in seminiferous tubules from GRTH-transgenic mice (dark zone, round spermatid rich) caused decreased GFP expression. Exposure of tubules to flutamide caused decrease in GCNF and GFP expression, whereas androgen exposure induced significant increase. Our studies provide evidence for actions of androgen on GCNF cell-specific regulation of GRTH expression in GC. GRTH associates with GCNF mRNA, its absence caused increase on GCNF expression and mRNA stability indicative of a negative autocrine regulation of GCNF by GRTH. These in vivo/in vitro models link androgen actions to GC through GCNF, as regulated transfactor that controls transcription/expression of GRTH.
| S-EPMC4664233 | biostudies-literature