{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:55:45Z","timestamp":1760147745390,"version":"build-2065373602"},"reference-count":49,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2023,2,27]],"date-time":"2023-02-27T00:00:00Z","timestamp":1677456000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009633","name":"National Institutes of Health Intramural Research Program","doi-asserted-by":"publisher","award":["1ZIAHD000150-44","1ZICHD008986-03"],"award-info":[{"award-number":["1ZIAHD000150-44","1ZICHD008986-03"]}],"id":[{"id":"10.13039\/100009633","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>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\u2013mRNA network. We identified increased levels of miRNAs such as miR146, miR122a, miR26a, miR27a, miR150, miR196a, and miR328 that are relevant to spermatogenesis. mRNA\u2013miRNA 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\u2013mRNA interactions.<\/jats:p>","DOI":"10.3390\/cells12050756","type":"journal-article","created":{"date-parts":[[2023,2,27]],"date-time":"2023-02-27T05:20:50Z","timestamp":1677475250000},"page":"756","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["miRNA Expression Profiles of Mouse Round Spermatids in GRTH\/DDX25-Mediated Spermiogenesis: mRNA\u2013miRNA Network Analysis"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7643-4479","authenticated-orcid":false,"given":"Rajakumar","family":"Anbazhagan","sequence":"first","affiliation":[{"name":"Section on Molecular Endocrinology, Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raghuveer","family":"Kavarthapu","sequence":"additional","affiliation":[{"name":"Section on Molecular Endocrinology, Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryan","family":"Dale","sequence":"additional","affiliation":[{"name":"Bioinformatics and Scientific Programming Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kiersten","family":"Campbell","sequence":"additional","affiliation":[{"name":"Bioinformatics and Scientific Programming Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7959-9842","authenticated-orcid":false,"given":"Fabio R.","family":"Faucz","sequence":"additional","affiliation":[{"name":"Molecular Genomics Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria L.","family":"Dufau","sequence":"additional","affiliation":[{"name":"Section on Molecular Endocrinology, Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/nature03368","article-title":"Chromatin remodelling and epigenetic features of germ cells","volume":"434","author":"Kimmins","year":"2005","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1007\/s004290100176","article-title":"Haploid spermatids exhibit translationally repressed mRNAs","volume":"203","author":"Steger","year":"2001","journal-title":"Anat. Embryol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1038\/nrm2081","article-title":"The chromatoid body: A germ-cell-specific RNA-processing centre","volume":"8","author":"Kotaja","year":"2007","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"580019","DOI":"10.3389\/fcell.2020.580019","article-title":"Role of phosphorylated gonadotropin-regulated testicular RNA helicase (GRTH\/DDX25) in the regulation of germ cell specific mRNAs in chromatoid bodies during spermatogenesis","volume":"8","author":"Anbazhagan","year":"2020","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"6373","DOI":"10.1073\/pnas.0401855101","article-title":"Gonadotropin regulated testicular RNA helicase (GRTH\/DDX25) is essential for spermatid development and completion of spermatogenesis","volume":"101","author":"Sheng","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"887","DOI":"10.1093\/molehr\/gam065","article-title":"Polymorphism of the GRTH\/DDX25 gene in normal and infertile Japanese men: A missense mutation associated with loss of GRTH phosphorylation","volume":"13","author":"Koh","year":"2007","journal-title":"Mol. Hum. Reprod."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2561","DOI":"10.1093\/hmg\/ddz079","article-title":"Targeted knock-in mice with a human mutation in GRTH\/DDX25 reveals the essential role of phosphorylated GRTH in spermatid development during spermatogenesis","volume":"28","author":"Kavarthapu","year":"2019","journal-title":"Hum. Mol. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Kavarthapu, R., Anbazhagan, R., Pal, R., and Dufau, M.L. (2023). Single cell transcriptomic profiling of testicular germ cells reveals important role of phosphorylated GRTH\/DDX25 in round spermatid differentiation and acrosome biogenesis during spermiogenesis. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24043127"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"613","DOI":"10.3390\/cells11040613","article-title":"Chromatoid Bodies in the Regulation of Spermatogenesis: Novel Role of GRTH","volume":"11","author":"Anbazhagan","year":"2022","journal-title":"Cells"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1016\/j.bbamcr.2010.02.004","article-title":"Relevance of gonadotropin-regulated testicular RNA helicase (GRTH\/DDX25) in the structural integrity of the chromatoid body during spermatogenesis","volume":"1803","author":"Sato","year":"2010","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"44306","DOI":"10.1074\/jbc.M111.282756","article-title":"Testis-specific miRNA-469 up-regulated in gonadotropin-regulated testicular RNA helicase (GRTH\/DDX25)-null mice silences transition protein 2 and protamine 2 messages at sites within coding region: Implications of its role in germ cell development","volume":"286","author":"Dai","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1186\/s40104-017-0166-4","article-title":"The roles of microRNAs in regulation of mammalian spermatogenesis","volume":"8","author":"Chen","year":"2017","journal-title":"J. Anim. Sci. Biotechnol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1530\/REP-14-0643","article-title":"MicroRNAs and DNA methylation as epigenetic regulators of mitosis, meiosis and spermiogenesis","volume":"150","author":"Yao","year":"2015","journal-title":"Reproduction"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.cell.2018.03.006","article-title":"Metazoan microRNAs","volume":"173","author":"Bartel","year":"2018","journal-title":"Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1093\/molehr\/gaaa016","article-title":"Using miRNAs as diagnostic biomarkers for male infertility: Opportunities and challenges","volume":"26","author":"Vashisht","year":"2020","journal-title":"Mol. Hum. Reprod."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1016\/j.semcdb.2021.05.009","article-title":"Regulation of mammalian spermatogenesis by miRNAs","volume":"121","author":"Walker","year":"2022","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1186\/s12958-022-00990-7","article-title":"Qualitative and quantitative assessment of sperm miRNAs identifies hsa-miR-9-3p, hsa-miR-30b-5p and hsa-miR-122-5p as potential biomarkers of male infertility and sperm quality","volume":"20","author":"Joshi","year":"2022","journal-title":"Reprod. Biol. Endocrinol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"250","DOI":"10.1016\/j.ydbio.2008.11.011","article-title":"Sertoli cell Dicer is essential for spermatogenesis in mice","volume":"326","author":"Papaioannou","year":"2009","journal-title":"Dev. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2647","DOI":"10.1073\/pnas.0509333103","article-title":"The chromatoid body of male germ cells: Similarity with processing bodies and presence of Dicer and microRNA pathway components","volume":"103","author":"Kotaja","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","first-page":"e60271","article-title":"Enrichment of pachytene spermatocytes and spermatids from mouse testes using standard laboratory equipment","volume":"151","author":"Lehtiniemi","year":"2019","journal-title":"J. Vis. Exp."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2184","DOI":"10.1093\/bioinformatics\/bts356","article-title":"RSeQC: Quality control of RNA-seq experiments","volume":"28","author":"Wang","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3047","DOI":"10.1093\/bioinformatics\/btw354","article-title":"MultiQC: Summarize analysis results for multiple tools and samples in a single report","volume":"32","author":"Ewels","year":"2016","journal-title":"Bioinformatics"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"10","DOI":"10.14806\/ej.17.1.200","article-title":"Cutadapt removed adapter sequences from high-throughput sequencing reads","volume":"17","author":"Martin","year":"2011","journal-title":"EMBnet. J."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1093\/bioinformatics\/bts635","article-title":"STAR: Ultrafast universal RNA-seq aligner","volume":"29","author":"Dobin","year":"2013","journal-title":"Bioinformatics"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1093\/bioinformatics\/btt656","article-title":"FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features","volume":"30","author":"Liao","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_26","first-page":"275","article-title":"False discovery rates: A new deal","volume":"18","author":"Stephens","year":"2016","journal-title":"Biostatistics"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.1923","article-title":"Fast gapped-read alignment with Bowtie2","volume":"9","author":"Langmead","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1120","DOI":"10.1261\/rna.055509.115","article-title":"Evaluation of microRNA alignment techniques","volume":"22","author":"Ziemann","year":"2016","journal-title":"RNA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e05005","DOI":"10.7554\/eLife.05005","article-title":"Predicting effective microRNA target sites in mammalian mRNAs","volume":"4","author":"Agarwal","year":"2015","journal-title":"eLife"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1261\/rna.2239606","article-title":"TarBase: A comprehensive database of experimentally supported animal microRNA targets","volume":"12","author":"Sethupathy","year":"2006","journal-title":"RNA"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1093\/nar\/gkz757","article-title":"miRDB: An online database for prediction of functional microRNA targets","volume":"48","author":"Chen","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"310","DOI":"10.3389\/fcell.2020.00310","article-title":"Linking Phospho-Gonadotropin Regulated Testicular RNA Helicase (GRTH\/DDX25) to Histone Ubiquitination and Acetylation Essential for Spermatid Development during Spermiogenesis","volume":"8","author":"Kavarthapu","year":"2020","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1446","DOI":"10.1038\/ncb3259","article-title":"The RNF138 E3 ligase displaces Ku to promote DNA end resection and regulate DNA repair pathway choice","volume":"17","author":"Ismail","year":"2015","journal-title":"Nat. Cell. Biol"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e2795","DOI":"10.1038\/cddis.2017.110","article-title":"Rnf138 deficiency promotes apoptosis of spermatogonia in juvenile male mice","volume":"8","author":"Xu","year":"2017","journal-title":"Cell Death Dis."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/j.bbagrm.2013.08.004","article-title":"Chromatin dynamics during spermiogenesis","volume":"1839","author":"Rathke","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"870695","DOI":"10.1155\/2014\/870695","article-title":"The role of histone ubiquitination during spermatogenesis","volume":"2014","author":"Sheng","year":"2014","journal-title":"Biomed. Res. Int."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1038\/s42003-020-0984-3","article-title":"The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells","volume":"3","author":"Fatima","year":"2020","journal-title":"Commun. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1262\/jrd.2014-018","article-title":"Histone H4 modification during mouse spermatogenesis","volume":"60","author":"Shirakata","year":"2014","journal-title":"J. Reprod. Dev."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1530\/REP-15-0562","article-title":"Epigenetic regulation of the histone-to-protamine transition during spermiogenesis","volume":"151","author":"Boa","year":"2016","journal-title":"Reproduction"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Vatin, M., Girault, M.-S., Firlej, V., Marchiol, C., and Ialy-Radio, C. (2020). Identification of a New QTL region on mouse chromosome 1 responsible for male hypofertility: Phenotype characterization and candidate genes. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21228506"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Girault, M.S., Dupuis, S., Ialy-Radio, C., Stouvenel, L., Viollet, C., Pierre, R., Favier, M., Ziyyat, A., and Barbaux, S. (2021). Deletion of the Spata3 gene induces sperm alterations and In vitro Hypofertility in Mice. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22041959"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1093\/biolreprod\/83.s1.177","article-title":"SPATA3 and SPATA6 Interact with KLHL10 and participate in spermatogenesis","volume":"83","author":"Wu","year":"2010","journal-title":"Biol. Reprod."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1095\/biolreprod.105.045484","article-title":"Cullin3 is a KLHL10-interacting protein preferentially expressed during late spermiogenesis","volume":"74","author":"Wang","year":"2006","journal-title":"Biol. Reprod."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1089\/scd.2016.0318","article-title":"The NOTCH ligand JAG1 regulates GDNF expression in Sertoli cells","volume":"26","author":"Garcia","year":"2017","journal-title":"Stem Cells Dev."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Ma, J., Fan, Y., Zhang, J., Feng, S., Hu, Z., Qiu, W., Long, K., Jin, L., Tang, Q., and Wang, X. (2018). Testosterone-Dependent miR-26a-5p and let-7g-5p Act as Signaling Mediators to Regulate Sperm Apoptosis via Targeting PTEN and PMAIP1. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19041233"},{"key":"ref_46","first-page":"961","article-title":"Hsa-miR-27a-3p overexpression in men with nonobstructive azoospermia: A case-control study","volume":"18","author":"Norioun","year":"2020","journal-title":"Int. J. Reprod. Biomed."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1038\/nature06236","article-title":"Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis","volume":"450","author":"Okada","year":"2007","journal-title":"Nature"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"492","DOI":"10.1111\/andr.12164","article-title":"Expression ratio of histone demethylase KDM 3A to protamine-1 mRNA is predictive of successful testicular sperm extraction in men with obstructive and non-obstructive azoospermia","volume":"4","author":"Javadirad","year":"2016","journal-title":"Andrology"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e2842","DOI":"10.1038\/cddis.2017.241","article-title":"Mitochondrial AKAP1 supports mTOR pathway and tumor growth","volume":"8","author":"Rinaldi","year":"2017","journal-title":"Cell Death Dis."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/5\/756\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:43:41Z","timestamp":1760121821000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/5\/756"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,2,27]]},"references-count":49,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2023,3]]}},"alternative-id":["cells12050756"],"URL":"https:\/\/doi.org\/10.3390\/cells12050756","relation":{},"ISSN":["2073-4409"],"issn-type":[{"type":"electronic","value":"2073-4409"}],"subject":[],"published":{"date-parts":[[2023,2,27]]}}}