{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:09:19Z","timestamp":1785434959333,"version":"3.56.0"},"posted":{"date-parts":[[2025,8,22]]},"group-title":"Cell Biology","reference-count":61,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,8,22]],"date-time":"2025-08-22T00:00:00Z","timestamp":1755820800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["Postdoctoral Fellowship"],"award-info":[{"award-number":["Postdoctoral Fellowship"]}],"id":[{"id":"https:\/\/ror.org\/03vpdge44","id-type":"ROR","asserted-by":"publisher"}]},{"award":["Biomedical Scholars Award"],"award-info":[{"award-number":["Biomedical Scholars Award"]}],"id":[{"id":"https:\/\/ror.org\/02xhk2825","id-type":"ROR","asserted-by":"publisher"}]},{"award":["F31 HD114433"],"award-info":[{"award-number":["F31 HD114433"]}],"id":[{"id":"https:\/\/ror.org\/04byxyr05","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["T32GM156697"],"award-info":[{"award-number":["T32GM156697"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,8,22]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>The epididymis plays a critical role in sperm maturation, including remodeling the sperm RNA payload. While small RNAs have been extensively studied in this context, the epididymal contribution to larger sperm RNAs, such as mRNAs, remains underexplored. mRNAs were among the first RNA species identified in sperm, yet their functional relevance has remained elusive, largely due to the translational quiescence of mature spermatozoa and the hypothesis that these RNAs are residual by-products of spermatogenesis. Yet, mRNAs carried by sperm have been detected in the zygote, indicating they could play a role beyond fertilization. However, if the soma, the epididymis, actively contributes mRNAs to sperm as it does small RNAs, has not been experimentally assessed. To investigate this, here, we provide a comprehensive analysis of the mRNA landscape of mouse sperm, epithelial cells and extracellular vesicles (EVs) isolated from the proximal (caput) and distal (cauda) epididymis. Through this analysis and sperm-EV co-incubation experiments we demonstrate the transfer of mRNAs from epididymal EVs to sperm. Further, through sperm RNA microinjection into zygotes we uncover gene regulation in the early embryo driven by the introduction of sperm RNAs, specific to large RNA species. These findings ultimately reveal the dynamic mRNA profile of sperm that is delivered to the egg and demonstrate that RNA species beyond small RNAs are capable of influencing preimplantation embryo gene expression.<\/jats:p>","DOI":"10.1101\/2025.08.18.670952","type":"posted-content","created":{"date-parts":[[2025,8,23]],"date-time":"2025-08-23T00:00:19Z","timestamp":1755907219000},"source":"Crossref","is-referenced-by-count":1,"title":["Epididymal extracellular vesicles harbor and convey mRNA to sperm for transfer to zygotes"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8999-5731","authenticated-orcid":false,"given":"Natalie A.","family":"Trigg","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Grace S.","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexis G.","family":"Leach","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Colin C.","family":"Conine","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025082507250528000_2025.08.18.670952v1.1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1019055108"},{"key":"2025082507250528000_2025.08.18.670952v1.2","doi-asserted-by":"crossref","unstructured":"Battistone, M. A. , Spallanzani, R. G. , Mendelsohn, A. C. , Capen, D. , Nair, A. V. , Brown, D. & Breton, S . 2019. Novel role of proton-secreting epithelial cells in sperm maturation and mucosal immunity. J Cell Sci, 133.","DOI":"10.1242\/jcs.233239"},{"key":"2025082507250528000_2025.08.18.670952v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/nrg3662"},{"key":"2025082507250528000_2025.08.18.670952v1.4","doi-asserted-by":"publisher","DOI":"10.1006\/excr.1995.1130"},{"key":"2025082507250528000_2025.08.18.670952v1.5","doi-asserted-by":"publisher","DOI":"10.1530\/ror.0.0040135"},{"key":"2025082507250528000_2025.08.18.670952v1.6","doi-asserted-by":"crossref","unstructured":"Cannarella, R. , Rando, O. J. , Condorelli, R. A. , Chamayou, S. , Romano, S. , Guglielmino, A. , Yin, Q. , Hans, T. G. , Mancuso, F. , Arato, I. , Bellucci, C. , Luca, G. , Lundy, S. D. , LA Vignera, S. & Calogero, A. E. 2024. Sperm-carried IGF2: towards the discovery of a spark contributing to embryo growth and development. Molecular Human Reproduction, 30.","DOI":"10.1093\/molehr\/gaae034"},{"key":"2025082507250528000_2025.08.18.670952v1.7","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1186\/s12915-022-01268-5","article-title":"The repertoire of testicular extracellular vesicle cargoes and their involvement in inter-compartmental communication associated with spermatogenesis","volume":"20","year":"2022","journal-title":"BMC Biology"},{"key":"2025082507250528000_2025.08.18.670952v1.8","doi-asserted-by":"crossref","unstructured":"Conine, C. C. , Krykbaeva, M. , Song, L. , Brewster, R. C. , Friedman, N. & Rando, O. J . 2020. Flexibility and constraint in preimplantation gene regulation in mouse. bioRxiv, 2020.03.13.990275.","DOI":"10.1101\/2020.03.13.990275"},{"key":"2025082507250528000_2025.08.18.670952v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.devcel.2018.06.024"},{"key":"2025082507250528000_2025.08.18.670952v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/aja.2010.97"},{"key":"2025082507250528000_2025.08.18.670952v1.11","doi-asserted-by":"publisher","DOI":"10.1093\/molehr\/gau018"},{"key":"2025082507250528000_2025.08.18.670952v1.12","doi-asserted-by":"crossref","unstructured":"Fang, P. , Zeng, P. , Wang, Z. , Liu, M. , Xu, W. , Dai, J. , Zhao, X. , Zhang, D. , Liang, D. , Chen, X. , Shi, S. , Zhang, M. , Wang, L. , Qiao, Z. & Shi, H . 2014. Estimated Diversity of Messenger RNAs in Each Murine Spermatozoa and Their Potential Function During Early Zygotic Development1. 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The microbiota and T cells non-genetically modulate inherited phenotypes transgenerationally. 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A. , Anderson, A. L. , Bromfield, E. G. , Bernstein, I. R. , Mulhall, J. E. , Schjenken, J. E. , Dun, M. D. , Humphrey, S. J. & Nixon, B . 2022. Global profiling of the proteomic changes associated with the post-testicular maturation of mouse spermatozoa. Cell Reports, 41.","DOI":"10.1016\/j.celrep.2022.111655"},{"key":"2025082507250528000_2025.08.18.670952v1.46","doi-asserted-by":"crossref","unstructured":"Sulzyk, V. , Curci, L. , Gonz\u00e1lez, L. N. , Rebagliati Cid, A. , WEIGEL Mu\u00f1oz, M. & Cuasnicu, P. S. 2025. 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N. , Dun, M. D. , Roman, S. D. , Eamens, A. L. & Nixon, B. 2021. Acrylamide modulates the mouse epididymal proteome to drive alterations in the sperm small non-coding RNA profile and dysregulate embryo development. Cell Reports, 37.","DOI":"10.1016\/j.celrep.2021.109787"},{"key":"2025082507250528000_2025.08.18.670952v1.54","first-page":"e240204","article-title":"A lack of commensal microbiota influences the male reproductive tract intergenerationally in mice","volume":"169","year":"2025","journal-title":"Reproduction"},{"key":"2025082507250528000_2025.08.18.670952v1.55","doi-asserted-by":"crossref","unstructured":"Trombetta, J. J. , Gennert, D. , Lu, D. , Satija, R. , Shalek, A. K. & Regev, A . 2014. Preparation of Single-Cell Rna-Seq Libraries for Next Generation Sequencing. 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