{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T05:00:04Z","timestamp":1790830804803,"version":"4.1.0"},"reference-count":38,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Natural Science Foundation of China","award":["32472903"],"award-info":[{"award-number":["32472903"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"crossref"}]},{"name":"National Natural Science Foundation of China","award":["32502910"],"award-info":[{"award-number":["32502910"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"crossref"}]},{"award":["32472903"],"award-info":[{"award-number":["32472903"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"award":["32502910"],"award-info":[{"award-number":["32502910"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Basic Research Program of Jiangsu","award":["BK20251531"],"award-info":[{"award-number":["BK20251531"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Melatonin is a hormone primarily secreted by the pineal gland, best known for regulating circadian rhythms. It also plays a critical role in ovarian physiology by modulating granulosa cell (GC) function during follicular development, although the underlying molecular mechanisms remain incompletely understood. Recent advances in ribosome sequencing (Ribo-seq) have revealed that many small open reading frames (sORFs), previously overlooked in conventional annotations, actually encode functional micropeptides. However, whether melatonin exerts its ovarian effects through such micropeptides has remained unexplored. In the present study, we identified a previously unannotated sORF within the 5\u2032untranslated region (UTR) of sheep IGF1R mRNA and demonstrated that this sORF encodes a 32-amino-acid micropeptide, which we named IGF1Raa. Knockdown and overexpression of IGF1Raa in sheep GCs, assessed by CCK-8, Edu, flow cytometry, and ELISA, revealed that IGF1Raa negatively regulates GC proliferation and E2 secretion and promotes apoptosis. Co-ip assays and RNA-seq show that IGF1Raa physically associates with PPP2R2B, a regulatory subunit of protein phosphatase 2A (PP2A), and modulates PP2A protein expression. Consequently, IGF1Raa regulates the phosphorylation of AMPK (Thr172) and its downstream effector Raptor (Ser792), thereby controlling the AMPK\/mTOR signaling cascade. Importantly, melatonin suppresses IGF1Raa expression, and overexpression of IGF1Raa partially abrogates melatonin\u2019s stimulatory effects on GC proliferation and steroidogenesis. Collectively, our findings identify IGF1Raa as a melatonin-responsive micropeptide that governs GC function through the PP2A\/AMPK\/mTOR axis, providing new insights into the regulatory network of ovarian physiology and offering potential therapeutic targets for reproductive disorders.<\/jats:p>","DOI":"10.3390\/cells15191771","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T08:12:42Z","timestamp":1790669562000},"page":"1771","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Melatonin Regulates Sheep Granulosa Cell Proliferation and Apoptosis Through a Novel Micropeptide, IGF1Raa"],"prefix":"10.3390","volume":"15","author":[{"given":"Weiming","family":"Zhong","sequence":"first","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"given":"Jianyu","family":"Ma","sequence":"additional","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8409-5534","authenticated-orcid":false,"given":"Zhibo","family":"Wang","sequence":"additional","affiliation":[{"name":"Shanghai Animal Disease Prevention and Control Center, Shanghai 201103, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Hua","family":"Yang","sequence":"additional","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-3404-1022","authenticated-orcid":false,"given":"Xiuyuan","family":"Wang","sequence":"additional","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"data-curation","vocabulary":"credit"}]},{"given":"Yang","family":"Gao","sequence":"additional","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"}]},{"given":"Shang","family":"Gao","sequence":"additional","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"}]},{"given":"Peiyong","family":"Chen","sequence":"additional","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"data-curation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6846-3755","authenticated-orcid":false,"given":"Yongjie","family":"Wan","sequence":"additional","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"formal-analysis","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7901-7774","authenticated-orcid":false,"given":"Yanli","family":"Zhang","sequence":"additional","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4965-836X","authenticated-orcid":false,"given":"Feng","family":"Wang","sequence":"additional","affiliation":[{"name":"Sanya Institute of Nanjing Agricultural University, Sanya 572024, China"},{"name":"Jiangsu Livestock Embryo Engineering Laboratory, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"547","DOI":"10.3390\/endocrines5040040","article-title":"Granulosa Cells: Central Regulators of Female Fertility","volume":"5","author":"Batalha","year":"2024","journal-title":"Endocrines"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Del Bianco, D., Gentile, R., Sallicandro, L., Biagini, A., Quellari, P.T., Gliozheni, E., Sabbatini, P., Ragonese, F., Malvasi, A., and D\u2019Amato, A. (2024). Electro-Metabolic Coupling of Cumulus\u2013Oocyte Complex. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25105349"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2972","DOI":"10.1007\/s43032-024-01643-3","article-title":"Inducers and Inhibitors of Pyroptotic Death of Granulosa Cells in Models of Premature Ovarian Insufficiency and Polycystic Ovary Syndrome","volume":"31","author":"Berkel","year":"2024","journal-title":"Reprod. Sci."},{"key":"ref_4","first-page":"541677","article-title":"A single-cell atlas of the single versus multiple parous Hu Sheep ovary","volume":"14","author":"Ge","year":"2023","journal-title":"J. bioRxiv"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1186\/s13048-023-01204-3","article-title":"Correlation between abnormal energy metabolism of ovarian granulosa cells and in vitro fertilization\u2013embryo transfer outcomes in patients with polycystic ovary syndrome and obesity","volume":"16","author":"Zhao","year":"2023","journal-title":"J. Ovarian Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"5541","DOI":"10.1007\/s12035-024-03915-0","article-title":"Significance of Melatonin in the Regulation of Circadian Rhythms and Disease Management","volume":"61","author":"Megha","year":"2024","journal-title":"Mol. Neurobiol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Reiter, R.J., Sharma, R., Tan, D.-X., Chuffa, L.G.d.A., da Silva, D.G.H., Slominski, A.T., Steinbrink, K., and Kleszczynski, K. (2024). Dual sources of melatonin and evidence for different primary functions. Front. Endocrinol., 15.","DOI":"10.3389\/fendo.2024.1414463"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"108534","DOI":"10.1016\/j.reprotox.2024.108534","article-title":"Melatonin-mediated actions and circadian functions that improve implantation, fetal health and pregnancy outcome","volume":"124","author":"Reiter","year":"2024","journal-title":"Reprod. Toxicol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"859","DOI":"10.1080\/07420528.2024.2353857","article-title":"The environment and the internal clocks: The study of their relationships from prehistoric to modern times","volume":"41","author":"Touitou","year":"2024","journal-title":"Chronobiol. Int."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e70171","DOI":"10.1111\/jsr.70171","article-title":"Sleep and Rhythmic Profile After Pineal Gland Removal in Humans","volume":"35","author":"Gobetti","year":"2025","journal-title":"J. Sleep Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"102659","DOI":"10.1016\/j.redox.2023.102659","article-title":"Reactive oxygen species and ovarian diseases: Antioxidant strategies","volume":"62","author":"Liang","year":"2023","journal-title":"Redox Biol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Sun, T.C., Liu, X.C., Yang, S.H., Song, L.L., Zhou, S.J., Deng, S.L., Tian, L., and Cheng, L.Y. (2020). Melatonin Inhibits Oxidative Stress and Apoptosis in Cryopreserved Ovarian Tissues via Nrf2\/HO-1 Signaling Pathway. Front. Mol. Biosci., 7.","DOI":"10.3389\/fmolb.2020.00163"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Xu, G., Dong, Y., Wang, Z., Ding, H., Wang, J., Zhao, J., Liu, H., and Lv, W. (2023). Melatonin Attenuates Oxidative Stress-Induced Apoptosis of Bovine Ovarian Granulosa Cells by Promoting Mitophagy via SIRT1\/FoxO1 Signaling Pathway. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241612854"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Zhai, B., Li, X., Zhao, Z., Cao, Y., Liu, X., Liu, Z., Ma, H., and Lu, W. (2023). Melatonin Protects the Apoptosis of Sheep Granulosa Cells by Suppressing Oxidative Stress via MAP3K8 and FOS Pathway. Genes, 14.","DOI":"10.3390\/genes14051067"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1016\/j.anireprosci.2016.07.015","article-title":"Melatonin modulates the functions of porcine granulosa cells via its membrane receptor MT2 in vitro","volume":"172","author":"He","year":"2016","journal-title":"Anim. Reprod. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Tharakan, R., and Sawa, A. (2021). Minireview: Novel Micropeptide Discovery by Proteomics and Deep Sequencing Methods. Front. Genet., 12.","DOI":"10.3389\/fgene.2021.651485"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1126\/science.1168978","article-title":"Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling","volume":"324","author":"Ingolia","year":"2009","journal-title":"Science"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"15664","DOI":"10.1038\/ncomms15664","article-title":"The microprotein Minion controls cell fusion and muscle formation","volume":"8","author":"Zhang","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_19","first-page":"ec48","article-title":"Calcium regulator hidden in a long \u201cnoncoding\u201d RNA","volume":"8","author":"Gough","year":"2015","journal-title":"Sci. Signal."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"135051","DOI":"10.1016\/j.ijbiomac.2024.135051","article-title":"Integrative analysis of RNA-seq and Ribo-seq reveals that lncRNA-GRN regulates chicken follicular atresia through miR-103-3p\/FBXW7 axis and encoding peptide","volume":"278","author":"Yu","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"106206","DOI":"10.1016\/j.psj.2025.106206","article-title":"LncRNA HAND2OT encoding micropeptide regulates the estradiol secretion, proliferation and apoptosis of ovarian follicular theca cells in chickens","volume":"105","author":"Shu","year":"2026","journal-title":"Poult. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e2001897118","DOI":"10.1073\/pnas.2001897118","article-title":"Identification of a micropeptide and multiple secondary cell genes that modulate Drosophila male reproductive success","volume":"118","author":"Immarigeon","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"122693","DOI":"10.1016\/j.lfs.2024.122693","article-title":"Melatonin regulates the antioxidant capacity of sheep granulosa cells through a novel uORF-Nrf2aa mediated Nrf2\/KEAP1 pathway","volume":"349","author":"Ma","year":"2024","journal-title":"Life Sci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e2404109","DOI":"10.1002\/advs.202404109","article-title":"Targeting ATAD3A Phosphorylation Mediated by TBK1 Ameliorates Senescence-Associated Pathologies","volume":"12","author":"He","year":"2025","journal-title":"Adv. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1016\/j.theriogenology.2023.11.010","article-title":"Melatonin promotes progesterone secretion in sheep luteal cells by regulating autophagy via the AMPK\/mTOR pathway","volume":"214","author":"Duan","year":"2024","journal-title":"Theriogenology"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Basini, G., and Grasselli, F. (2024). Role of Melatonin in Ovarian Function. Animals, 14.","DOI":"10.3390\/ani14040644"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1007\/s00404-025-08079-3","article-title":"Melatonin: A potential target for regulating ovarian function","volume":"312","author":"He","year":"2025","journal-title":"Arch. Gynecol. Obstet."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1002\/mrd.23639","article-title":"Immunolocalization of melatonin receptor type 1 in the sheep ovary and involvement of the PI3K\/Akt\/FOXO3a signaling pathway in the effects of melatonin on survival and in vitro activation of primordial follicles","volume":"89","author":"Barberino","year":"2022","journal-title":"Mol. Reprod. Dev."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Ryczek, N., \u0141y\u015b, A., and Maka\u0142owska, I. (2023). The Functional Meaning of 5\u2032UTR in Protein-Coding Genes. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24032976"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"EMBR202153140","DOI":"10.15252\/embr.202153140","article-title":"lncRNA-encoded pep-AP attenuates the pentose phosphate pathway and sensitizes colorectal cancer cells to Oxaliplatin","volume":"23","author":"Wang","year":"2021","journal-title":"EMBO Rep."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Bottorff, T.A., Park, H., Geballe, A.P., and Subramaniam, A.R. (2022). Translational buffering by ribosome stalling in upstream open reading frames. PLoS Genet., 18.","DOI":"10.1101\/2022.01.06.475296"},{"key":"ref_32","first-page":"e2610782123.","article-title":"An upstream open reading frame represses translation of the neuronal potassium channel KCNQ2","volume":"123","author":"Huey","year":"2026","journal-title":"J. bioRxiv"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1007\/s00018-022-04145-0","article-title":"Upstream open reading frames regulate translation of cancer-associated transcripts and encode HLA-presented immunogenic tumor antigens","volume":"79","author":"Nelde","year":"2022","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Reiter, R.J., Sharma, R., Romero, A., Manucha, W., Tan, D.-X., Zuccari, D.A.P.d.C., and Chuffa, L.G.d.A. (2023). Aging-Related Ovarian Failure and Infertility: Melatonin to the Rescue. Antioxidants, 12.","DOI":"10.3390\/antiox12030695"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"145","DOI":"10.33069\/cim.2024.0022","article-title":"Melatonin in Female Fertility: Multifaceted Role From Reproductive Physiology to Therapeutic Potential in Polycystic Ovary Syndrome, Endometriosis, and Ovarian Failure","volume":"6","author":"Parua","year":"2024","journal-title":"Chronobiol. Med."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Zhang, X., Zhang, D., Li, H., Liu, Z., Yang, Y., Li, J., Tang, L., Tao, J., Liu, H., and Shen, M. (2023). Melatonin-Mediated Suppression of mtROS-JNK-FOXO1 Pathway Alleviates Hypoxia-Induced Apoptosis in Porcine Granulosa Cells. Antioxidants, 12.","DOI":"10.3390\/antiox12101881"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"de Lima, A.V., da Silva, H.M., de S\u00e1 Carvalho Filho, A., Maia, M.I.L., do Nascimento, C.H., de Barros, M.M.L., de Moura, B.R., dos Santos Silva, R., da Cruz J\u00fanior, C.B., and da Silva Gomes, D. (2026). Photoperiodic regulation of reproduction in livestock: Chronobiological mechanisms, management implications and future perspectives. Biol. Rhythm Res., 1\u201327.","DOI":"10.1080\/09291016.2026.2720341"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"eabh3831","DOI":"10.1126\/scitranslmed.abh3831","article-title":"Hepatic IRF3 fuels dysglycemia in obesity through direct regulation of Ppp2r1b","volume":"14","author":"Patel","year":"2022","journal-title":"Sci. Transl. Med."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/19\/1771\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T04:23:13Z","timestamp":1790828593000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/19\/1771"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,29]]},"references-count":38,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["cells15191771"],"URL":"https:\/\/doi.org\/10.3390\/cells15191771","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,29]]}}}