{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,3]],"date-time":"2026-04-03T06:35:52Z","timestamp":1775198152673,"version":"3.50.1"},"reference-count":42,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T00:00:00Z","timestamp":1775001600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>The brown trout (Salmo trutta) is a commercially and ecologically significant salmonid fish, yet its hepatic cellular and functional dynamics throughout the reproductive cycle remain poorly characterised, particularly in males. This study investigated seasonal and sex-specific liver plasticity across four reproductive stages: spawning capable (December), regressing (March), regenerating (July), and developing (November). We quantified mRNA and protein abundance of key oestrogen-responsive targets\u2014vitellogenin (VtgA) and zona pellucida (ZP) proteins\u2014alongside cell turnover markers, caspase 3 (Casp3) and proliferating cell nuclear antigen (PCNA). These molecular endpoints were integrated with stereological analyses to estimate hepatocyte, nuclear, and cytoplasmic volumes. Results revealed stage-dependent mobilisation and transient hepatic retention of reproductive proteins; females exhibited stronger vitellogenic signatures and more pronounced seasonal shifts than males. Although VtgA and ZP mRNA levels peaked during the developing and spawning-capable stages, males maintained low but consistent levels throughout the cycle, indicating constitutive hepatic oestrogen sensitivity. Regarding cell turnover, PCNA protein data indicated heightened proliferative activity during the spawning-capable and regressing stages. In contrast, while Casp3 mRNA levels remained stable across all stages, protein detection suggested a post-transcriptional increase in apoptotic signalling during the developing phase, consistent with controlled tissue remodelling rather than extensive cell loss. Stereological data confirmed enlarged hepatocyte and nuclear volumes during periods of high secretory and proliferative demand. Overall, these findings demonstrate significant stage-dependent and sex-specific plasticity in brown trout liver, providing a robust reference framework for ecological monitoring, endocrine disruption assessments, and studies of teleost reproductive physiology.<\/jats:p>","DOI":"10.3390\/ani16071073","type":"journal-article","created":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T10:09:21Z","timestamp":1775038161000},"page":"1073","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Seasonal and Sex-Specific Liver Plasticity in Brown Trout: Estrogen-Responsive Targets and Cell Turnover Dynamics"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-1260-9082","authenticated-orcid":false,"given":"Am\u00e2ndio","family":"de Barros","sequence":"first","affiliation":[{"name":"Group of Animal Morphology and Toxicology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR\/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leix\u00f5es, Av. General Norton de Matos s\/n, 4450-208 Matosinhos, Portugal"},{"name":"Laboratory of Histology and Embryology, Department of Microscopy, ICBAS\u2014School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0912-3379","authenticated-orcid":false,"given":"Diana","family":"Santos","sequence":"additional","affiliation":[{"name":"Laboratory of Histology and Embryology, Department of Microscopy, ICBAS\u2014School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8330-7419","authenticated-orcid":false,"given":"Tiago","family":"Louren\u00e7o","sequence":"additional","affiliation":[{"name":"Group of Animal Morphology and Toxicology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR\/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leix\u00f5es, Av. General Norton de Matos s\/n, 4450-208 Matosinhos, Portugal"},{"name":"Laboratory of Histology and Embryology, Department of Microscopy, ICBAS\u2014School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4096-6507","authenticated-orcid":false,"given":"C\u00e9lia","family":"Lopes","sequence":"additional","affiliation":[{"name":"Group of Animal Morphology and Toxicology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR\/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leix\u00f5es, Av. General Norton de Matos s\/n, 4450-208 Matosinhos, Portugal"},{"name":"Laboratory of Histology and Embryology, Department of Microscopy, ICBAS\u2014School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal"}]},{"given":"T\u00e2nia Vieira","family":"Madureira","sequence":"additional","affiliation":[{"name":"Group of Animal Morphology and Toxicology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR\/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leix\u00f5es, Av. General Norton de Matos s\/n, 4450-208 Matosinhos, Portugal"},{"name":"Laboratory of Histology and Embryology, Department of Microscopy, ICBAS\u2014School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9813-4389","authenticated-orcid":false,"given":"Eduardo","family":"Rocha","sequence":"additional","affiliation":[{"name":"Group of Animal Morphology and Toxicology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR\/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leix\u00f5es, Av. General Norton de Matos s\/n, 4450-208 Matosinhos, Portugal"},{"name":"Laboratory of Histology and Embryology, Department of Microscopy, ICBAS\u2014School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1034\/j.1600-0633.2003.00010.x","article-title":"Atlantic salmon Salmo salar L., brown trout Salmo trutta L. and Arctic charr Salvelinus alpinus (L.): A review of aspects of their life histories","volume":"12","author":"Klemetsen","year":"2003","journal-title":"Ecol. Freshw. Fish"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Domingues, A., Alexandre, C.M., Mateus, C.S., Silva, S., Pereira, J., and Almeida, P.R. (2022). Into the wild: A new approach to the aquaculture production of brown trout (Salmo trutta L.) to enhance restocking success. Biol. Life Sci. Forum, 13.","DOI":"10.3390\/blsf2022013115"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"124661","DOI":"10.1016\/j.envpol.2024.124661","article-title":"A critical review of pollution active biomonitoring using sentinel fish: Challenges and opportunities","volume":"360","author":"Bancel","year":"2024","journal-title":"Environ. Pollut."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1007\/BF00002633","article-title":"Use of space and food by resident and migrant brown trout, Salmo trutta","volume":"14","author":"Jonsson","year":"1985","journal-title":"Environ. Biol. Fishes"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1039\/B315886A","article-title":"Brown trout as a sentinel organism for organic pollution in the field using catalytic and immunochemical assays of cytochrome P-450 1A","volume":"6","year":"2004","journal-title":"J. Environ. Monit."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1002\/jez.a.10278","article-title":"Oogenesis and plasma levels of sex steroids in cultured females of brown trout (Salmo trutta Linnaeus, 1758) in Chile","volume":"298","author":"Estay","year":"2003","journal-title":"J. Exp. Zool. Part A Comp. Exp. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1016\/j.ygcen.2009.05.004","article-title":"Control of puberty in farmed fish","volume":"165","author":"Taranger","year":"2010","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1111\/j.1439-0426.2003.00455.x","article-title":"Reproductive biology of brown trout, Salmo trutta macrostigma Dumeril 1858, in a tributary of the Ceyhan River which flows into the eastern Mediterranean Sea","volume":"19","author":"Alp","year":"2003","journal-title":"J. Appl. Ichthyol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1080\/11250000802589576","article-title":"Pattern of gonad maturation in a highly stocked population of brown trout (Salmo trutta L., 1758) from Central Italy","volume":"77","author":"Caputo","year":"2010","journal-title":"Ital. J. Zool."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Santos, D., Rocha, E., Malhao, F., Lopes, C., Gon\u00e7alves, J.F., and Madureira, T.V. (2021). Multi-parametric portfolio to assess the fitness and gonadal maturation in four key reproductive phases of brown trout. Animals, 11.","DOI":"10.3390\/ani11051290"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1186\/1476-5926-2-4","article-title":"Eggshell and egg yolk proteins in fish: Hepatic proteins for the next generation: Oogenetic, population, and evolutionary implications of endocrine disruption","volume":"2","author":"Arukwe","year":"2003","journal-title":"Comp. Hepatol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1007\/s12562-015-0957-5","article-title":"Vitellogenesis and choriogenesis in fishes","volume":"82","author":"Hara","year":"2016","journal-title":"Fish. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"105586","DOI":"10.1016\/j.aquatox.2020.105586","article-title":"Disruption of classical estrogenic targets in brown trout primary hepatocytes by the model androgens testosterone and dihydrotestosterone","volume":"227","author":"Lopes","year":"2020","journal-title":"Aquat. Toxicol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.cbpc.2018.06.001","article-title":"Sex-steroids and hypolipidemic chemicals impacts on brown trout lipid and peroxisome signaling\u2014Molecular, biochemical and morphological insights","volume":"212","author":"Madureira","year":"2018","journal-title":"Comp. Biochem. Physiol. C Toxicol. Pharmacol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1016\/0305-0491(86)90400-1","article-title":"Vitellogenin levels in male and female rainbow trout (Salmo gairdneri Richardson) at various stages of the reproductive cycle","volume":"83","author":"Copeland","year":"1986","journal-title":"Comp. Biochem. Physiol. Part B Biochem. Mol. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1007\/s10695-010-9464-x","article-title":"Global proteomics analysis of testis and ovary in adult zebrafish (Danio rerio)","volume":"37","author":"Groh","year":"2011","journal-title":"Fish Physiol. Biochem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"805","DOI":"10.1095\/biolreprod64.3.805","article-title":"Cloning of rainbow trout egg envelope proteins: Members of a unique group of structural proteins","volume":"64","author":"Hyllner","year":"2001","journal-title":"Biol. Reprod."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1016\/j.cbpb.2009.11.016","article-title":"Molecular characterization and expression pattern of two zona pellucida genes in half-smooth tongue sole (Cynoglossus semilaevis)","volume":"155","author":"Sun","year":"2010","journal-title":"Comp. Biochem. Physiol. Part B Biochem. Mol. Biol"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1002\/jemt.20820","article-title":"The hepatocytes of the brown trout (Salmo trutta fario): A stereological study of some cytoplasmic components with the breeding cycle","volume":"73","author":"Rocha","year":"2010","journal-title":"Microsc. Res. Tech."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1007\/s10228-008-0066-x","article-title":"The hepatocytes of the brown trout (Salmo trutta f. fario): A stereological study of their number and size during the breeding cycle","volume":"56","author":"Rocha","year":"2009","journal-title":"Ichthyol. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1152\/physiolgenomics.00123.2014","article-title":"Identification of conserved hepatic transcriptomic responses to 17\u03b2-estradiol using high-throughput sequencing in brown trout","volume":"47","author":"Webster","year":"2015","journal-title":"Physiol. Genom."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"198","DOI":"10.1186\/1756-3305-5-198","article-title":"Proliferative cell nuclear antigen (PCNA) expression in the intestine of Salmo trutta trutta naturally infected with an acanthocephalan","volume":"5","author":"Dezfuli","year":"2012","journal-title":"Parasite Vector"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1016\/j.tice.2014.09.001","article-title":"Apoptotic effects and glucose-6-phosphate dehydrogenase responses in liver and gill tissues of rainbow trout treated with chlorpyrifos","volume":"46","author":"Topal","year":"2014","journal-title":"Tissue Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1038\/sj.onc.1200886","article-title":"PCNA: Structure, functions and interactions","volume":"14","author":"Kelman","year":"1997","journal-title":"Oncogene"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1007\/s00418-005-0003-2","article-title":"Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis","volume":"124","author":"Leung","year":"2005","journal-title":"Histochem. Cell Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1104","DOI":"10.1038\/cdd.2009.180","article-title":"Neuronal caspase-3 signaling: Not only cell death","volume":"17","author":"Cavallucci","year":"2010","journal-title":"Cell Death Differ."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1111\/j.1095-8649.2007.01665.x","article-title":"Caspases and apoptosis in fish","volume":"71","author":"Takle","year":"2007","journal-title":"J. Fish Biol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Varghese, F., Bukhari, A.B., Malhotra, R., and De, A. (2014). IHC profiler: An open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0096801"},{"key":"ref_29","first-page":"630","article-title":"E. R. Weibel. Stereological methods, Vol. 1: Practical methods for biological morphometry","volume":"21","author":"Fritsch","year":"1981","journal-title":"Z. Allg. Mikrobiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1002\/jemt.23228","article-title":"Estimating volumes from common carp hepatocytes using design-based stereology and examining correlations with profile areas: Revisiting a nutritional assay and unveiling guidelines to microscopists","volume":"82","author":"Raskovic","year":"2019","journal-title":"Microsc. Res. Tech."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1111\/j.1365-2818.1981.tb01199.x","article-title":"Optimizing sampling efficiency of stereological studies in biology: Or \u2018do more less well!\u2019","volume":"121","author":"Gundersen","year":"1981","journal-title":"J. Microsc."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e45","DOI":"10.1093\/nar\/29.9.e45","article-title":"A new mathematical model for relative quantification in real-time RT-PCR","volume":"29","author":"Pfaffl","year":"2001","journal-title":"Nucleic Acids Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.aquatox.2008.08.012","article-title":"Water temperature and concomitant waterborne ethinylestradiol exposure affects the vitellogenin expression in juvenile brown trout (Salmo trutta)","volume":"90","author":"Kohno","year":"2008","journal-title":"Aquat. Toxicol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1186\/s40064-015-1432-6","article-title":"Hydrolyzed fish proteins reduced activation of caspase-3 in H2O2 induced oxidative stressed liver cells isolated from Atlantic salmon (Salmo salar)","volume":"4","author":"Espe","year":"2015","journal-title":"SpringerPlus"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.aquatox.2012.01.019","article-title":"Early stress responses in Atlantic salmon (Salmo salar) exposed to environmentally relevant concentrations of uranium","volume":"112","author":"Song","year":"2012","journal-title":"Aquat. Toxicol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1016\/j.cell.2016.03.014","article-title":"On the dependency of cellular protein levels on mRNA abundance","volume":"165","author":"Liu","year":"2016","journal-title":"Cell"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Chen, H.Q., Bi, B.L., Kong, L.F., Rong, H., Su, Y.H., and Hu, Q. (2021). Seasonal changes in plasma hormones, sex-related genes transcription in brain, liver and ovary during gonadal development in female rainbow trout (Oncorhynchus mykiss). Fishes, 6.","DOI":"10.3390\/fishes6040062"},{"key":"ref_38","first-page":"173","article-title":"Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment","volume":"103","author":"Sumpter","year":"1995","journal-title":"Environ. Health Perspect."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1177\/41.1.7678022","article-title":"Evaluation of proliferating cell nuclear antigen (PCNA) as an endogenous marker of cell-proliferation in rat-liver\u2014A dual-stain comparison with 5-Bromo-2\u2019-Deoxyuridine","volume":"41","author":"Connolly","year":"1993","journal-title":"J. Histochem. Cytochem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1136\/jcp.43.3.184","article-title":"Review\u2014Assessment of cell-proliferation in histological material","volume":"43","author":"Hall","year":"1990","journal-title":"J. Clin. Pathol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"e44700","DOI":"10.7554\/eLife.44700","article-title":"Mammalian cell growth dynamics in mitosis","volume":"8","author":"Miettinen","year":"2019","journal-title":"eLife"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1007\/s00294-019-00999-3","article-title":"Unravelling nuclear size control","volume":"65","author":"Cantwell","year":"2019","journal-title":"Curr. Genet."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/7\/1073\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,3]],"date-time":"2026-04-03T04:27:06Z","timestamp":1775190426000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/7\/1073"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,1]]},"references-count":42,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["ani16071073"],"URL":"https:\/\/doi.org\/10.3390\/ani16071073","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,1]]}}}