Tissue and maturation specific DNA methylation dynamics of gonadotropin genes in chub mackerel (Scomber japonicus) using cost-effective targeted bisulfite sequencing

tissue-and-maturation-specific-dna-methylation-dynamics-of-gonadotropin-genes-in-chub-mackerel-(scomber-japonicus)-using-cost-effective-targeted-bisulfite-sequencing
Tissue and maturation specific DNA methylation dynamics of gonadotropin genes in chub mackerel (Scomber japonicus) using cost-effective targeted bisulfite sequencing

Scientific Reports , Article number:  (2026) Cite this article

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Abstract

Fish reproduction is tightly regulated by hormonal pathways, and epigenetic mechanisms may modulate key reproductive genes such as the gonadotropins (GtHs). Determining whether DNA methylation influences GtH expression during maturation is a crucial step before exploring how these regulatory pathways might respond to environmental cues. Chub mackerel (Scomber japonicus) is a key species in fisheries and aquaculture, where improving reproductive performance is a priority. In this study, we used an optimized Targeted bisulfite sequencing (BS-seq) protocol, to investigate methylation patterns in GtH promoters across tissues and age groups. We complemented this analysis with RT-qPCR and functional validation of lhb promoter activity using reporter assays in cultured cells. The modified protocol allowed analysis of 2880 targets, exceeding Sanger sequencing capacity and improving efficiency and cost-effectiveness for larger studies. The DNA methylation patterns exhibited tissue- and maturation-specific dynamics. Fshb promoter methylation was stable in most tissues but varied in the gonad and pituitary: immature gonads had lower methylation, while mature pituitaries showed hypomethylated CpG sites and the lowest overall methylation. In contrast, methylation in the gene body of lhb showed little variation across tissues but was higher in the mature pituitary. In vitro assays identified a silencing regulatory element (222–232 bp) overlapping a putative Sp1 binding site and methylation at this position was low (< 10%). Furthermore, tissue- and maturation-specific variations in DNA methylation enzymes expression (dnmt1, tet1, tet2, tet3) suggest changes linked to reproductive maturation rather than age-related epigenetic drift. This study reveals maturation-related epigenetic regulation of gonadotropins in fish. By optimizing a Targeted BS-seq method, we identified variation in fshb and lhb methylation that appears linked to changes in gene expression during reproductive development, and we found that a regulatory element may influence lhb expression through Sp1 binding. These findings contribute to a clearer understanding of how DNA methylation regulates fish reproductive physiology.

Data availability

The genomic sequences used in this study are deposited in the NCBI repository https://www.ncbi.nlm.nih.gov under accession numbers PX025811 and PV798563.

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Acknowledgements

The authors express their gratitude to Dr. Marie Saitou from the Center for Integrative Genetics, Norwegian University of Life Sciences, Oslo, Norway, for her valuable support throughout the study. They also thank the staff of the Aqua-BioResource Innovation Center (ABRIC) Karatsu Satellite, Kyushu University, for their assistance with fish maintenance and sampling.

Funding

This research was financially supported by the Research and Implementation Promotion Program through Open Innovation Grants (JPJ011937) from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN), as well as by the Grants-in-Aid for Scientific Research (KAKENHI) program administered by the Japan Society for the Promotion of Science (JSPS) [grant numbers: JP16H04981, JP19H03049, JP22H00386, JP22K05832, and JP22K19211].

Author information

Authors and Affiliations

  1. Laboratory of Marine Biology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

    Mariel Galotta, Tapas Chakraborty & Kohei Ohta

  2. Laboratory of Aquatic Molecular Developmental Biology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

    Yukiko Ogino

  3. Laboratory of Aquaculture and Nutrient Chemistry, Faculty of Agriculture, Miyazaki University, Miyazaki, Japan

    Naoki Nagano

  4. School and Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan

    Takehiko Itoh

  5. Karatsu satellite of Aqua-Bioresource Innovation Center, Kyushu University, Karatsu, Japan

    Michiya Matsuyama, Tapas Chakraborty & Kohei Ohta

Authors

  1. Mariel Galotta
  2. Yukiko Ogino
  3. Naoki Nagano
  4. Takehiko Itoh
  5. Michiya Matsuyama
  6. Tapas Chakraborty
  7. Kohei Ohta

Contributions

MG conceived and designed the study, performed the experiments, and acquired and analyzed the data. TI provided the aligned genomic data. YO, NN, TC, MM, and KO contributed to manuscript writing. All authors revised and approved the final version of the manuscript.

Corresponding authors

Correspondence to Tapas Chakraborty or Kohei Ohta.

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The authors declare no competing interests.

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Galotta, M., Ogino, Y., Nagano, N. et al. Tissue and maturation specific DNA methylation dynamics of gonadotropin genes in chub mackerel (Scomber japonicus) using cost-effective targeted bisulfite sequencing. Sci Rep (2026). https://doi.org/10.1038/s41598-026-40580-w

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