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

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.