Sexual dimorphism in the DNA methylation pattern after immune stimulation in the zebrafish (Danio rerio) gonads

Marcela Salazar¹Marta Caballero-Huertas^2,3Tosca Agneta van Gelderen¹Myrthe Naomi Krabbe¹Marta Gut⁴Simon Charles Heath⁴Anna Esteve^4,5Laia Ribas^1*

• ¹Institute of Marine Sciences, Spanish National Research Council (CSIC), Barcelona, Spain
• ²CIRAD Direction Regionale Montpellier-Occitanie, Montpellier, France
• ³ISEM, CNRS, IRD, CIRAD,, Universite de Montpellier, Montpellier, France
• ⁴Centre Nacional d'Analisi Genomica, Barcelona, Spain
• ⁵Universitat Autonoma de Barcelona, Barcelona, Spain

In fish, epigenetic modifications play a key role in regulating development, growth, and adaptation to environmental factors. Interestingly, emerging evidence suggests that epigenetic mechanisms may also play a crucial role in modulating the response of the fish gonads to infectious agents. Gonadal factors, including reproductive hormones and cytokines, can modulate immune cell activities, regulate the production of immune molecules, influence the overall immune response, and play a role during gonadal sex differentiation. Although the existence of the reproductive-immune system interaction is well-known, the underlying epigenetic mechanisms require further elucidation, not only in fish but also in mammals. To study the epigenetic events involved in the interaction between the immune-reproduction system, two approaches were performed by immune-stimulating the animal model zebrafish (Danio rerio) with lipopolysaccharide (LPS) with the following aims: 1) to study the resulting sex ratio after immune stimulating fish during gonadal development (17–30 dpf); 2) to decipher methylation patterns in two key innate immune genes caspase 9 (Casp9) and interleukin 1𝛽 (Il1𝛽) in immune-stimulated adult fish. For the study, larvae were bathed for the first approach while adults were intraperitoneally injected for the second. The DNA methylation of the two relevant innate immune genes was studied by a candidate gene approach at single-nucleotide resolution using sequencing strategies. Results showed no significant sex ratio difference, although a feminization tend was observed after immune-stimulating fish during gonadal development. DNA methylation differences of the Casp9 gene was significant for the interaction of treatment and sex. In particular, 8 CpG islands in treated females were identified as significant, while 3 CpGs in treated males. The Il1𝛽 gene presented a sexually dimorphic difference, but not due to immunostimulant treatment. Overall data suggested the existence of an interplay between sex and immune response in the fish gonads at the epigenetic level. Data shown here help in the understanding of these interactions, which are crucial venues for unraveling epigenetic mechanisms underlying sexually dimorphic immune responses in reproductive tissues.