Oral Presentation 47th Lorne Genome Conference 2026

Chronic corticosterone supplementation alters sperm DNA methylation and hydroxymethylation in exposed mice and their male offspring (133316)

Coralina Collar Fernández 1 , Terence Pang 2 , Lucas B Hoffmann 3 , Quentin Gouil 4 5 , Ricardo De Paoli-Iseppi 3 , Sefi Y D J Prawer 1 3 , Rebekah V Harris 6 , Mirana Ramialison 1 7 8 , Fernando J Rossello 1 7 9 , Michael B Clark 3 , Anthony J Hannan 3 6
  1. MCRI, North Melbourne, VIC, Australia
  2. Institute for Health & Sport, Victoria University, Melbourne, VIC, Australia
  3. Anatomy and phisiology, University of Melbourne, Parkville, VIC, 3052
  4. Olivia Newton-John Cancer Research Institute, Heidelberg, VIC, Australia
  5. School of Cancer Medicine, La Trobe University, Bundoora, VIC, Australia
  6. The Florey Institute, Parkville, VIC, Australia
  7. Novo Nordisk Foundation Center for Stem Cell Medicine, Parkville, VIC, Australia
  8. Australian Regenerative Medicine Institute, System Biology Institute, Monash University, Clayton, VIC, Australia
  9. Department of Clinical Pathology, University of Melbourne, Parkville, VIC, Australia

Stress and trauma have been reported to exert long-lasting psychological and physiological effects that persist across multiple generations. To model hypothalamic–pituitary–adrenal (HPA) axis activation under chronic stress conditions, we employed a mouse model involving corticosterone (CORT) supplementation in drinking water. Our group had previously demonstrated that Paternal CORT treatment leads to transgenerational molecular and behavioural modifications. Knowing how stress effects are being inherited is the first step towards stopping this transmission.

In the present study, we sequenced mouse sperm DNA with  Oxford Nanopore Technologies (ONT) long-read sequencing to determine the impact of CORT treatment on DNA methylation. We sequenced both sperm DNA from CORT treated fathers (F0) and their male offspring (F1). This technique allowed direct characterization of the methylation (5mC) and hydroxymethylation (5hmC) status of more than 20 million CpG in the genome without bisulphite conversion.

Differentially methylated region (DMR) analysis revealed that PatCORT exposure resulted in predominately decreased 5mC levels at specific loci accompanied by localized increases in 5hmC, although the magnitude of the 5hmC changes was smaller. F1 PatCORT showed the same pattern of increased 5hmC but less bias towards demethylation. Notably, 17 DMRs were consistent between F0 and F1 sperm suggesting inheritance of PatCORT effects. There were no common  differentially hydroxymethylated regions (DhMRs) to both generations. Intra-generational comparisons of DMRs and DhMRs revealed an overlap of  up to 20%.

Motif enrichment analysis of overlapping DMRs identified an overrepresentation of transcription factor binding motifs implicated in embryonic development, suggesting potential functional relevance of these regions.

In summary, we have demonstrated alterations in sperm cytosine modification patterns in both F0 and F1 generations in our model of paternal chronic stress. Collectively, these findings contribute to the growing body of evidence on epigenetic inheritance and provides further insight into the mechanisms underlying the intergenerational transmission of stress effects.