Liver Tissue of adult mice conceived by IVF does not show changes in Histone Lactylation

V. Giritharan^{1; 2; 3}, X. Liu³, S.H. Lee³, R. Simbulan³, J.F. Costello⁴, P. F. Rinaudo³

1. UBC-BCIT Biotechnology Program, University of British Columbia, Vancouver, Canada


2. UBC-BCIT Biotechnology Program, British Columbia Institute of Technology, Burnaby, Canada


3. Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, USA


4. Department of Neurological Surgery, University of California San Francisco, San Francisco, USA

IVF-generated embryos show evidence of metabolic stress.  We have found that IVF-conceived mouse blastocysts display accentuation of glycolysis even in the presence of high oxygen (Warburg metabolism), have higher intracellular and lower extracellular lactate levels and increased histone lactylation.  Importantly, embryos and tissues of IVF-conceived mice show decreases in protein expression of the lactate metabolism enzymes LDH-B and MCT1, while lactate levels in adult mouse offspring trend lower. However, it is unknown what epigenetic changes in adult tissues might follow from the observed changes in blastocysts and in particular if patterns of histone lactylation are   altered in tissues of adult IVF offspring. Lactate-derived lysine lactylation in histones is a newly discovered histone mark; lactylation sites on core histones have been identified, including H3K4, H3K18, and H4K5.  For example, Zhang et al, using a pan anti-lysine antibody (pan anti-Kla) have shown an increase in global histone lactylation when MCF-7 cells were exposed to progressively increasing glucose concentration.  Further they found increased H3K18la histone lactylation in the promoter regions of selected genes, associated with increased gene expression.

In this study we aimed to investigate if liver tissue of IVF conceived mice, compared to control mice have different patterns of H3K18 lactylation. Blastocysts (CF1 x B6D2F1) were generated by IVF using KSOM medium with amino acids or by natural mating (control) were flushed out of the uterus and transferred to recipient mice. The resulting offspring were raised to maturity.  At one year of life, mice were euthanized, and liver was obtained and frozen. To identify if changes in lactylation patterns occur in IVF versus control mouse offspring, Histone 3 lysine 18 lactylation (H3K18la), the most common lactylation marker, was profiled in liver. H3K18 acetylation (H3K18ac) was used as an internal control. Cleavage Under Targets and Tagmentation (CUT & Tag) analysis of liver tissues harvested from three adult offspring per group (IVF and control) was used.

Unsupervised analysis showed that discrete regions in 6 genes (Gm7550, Vcan, Fkbp5, Msi2, Platr4, & Mast4) showed significantly (FDR < 0.05) altered H3K18la levels between IVF and control groups; visual inspection of normalized genome browser tracks showed that there was decreased lactylation in the IVF group compared to the control group.  Four genes (Gm7550, Dchs1, Ly6f, & 1700123O12Rik) showed altered H3K18ac between groups. The gene Gm7550 showed changes in both histone marks.

Selective analysis of lactylation levels in genes involved in lactate metabolism (Lactate dehydrogenase A and B and Monocarboxylate transporter 1 -Mct1, also known as Slc16A1) showed no differences between groups.

In summary we did not find significant changes in H3K18la patterns in liver of adult IVF offspring compared to naturally conceived offspring.  While additional lactylation marks and different tissues will be further tested, these results suggest that the epigenetic reprogramming caused by increased lactate in early embryo is not persistent in liver of adult mice.  Given that IVF results in phenotypic changes in adulthood, future studies should investigate what epigenetic marks are responsible for these changes.