4 Oxygen concentrations alter histone lactylation levels in bovine preimplantation embryos

Glucose metabolism by the glycolytic pathway leading to the production of lactate (known as the Warburg effect), although present since the beginning of development, increases in bovine embryos from the morula stage, even in the presence of oxygen. In high-proliferating cells at the end of the glycolytic pathway, the pyruvate is metabolised to lactate by the lactate dehydrogenase (LDH), thus, the lactate production is determined by the balance between glycolysis and mitochondrial metabolism. Besides its metabolic function, lactate has recently been described as an important source for a new epigenetic mark: the histone lysine lactylation. Although there are still no studies on the lactylation of histone in the bovine model, it is already known that the balance between glycolysis and mitochondrial metabolism is strongly dependent on the oxygen concentration, as is the lactate production. The objectives of this work were (1) to verify the presence of lysine histone lactylation in bovine embryos, and (2) to evaluate the global levels of histone lactylation in bovine morulae cultured in 5% O₂ or 20% O₂. To this end, embryos were in vitro-produced by standard protocols. After fertilisation, the presumptive zygotes were randomly divided into groups, 5% O₂ or 20% O₂, and cultured in embryonic culture system 50 medium, supplemented with 4 mg/mL⁻¹ bovine serum albumin until Day 4 (as embryos increase the glycolytic activity from the morula stage). The mitochondrial membrane potential (MMP) was determined by the Mitotracker™ Red CMXRos dye (ThermoFisher) (n = 30 morulae/group), and the global levels of lactylation were determined by immunostaining using the polyclonal antibody pan histone lactylation (PTM-1401) – n = 30 morulae/group. The images were acquired using a fluorescence microscope and analysed by ImageJ software (National Institutes of Health). Data were analysed by Student’s t-test considering P < 0.05 and Pearson correlation analysis. As expected, higher levels of MMP were found in 5% O₂ versus 20% O₂ (3.8 ± 0.5 and 2.4 ± 0.8, respectively; P < 0.001), together with lower global levels of histone lactylation in 5% O₂ than 20% O₂ (1.7 ± 0.5 and 2.4 ± 0.9, respectively; P = 0.0001). A negative correlation was also found regarding MMP and lactylation global levels (P = 0.0443, R-squared 0.06). These data suggest that the oxygen tension controls the glycolysis/oxidative phosphorylation balance and, by favouring glycolysis, histone lactylation global levels also increase. In conclusion, as far as we know, this work describes, for the first time, the lysine histone lactylation in a bovine model and suggests that different availability of O₂ in in vitro culture induces changes in the oxidative metabolism, consequently shaping the epigenetic mechanisms that act by regulating the embryonic transcriptional machinery.

This research was supported by FAPESP (19/25982-7).