70 Glycolytic substrates influence intracellular movement of PKM2 and OCT4 expression in bovine preimplantation embryos

Despite decades of research and improvements, in vitro-produced (IVP) embryos are still not optimal compared with their in vivo counterparts. Current practices to select high-quality embryos are subjective or highly invasive, but metabolic assessment presents a noninvasive means to evaluate embryo health. Metabolism and the flux of nutrients through energy production pathways are determined by protein function. Pyruvate kinase type M2 (PKM2) is a key glycolytic enzyme that has been shown to translocate to the nucleus in cancer cells and serve as a transcription factor for OCT4, which plays a role in maintaining pluripotency. This additional role of PKM2 could be influenced by the culture environment, and may affect such events as lineage differentiation and maternal recognition of pregnancy. The objective of this study is to determine how substrate availability affects PKM2 localisation in embryos at the blastocyst stage. We hypothesised that altering substrate availability would affect metabolic flux and in turn affect PKM2 localisation. In conjunction with ongoing flux studies, IVP bovine embryos (ART Inc.) of similar grade and stage were preconditioned in standard growth media (SOFBE1) in groups of 10 to 12 on culture Day 6 for 24 h (10 replicates). Resulting blastocysts were moved in groups of five to SOFBE1 (control) or SOFBE1 supplemented with 1.5 mM glycolytic substrate (SOFBE1 + 1.5 mM glucose = SOFGlc) in 40-µL microdroplets under mineral oil in 5% CO₂, 5% O₂, and 90% N₂ at 37.5°C. After 24 h of incubation, embryos were fixed, permeabilised, then stained with PKM2 and OCT4 antibodies (R&D Systems) using the MAXstain system (Active Motif). Embryos were imaged using a Leica DMI6000b microscope and analysed using ImageJ (National Institutes of Health). The percent of embryos scored as grade 1 (G1) or G2 after incubation was not significantly different between SOFBE1 and SOFGlc (26% and 29%, respectively; chi-squared test, P > 0.05). Fluorescence intensity (FI) was quantified in G1 embryos along two 3-cell transect lines per embryo: one in the inner cell mass (ICM) and one in the trophectoderm (TE). FI was analysed for shifts in protein localisation and identified as one of three patterns: (A) no difference between cytoplasm and nucleus, (B) higher FI in the nucleus than in the cytoplasm, and (C) higher FI in the cytoplasm than in the nucleus. Statistical analysis of staining patterns was performed using a chi-squared test. In the ICM, nuclear PKM2 staining (pattern B) was observed in 38% of SOFBE1 embryos (n = 24) compared with 5% of embryos in SOFGlc (n = 22; P < 0.05). In the TE, 41% of embryos in SOFGlc (n = 22) showed cytoplasmic PKM2 staining (pattern C) compared with 20% of embryos in SOFBE1 (n = 25). Furthermore, 44% of embryos in SOFBE1 showed nuclear OCT4 staining in the TE (pattern B) compared with 64% of embryos in SOFGlc. Together, these data support our hypothesis that glycolytic substrates influence the intracellular movement of PKM2 between the cytoplasm and the nucleus in preimplantation embryos, and their absence may influence its translocation to the nucleus.