Glucose Exposure After Fertilization Shifts Preimplantation Mouse Embryos Towards a More Autophagy-Dependent and Less...

Optimizing culture conditions is essential for enhancing embryonic developmental potential. During the early developmental stages of preimplantation embryos, pyruvate is preferentially consumed in the lower-glucose environment of the fallopian tube, whereas glucose becomes the primary energy substrate in the higher-glucose uterine environment following compaction. However, the specific effects of glucose exposure on autophagy during early development of preimplantation mouse embryos remained unclear. This study used chloroquine (CQ), an autophagy inhibitor in preimplantation embryos. Furthermore, the effects of glucose exposure on the early embryo were investigated, with a focus on changes in autophagy and mitochondrial dependency during the process of embryo development.

We examined the sensitivity of in vivo-fertilized embryos (1-cell and 2-cell flushed embryos; 1-CF and 2-CF, respectively) and in vitro-fertilized (IVF) embryos to CQ, and to rotenone, a mitochondrial respiration inhibitor, from the 2-cell stage. Furthermore, using glucose-free CZB medium and glucose-containing modified CZB (mCZB), comparisons were made of development rates, autophagy activity measured with DAPGreen, and mitochondrial activity measured with MitoTracker. Additionally, we examined the effects of O-GlcNAc transferase (OGT) inhibition using the OGT inhibitor OSMI-1.

Under CQ treatment, the blastocyst formation rate decreased significantly in IVF embryos and 1-CF embryos cultured in mCZB compared to 2-CF embryos, starting at 2.0 μM CQ treatment and this was accompanied by reduced cell numbers. Interestingly, autophagy activity detected by DAPGreen was significantly higher at the morula stage in IVF embryos compared with in 2-CF embryos. Oppositely, under rotenone treatment, the blastocyst formation rate increased significantly in IVF embryos and 1-CF embryos compared to 2-CF embryos, starting at 1.0 μM rotenone treatment. Next, glucose-deprived IVF embryos, which cultured in CZB for 24 hours starting at 2 hours after insemination, exhibited increased sensitivity to rotenone during development and elevated mitochondrial activity at the 2-cell stage, followed by decreased autophagy activity at the 4/8-cell stage. Finally, OSMI-1 treatment in mCZB for 24 hours starting at 2 hours after insemination showed an increased mitochondrial activity at the 2-cell stage but, unexpectedly, these embryos showed a tendency toward increased CQ sensitivity.

Our data indicate that, regardless of fertilization conditions, glucose exposure immediately after fertilization begins to shift preimplantation mouse embryos towards a more autophagy-dependent and less mitochondria-dependent mode of development. This shift is accompanied by increased autophagy activity and reduced mitochondrial activity, potentially mediated in part by O-GlcNAc modification.