76 Investigating apoptosis and autophagy in hyperglycemic embryo culture

Pre-implantation mouse embryo culture under hyperglycemic conditions increases embryo oxidative stress and apoptosis and reduces blastocyst development. Autophagy is a vital cellular degradation pathway active during mammalian pre-implantation development. We examined hyperglycemic culture exposure on autophagic and apoptotic responses in pre-implantation embryos. We hypothesised that autophagic and apoptotic responses are dependent on the timing of hyperglycemic culture initiation, and that altered autophagy is a primary response. Weekly experimental replicates included embryo collection from oviducts of 15 plugged CD1 females (4–6 weeks old) mated with CD1 males (4–6 months old); (total 160–300 embryos/week). Collections were at 36 h or 48 h post-human chorionic gonadotrophin (hCG) injection (early and late two-cell stage). Pools of 40 embryos were allocated to culture groups of KSOMaa medium supplemented with 0.2 mM (control) or 25 mM (experimental) D-glucose, under a 5%O₂/5%CO₂/90%N₂ atmosphere at 37°C to assess blastocyst development (96 h post-hCG for all experiments). Outcome measures included blastocyst formation (all 18 replicates; 1,280 embryos), blastocyst total cell number (TCN; DAPI-stained nuclei; 14 replicates; 300 embryos), apoptosis (TUNEL-positive nuclei; 6 replicates; 300 embryos), autophagy (LC3 puncta quantification; 3 replicates; 150 embryos and p62 fluorescence intensity; 4 replicates; 200 embryos). Statistical analysis was performed using a Student’s t-test with P < 0.05 representing significance. Embryos flushed at 48 h showed no effect on blastocyst formation (P = 0.4381) or TUNEL-positive nuclei (P = 0.825), but TCN was significantly decreased from controls (P = 0.011). Embryos flushed at 36 h displayed a significant decrease in blastocyst formation (P = 0.020), in TCN (P = 0.023), and a significant increase in LC3 puncta (P = 0.046) relative to controls. TUNEL-positive nuclei (P = 0.959) and p62 fluorescence (P = 0.212) were unchanged. Hyperglycemia is known to downregulate embryonic glucose transporters, thus may induce autophagy in response to nutrient deprivation. We uncovered a 12 h window during which hyperglycemia induces elevated effects on early two-cell embryos, possibly due to zygotic genome activation in the late 2–4 cell stage. The JNK/SAPK (C-JUN N-terminal kinase) signalling pathway is activated by cellular stress and is an upstream regulator of both apoptosis and autophagy in many cell systems, so further experiments will quantify JNK activation and efficacy of JNK and autophagy culture inhibitors to study their role in pre-implantation embryonic hyperglycemic responses.

Thanks to Michele, Zuleika, and Andrew for technical support and advice. Research was supported by the Canadian Institutes of Health Research, an Obstetrics and Gynaecology Graduate Scholarship, and a Children’s Health Research Institute Traineeship to VW.