Two-cell embryos are more sensitive than blastocysts to AMPK-dependent suppression of anabolism and stemness by commonly used fertility drugs, a diet supplement, and stress

Abstract

Purpose: This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos.

Methods: Culture mouse zygotes to two-cell embryos and test effects after 0.5-1 h AMPK agonists' (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells.

Result(s): Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~ 20-fold, and BR-DIM caused a ~ 30-fold increase over two-cell embryos cultured for 1 h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40-85% in two-cell embryos that was ~ 60-90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30-50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease.

Conclusion: Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

Keywords: Apoptosis; Blastocyst; Cell number; Diet supplement; Embryo culture; Embryo development; Embryo quality; Fertility drugs; Mouse embryo; Oxygen.

Fig. 1

a, b Two-cell stage embryos undergo rapid diet supplement- or drug-induced 20- to 30-fold increases and blastocysts undergo rapid 5- to 6-fold increases in pACC ser79P in an AMPK-dependent (CC-sensitive) manner. Zygotes or morulae were acclimated by overnight culture and to two-cell or blastocyst stage, respectively, preloaded with CC (5 μM) for 2 h, continued +/− CC with BR-DIM (20 μM), metformin (40 μM), aspirin (10 μM), or sorbitol (200 mM) for an additional 1 h and then fixed, stained for pACC ser79P and Hoechst, micrographed quantitated and graphed. Triplicate biological experiments using 163 2-cell embryos or 267 morulae were performed, tested for normal distribution by ANOVA and significance by Tukey’s post hoc t test. Significance compared with KSOM (a) and significance for each stimulus with CC compared with the stimulus alone (b). White bar in first panel shows 25 μm

Fig. 2

a, b. Blastocysts undergo rapid ~ 30–50% decrease in Oct4 in and AMPK-dependent (CC-sensitive) manner. Morulae were cultured overnight to acclimate to culture to blastocyst stage, preloaded with CC (5 μM) for 2 h and then continued +/− CC with stimuli BR-DIM (20 μM) or metformin (40 μM), for an additional 1 h and then fixed, stained for Oct4 and Hoechst, micrographed quantitated, and graphed. Triplicate biological experiments were performed using 190 morulae, tested for normal distribution by ANOVA and significance by Tukey’s post hoc test. Significance compared with KSOM (a) and lack of significance for each stimulus with CC compared with the stimulus alone (b). White bar in first panel shows 25 μm

Fig. 3

Two-cell stage embryos have AMPK agonist-induced (AICAR) and sorbitol-induced AMPK-dependent (compound C-sensitive) Oct4 losses. Compared with embryo cultured in KSOMaa (a, b), nuclear Oct4 is lost after 1 h of 200 mM (c, d) sorbitol but loss if largely reversed in the presence of 5 μM CC (e, f). AMPK agonist AICAR (0.5 mM) for 0.5 h (g, h) is sufficient to cause Oct4 loss without added stress). Eighty-three embryos were tested in triplicate experiments

Fig. 4

Blastocysts have significantly more cells after 24 h of culture, but BR-DIM or metformin significantly decreases cell growth compared with unstimulated embryos. Morulae were cultured overnight to acclimate to culture to blastocyst stage, one group was fixed and counted for cells and the rest were preloaded with CC (5 μM) for 2 h and then continued +/− CC with stimuli BR-DIM (20 μM) or metformin (40 μM), for an additional 24 h, and then fixed cells were counted and graphed. Triplicate biological experiments were performed using 174 embryos, tested for normal distribution by ANOVA and significance by Dunnett’s post hoc t test. Significance compared with KSOM (a) and lack of significance for each stimulus with CC compared with the stimulus alone (b)