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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

  • Embryo Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

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.

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Acknowledgements

Thanks to Dr. Erica Louden and Dr. Yu Yang for comments on the manuscript.

Funding

DAR and EEP from the Office of the Vice President for Research at Wayne State University and an R03 to DAR 1R03HD061431, from the REI fellows’ fund (AB), and from the funding of the Mary Iacobell and Kamran Moghissi Endowed Chairs.

Author information

Author notes

  1. Yufen Xie

    Present address: Fertility and Surgical Associates of California, Thousand Oaks, CA, 91361, USA

Authors and Affiliations

  1. CS Mott Center for Human Growth and Development, Department of Ob/Gyn, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, 275 East Hancock, Detroit, MI, 48201, USA

    Alan Bolnick, Mohammed Abdulhasan, Brian Kilburn, Jing Dai, Elizabeth E. Puscheck & Daniel A. Rappolee

  2. EmbryoTech Laboratories, 140 Hale Street, Haverhill, MA, 01830, USA

    Mindie Howard

  3. Wayne State University School of Medicine, Ob/Gyn, IVF Clinic, University Physician Group, 26400 W 12 Mile Road, Suite 140, Southfield, MI, 48034, USA

    Paul Andresen

  4. University of Utah, 201 Presidents Circle, Salt Lake City, UT, 84112, USA

    Alexandra M. Shamir

  5. Department of Medicine, Hartford Hospital and University of Connecticut, Hartford, CT, USA

    Elizabeth E. Puscheck

  6. Program for Reproductive Sciences and Department of Physiology, Wayne State University School of Medicine, Detroit, MI, 48201, USA

    Eric Secor

  7. Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USA

    Daniel A. Rappolee

  8. Institutes for Environmental Health Science, Wayne State University School of Medicine, Detroit, MI, USA

    Daniel A. Rappolee

  9. Department of Biology, University of Windsor, Windsor, ON, N9B 3P4, Canada

    Daniel A. Rappolee

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  1. Alan Bolnick
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Corresponding author

Correspondence to Alan Bolnick.

Electronic supplementary material

Supplemental Figure 1

Blastocysts have significantly more cells after 24hr of culture, but BR-DIM or Metformin significantly decrease cell growth compared with unstimulated embryos at the end of culture. 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 (5uM) for 2h and then continued +/-CC with stimuli BR-DIM (20uM) or Metformin (40uM), for an additional 24hr and then fixed cells were counted and graphed. Triplicate biological experiments using 174 embryos were performed, tested for normal distribution by ANOVA and significance by Dunnett post hoc t-test. (a) Shows significance compared with KSOM and b shows lack of significance for each stimulus with CC compared with the stimulus alone. (JPEG 224 kb)

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Bolnick, A., Abdulhasan, M., Kilburn, B. et al. 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. J Assist Reprod Genet 34, 1609–1617 (2017). https://doi.org/10.1007/s10815-017-1028-x

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  • DOI: https://doi.org/10.1007/s10815-017-1028-x

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