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Published on: October 21, 2011
Treatment with AICAR inhibits blastocyst development, trophectoderm differentiation and tight junction formation and
Michele D Calder1,2, Nicole A Edwards1, Dean H Betts1,2,3
1Departments of Physiology and Pharmacology, Western University, London, Ontario, Canada.
Study Question:
What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function?
Summary Answer:
AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur.
What Is Known Already:
AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK.
Study Design, Size, Duration:
Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 μM) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 μM AICAR.
Participants/Materials, Setting, Methods:
Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR.
Main Results And The Role Of Chance:
Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible.
Large Scale Data:
Not applicable.
Limitations, Reasons For Caution:
Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin.
Wider Implications Of The Findings:
Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles.
Study Funding And Competing Interest(S):
Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.
Insights
AMPK activation by AICAR disrupts early embryo development, decreasing blastocyst formation and altering gene expression. Tight regulation of AMPK is crucial for successful preimplantation development.
Area of Science:
- Reproductive biology and developmental science.
- Molecular and cellular biology.
- Biochemistry and metabolism.
Background:
- Adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in cellular energy homeostasis.
- AMPK activity is essential for normal preimplantation embryonic development and blastocyst formation.
- AMPK isoforms are present in oocytes, cumulus cells, and early embryos, which can be activated by various stresses.
Purpose of the Study:
- To investigate the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation.
- To examine the effects of AMPK activation on gene expression, including blastocyst formation genes.
- To assess the influence of AMPK activation on tight junction formation and function in developing embryos.
Main Methods:
- Two experiments involved treating mouse embryos (2-cell stage and expanded blastocysts) with AICAR, an AMPK activator.
- Assessed outcomes included blastocyst formation, cell number, gene expression (mRNA and protein), and tight junction integrity.
- Compound C, an AMPK inhibitor, was used to assess the reversibility of AICAR effects.
Main Results:
- Prolonged AICAR treatment from the 2-cell stage impaired blastocyst formation, reduced cell number, and disrupted trophectoderm cell contacts.
- Short-term AICAR treatment of blastocysts induced collapse, altered gene expression, increased tight junction permeability, and decreased CDX2 expression.
- AICAR treatment affected mRNA and protein levels of key blastocyst formation genes, with some effects being reversible.
Conclusions:
- AMPK activation by AICAR negatively impacts early embryonic development, including blastocyst formation and integrity.
- Tight regulation of AMPK activity is critical for successful preimplantation development.
- The findings suggest careful consideration of metformin use during fertility treatments due to its AMPK-activating properties.
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