Treatment with AICAR inhibits blastocyst development, trophectoderm differentiation and tight junction formation and function in mice

Abstract

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.

Keywords: AMPK; assisted reproductive technologies; blastocyst formation; embryo culture; preimplantation; stress pathways.

Figure 1

AICAR treatment at the 2-cell stage decreased development, blastocyst diameter and total cell counts. (A) Development to blastocyst after treatment with various doses of AICAR, n = 5, 133–148 embryos per dose. a,b,c,d Groups without letters in common are significantly different, P < 0.001. (B) Cell counts after culture for 48 h with 0 or 1000 μM AICAR, n = 5, with 92 embryos for control and 65 AICAR-treated embryos. ***P < 0.001. (C) Embryo diameter after culture for 48 h with 0 or 1000 μM AICAR, n = 9, with 317 for control and 333 for AICAR-treated embryos. ***P < 0.001.

Figure 2

AICAR treatment increased detection of p-AMPK and decreased ZO1 (TJP1) localization to the cell membrane. (A) phospho-AMPK after 48 h culture of 2-cell embryos. Green panel shows p-AMPK fluorescence. Red is rhodamine-phalloidin staining of F-actin. Blue is DAPI-stained DNA. Merged figure shows color overlap. Arrowheads show evidence of loss of cell-to-cell contacts. Arrows show examples of condensed nuclei. Scale bar demonstrates 50 μM. Inset shows negative control with no primary antibody. (B) ZO1 (TJP1) after 48 h culture of 2-cell embryos. Green panel shows ZO1 fluorescence. Red is rhodamine-phalloidin staining of F-actin. Blue is DAPI-stained DNA. Yellow demonstrates overlap of ZO-1 and actin in the merged figure of the control embryos, which is missing in the AICAR-treated embryos. Scale bar demonstrates 50 μM. Inset shows negative control with no primary antibody.

Figure 3

AICAR treatment decreased AQP9 but did not affect E-cadherin (CDH1) protein. (A) Aquaporin 9 (AQP9) after 48 h culture of 2-cell embryos. Green panel shows AQP9 fluorescence. Red is rhodamine-phalloidin staining of F-actin. Blue is DAPI-stained DNA. Merged figure shows color overlap, there is less overlap of AQP9 and actin in AICAR-treated compared to control embryos. Scale bar demonstrates 50 μM. Inset shows negative control with no primary antibody. (B) E-cadherin (CDH1) after 48 h culture of 2-cell embryos. Green panel shows CDH1 fluorescence. Red is rhodamine-phalloidin staining of F-actin. Blue is DAPI-stained DNA. Yellow demonstrates overlap of CDH1 and actin in the merged figure. There is a similar extent of overlap in both control embryos and AICAR-treated embryos. Scale bar demonstrates 50 μM. Inset shows negative control with no primary antibody.

Figure 4

AICAR treatment increased detection of p-AMPK and decreased CDX2 by Western blot. Top panel shows Western blots, Control (01, 02, 03) and AICAR-treated (A1, A2, A3) embryos. β-actin shows equal protein loading, not significantly different between control and AICAR-treated embryos. (A) p-AMPK after 48 h culture of 2-cell embryos. There was n = 3 independent sample pools (30 embryos per pool). Control (01, 02, 03) and AICAR-treated (A1, A2, A3) embryos were significantly different, *P < 0.05. (B) CDX2 after 48 h culture of 2-cell embryos. Control (01, 02, 03) and AICAR-treated (A1, A2, A3) embryos were significantly different, **P < 0.01. (C) CDH1 after 48 h culture of 2-cell embryos. Control (01, 02, 03) and AICAR-treated (A1, A2, A3) embryos were not significantly different.

Figure 5

Real-time PCR analysis of mRNA levels of 2-cell embryos cultured for 48 h with AICAR. Cdx2, Cdh1, Aqp9, Atp1b1, Atp1a1, Actb, Tjp1, Ocln and Gadd45a are shown. Significant differences between control and AICAR-treated embryos are signified by *P < 0.05. Mean ± SEM are depicted. There was n = 3–5 independent samples.

Figure 6

Recovery of Blastocysts following 9 h of AICAR Treatment. (A) Embryo diameter at time 0, 9 h of treatment or 24 h of recovery. n = 5–8 with 96–563 embryos per group. a,b,c,d,e Groups that do not have a letter in common are significantly different, P < 0.05. (B) A photomicrograph of control or AICAR-treated embryos of one experiment is representatively shown after 9 h treatment and then after 24 h of recovery in drug free medium (Con 24 h and ai 24 h). At 9 h, AICAR embryos were divided into those that remained blastocysts and those that collapsed. AICAR-treated embryos are darker and less expanded than controls. Examples of collapsed embryos are shown with red asterisks. Scale bar demonstrates 50 μM. Time 0, (control at start of experiment); Con 9 h, untreated controls cultured for 9 h; AICAR 9 h, (blastocysts treated with AICAR for 9 h); Con 24 h, (controls cultured for 24 h in drug free medium); aibl 24 h, (AICAR treated embryos that did not collapse after 9 h treatment); ai rec 24 h, (AICAR treated embryos which initially collapsed that did recover after 24 h in drug free medium); aicoll 24 h, (AICAR treated collapsed blastocysts that did not recover after 24 h in drug free medium).

Figure 7

AICAR treatment of blastocysts decreased immunofluorescent detection of CDX2 protein. (A) CDX2 after 48 h culture of 2-cell embryos. Green panel shows CDX2 fluorescence. Red is rhodamine-phalloidin staining of F-actin. Blue is DAPI-stained DNA. Merged figure shows color overlap. CDX2 is confined to the nuclei of TE in control embryos. (B) Quantification of CDX2 staining in relative fluorescence units, n = 3 with a total of 49 and 50 embryos. CDX2 protein is lower in AICAR-treated embryos, P < 0.05. (C) Quantification of F-actin staining in relative fluorescent units, n = 3 with a total of 49 and 50 embryos. F-actin intensity is not different between control and AICAR-treated embryos. Scale bar demonstrates 50 μM. Inset shows negative control with no primary antibody.

Figure 8

Real-time PCR analysis of blastocyst mRNA levels at time 0, 9 h of treatment or 24 h of recovery. Cdx2, Cdh1, Aqp9, Atp1b1, Atp1a1, Actb, Tjp1, Ocln and Gadd45a are shown. Significant differences between control and AICAR-treated embryos are signified by a,b,c,d P < 0.05. There were n = 4 replicates. Time 0, (control at start of experiment); Con 9 h, untreated controls; AICAR 9 h, (blastocysts treated with AICAR for 9 h); Con 24 h, (sham controls cultured for 24 h in drug free medium); aibl 24 h, (AICAR treated embryos that did not collapse after 9 h treatment after a further 24 h in drug free medium); ai rec 24 h, (AICAR treated collapsed blastocyst that did recover after 24 h in drug free medium); aicoll 24 h, (AICAR treated collapsed blastocysts that did not recover after 24 h in drug free medium).

Figure 9

AICAR treatment increased uptake of 40 kDa FITC-Dextran in blastocysts. (A) Significant differences in dye uptake between control, EGTA and AICAR-treated embryos are signified by a,b P < 0.05. There was n = 5 replicates. (B) Representative bright-field and FITC-filter photographs for control, EGTA and AICAR. Scale bar demonstrates 50 μM.