Stress induces AMPK-dependent loss of potency factors Id2 and Cdx2 in early embryos and stem cells [corrected]

Abstract

The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id)2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the blastocyst stage. The caudal-related homeodomain protein (Cdx)2 is a regulatory gene for determining TSC, the earliest placental lineage in the preimplantation mouse embryo, but is expressed in the oocyte and in early cleavage stage embryos before TSC arise. We assayed the expression of putative potency-maintaining phosphorylated Cdx2 ser60 in the oocyte, two-cell stage embryo, blastocyst, and in TSC. We studied the loss of Cdx2 phospho ser60 expression induced by hyperosmolar stress and its underlying mechanisms. Hyperosmolar stress caused rapid loss of nuclear Cdx2 phospho ser60 and Id2 in the two-cell stage embryo by 0.5 h. Stress-induced Cdx2 phospho ser60 and Id2 loss is reversed by the AMPK inhibitor compound C and is induced by the AMPK agonist 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide in the absence of stress. In the two-cell stage embryo and TSC hyperosmolar, stress caused AMPK-mediated loss of Cdx2 phospho ser60 as detected by immunofluorescence and immunoblot. We propose that AMPK may be the master regulatory enzyme for mediating stress-induced loss of potency as AMPK is also required for stress-induced loss of Id2 in blastocysts and TSC. Since AMPK mediates potency loss in embryos and stem cells it will be important to measure, test mechanisms for, and manage the AMPK function to optimize the stem cell and embryo quality in vitro and in vivo.

FIG. 1.

The caudal-related homeobox protein 2 (Cdx2) protein is detected in oocytes, two-cell stage embryos, blastocysts, and trophoblast stem cells (TSC), and Cdx2 phospho ser60 is detected in the nuclei of both blastomeres in the two-cell stage embryo and all trophoblast nuclei in blastocysts and TSC. Cdx2 mRNA is detected throughout these developmental stages, but at similar levels in oocytes and two-cell stage embryos and >100-fold higher in blastocysts. Part 1: The Cdx2 phospho ser60 protein (pCdx2 in the figure) is expressed in the cytoplasm and nuclear DNA (arrowhead) of an oocyte. Oocytes were flushed from the oviduct 15 h after human chorionic gonadotropin (hCG) (A–C) fixed and stained for Cdx2 phospho ser60 protein, Hoechst stain (B), or (C) merge, or oocytes were lysed and tested using western blot (D). Bar in (A) shows 25 μM. Part 2: Late two-cell stage embryos from MF1 (outbred mice from a cross of Laboratory Animal Centre strain A and CS1 from Scientific Products farm) and CF1 [Carworth farms mice (not Swiss-derived)] mouse strains were collected at E1.5, fixed, and stained with six antibodies for Cdx2. At top is immunofluorescence adjusted to the same intensity to show location of Cdx2. In A–F six antibodies to Cdx2 were tested in 2-cell stage embryos from MF1 mice and in G–L the same antibodies were tested in 2-cell stage embryos from CF1 mice. Four antibodies were also tested using western blots, producing single bands at 36 kDa for three and a single band at 90 kDa for the fourth. The epitope of each antibody is also shown at bottom. Note there are no tyrosine or threonine residues that are possible alternate phosphorylation sites, but that there are other possible serine residues besides the one at position 60. The only overt phospho-specific antibody is indicated by a large S at the far right. Part 3: TSC were cultured, fixed, and stained with three antibodies for Cdx2. At top is immunofluorescence adjusted to the same intensity to show location of Cdx2. Four antibodies were also tested using immunoblots, and all detected bands at 36 kDa. Three antibodies were tested by using immunofluorescence, and all detected nuclear localization of all forms of Cdx2. Arrows in (A, D, G) show nuclear Cdx2 and arrowheads in (B, C, E, F, H, I) show nuclei. Bars in micrograph (A) in Parts 1, 2 and Part 3, show 25, 50, and 10 μm, respectively. Part 4: Cdx2 mRNA was tested using real-time quantitative polymerase chain reaction for a dilution series of TSC from 10 pg–50 ng, and 25 oocytes, 25 two-cell stage embryos, and 25 blastocysts assayed ex vivo. All biological experiments were repeated at least three times. Color images available online at www.liebertpub.com/scd

FIG. 2.

Hyperosmolar stress induced Cdx2 phospho ser60 protein loss from the nucleus after 0.5 h in E1.5 and E3.5 embryos and this was AMP-activated protein kinase (AMPK)-dependent (inhibitor sensitive) and AMPK sufficient [5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR), AMPK agonist, without stress] in E1.5 embryos. Embryos were incubated for 0.5 h with/without 400 mM sorbitol, fixed, and stained for Cdx2 phospho ser60. Bar in (A) is 50 μM. Part 1: E1.5 embryos were cultured without stress (A, B), stressed for 0.5 h without (C, D) or with (E, F) compound C (an AMPK inhibitor) or without stress and with AICAR for 0.5 h (an AMPK agonist) (G, H). Part 2: E3.5 embryos were cultured without (A–C) or with stress (D–F) and stained for the Cdx2 phospho ser60 protein. Bar in (A) is 25 μM. Color images available online at www.liebertpub.com/scd

FIG. 3.

Hyperosmolar stress-induced inhibitor of differentiation 2 (Id2) protein loss from the nucleus after 0.5 h in E1.5 and. E3.5 embryos and this was AMPK-dependent (AMPK inhibitor sensitive) and AMPK-sufficient (AICAR, AMPK agonist, without stress) in E1.5 embryos. Embryos were incubated for 0.5 h with/without 400 mM sorbitol, fixed, and stained for phosphorylated Id2. Part 1: E1.5 embryos were cultured without stress (A, B), stressed for 0.5 h without (C, D) or with (E, F) compound C (an AMPK inhibitor) or without stress and with AICAR (an AMPK agonist) for 0.5 h (G, H). Bar in (A) is 50 μM. Part 2: E3.5 embryos were cultured without (A–C) or with stress (D–F) and stained for the Id2 protein. Bar in (A) is 25 μM. Color images available online at www.liebertpub.com/scd

FIG. 4.

Cdx2 phospho ser60 in TSC undergoes hyperosmolar stress-induced, AMPK-dependent (AMPK antagonist sensitive) loss that is AMPK sufficient without stress (AMPK agonist sensitive). Part 1: TSC were stimulated with 200 mM sorbitol for 4 h with or without compound C, or with AICAR, lysed and the immunoblot stained for Cdx2 phospho ser60. Part 2: TSC were cultured without stress (A–C) or with 400 mM sorbitol for 0.5 h (D–F) stained using Cdx2 total (C terminus) antibody and immunofluorescence was detected. All experiments were repeated three times. Part 3: TSC were cultured without stress (A–C) or with 400 mM sorbitol for 0.5 h (D–F) stained using the Cdx2 phospho ser60 antibody and immunofluorescence was detected. All experiments were repeated three times. Color images available online at www.liebertpub.com/scd

FIG. 5.

Unlike small intestine, phosphorylation of nuclear CDX2 phospho ser60 (pCDX2) is not mitogen-activated protein kinase kinase (MEK)1/2-dependent in two-cell stage embryos, although phosphorylated (p) mitogen-activated protein kinase (MAPK)1/3 and (p) retinoblastoma (Rb) are MEK1/2 dependent. Two-cell stage embryos were cultured with or without MEK1/2 inhibitor U0126 (1 μM) for 4 h, fixed, permeabilized, and stained for CDX2 phospho ser60 (A, C), phosphorylated (p)MAPK 1/3 Thr/Tyr 202/204 (E, G), and phosphorylated (p)Rb Ser795 (I, K). Hoechst-stained nuclei (B, D, F, H, J, L) complement antigen staining (A, C, E, G, I, K), respectively. Arrows show antigen staining and arrowheads show nuclear position. Color images available online at www.liebertpub.com/scd

FIG. 6.

Summary diagram for stress-induced AMPK-dependent and -sufficient loss of Cdx2 phospho ser60 and Id2 in two-cell stage embryos, blastocysts, and TSC are shown in the two columns to the left. Cellular localization of Cdx2 phospho ser60, Ser60 nonphospho ser60, and total Cdx2 are shown in the column on the right. Putative functions mediated by stress-induced AMPK are shown in the column next to one on the right. Black boxes show data from this report, red boxes show data from Zhong et al. [5], orange box shows data from Awonuga et al. [4], and purple box shows data from LaRosa and Downs [2]. Blue arrows indicate outcomes induced by AICAR and black arrows indicate outcomes induced by stress in an AMPK-dependent manner. The green line separates the earlier oocyte and two-cell embryo stages of development that are totipotent from the cultured TSC and the TSC in the blastocysts that are multipotent. Color images available online at www.liebertpub.com/scd