Regulation of endoplasmic reticulum stress and trophectoderm lineage specification by the mevalonate pathway in the mouse preimplantation embryo

Abstract

Early embryos are vulnerable to environmental insults, such as medications taken by the mother. Due to increasing prevalence of hypercholesterolemia, more women of childbearing potential are taking cholesterol-lowering medications called statins. Previously, we showed that inhibition of the mevalonate pathway by statins impaired mouse preimplantation development, by modulating HIPPO signaling, a key regulator for trophectoderm (TE) lineage specification. Here, we further evaluated molecular events that are altered by mevalonate pathway inhibition during the timeframe of morphogenesis and cell lineage specification. Whole transcriptome analysis revealed that statin treatment dysregulated gene expression underlying multiple processes, including cholesterol biosynthesis, HIPPO signaling, cell lineage specification and endoplasmic reticulum (ER) stress response. We explored mechanisms that link the mevalonate pathway to ER stress, because of its potential impact on embryonic health and development. Upregulation of ER stress-responsive genes was inhibited when statin-treated embryos were supplemented with the mevalonate pathway product, geranylgeranyl pyrophosphate (GGPP). Inhibition of geranylgeranylation was sufficient to upregulate ER stress-responsive genes. However, ER stress-responsive genes were not upregulated by inhibition of ras homolog family member A (RHOA), a geranylgeranylation target, although it interfered with TE specification and blastocyst cavity formation. In contrast, inhibition of Rac family small GTPase 1 (RAC1), another geranylgeranylation target, upregulated ER stress-responsive genes, while it did not impair TE specification or cavity formation. Thus, our study suggests that the mevalonate pathway regulates cellular homeostasis (ER stress repression) and differentiation (TE lineage specification) in preimplantation embryos through GGPP-dependent activation of two distinct small GTPases, RAC1 and RHOA, respectively. Translation of the findings to human embryos and clinical settings requires further investigations.

Keywords: blastocyst; cell lineage; geranylgeranylation; hypercholesterolemia; unfolded protein response.

Figure 1.

Upregulation of the ER stress-responsive genes in mouse preimplantation embryos by inhibition of the mevalonate pathway. A, a schematic diagram of the mevalonate pathway, highlighting the key intermediates and pharmacological agents that interfere with specific steps. B, bright-field images of E3.5 embryos that have been treated with lovastatin (1 μM) or with lovastatin plus MVA (100 μM). Scale bar, 100 μm. C, relative gene expression levels of the YAP/TEAD targets, lineage specification regulators and ER stress-responsive genes. Means ± standard deviations (n = 3) are shown in bar graphs, in which the means for the control samples (vehicle only) are set as 1. Asterisks indicate significant differences between the two groups marked by horizontal bars.

Figure 2.

Upregulation of the ER stress-responsive genes by tunicamycin. A, bright-field images of E3.5 embryos treated with tunicamycin (0.1 μg/ml). Scale bar, 100 μm. B, relative expression levels of the ER stress-responsive genes. Means ± standard deviations (n = 3) are shown in bar graphs, in which the means for the control samples (vehicle only) are set as 1. Asterisks indicate significant differences.

Figure 3.

Suppression of lovastatin-induced upregulation of the ER stress-responsive genes by the supplementation of FPP and GGPP. A, bright-field images of E3.5 embryos treated with lovastatin (1 μM) plus FPP (10 μM) or GGPP (10 μM). Scale bar, 100 μm. B, relative expression levels of the YAP/TEAD targets, lineage specification regulators and ER stress-responsive genes. Means ± standard deviations (n = 3) are shown in bar graphs, in which the means for the control samples (vehicle only) are set as 1. Asterisks indicate significant differences between the two groups marked by horizontal bars.

Figure 4.

Upregulation of the ER stress-responsive genes by pharmacological inhibition of geranylgeranyltransferase. A, bright-field images of E3.5 embryos treated with B581 (10 μM; farnesyltransferase inhibitor) or GGTI-298 (10 μM; geranylgeranyltransferase inhibitor). Scale bar, 100 μm. B, relative expression levels of the YAP/TEAD targets, lineage specification regulators and ER stress-responsive genes. Means ± standard deviations (n = 3) are shown in bar graphs, in which the means for the control samples (vehicle only) are set as 1. Asterisks indicate significant differences between the two groups marked by horizontal bars.

Figure 5.

Impact of small GTPase inhibition on embryo morphology and gene expression patterns. A, bright-field images of E3.5 embryos treated with ras homolog family member A inhibitor (RHOi; 1 μg/ml). B, relative expression levels of the YAP/TEAD targets, lineage specification regulators and ER stress-responsive genes. Embryos treated with lovastatin (1 μM) are also examined as a comparison. C, bright-field images of E3.5 embryos treated with EHop-016 (0.5 μM). D, relative expression levels of the YAP/TEAD targets, lineage specification regulators and ER stress-responsive genes. A and C, scale bars, 100 μm. B and D, Means ± standard deviations (n = 3) are shown in bar graphs, in which the means for the control samples (vehicle only) are set as 1. Asterisks indicate significant differences between the two groups marked by horizontal bars.

Figure 6.

Distribution of the cell lineage marker proteins in embryos with upregulated ER stress-responsive genes. A, projected confocal images of embryos at E3.5 that were immunostained for CDX2 (green) and SOX2 (red). Nuclei are stained with DAPI. Images of representative embryos from the control (n = 14), lovastatin-treated (n = 13) and EHop-016-treated (n = 15) groups are shown. B, relative gene expression levels for the YAP/TEAD targets and lineage specification regulators in embryos treated with tunicamycin (0.1 μg/ml). Asterisk indicates significant difference. C, projected confocal images of tunicamycin-treated embryos that were stained for CDX2 (green), SOX2 (red) and nuclei. Images of representative embryos from the control (n = 18) and tunicamycin-treated (n = 18) groups are shown. A and C, scale bars, 50 μm.

Figure 7.

Model of mevalonate pathway regulation of trophectoderm specification and ER stress repression through GGPP-dependent ras homolog family small GTPases in mouse blastocyst formation. See Discussion section for details. Solid arrows, direct relationship; broken arrow, several intervening steps.