Effects of thyroid hormone on mitochondria and metabolism of human preimplantation embryos

Abstract

Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10^-7 M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0^{v2018 JUN} platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes-10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes-0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.

Keywords: T3; embryo development; mitochondria; oxidative phosphorylation; thyroid hormone.

Figure 1

Biopsy of inner cell mass (ICM). A‐I, Images illustrate mechanical separation of ICM from trophectoderm (TE). AP, aspiration pipette; HP, holding pipette; ZP, zona pellucida

Figure 2

Quality control of RNAseq data. A, Mapped read annotations. Read annotations are homogeneous distributed and have a high proportion of “usable reads” for all samples. B, Estimation of insert size. Mapping statistics shows medium/high proportion of mapped reads (cca 84%). No or few overlap between forward and reverse reads. No bias regarding 5′3′ coverage of transcripts has been detected

Figure 3

Effects of T3 exposure on transcriptome of human preimplantation embryos. Human 2pn embryos were cultured to the blastocyst stage either in the standard culture medium or in the medium supplemented with 100 nM T3. Inner cell mass was separated mechanically from trophectoderm (TE) (Figure 1), and the transcriptome has been analyzed with RNAseq using Illumina's HiSeq platform (Figure 2). A, Principal component analysis of samples: untreated control (CTL) ICM (n = 3) and TE (n = 4) and T3‐treated ICM (n = 4) and TE (n = 6). Cumulative proportion of variance: 82.4%. B, Analysis of differential gene expression between ICM of embryos cultured in a standard medium and in the medium supplemented with 100 nM T3 has identified 1178 genes (fold change ≥1.5, P ≤ .05). Kyoto Encyclopedia of Genes and Genomes database singled out 11 pathways regulated by T3. The most significantly upregulated are genes in Ribosome and Oxidative phosphorylation pathways (red arrow heads) and downregulated genes in Glyoxylate and dicarboxylate metabolism and Citrate (TCA) cycle (green arrowheads). Taken together, the data suggest a metabolic switch from glycolysis to oxidative phosphorylation accompanied with an increase in protein synthesis (Ribosome pathway). C, Analysis of differential gene expression between TE of embryos cultured in a standard medium and in the medium supplemented with 100 nM T3 has identified only 38 genes (fold change ≥1.5, P ≤ .05) suggesting that TE cells do not respond to thyroid hormone in the same way as the cells from ICM

Figure 4

In silico analysis of mitochondrial bioenergetic differential events. A, Network of mitochondrial genes changed on T3 exposure. The two dominant clusters of the k‐means network are composed of protein subunits involved in respiratory chain architecture (red circles) and the mitoribosome (blue circles). Upregulated mitochondrial genes that are not directly part of either respiratory chain or mitoribosome are in purple. Green and orange circles represent mitochondrial genes that are significantly downregulated. B, Network of upregulated mitochondrial genes suggest shift in ribosome and oxidative phosphorylation activity. Red circles, respiratory chain proteins; blue circles, mitoribosome; purple circles, other upregulated mitochondrial genes. C, Changes of individual genes involved in respiratory chain architecture (correspond to red circles in A and B). D, Changes of individual genes involved in mitoribosome architecture (correspond to blue circles in A and B). E, Network of downregulated mitochondrial genes is primarily limited to the tricarboxylic acid cycle (TCA; green circles). Orange circles, other downregulated mitochondrial genes. F, Changes of individual genes affecting TCA cycle energy production (correspond to green circles in A and E)

Figure 5

T3 effect on transcription of mtDNA encoded genes and mitochondrial biogenesis. A, Changes of individual mtDNA encoded genes. B, mtDNA content of single inner cell mass (ICM) and trophectoderm (TE) cells from five embryos in vivo. Each data point represents the mtDNA content in a single cell corresponding to the mean value from the independent qPCR measurements. Wilcoxon rank sum test has shown significant differences between the untreated control (CTL) and T3‐treated samples ICM cells (W = 143; P = .0087), whereas TE cells lacked the statistically significant difference between the control and T3‐treated groups (W = 60, P = .7021)