Alpha-ketoglutarate affects murine embryo development through metabolic and epigenetic modulations

in Reproduction
Authors:
Zhenzhen Zhang National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China

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Changjiu He National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, China

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Lu Zhang National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China

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Tianqi Zhu National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China

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Dongying Lv National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China

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Guangdong Li National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China

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Yukun Song National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China
College of Animal Science and Technology, Xinjiang Agricultural University, Wulumuqi, China

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Jing Wang National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China

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Hao Wu National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China

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Pengyun Ji National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China

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Guoshi Liu National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing, China

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Correspondence should be addressed to G Liu; Email: gshliu@cau.edu.cn

*(Z Zhang and C He contributed equally to this work)

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α-Ketoglutarate (α-KG) is an intermediary metabolite in the tricarboxylic acid (TCA) cycle and functions to inhibit ATPase and maintain the pluripotency of embryonic stem cells (ESCs); however, little is known regarding the effects of α-KG on the development of preimplantation embryos. Herein, we report that α-KG (150 μM) treatment significantly promoted the blastocyst rate, the number of inner cell mass (ICM) cells and foetal growth after embryo transfer. Mechanistic studies revealed two important pathways involved in the α-KG effects on embryo development. First, α-KG modulates mitochondria function by inducing relatively low ATP production without modification of mitochondrial copy number. The relatively low energy metabolism preserves the pluripotency and competence of the ICM. Second, α-KG modifies epigenetics in embryos cultured in vitro by affecting the activity of the DNA demethylation enzyme TET and the DNA methylation gene Dnmt3a to increase the ratio of 5hmC/5mC ratio. Elevation of the 5hmC/5mC ratio not only promotes the pluripotency of the ICM but also leads to a methylation level in an in vitro embryo close to that in an in vivo embryo. All these functions of α-KG collectively contribute to an increase in the number of ICM cells, leading to greater adaptation of cultured embryos to in vitro conditions and promoting foetal growth after embryo transfer. Our findings provide basic knowledge regarding the mechanisms by which α-KG affects embryo development and cell differentiation.

Supplementary Materials

    • S_Fig. 1 (a) Chromosome distribution of upregulated genes; (b-c) Relative FPKM expression levels of Tet1/2/3 in the RNAseq analysis.
    • S_Fig. 2 Effects of α-KG on TUNEL staining and the ROS level in embryos. (a) Representative images of apoptosis detected by TUNEL staining in blastocysts. Green: TUNEL-positive apoptotic nuclei; blue: DAPI-stained nuclei in blastocysts. (b) Statistical comparisons of the apoptotic rates in blastocysts from the control and α-KG groups. Each circle represents the apoptotic rates detected in a single embryo (control, n=36; α-KG, n=18). (c-f) Quantification of the ROS levels determined by the mean 2’,7’-dichlorohydrofluorescein diacetate (DCHFDA) fluorescence intensity and analysed using ImageJ software in blastocysts in the control and α-KG groups (2-cell: control, n=12, and α-KG, n=11; 4-cell: control, n=14, and α-KG, n=15; morula: control, n=13, and α-KG, n=11; blastocyst: control, n=17, and α-KG, n=18). (g) Representative fluorescence images of ROS levels in 2-cell, 4-cell, morula and blastocyst stage embryos from the control and α-KG groups. *p<0.05. Scale bars, 20 µm (A) and 200 µm (G).
    • Table 1 Sequence of primers
    • Mitochondria related genes

 

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