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Embryonic MTHFR contributes to blastocyst development

  • Embryo Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Reduction in methylenetetrahydrofolate reductase (MTHFR) activity due to genetic variations in the MTHFR gene has been controversially implicated in subfertility in human in vitro fertilization. However, there is no direct gene-knockdown study of embryonic MTHFR to assess its involvement in mammalian preimplantation development. The purpose of this study is to investigate expression profiles and functional roles of MTHFR in bovine preimplantation development.

Methods

Reverse transcription-quantitative PCR (RT-qPCR) and analysis of publicly available RNA-seq data were performed to reveal expression levels of MTHFR during bovine preimplantation development. We knocked down MTHFR by siRNA-mediated RNA interference from the 8- to 16-cell stage and assessed the effects on preimplantation development.

Results

The RT-qPCR analysis showed relatively high MTHFR expression at the GV oocyte stage, which was decreased toward the 8- to 16-cell stage and then slightly restored at the blastocyst stage. Public data–based analysis also showed the similar pattern of expression with substantial embryonic expression at the blastocyst stage. MTHFR knockdown reduced the blastocyst rate (P < 0.01) and the numbers of total (P < 0.0001), trophectoderm (P < 0.0001), and inner cell mass (P < 0.001) cells.

Conclusion

The results indicate that embryonic MTHFR is indispensable for normal blastocyst development. The findings provide insight into the debatable roles of MTHFR in fertility and may be applicable for the improvement of care for early embryos via modulation of surrounding folate-related nutritional conditions in vitro and/or in utero, depending on the parental and embryonic MTHFR genotype.

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Acknowledgments

The authors deeply thank the staff at the Kyoto-Meat-Market for allowing us access to bovine ovaries.

Funding

This work was supported in part by the Kieikai Research Foundation and JSPS KAKENHI Grant Number 15K07779 and 19H03104.

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Authors and Affiliations

  1. Laboratory of Animal Physiology and Functional Anatomy, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan

    Hiroki Ishitani, Kai Egashira, Miki Sugimoto, Shinichi Kume & Takeshi Ohta

  2. Laboratory of Reproductive Biology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan

    Shuntaro Ikeda & Naojiro Minami

Authors
  1. Hiroki Ishitani
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  2. Shuntaro Ikeda
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  3. Kai Egashira
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  4. Miki Sugimoto
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  5. Shinichi Kume
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  6. Naojiro Minami
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  7. Takeshi Ohta
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Corresponding author

Correspondence to Shuntaro Ikeda.

Ethics declarations

This study was approved by the Animal Research Committee of Kyoto University (Permit Numbers 29-10, 30-10, and 31-10).

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The authors declare that they have no conflict of interest.

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ESM 1

(A) Expression levels of MTHFR mRNA based on RT-qPCR with normalization by the geometric mean of three traditional housekeeping genes (GAPDH, SDHA, and YWHAZ), that has been recommended by a previous study [17]. Data are expressed as mean ± standard error relative to the GV stage. Data were obtained from two independent sample sets containing GV oocytes (GV), MII oocytes (MII), 1-cell- (1C), 2-cell- (2C), 8- to 16-cell- (8-16C), morula- (MO), and blastocyst- (BL) stage embryos. (B) Expression levels of mRNA of the three housekeeping genes calculated from Ct values as in the Fig. 2A. (PPTX 53 kb)

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Ishitani, H., Ikeda, S., Egashira, K. et al. Embryonic MTHFR contributes to blastocyst development. J Assist Reprod Genet 37, 1807–1814 (2020). https://doi.org/10.1007/s10815-020-01898-0

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  • DOI: https://doi.org/10.1007/s10815-020-01898-0

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