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Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism

  • Embryo Biology
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Abstract

Purpose

This study was designed to determine if DMO limits in vitro development of aneuploid-enriched mouse embryos by activating a Trp53-dependent mechanism.

Methods

Mouse cleavage-stage embryos were treated with reversine to induce aneuploidy or vehicle to generate controls, and then cultured in media supplemented with DMO to reduce the pH of the culture media. Embryo morphology was assessed by phase microscopy. Cell number, mitotic figures, and apoptotic bodies were revealed by staining fixed embryos with DAPI. mRNA levels of Trp53, Oct-4, and Cdx2 were monitored by quantitative polymerase chain reactions (qPCRs). The effect of Trp53 on the expression of Oct-4 and Cdx2 was assessed by depleting Trp53 using Trp53 siRNA.

Results

Aneuploid-enriched late-stage blastocysts were morphologically indistinguishable from control blastocysts but had fewer cells and reduced mRNA levels of Oct-4 and Cdx2. Adding 1 mM DMO to the culture media during the 8-cell to blastocyst transition reduced the formation of aneuploid-enriched late-stage blastocysts but not control blastocysts and further suppressed the levels of Oct-4 and Cdx2 mRNA. Trp53 RNA levels in aneuploid-enriched embryos that were exposed to DMO were > twofold higher than controls, and Trp53 siRNA levels reduced the levels of Trp53 and increased levels of Oct-4 and Cdx2 mRNA by > twofold.

Conclusion

These studies suggest that the development of morphologically normal aneuploid-enriched mouse blastocysts can be inhibited by adding low amounts of DMO to the culture media, which results in elevated levels of Trp53 mRNA that suppresses Oct-4 and Cdx2 expression.

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Data Availability

All the data from this study is available in the supplemental tables. As such all the data is available to the public.

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Author information

Authors and Affiliations

  1. Department of Cell Biology, University of Connecticut Health Center, 263 Farmington Ave., CT, 06030, Farmington, USA

    Katie M. Lowther & John J. Peluso

  2. Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, CT, 06030, USA

    Alison F. Bartolucci & John J. Peluso

  3. The Center for Advanced Reproductive Services, University of Connecticut Health Center, Farmington, CT, 06030, USA

    Alison F. Bartolucci

  4. Institute for Systems Genomics, UCONN, Storrs, CT, 06268, USA

    Rachael E. Massey & Judy Brown

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Correspondence to John J. Peluso.

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Supplemental Figure 1.

The effect of reversine (5 μM) on the induction of aneuploidy as revealed by FISH analysis. The upper panel shows a typical diploid blastomere with two red dots and two green dots that represent chromosome 11q and 2q, respectively as well as examples of various aneuploid blastomeres. The table in the lower panel presents the ploidy status of all the blastomeres that were analyzed after DMSO (control) and reversine treatment as revealed by the number of 11q and 2q chromosomes present. Ploidy status for each blastomeres is indicated by a D (diploid) or A (aneuploid). (PNG 2491 kb)

High resolution image (TIFF 3574 kb)

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Lowther, K.M., Bartolucci, A.F., Massey, R.E. et al. Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism. J Assist Reprod Genet 40, 1215–1223 (2023). https://doi.org/10.1007/s10815-023-02788-x

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  • DOI: https://doi.org/10.1007/s10815-023-02788-x

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