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Updated: Apr 16, 2026
Single Oocyte Bisulfite Mutagenesis
Published on: June 27, 2012
Impaired active DNA demethylation in zygotes generated by round spermatid injection
Yoko Kakino Kurotaki1, Yuki Hatanaka2, Satoshi Kamimura1
1RIKEN BioResource Center, 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Study Question:
Is the poor development of embryos generated from round spermatid injection (ROSI) in humans and animals associated with abnormal active DNA demethylation?
Summary Answer:
A significant proportion of ROSI-derived embryos failed to undergo active DNA demethylation.
What Is Known Already:
Active DNA demethylation is initiated by the conversion of 5-methylcytosine (5mC) to 5-hydroxycytosine (5hmC) by the Tet3 enzyme. Active demethylation proceeds in a more pronounced manner in the male pronucleus than in the female one.
Participants/Materials, Setting, Methods:
Mouse zygotes generated by ICSI or ROSI were analyzed for active DNA methylation by quantification of 5mC and 5hmC using specific antibodies. Some ROSI-derived embryos were subjected to time-lapse imaging for DNA methylation levels and were transferred into recipient pseudo-pregnant female mice.
Main Results And The Role Of Chance:
In ICSI-derived embryos, the male:female pronucleus (M/F) ratio of 5mC immunostaining intensity was decreased while that of 5hmC was increased. However, a significant proportion of ROSI-derived embryos showed unchanged M/F ratios for 5mC and 5hmC even at the late zygotic period, indicating that they failed to undergo asymmetric active DNA demethylation. Consistent with this, some ROSI-derived embryos did not show preferential localization of Tet3 to the male pronucleus. ROSI-derived embryos were classified into 'demethylated' or 'non-demethylated' groups by time-lapse imaging and transferred into recipient female mice separately. More normal-sized fetuses were retrieved from the 'demethylated' group than 'non-demethylated' group at Day 11.5 of pregnancy.
Limitations, Reasons For Caution:
A causal relationship between impaired active DNA demethylation and the poor developmental ability of ROSI-derived embryos remains to be determined.
Wider Implications Of The Findings:
We identified two types of ROSI-derived embryos in terms of the degree of active DNA demethylation. Induction of normal DNA demethylation at the zygotic stage might help in the technical improvement of ROSI.
Study Funding/Competing Interests:
The work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the RIKEN Epigenetics Program. The authors have no competing interests to declare.
