Impaired active DNA demethylation in zygotes generated by round spermatid injection.

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.