Yoko Kakino Kurotaki1, Yuki Hatanaka2, Satoshi Kamimura1, Mami Oikawa2, Hiroki Inoue3, Narumi Ogonuki2, Kimiko Inoue1, Atsuo Ogura4. 1. RIKEN BioResource Center, 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba, Ibaraki, Japan. 2. RIKEN BioResource Center, 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan. 3. RIKEN BioResource Center, 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan Laboratory of Animal Reproduction and Development, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan. 4. RIKEN BioResource Center, 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba, Ibaraki, Japan Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan ogura@rtc.riken.go.jp.
Abstract
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.
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.
Authors: Mami Oikawa; Angela Simeone; Eva Hormanseder; Marta Teperek; Vincent Gaggioli; Alan O'Doherty; Emma Falk; Matthieu Sporniak; Clive D'Santos; Valar Nila Roamio Franklin; Kamal Kishore; Charles R Bradshaw; Declan Keane; Thomas Freour; Laurent David; Adrian T Grzybowski; Alexander J Ruthenburg; John Gurdon; Jerome Jullien Journal: Nat Commun Date: 2020-07-13 Impact factor: 14.919
Authors: Martin J Blythe; Ayhan Kocer; Alejandro Rubio-Roldan; Tom Giles; Abdulkadir Abakir; Côme Ialy-Radio; Lee M Wheldon; Oxana Bereshchenko; Stefano Bruscoli; Alexander Kondrashov; Joël R Drevet; Richard D Emes; Andrew D Johnson; John R McCarrey; Daniel Gackowski; Ryszard Olinski; Julie Cocquet; Jose L Garcia-Perez; Alexey Ruzov Journal: Commun Biol Date: 2021-06-07