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Mol. Hum. Reprod. Advance Access originally published online on February 4, 2005
Molecular Human Reproduction 2005 11(3):167-171; doi:10.1093/molehr/gah145
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Molecular Human Reproduction Vol. 11 No. 3 © European Society of Human Reproduction and Embryology 2005; all rights reserved

DNA methylation patterns in human tripronucleate zygotes

Yanwen Xu1,2, John J. Zhang1, James A. Grifo1 and Lewis C. Krey1,3

1Program for In Vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, NY10016, USA and 2Current address: Reproductive Medical Research Center, The 1st Affiliated Hospital of Sun Yat-sen University, Guangzhou, China 510080

3 To whom correspondence should be addressed at: 660 First Avenue 5th Floor, New York, NY 10016, USA. Email: kreyivf{at}yahoo.com

In mammals, the dynamic reprogramming of DNA methylation begins during gametogenesis and continues through embryogenesis. Recently, immunofluorescence staining with an antibody against 5-methylcytosine (anti-5-MeC) has revealed active demethylation of the male pronucleus in zygotes beginning at 4–6 h after fertilization. In this study, we characterized the DNA methylation patterns in mouse zygotes and in human tripronucleate (3 PN) zygotes discarded after conventional fertilization or following ICSI. Pronuclei were subjected to fluorescence in-situ hybridization to identify the X and/or Y chromosomes and then stained with anti-5-MeC. In diandric 3 PN zygotes from conventional IVF, we consistently observed one strongly and two weakly stained pronuclei. In contrast, the majority of 3 PN ICSI zygotes, mainly digynic zygotes, displayed two strongly and one weakly stained pronuclei. Two zygotes from ICSI failed to show any staining difference among the three pronuclei. Our results indicate that the active demethylation of male pronuclei occurs in both mouse and human zygotes. It is possible that the abnormal methylation patterns resulting from a dysfunctional cytoplasm may occur in a small number of oocytes and may affect embryonic viability.

Key words: DNA methylation immunostaining/FISH/tripronucleate zygotes


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