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Effect of Zona Pellucida Removal on DNA Methylation in Early Mouse Embryos¹

Roslin Institute,⁴ Roslin, Midlothian EH25 9PS, United Kingdom Centro de Estudos de Ciência Animal (CECA)-Instituto de Ciências e Tecnologicas Agrárias e Agro-Alimentares (ICETA),⁵ Campus Agrário de Vairão, 4485–661 Vairão, Portugal Instituto Ciências Biomédicas Abel Salazar (ICBAS),⁶ 4099–003 Porto, Portugal

ABSTRACT

Removal of the zona pellucida is known to affect mouse development to term. Zygotes were recovered immediately after fertilization and their zona pellucida removed by exposure to pronase before culture and comparison with zona-intact embryos. The effect of removing the zona pellucida was assessed in terms of embryo development to blastocyst, DNA methylation, histone acetylation, and expression of three developmentally regulated genes. No significant differences were seen in percentage of embryos that developed to the blastocyst stage. However, zona-free embryos showed a significant reduction in the DNA methylation level at two-cell and four-cell stages, but no differences at pronuclear, morula, and blastocyst stages, as observed by immunofluorescence. Mechanical or enzymatic removal of the zona pellucida showed similar DNA methylation staining patterns at the two-cell stage. The time when the zona pellucida was removed appears to influence the levels of DNA methylation. When zona removal was delayed for 8 h, there was no difference in DNA methylation levels between zona-free and zona-intact two-cell embryos, indicating that the critical time is early on, between 1 and 8 h postfertilization. In contrast, when immunofluorescence analysis of histone acetylation was performed, no significant differences were seen between zona-free and zona-intact embryos at any of the developmental stage. Similarly, no differences were found regarding the onset of transcription of Dnmt1s, Nanog, and Fgf4 genes.

developmental biology, early development, embryo, gene regulation

¹ Supported by Fundação para a Ciência e Tecnologia (FCT), Ministério da Ciência e do Ensino Superior (MCES), Portugal.

² Correspondence: FAX: 0044 131 242 6629; ricardo.ribas{at}icr.ac.uk

³ Current address: The Queen's Medical Research Institute, Centre for Reproductive Biology, Reproductive and Developmental Sciences, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom.

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