Effect of IVF and laser zona dissection on DNA methylation pattern of mouse zygotes.

Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
Mammalian Genome (Impact Factor: 2.42). 10/2009; 20(9-10):664-73. DOI:10.1007/s00335-009-9227-0
Source: PubMed

ABSTRACT In vitro fertilization (IVF) and zona pellucida laser microdissection-facilitated IVF (Laser-IVF) are presently routine procedures in human assisted reproduction. The safety of these methods at the epigenetic level is not fully understood. Studies on mouse Laser-IVF embryos provide evidence that the use of Laser-IVF leads to reduced birth rate, indicating a potential harm of this technique for the embryo. Hence, the aim of this study was to examine the difference in DNA methylation pattern between IVF- and Laser-IVF-derived mouse zygotes. We examined two experimental groups of C3HeB/FeJ oocytes: (1) zona-intact and (2) laser-microdissected oocytes that were fertilized in vitro with freshly collected spermatozoa. Zygotes were fixed 5, 8, and 12 h after fertilization, and indirect immunofluorescence staining was studied using an anti-5-methylcytidine (5-MeC) antibody. The fluorescence intensities of paternal and maternal pronuclei were evaluated using the computer-assisted analysis of digital images. In addition, we performed a semiquantitative RT-PCR analysis of the presence of transcripts of three developmental marker genes, Oct4, Dab2, and Dnmt3b, in IVF- and Laser-IVF-derived blastocysts. We observed no significant differences in methylation status of the paternal genome and in the transcripts of the developmental marker genes after IVF and Laser-IVF. In conclusion, epigenetic patterns and early embryonic development are not altered by laser-assisted IVF techniques and another explanation must be sought for the poor implantation rates observed in mice.

0 0
  • [show abstract] [hide abstract]
    ABSTRACT: DNA methylation and demethylation are crucial for modulating gene expression and regulating cell differentiation. Functions and mechanisms of DNA methylation/demethylation in mammalian embryos are still far from being understood clearly. In this review we firstly describe new insights into DNA demethylation mechanisms, and secondly introduce the differences in active DNA methylation patterns in zygotes and early embryos in various mammalian species. Thirdly, we attempt to clarify the functions of DNA demethylation in early embryos. Most importantly we summarize the importance of active DNA demethylation and its possible relevance to human IVF clinics. Finally research perspectives regarding DNA demethylation are also discussed.
    Molecular Human Reproduction 03/2012; 18(7):333-40. · 4.54 Impact Factor
  • [show abstract] [hide abstract]
    ABSTRACT: In the present report, we studied the safety, efficacy, and efficiency of using an infrared laser to facilitate in vitro fertilization (IVF) by assessing fertilization, development, and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate 4 to 10 times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved 2-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen-thawed embryos derived by regular IVF. Surprisingly, after surgical transfer to pseudopregnant recipients, the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However, this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space (PVS). By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious, and efficient assisted reproductive technology (ART) for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm-zona penetration defects.
    Reproduction 01/2013; · 3.56 Impact Factor
  • [show abstract] [hide abstract]
    ABSTRACT: The large-scale mutagenesis programmes underway around the world are generating thousands of novel GA mouse strains that need to be securely archived. In parallel with advances in mutagenesis, the procedures used to cryopreserve mouse stocks are being continually refined in order to keep pace with demand. Moreover, the construction of extensive research infrastructures for systematic phenotyping is fuelling demand for these novel strains of mice and new approaches to the distribution of frozen and unfrozen embryos and gametes are being developed in order to reduce the dependency on the transportation of live mice. This article highlights some contemporary techniques used to archive, rederive, and transport mouse strains around the world.
    Mammalian Genome 08/2012; 23(9-10):572-9. · 2.42 Impact Factor

Full-text (2 Sources)

Available from
Apr 17, 2013