Single Oocyte Bisulfite Mutagenesis (original) (raw)

Summary

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Abstract

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.

One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.

Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Protocol

DAY 1

Prepare the following solutions fresh on the day of oocyte collection with sterile, distilled water such as GIBCO water. To reduce the chance of DNA contamination, change gloves often and use filter tips. Keep tubes angled away when open, and recap all tubes when not in use. We recommend that solutions are made as n+1.

3% LMP Agarose

30 mg Low Melting Point (LMP) Agarose
up to 1 ml GIBCO H2O
dissolve @ 70 °C

Lysis Solution

8 μl lysis buffer
1 μl proteinase K
1 μl 10% IGEPAL
place ....

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Discussion

This single oocyte assay contains many steps with a number that are critical and require special care. The first is oocyte washing. It is particularly important to wash each oocyte multiple times in fresh medium drops following hyaluronidase treatment to remove as many cumulus cells as possible. Moreover, when transferring oocytes to acidic tyrode's solution for zona pellucida removal make sure surrounding medium is clear of cumulus cells. The oocyte is very sticky following zona removal, and any surrounding cumulus cell.......

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the University of Western Ontario, the Department of Obstetrics and Gynaecology; and a grant ER06-02-188 from the Ministryof Research and Innovation, Early Researcher Award. MMD was supported by a CIHR Training Program in Reproduction, Early Development and the Impact on Health (REDIH) Graduate Scholarship.

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