Expression profiling of DNA repair genes in human oocytes and blastocysts using microarrays

Human Reproduction, Vol.24, No.10 pp. 2649–2655, 2009 Advanced Access publication on June 20, 2009 doi:10.1093/humrep/dep224 ORIGINAL ARTICLE Reproductive genetics Expression profiling of DNA repair genes in human oocytes and blastocysts using microarrays Souraya Jaroudi 1, Georgia Kakourou 1, Suzanne Cawood 2, Alpesh Doshi 2, Domenico M. Ranieri 2, Paul Serhal 2, Joyce C. Harper 1, and Sioban B. SenGupta 1,3 1 UCL Centre for PGD, Institute for Women’s Health, University College London, 86-96 Chenies Mews, London WC1E 6HX, UK 2The Assisted Conception Unit, University College Hospital, The New Wing Eastman Dental Hospital, 256 Gray’s Inn Road, London WC1X 8LD, UK 3 Correspondence address. E-mail: background: The early preimplantation embryo relies on mRNA and protein from the oocyte to detect DNA damage and activate DNA repair, cell cycle arrest or apoptosis. Expression of some repair genes has been detected in mammalian oocytes and embryos; however, little is known about DNA repair gene expression in human blastocysts. In this study, DNA repair gene expression was investigated in human oocytes and blastocysts to identify the pathways involved at these stages and detect potential differences in repair mechanisms preand post-embryonic genome activation. methods: Triplicate sets of pooled metaphase II oocytes or blastocysts were processed for analysis using the Human Genome Survey Microarrays V2.0 (Applied Biosystems). results: Of 154 DNA repair genes investigated, 109 were detected in blastocysts and 107 in oocytes. Among differentially expressed DNA repair genes, 40/55 (73%) had lower expression levels in blastocysts compared with oocytes (P , 0.05, fold change .3). conclusion: Despite experimental limitations due to culture or freezing and thawing of samples, large numbers of repair genes were detected indicating that all DNA repair pathways are potentially functional in human oocytes and blastocysts. The higher mRNA level for most repair genes in oocytes compared with blastocysts ensures sufficient availability of template until embryonic genome activation. Key words: DNA repair / gene expression / human blastocyst embryo / human oocyte / microarrays Introduction DNA repair is responsible for protecting the genome of a cell or organism from endogenous metabolites or exogenous agents causing DNA damage. Various DNA repair proteins act together in elaborate cellular pathways in order to detect and repair different types of DNA lesions, e.g. double strand breaks (DSBs), errors during replication etc. The main DNA repair pathways active in mammalian cells are: base excision repair (BER), direct reversal of damage, double strand break repair (DSBR), mismatch repair (MMR) and nucleotide excision repair (NER). The mechanisms of mammalian DNA repair, overall involving DNA lesion recognition, DNA exonuclease, DNA polymerase and DNA ligase activities, have been reviewed extensively (Christmann et al., 2003; Sancar et al., 2004; Hakem, 2008). Following DNA damage detection, repair pathways are activated via transient cell cycle arrest (Branzei and Foiani, 2008); when a lesion cannot be repaired, cell cycle arrest is either permanently sustained or the apoptotic pathways are triggered to eliminate the cell. There is not much information available regarding DNA repair in early development of totipotent cells. Some studies on human and murine embryonic stem cells have reported lower mutation frequencies and different mutation types compared with somatic cells (Maynard et al., 2008; Tichy and Stambrook, 2008), underlining the potential differences of DNA repair mechanisms at these most critical stages of early development. Overall, DNA repair has been considered a maternal trait. The DNA repair transcripts that have accumulated in the human oocyte play a role during fertilization in controlling changes in chromatin remodelling and maintaining chromatin integrity; experiments on rat and mouse zygotes have indicated recognition of DNA lesions and repair in the paternal chromatin after fertilization (Barton et al., 2007; Derijck et al., 2008). Subsequent to this, DNA repair is expected to have a & The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: 2650 major impact on embryo development. It has been shown that human oocytes express DNA repair genes at high levels allowing low tolerance for DNA decays (Menezo et al., 2007). The maternal transcripts and proteins are expected to support the zygote’s development until embryonic genome activation (EGA). Gene expression profiling of mouse preimplantation embryos showed characteristic patterns of maternal RNA depletion and revealed that EGA happens in two phases: an initial weak transcription from the new zygotic genome followed by major EGA allowing dramatic reprogramming of expression patterns (reviewed by Hamatani et al., 2006). The factors controlling the reprogramming of the embryonic genome, including DNA methylation and histone acetylation, have been investigated by a large number of studies in recent years and are described in great detail by Bell et al. (2008) and Duranthon et al. (2008). In humans, EGA occurs at the 4–8-cell stage (Tesarik et al., 1986, 1987, 1988; Braude et al., 1988; Telford et al., 1990). Although the expression of some DNA repair genes has been detected in mammalian embryos at different stages of development (as reviewed by Jaroudi and SenGupta, 2007), the ability of the human preimplantation embryo to detect and repair DNA damage has not yet been well described. Recent developments in RNA amplification methods and microarray technologies make it possible to detect the expression of virtually every gene in the genome from a single sample. In this study, Human Genome Survey Microarrays V2.0 (Applied Biosystems, UK), which interrogate 29 098 genes, were used to investigate human metaphase II (MII) oocytes and blastocysts for the expression of a comprehensive list of genes obtained from a Supplement Table to a review by Wood et al. (2005). This list included genes coding for DNA repair enzymes, cellular response to DNA damage genes or genes known to be mutated in human diseases associated with DNA sensitivity. Based on previous expression studies on germinal vesicle (GV) oocytes (Menezo et al., 2007), we hypothesized that the human oocyte expresses most DNA repair genes to support the early preimplantation embryo and limit DNA damage. There is no evidence indicating whether the blastocyst has a supply of mRNA for DNA repair under routine IVF conditions, without the induction of specific DNA lesions. Due to the high rate of replication and the onset of differentiation in the blastocyst, the expression profile of DNA repair genes may be different to oocytes. Our main aim was to identify the DNA repair pathways that may be active pre- and post-EGA by investigating mRNA in human in v (...truncated)