Epigenetically Immature Oocytes Lead to Loss of Imprinting During Embryogenesis

Abstract

Abstract. Loss of imprinting (LOI) is occasionally observed in human imprinting disorders. However, the process behind the LOI is not fully understood. To gain a better understanding, we produced embryos and pups from mouse oocytes that lacked a complete methylation imprint using a method that involved transferring the nuclei of growing oocytes into the cytoplasm of enucleated fully grown oocytes following in vitro fertilization (IVF). We then analyzed the imprinting statuses. Our findings show that the incomplete methylation imprint derived from growing oocytes results in epigenetic mosaicism or a loss of methylation imprint (LOM) at maternal alleles in embryos. In some embryos, both hypo-and hypermethylated maternal Kcnq1ot1 alleles were detected, whereas either hypo-or hypermethylated maternal Kcnq1ot1 alleles were detected in others. Such tendencies were also observed at the Igf2r and Mest loci. Gene expression levels of imprinted genes were linked with their methylation statuses in some but not all embryos. Possible explanations of the inconsistency between the data from DNA methylation and gene expression include epigenetic mosaicism in embryos. Pups were successfully produced from growing oocytes at a quite low frequency. They exhibited an obese phenotype and LOI with respect to Igf2r, Snrpn and Mest. Our finding suggests the possibility that LOI/LOM at maternal alleles in human concepti could be derived from epigenetically immature/mutated oocytes. Key words: DNA methylation, Gene expression, Genomic imprinting, Growing oocyte, Loss of imprinting (LOI) (J. Reprod. Dev. 57: [327][328][329][330][331][332][333][334] 2011) enomic imprinting is an epigenetic modification specifically imposed on male and female gametes that leads to the parental origin-specific gene expression in embryos. DNA methylation is the primary mechanism behind genomic imprinting [1,2]. Gamete-specific DNA methylation is established by DNA methyltransferase (DNMT) 3A and DNMT3L and is maintained after fertilization by DNMT1. Embryos derived from oocytes with defective Dnmt3a, Dnmt3L or Dnmt1 genes exhibit a LOM and the aberrant expression of imprinted genes (referred to LOI). In turn, this causes embryonic death [3][4][5]. Therefore, the regulatory expression of imprinted genes via DNA methylation is essential for normal ontogeny and biological phenomena in mammals.So far, we and other researchers have reported on the stage of the establishment of methylation imprints during mouse oocyte growth [6,7]. The results show that the Impact, Snrpn, Mest and Grb10 genes are hypermethylated in growing oocytes with diameters of 60-65 μm; the Igf2r, Kcnq1ot1 and Plagl1 genes are hypermethylated in growing oocytes with diameters of 55-60 μm. The first appearance of oocytes with complete methylation imprints at all of the examined loci is at an age of approximately 15 days in mice. Because fertilized eggs containing genomes from growing oocytes of mice 15 days old or younger do not develop into offspring, establishment of genomic imprinting is r...

Methylation levels at imprinting control regions are not altered with ovulation induction or in vitro fertilization in a birth cohort

“…H19 and Snrpn; Fortier et al, 2008). Perhaps hormone stimulation brings forth the presence of oocytes which are epigenetically immature, carrying improper marks, resulting in LOI (Obata et al, 2011). However, we did not observe a disrupted methylation pattern at the imprinted loci studied (GRB10, MEST, H19/IGF2, SNRPN and KCNQ1) and it is possible that our OI group represents examples of successful conception due to mature oocytes with the proper epigenetic marks which may be preferentially fertilized.…”

“…It is possible that the use of such ovulation inducing hormones may result in improper establishment of key epigenetic marks (e.g. DNA methylation) in the germline during oogenesis, potentially resulting in immature oocytes and/or incomplete methylation patterns (Santos et al, 2010;Obata et al, 2011). The possible effect of OI on imprinting patterns has not been studied in humans.…”

Epigenetic reprogramming in somatic cells induced by extract from germinal vesicle stage pig oocytes

“…This is accompanied by very high levels of RNA polymerase I and II transcription that are indispensable for the production of ribosomes and specific mRNAs (Picton et al, 1998). In the mouse, immature oocytes can reprogram the immature nucleus, whereas MII stage oocytes cannot (Bao et al, 2000;Obata et al, 2002), and pups can be produced by transferring the nuclei of growing oocytes into the cytoplasm of enucleated fully grown oocytes following IVF (Obata et al, 2011). Therefore, there must be several reprogramming factors from the cytoplasm of growing and fully grown oocytes in our GVcyto-extract.…”

Variable imprinting of the MEST gene in human preimplantation embryos

“…In mice, elevated expression of Mest has been associated with fat mass expansion 4,51,52 and obese phenotypes with LOI of Mest have been obtained from epigenetically immature oocytes. 53 Finally, it remains possible that uncharacterized MEST transcripts, such as human equivalents of the longer MestXL variants that were recently described in the mouse, 54 may have been amplified in our non-isoform-specific PCR assays. These observations suggest that further characterisation of the causes and effects of contrasting embryonic imprinting states during in vitro conception are required.…”