Methylation In Oocytes Research Articles

DNA methylation establishment during oocyte growth: mechanisms and significance

DNA methylation in the oocyte has a particular significance: it may contribute to gene regulation in the oocyte and marks specific genes for activity in the embryo, as in the case of imprinted genes. Despite the fundamental importance of DNA methylation established in the oocyte, knowledge of the mechanisms by which it is conferred and how much is stably maintained in the embryo has remained very limited. Next generation sequencing approaches have dramatically altered our views on DNA methylation in oocytes. They have revealed that most methylation occurs in gene bodies in the oocyte. This observation ties in with genetic evidence showing that transcription is essential for methylation of imprinted genes, and is consistent with a model in which DNA methyltransferases are recruited by the histone modification patterns laid down by transcription events. These findings lead to a new perspective that transcription events dictate the placing and timing of methylation in specific genes and suggest a mechanism by which methylation could be coordinated by the events and factors regulating oocyte growth. With these new insights into the de novo methylation mechanism and new methods that allow high resolution profiling of DNA methylation in oocytes, we should be in a position to investigate whether and how DNA methylation errors could arise in association with assisted reproduction technologies or in response to exposure to environmental toxins.

Compromised fertility disrupts Peg1 but not Snrpn and Peg3 imprinted methylation acquisition in mouse oocytes.

Growth and maturation of healthy oocytes within follicles requires bidirectional signaling and intercellular gap junctional communication. Aberrant endocrine signaling and loss of gap junctional communication between the oocyte and granulosa cells leads to compromised folliculogenesis, oocyte maturation, and oocyte competency, consequently impairing fertility. Given that oocyte-specific DNA methylation establishment at imprinted genes occurs during this growth phase, we determined whether compromised endocrine signaling and gap junctional communication would disrupt de novo methylation acquisition using ERβ and connexin37 genetic models. To compare mutant oocytes to control oocytes, DNA methylation acquisition was first examined in individual, 20–80 μm control oocytes at three imprinted genes, Snrpn, Peg3, and Peg1. We observed that each gene has its own size-dependent acquisition kinetics, similar to previous studies. To determine whether compromised endocrine signaling and gap junctional communication disrupted de novo methylation acquisition,individual oocytes from Esr2- and Gja4-deficient mice were also assessed for DNA methylation establishment. We observed no aberrant or delayed acquisition of DNA methylation at Snrpn, Peg3, or Peg1 in oocytes from Esr2-deficient females, and no perturbation in Snrpn or Peg3 de novo methylation in oocytes from Gja4-null females. However, Gja4 deficiency resulted in a loss or delay in methylation acquisition at Peg1. One explanation for this difference between the three loci analyzed is the late establishment of DNA methylation at the Peg1 gene. These results indicate that compromised fertility though impaired intercellular communication can lead to imprinting acquisition errors. Further studies are required to determine the effects of subfertility/infertility originating from impaired signaling and intercellular communication during oogenesis on imprint maintenance during preimplantation development.

Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes

Mammalian imprinted genes are associated with differentially methylated regions (DMRs) that are CpG methylated on one of the two parental chromosomes. In mice, at least 21 DMRs acquire differential methylation in the germline and many of them act as imprint centres. We previously reported the physical extents of differential methylation at 15 DMRs in mouse embryos at 12.5 days postcoitum. To reveal the ontogeny of differential methylation, we determined and compared methylation patterns of the corresponding regions in sperm and oocytes. We found that the extent of the gametic DMRs differs significantly from that of the embryonic DMRs, especially in the case of paternal gametic DMRs. These results suggest that the gametic DMR sequences should be used to extract the features specifying methylation imprint establishment in the germline: from this analysis, we noted that the maternal gametic DMRs appear as unmethylated islands in male germ cells, which suggests a novel component in the mechanism of gamete-specific marking. Analysis of selected DMRs in blastocysts revealed dynamic changes in allelic methylation in early development, indicating that DMRs are not fully protected from the major epigenetic reprogramming events occurring during preimplantation development. Furthermore, we observed non-CpG methylation in oocytes, but not in sperm, which disappeared by the blastocyst stage. Non-CpG methylation was frequently found at maternally methylated DMRs as well as non-DMR regions, suggesting its prevalence in the oocyte genome. These results provide evidence for a unique methylation profile in oocytes and reveal the surprisingly dynamic nature of DMRs in the early embryo.

Size-Dependent Acquisition of Global DNA Methylation in Oocytes Is Altered by Hormonal Stimulation.

The normal progression of acquisition of global DNA methylation in oocytes of adult female mice is not known. Assisted reproductive technologies (ART) are commonly used for the treatment of infertility problems. Several studies have shown that ART procedures alter the expression of genes, causing delayed embryonic development, increased abnormal blastocyst formation, pronounced fetal growth retardation and is associated with increased incidence of loss-of-imprinting syndromes. A previous study from our laboratory showed that the maternal pronuclei of one-cell mouse embryos formed after superovulation (SO) had 50% less methylation when compared to those formed after natural ovulation (NO). We hypothesize that oocytes in ovaries of females undergoing a SO scheme will have different levels of global DNA methylation than oocytes from naturally cycling animals. First, we determined the progression of global DNA methylation during oocyte growth in naturally cycling animals and then compared this to the levels of DNA methylation in oocytes from females undergoing a SO scheme. A group of 14 cyclic females were selected at diestrus and divided into seven groups. Two females were sacrificed and collected ovaries at diestrus (0h). Six females underwent a SO scheme while the remaining six did not. In brief, SO females were injected with 5 IU of eCG followed 44 h later by 5 IU of hCG. Ovaries were collected at 24- and 44 h post-eCG and 10 h post-hCG (data not discussed). Ovaries from their NO counterparts were collected at the same time as the SO females. Ovarian sections were stained with an antibody that recognizes methylated cytosines (5MeC). All oocytes in a section were photographed and the largest cross section was used for analysis. Measurements related to oocyte growth were; 1) oocyte size in micrometers, 2) area of germinal vesicle, 3) size and type of enclosing follicle. Global DNA methylation was determined using the background corrected density macro of ImageJ. The 5MeC staining intensity level of the GV was normalized to the staining of the surrounding granulosa cells. For analyses, correlations were made between oocyte size and level of methylation and GV size and level of methylation. These numbers were then compared between similar size oocytes from SO and NO females. Results show that at diestrus the level of methylation increases in a linear manner form 10 to 80 µm diameter. The curve becomes exponential at 24 and 44 h in the NO group but is sigmoid in both SO groups. Analysis of full-grown oocytes (>70 µm) show that global methylation increases from diestrus (0 h) to 24 h and again to 44 h in the NO group but not in the SO groups. Interestingly, the levels of methylation remain low until ~ 40 µm diameter and start increasing exponentially in oocytes > 41 µm. This is coincident with the initiation of the formation of the second layer of granulosa cells. At 44 h, levels of DNA methylation were different in full-grown oocytes of NO vs. SO groups (P < 0.02). These full-grown oocytes are expected to ovulate upon the LH surge. Our results show that acquisition of DNA methylation continues in full grown oocytes until the presumed LH surge and we speculated that this is necessary to attain full developmental competence. On the other hand, SO full-grown oocytes do not attained full methylation before ovulation and this may in part be responsible for the lower developmental competence observed in embryos produced from SO oocytes. (poster)

The global DNA methylation of mouse oocytes following vitrification

OBJECTIVE: Recently it has been reported that oocyte survival rate was improved tremendously by vitrification method following cryopreservation. However, there is paucity of information about oocyte methylation following vitrification. Therefore, the purpose of this study is to determine the global DNA methylation following vitrification in order to ensure the safe protocol for oocyte vitrification.

Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos.

Epigenetic modifications of the genome, such as covalent modification of histone residues, ensure appropriate gene activation during pre-implantation development, and are probably involved in the asymmetric reprogramming of the parental genomes after fertilization. We investigated the methylation patterns of histone H3 at lysine 9 (H3/K9), and the regulatory mechanism involved in the asymmetric remodeling of parental genomes during early preimplantation development in mice. Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 showed a very weak or absent methylation signal in the male pronucleus, whereas a distinct methylation signal was detected in the female pronucleus. This asymmetric H3/K9 methylation pattern in the different parental genomes persisted until the two-cell stage. However, de novo methylation of H3/K9 occurred and the asymmetry was lost during the four-cell stage. The unmethylated male pronucleus underwent de novo methylation when it was transferred into enucleated GV- or MII-stage oocytes, which suggests that histone H3 methylase is active before fertilization, but not afterwards, and that the asymmetric methylation pattern is generated by this change in methylase activity in the cytoplasm after fertilization. Thus, histone H3 is methylated only in the maternal chromosomes, which are present in the oocytes before fertilization, and is not methylated in the paternal chromosomes, which are absent. The maintenance of asymmetric H3/K9 methylation patterns in early embryos is an active process that depends on protein synthesis and zygotic transcription, as de novo methylation in the male pronucleus occurred when either protein synthesis or gene expression was inhibited by cycloheximide or alpha-amanitin, respectively. In addition, corresponding de novo methylation of H3/K9 and DNA occurred when the male pronucleus was transferred to an enucleated GV oocyte. Our results suggest that H3/K9 methylation is an epigenetic marker of parental genome origin during early preimplantation development.

Methylation of Microinjected Isoaspartyl Peptides in Xenopus oocytes: Competition with Protein Carboxyl Methylation Reactions

Xenopus oocytes possess a highly conserved protein carboxyl methyltransferase postulated to function in the repair or metabolism of age-damaged protein aspartyl residues (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). Three hexapeptides of the general sequence Val-Tyr-Pro-isoAsp-X-Ala, in which isoAsp represents an L-isoaspartyl residue and X represents Gly, Ser, or Ala, are methylated with the same order of preference following their microinjection into oocytes as in a purified system containing bovine brain protein carboxyl methyltransferase and S-adenosyl-L-[methyl-3H]methionine. The affinities of the enzyme for the glycyl, seryl, and alanyl variants of the peptides in vitro are 4.25, 3.04, and 1.67 microM, respectively. A nonapeptide of the sequence Lys-Ala-Ser-Ala-isoAsp-Leu-Ala-Lys-Tyr is a higher affinity substrate for the methyltransferase in vitro, characterized by a Km of 0.88 microM, but it is modified to a lesser extent in oocytes, partially because of its reduced stability in cytoplasm. The hexapeptide Val-Tyr-Pro-Asp-Gly-Ala, which contains an aspartyl residue in the usual stereoconfiguration, is not methylated either in vitro or in intact oocytes. Microinjection of any of the four isoaspartyl-containing peptides greatly stimulates total carboxyl methylation in oocytes, with rate increases ranging from 19- to 51-fold after the injection of 30 pmol of peptide. The protein ovalbumin is also modified following its microinjection into oocytes to near its calculated methyl-accepting capacity. Each of the isoaspartyl peptides can act as a competitive inhibitor of ovalbumin methylation both in vitro and in microinjected oocytes. The inhibitory potencies of the peptides parallel their specific methyl-accepting activities. The results demonstrate that the oocyte may be a useful model for studying the significance of protein carboxyl methylation because of the large functional excess of methylation capacity and the fidelity of the reactions compared to those observed in purified systems. This excess capability may have physiological significance when structurally abnormal proteins accumulate as a result of cellular stress and or aging.