Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6-8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6-8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation. © 2013 Dobbs et al

Optimal Single-embryo Mass Spectrometry Fingerprinting.

In pre-implantation embryos, lipids play key roles in determining viability, cryopreservation and implantation properties, but often their analysis is analytically challenging because of the few picograms of analytes present in each of them. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) allows obtaining individual phospholipid profiles of these microscopic organisms. This technique is sensitive enough to enable analysis of individual intact embryos and monitoring the changes in membrane lipid composition in the early stages of development serving as screening method for studies of biology and biotechnologies of reproduction. This article introduces an improved, more comprehensive MALDI-MS lipid fingerprinting approach that considerably increases the lipid information obtained from a single embryo. Using bovine embryos as a biological model, we have also tested optimal sample storage and handling conditions before the MALDI-MS analysis. Improved information at the molecular level is provided by the use of a binary matrix that enables phosphatidylcholines, sphingomyelins, phosphatidylserines, phosphatidylinositols and phosphoethanolamines to be detected via MALDI(±)-MS in both the positive and negative ion modes. An optimal MALDI-MS protocol for lipidomic monitoring of a single intact embryo is therefore reported with potential applications in human and animal reproduction, cell development and stem cell research.48844-

Developmental changes in labeling of immunoreactive 5-methylcytosine from the 2-cell to blastocyst stages of development.

<p>Data represent the ratio of fluorescent intensity for anti-5-methylcytosine to that for propidium iodide. There was an overall effect of stage on labeling intensity (P<0.0001). Means with different superscripts differ (P<0.05). Data are least-squares means±SEM of results from 11–24 embryos per stage.</p

Effects of gender and cell type [inner cell mass (ICM) and trophectoderm (TE)] on immunoreactive 5-methylcytosine in blastocysts.

<p>Data represent the ratio of fluorescent intensity for anti-5-methylcytosine to that for propidium iodide. Data are least-squares means±SEM of results from 14–26 embryos per gender. Amounts of DNA methylation were affected by gender (P<0.02), and cell type (P<0.0001) but not the gender × cell type interaction (P<0.25).</p

Developmental changes in steady-state mRNA for <i>DNMT3B</i>.

<p>Data for mRNA are expressed as fold-change relative to amounts for zygotes and represent the least-squares means±SEM of results from 5 pools of 30 embryos per stage. Means with different superscripts differ (P<0.05).</p

Characteristics of the high resolution melt analysis for methylation of an intronic region of <i>DNMT3B</i>.

<p>Panel A shows the location of the intronic region between exons 7 and 8 that was analyzed. Note the presence of five CpG eligible for methylation in the 81 bp region amplified by PCR. Panel B is a representative result for the standard curve generated by analysis of a mix of control DNA at 0% and 100% methylation. The graph is a plot of the difference relative fluorescence units (RFU) as a function of shifted temperature (X-axis normalization to reduce variation between wells).</p

Representative images of embryos labeled for immunoreactive 5- methylcytosine at the 2 cell, 4 cell, 6–8 cell, 9–25 cell, 32 cell-morula and blastocyst stages of development.

<p>The scale bar represents 26.75 µm.</p

Effects of gender on immunoreactive 5-methylcytosine in embryos at the 6–8 cell and blastocyst stages of development.

<p>Data represent the ratio of fluorescent intensity for anti-5-methylcytosine to that for propidium iodide. The solid line represents female embryos while the hashed line represents male embryos. Data are least-squares means±SEM of results from 11–21 embryos per stage. Amounts of DNA methylation were affected by gender (P = 0.0029), stage of development (P<0.0001) and the stage × gender interaction (P = 0.0007).</p

Representative images of an embryo in which labeling with CDX2 (blue) was used to evaluate immunoreactive 5-methylcytosine (green) in ICM (CDX2-negative) and TE (CDX2-positive).

<p>Nuclei of all cells were labeled with propidium iodide (red). Panels represent merged images for all three fluorescent labels (A), 5-methylcytosine and PI (B), CDX2 and PI (C), CDX2 and 5-methylcytosine (D) and control antibodies for anti-5methylcytosine and anti-CDX2 as well as PI (E). The scale bar is 20 µm.</p