Loss of genomic imprinting in mouse embryos with fast rates of preimplantation development in culture

Currently, the stage of embryo development has been proposed as one of many criteria for identifying healthy embryos in infertility clinics with the fastest embryos being highlighted as the healthiest. However the validity of this as an accurate criterion with respect to genomic imprinting is unknown. Given that embryo development in culture generally requires an extra day compared to in vivo development, we hypothesized that loss of imprinting correlates with slower rates of embryonic development. To evaluate this, embryos were recovered at the 2-cell stage, separated into four groups based on morphological stage at two predetermined time points, and cultured to blastocysts. We examined cell number, embryo volume, embryo sex, imprinted Snrpn and H19 methylation, imprinted Snrpn, H19, and Cdkn1c expression, and expression of genes involved in embryo metabolism-Atp1a1, Slc2a1, and Mapk14-all within the same individual embryo. Contrary to our hypothesis, we observed that faster developing embryos exhibited greater cell numbers and embryo volumes as well as greater perturbations in genomic imprinting and metabolic marker expression. Embryos with slower rates of preimplantation development were most similar to in vivo derived embryos, displaying similar cell numbers, embryo volumes, Snrpn and H19 imprinted methylation, H19 imprinted expression, and Atp1a1 and Slc2a1 expression. We conclude that faster development rates in vitro are correlated with loss of genomic imprinting and aberrant metabolic marker expression. Importantly, we identified a subset of in vitro cultured embryos that, according to the parameters evaluated, are very similar to in vivo derived embryos and thus are likely most suitable for embryo transfer.     

Genomic imprinting in the placenta

Genomic imprinting is an epigenetic mechanism that is important for the development and function of the extra-embryonic tissues in the mouse. Remarkably all the autosomal genes which were found to be imprinted in the trophoblast (placenta) only are active on the maternal and repressed on the paternal allele. It was shown for several of these genes that their paternal silencing is not dependent on DNA methylation, at least not in its somatic maintenance. Rather, recent studies in the mouse suggest that placenta-specific imprinting involves repressive histone modifications and non-coding RNAs. This mechanism of autosomal imprinting is similar to imprinted X chromosome inactivation in the placenta. Although the underlying reasons remain to be explored, this suggests that imprinting in the placenta and imprinted X inactivation are evolutionarily related.     

A culture system for the development of the preimplantation rat embryo

We describe a system for the culture of 1-cell rat embryos through to the blastocyst stage, using co-culture on specific feeder cell layers and particular defined media. We show that the use of rat uterine epithelial cells as a feeder layer, together with either M16M, CZB or HECM-1 media at 38.5 degrees C can improve the in vitro development of cultured rat embryos. There was considerable variation in the culture conditions, which were optimal for each strain of rat tested. We show that the 4 to 8-cell embryos are viable after reimplantation and that the second 4 to 8-cell block in the rat can be overcome using HECM-1 in a co-culture system, thus enabling the in vitro culture of rat embryos up to the blastocyst stage.     

Regulation of proliferation and apoptosis during development of the preimplantation embryo and the placenta

The preimplantation embryo starts as a single cell, the zygote. The first cell divisions do not lead to volume expansion, but rather to an increasing number of small cells. At the morula stage the first two cell lineages differentiate into the trophoblast and the inner cells mass/embryoblast. During development of the preimplantation embryo, apoptosis occurs only after the onset of the embryonic genome. It has become clear that the development of a healthy child requires not only very high rates of proliferation and differentiation, but also apoptosis, which is a crucial mechanism for morphogenesis and the development of the inner organs. Furthermore, the generation of specific cell types, such as lens cells, erythrocytes, and thrombocytes, depends on the apoptosis pathways. This is also true later in gestation, when the trophoblasts form the placenta and provide the epithelial cover of the villous trees of the placenta. This layer is in direct contact with maternal blood and, as do all epithelia, displays a continuous turnover of cells. Thus, apoptosis is a normal constituent of survival in this layer as well, and changes in the regulation and rate of apoptosis have deleterious effects on the trophoblast and consequently the developing embryo or fetus. Here we present a very brief overview of the importance of apoptosis for the development of the preimplantation embryo and the maintenance of placental trophoblasts. Furthermore, we highlight what happens when regulation of proliferation or apoptosis fails in these systems, and attempt to show that apoptosis is only the consequence of poor embryo or trophoblast development -- not its cause.     

X chromosome imprinting and inactivation in preimplantation mammalian embryos

Dosage compensation for the mammalian X chromosome involves the silencing of one X chromosome to achieve equal X-linked gene expression between males and females. X chromosome inactivation (XCI) is controlled by a complex set of genetic elements located in a region known as the X chromosome inactivation center, and is regulated by a combination of genomic imprinting, cell lineage-dependent erasure of imprinting, an unidentified mechanism of X chromosome counting, an incompletely understood means of selection of one X chromosome for inactivation and developmentally regulated changes in X chromosome chromatin. A detailed understanding of when and how these components of XCI occur is essential for elucidating the operative mechanisms. A model accounting for early events related to XCI, including observations in uniparental and aneuploid embryos, is presented.     

Loss of Xist imprinting in diploid parthenogenetic preimplantation embryos.

We have analysed Xist expression patterns in parthenogenetic and control fertilised preimplantation embryos by using RNA FISH. In normal XX embryos, maternally derived Xist alleles are repressed throughout preimplantation development. Paternal alleles are expressed as early as the 2-cell stage. In parthenogenetic embryos, we observed Xist RNA expression and accumulation from the morula stage onwards, indicating loss of maternal imprinting. In the majority of cells, expression was from a single allele, indicating that X chromosome counting occurs to establish appropriate monoallelic Xist expression. We discuss these data in the context of models for regulation of imprinted and random X inactivation.     

Establishment and maintenance of H19 imprinting in the germline and preimplantation embryo

The mouse H19 and Igf2 genes are oppositely imprinted and share enhancers that reside 3' to the genes. The imprinted expression of these genes is coordinated by a 2-kb regulatory element, the differentially methylated domain (DMD), positioned between the two genes. The methylation status of this region determines the ability of the insulator factor CTCF to bind to its sites in the DMD. Deletions and mutations of the DMD that affect imprinting in the soma have little effect on the methylation pattern of H19 in the germline, suggesting that additional sequences and factors contribute to the earliest stages of imprinting regulation at this locus. Less is known about these initial steps, which include the marking of the parental alleles, the onset of allele-specific expression patterns and maintenance of the imprints in the preimplantation embryo. Here, we will focus on these early steps, summarizing what is known and what questions remain to be addressed.     

Selective abortion and the evolution of genomic imprinting

Mothers can determine which genotypes of offspring they will produce through selective abortion or selective implantation. This process can, at some loci, favour matching between maternal and offspring genotype whereas at other loci mismatching may be favoured (e.g. MHC, HLA). Genomic imprinting generally renders gene expression monoallelic and could thus be adaptive at loci where matching or mismatching is beneficial. This hypothesis, however, remains unexplored despite evidence that loci known to play a role in genetic compatibility may be imprinted. We develop a simple model demonstrating that, when matching is beneficial, imprinting with maternal expression is adaptive because the incompatible paternal allele is not detected, protecting offspring from selective abortion. Conversely, when mismatching is beneficial, imprinting with paternal expression is adaptive because the maternal genotype is more able to identify the presence of a foreign allele in offspring. Thus, imprinting may act as a genomic ‘cloaking device’ during critical periods in development when selective abortion is possible.     

Embryo sac development during the culture of placenta attached ovules ofMelandrium album

Ovules ofMelandrium album, attached to the placentas and containing immature embryo sacs, were culturedin vitro. The embryo sacs developed according to thePolygonum type but about 70% of them degenerated during the culture. Gametophytes of a non-typical structure were found in a few ovules,i.e. there appeared more nuclei or cells as in thePolygonum type and the arrangement of nuclei or cells was not true to type. Several-celled structures observed in some embryo sacs were recognized as gynogenetic embryoids.     

Differential regulation of imprinting in the murine embryo and placenta by the Dlk1-Dio3 imprinting control region

Genomic imprinting is an epigenetic mechanism controlling parental-origin-specific gene expression. Perturbing the parental origin of the distal portion of mouse chromosome 12 causes alterations in the dosage of imprinted genes resulting in embryonic lethality and developmental abnormalities of both embryo and placenta. A 1 Mb imprinted domain identified on distal chromosome 12 contains three paternally expressed protein-coding genes and multiple non-coding RNA genes, including snoRNAs and microRNAs, expressed from the maternally inherited chromosome. An intergenic, parental-origin-specific differentially methylated region, the IG-DMR, which is unmethylated on the maternally inherited chromosome, is necessary for the repression of the paternally expressed protein-coding genes and for activation of the maternally expressed non-coding RNAs: its absence causes the maternal chromosome to behave like the paternally inherited one. Here, we characterise the developmental consequences of this epigenotype switch and compare these with phenotypes associated with paternal uniparental disomy of mouse chromosome 12. The results show that the embryonic defects described for uniparental disomy embryos can be attributed to this one cluster of imprinted genes on distal chromosome 12 and that these defects alone, and not the mutant placenta, can cause prenatal lethality. In the placenta, the absence of the IG-DMR has no phenotypic consequence. Loss of repression of the protein-coding genes occurs but the non-coding RNAs are not repressed on the maternally inherited chromosome. This indicates that the mechanism of action of the IG-DMR is different in the embryo and the placenta and suggests that the epigenetic control of imprinting differs in these two lineages.     

Cytoskeleton in preimplantation mouse development

This paper reviews the constituents of the cytoskeleton in the cells of the preimplantation mouse embryo and how they change as the development proceeds. The cytoskeleton can be divided into two distinct groups, that in the cytosplasm and that associated with the membrane. The first and better-known group contains microfilaments, microtubules and intermediate filaments, the second such components of the cell and nuclear membrane as spectrin-like protein and nuclear lamin. The filamentous components of the cytoplasmic cytoskeleton adhere to the nuclear and cell membrane at attachment points where specific proteins such as vinculin may mediate the interaction. Each cell of the early embryo has all of these components, but their morphological organization and molecular constitution alter as the embryo develops. These modifications are especially pronounced when the cleavage-stage embryo compacts and when the blastocysts forms and differentiates. These events represent the most critical stages of morphogenesis and cytodifferentiation in the preimplantation embryo. The cytoskeleton may thus have an important role in the control of the early mammalian development.     

G9a histone methyltransferase contributes to imprinting in the mouse placenta

Whereas DNA methylation is essential for genomic imprinting, the importance of histone methylation in the allelic expression of imprinted genes is unclear. Imprinting control regions (ICRs), however, are marked by histone H3-K9 methylation on their DNA-methylated allele. In the placenta, the paternal silencing along the Kcnq1 domain on distal chromosome 7 also correlates with the presence of H3-K9 methylation, but imprinted repression at these genes is maintained independently of DNA methylation. To explore which histone methyltransferase (HMT) could mediate the allelic H3-K9 methylation on distal chromosome 7, and at ICRs, we generated mouse conceptuses deficient for the SET domain protein G9a. We found that in the embryo and placenta, the differential DNA methylation at ICRs and imprinted genes is maintained in the absence of G9a. Accordingly, in embryos, imprinted gene expression was unchanged at the domains analyzed, in spite of a global loss of H3-K9 dimethylation (H3K9me2). In contrast, the placenta-specific imprinting of genes on distal chromosome 7 is impaired in the absence of G9a, and this correlates with reduced levels of H3K9me2 and H3K9me3. These findings provide the first evidence for the involvement of an HMT and suggest that histone methylation contributes to imprinted gene repression in the trophoblast.     

Abnormal development and transport and increased sister-chromatid exchange in preimplantation embryos following superovulation in mice

Both sister-chromatid exchange (SCE) response and embryonic development and transport in preimplantation embryos were evaluated on day 3 of gestation (vaginal plug = 1) of superovulated Swiss mice. Superovulation was found to have significant effects on number of preimplantation embryos (increase), embryo localization (accelerated transport), cleavage rate (advanced development) and abnormality rate (misshaped, fragmented, dead embryos). Superovulated 4- and 8-cell embryos collected from oviducts and uteri and incubated in vitro with 5-bromodeoxyuridine (BrdU) displayed up to 4 times higher SCE frequency than spontaneously ovulated embryos. This increase is independent of stage of development and location at the time of embryo collection. The results indicate that superovulated embryos may have induced DNA lesions.     

Metabolism of progesterone by preimplantation mouse blastocysts in culture

This study examined the question whether or not preimplantation mouse blastocysts can metabolize progesterone (P). When young (Day 4) and implanting (Day 5) blastocysts were cultured in supplemented Eagle's minimum essential medium containing 0.4 microM [3H]P, metabolism of P and formation of metabolites were noticed at 10 h of culture. The metabolites accumulated in medium as the culture continued to 118 h. Three of the four metabolite fractions were identified, by crystallization to constant sp. act., to be 5 alpha-pregnane-3,20-dione and 3 beta-hydroxy-5 alpha-pregnan-20-one (or allopregnanolone), accounting for 22 and 57% of radioactivity, respectively, and a small amount (1-10%) of 3 alpha-hydroxy-5 alpha-pregnan-20-one. This suggests that both delta 4-5 alpha-reductase and 3 alpha- and 3 beta-hydroxysteroid dehydrogenase are active. Day 5 blastocysts were much more active than Day 4 blastocysts in P metabolism. It is suggested that the ability of blastocysts to metabolize P could produce the following effects in the adjacent endometrium: a lessening of P effects; and consequently a change in P-estrogen interaction; and possible effects from the metabolites. These local effects of embryos on the endometrium may be important for embryonic development and implantation.     

Genomic imprinting in fetal growth and development

Each somatic cell of the human body contains 46 chromosomes consisting of two sets of 23; one inherited from each parent. These chromosomes can be categorised as 22 pairs of autosomes and two sex chromosomes; females are XX and males are XY. Similarly, at the molecular level, two copies of each autosomal gene exist; one copy derived from each parent. Until the mid-1980s, it was assumed that each copy of an autosome or gene was functionally equivalent, irrespective of which parent it was derived from. However, it is now clear from classical experiments in mice and from examples of human genetic disease that this is not the case. The functional activity of some genes or chromosomal regions is unequal, and dependent on whether they have been inherited maternally or paternally. This phenomenon is termed 'genomic imprinting' and the activity or silence of an imprinted gene or chromosomal region is set during gametogenesis. Genomic imprinting involving the autosomes appears to be restricted to eutherian mammals, and has most likely evolved as a result of the conflicting concerns of the parental genomes in the growth and development of their offspring. When the normal pattern of imprinting is disrupted, the phenotypes observed in humans and mice are generally associated with abnormal fetal growth, development and behaviour, illustrating its importance for a normal intrauterine environment. The characteristics of imprinted genes, their regulation and the phenotypes associated with altered imprinting are discussed.     

Igf2 imprinting in development and disease

Igf2 is one of the first imprinted genes discovered and occupies a centre stage in the study of imprinting. This is because it has dramatic effects on the control of fetal growth, it is involved in growth disorders and in cancer, it interacts with products of other imprinted genes, and its imprinting status is under complex regulation in a cluster of tightly linked imprinted genes. Here we review briefly the key features of Igf2 imprinting in normal development and in disease, and hope to show what a fascinating subject of study this gene and its biology provides.