Mammalian embryo culture media: now and into the future.

For over 70years, since the culture of the first mammalian embryo in vitro , scientists have undertaken studies to devise and optimise media to support the manipulation and culture of gametes and embryos. This area of research became especially active in the late 1970s onwards following the successful birth of the first human in vitro fertilised embryo. This review summarises some of the key advances in mammalian embryo culture media over time based on a greater understanding of the biochemical milieu of the reproductive tract. It highlights how learnings from studies in mice and agricultural species have informed human culture media compositions, in particular the inclusion of albumin, growth factors, cytokines, and antioxidants into contemporary culture media formulations, and how these advances may then in turn help to inform and guide development of in vitro culture systems used in other arenas, in particular agriculture. Additionally, it will highlight how the introduction of new technologies, such as timelapse, can influence current trends in media composition and usage that may see a return to a single step medium.

Reduced levels of two modifiers of epigenetic gene silencing, Dnmt3a and Trim28, cause increased phenotypic noise.

BACKGROUND: Inbred individuals reared in controlled environments display considerable variance in many complex traits but the underlying cause of this intangible variation has been an enigma. Here we show that two modifiers of epigenetic gene silencing play a critical role in the process. RESULTS: Inbred mice heterozygous for a null mutation in DNA methyltransferase 3a (Dnmt3a) or tripartite motif protein 28 (Trim28) show greater coefficients of variance in body weight than their wild-type littermates. Trim28 mutants additionally develop metabolic syndrome and abnormal behavior with incomplete penetrance. Genome-wide gene expression analyses identified 284 significantly dysregulated genes in Trim28 heterozygote mutants compared to wild-type mice, with Mas1, which encodes a G-protein coupled receptor implicated in lipid metabolism, showing the greatest average change in expression (7.8-fold higher in mutants). This gene also showed highly variable expression between mutant individuals. CONCLUSIONS: These studies provide a molecular explanation of developmental noise in whole organisms and suggest that faithful epigenetic control of transcription is central to suppressing deleterious levels of phenotypic variation. These findings have broad implications for understanding the mechanisms underlying sporadic and complex disease in humans.

The role of epigenetics in mediating environmental effects on phenotype.

Epigenetics is being suggested as a possible interface between the genetic and environmental factors that together give rise to phenotype. In mice there exists a group of genes, known as metastable epialleles, which are sensitive to environmental influences, such as diet, and undergo molecular changes that, once established, remain for the life of the individual. These modifications are epigenetic and in some cases they survive across generations, that is, through meiosis. This is termed transgenerational epigenetic inheritance. These findings have led to the idea that similar processes might occur in humans. Although it is clear that the lifestyle of one generation can significantly influence the health of the next generation in humans, in the absence of supporting molecular data it is hard to justify the notion that this is the result of transgenerational epigenetic inheritance. What is required first is to ascertain whether genes of this type, that is genes that are sensitive to the epigenetic state, even exist in humans.

A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development.

BACKGROUND: Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design. RESULTS: We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome. CONCLUSIONS: These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.

The case for transgenerational epigenetic inheritance in humans.

Work in the laboratory mouse has identified a group of genes, called metastable epialleles, that are informing us about the mechanisms by which the epigenetic state is established in the embryo. At these alleles, transcriptional activity is dependent on the epigenetic state and this can vary from cell to cell in the one tissue type. The decision to be active or inactive is probabilistic and sensitive to environmental influences. Moreover, in some cases the epigenetic state at these alleles can survive across generations, termed transgenerational epigenetic inheritance. Together these findings raise the spectre of Lamarckism and epigenetics is now being touted as an explanation for some intergenerational effects in human populations. In this review we will discuss the evidence so far.

Multiplex detection of surface molecules on colorectal cancers.

A technique of fluorescence multiplexing is described for analysis of the plasma membrane proteome of colorectal cancer cells from surgically resected specimens, enabling detection and immunophenotyping when the cancer cells are in the minority. A single-cell suspension was prepared from a colorectal tumour, and the mixed population of cells was captured on a CD antibody microarray. The cancer cells were detected using a fluorescently tagged antibody for carcinoembryonic antigen (CEA-Alexa647) or epithelial cell adhesion marker (EpCAM-Alexa488). Using this multiplexing procedure, dot patterns from colorectal cancers were distinct from those of adjacent normal tissue. Subtraction of the expression levels for each antigen from normal tissue from those for the cancer shows differential expression in the cancer of CD66c, CD15s, CD55, CD45, CD71, CD45RO, CD11b and CEA, in descending order. Cells captured on the same microarray were also labelled with fluorescent CD3-phycoerythrin antibody revealing the presence of tumour-infiltrating lymphocytes. The immunophenotypes of T lymphocytes from the tumour samples showed differential expression of HLA-DR, TCR alpha/beta, CD49d, CD52, CD49e, CD5, CD95, CD28, CD38 and CD71, in descending order. Fluorescence multiplexing of mixed cell populations captured on a single antibody microarray enables expression profiling of multiple sub-populations of cells within a tumour sample.