Epigenetic disorders and altered gene expression after use of Assisted Reproductive Technologies in domestic cattle

Abstract

The use of Assisted Reproductive Technologies (ARTs) in modern cattle breeding is an important tool for improving the production of dairy and beef cattle. A frequently employed ART in the cattle industry is in vitro production of embryos. However, bovine in vitro produced embryos differ greatly from their in vivo produced counterparts in many facets, including developmental competence. The lower developmental capacity of these embryos could be due to the stress to which the gametes and/or embryos are exposed during in vitro embryo production, specifically ovarian hormonal stimulation, follicular aspiration, oocyte in vitro maturation in hormone supplemented medium, sperm handling, gamete cryopreservation, and culture of embryos. The negative effects of some ARTs on embryo development could, at least partially, be explained by disruption of the physiological epigenetic profile of the gametes and/or embryos. Here, we review the current literature with regard to the putative link between ARTs used in bovine reproduction and epigenetic disorders and changes in the expression profile of embryonic genes. Information on the relationship between reproductive biotechnologies and epigenetic disorders and aberrant gene expression in bovine embryos is limited and novel approaches are needed to explore ways in which ARTs can be improved to avoid epigenetic disorders.

Keywords: DNA methylation; embryo culture; genome imprinting; in vitro maturation; superovulation.

Figure 1. Epigenetic landscape during embryo development. Several epigenetic changes occurring during gamete formation and early embryo development could alter gene expression which in turn negatively affects embryo production. Histone acetylation in specific lysine residues is mostly associated with transcriptional activity, whereas methylation of other histone amino acids and DNA methylation tends to be linked with transcriptional repression.

Figure 2. Schematic drawing of the main steps of limiting dilution bisulfite sequencing. (A) Immature and mature oocytes are collected by OPU or after IVM. Ten oocytes of a defined group are pooled. (B) DNA isolation and bisulfite conversion. (C) Dilution of the converted oocyte DNA. (D) The diluted DNA is distributed over 20 wells on a microtiter plate. Most wells contain either no or a single DNA target molecule (ideograms); few wells may contain two or more copies. In addition, six negative controls (N) are added. First-round multiplex PCR is performed with outer primers for the PRDX1, ZAR1, and SLC2A1 genes.(E) Second-round singleplex PCRs of the three studied genes in individual microtiter plates (indicated by different colors) using 1 ml multiplex PCR product as template and gene-specific inner primers. (F) Second-round PCR products are visualized on agarose gels. The color code of each lane indicates the plate (gene), numbers, and Ns of the specific well on that plate. DNA from wells containing a PCR product is analyzed by direct bisulfite sequencing.,,

Figure 3. Relative poly(A) mRNA abundance of imprinted genes, methyltransferases, based on single cell preparations of 8–15 oocytes per group: Immature (shaded), mSOF (black), TCM (vertically lined), and in vivo (white).

Figure 4. Effects of SOV, IVM, sperm manipulation, IVC, and cryopreservation on epigenetic marks and changes in expression of genes in oocytes, sperm, embryos, organs and tissues. Genes with aberrant pattern are marked in red; genes with normal pattern are marked black.

Figure 5. Factors inducing epigenetic disorders and changes in gene expression in the in vitro embryo production. Protocols for superovulation, substances with ambiguous effects such as fetal calf serum (FCS), culture conditions including changes in pH, osmolality, temperature and various basic culture media may affect the normal epigenetic phenotype during early development, and thereby decrease the quality of the embryos.