Experimental design - MATERIALS AND METHODS

RES ETT ING

3 MATERIALS AND METHODS

3.10 Experimental design

This research project can be divided into four major parts (a schematic overview on the entire project is given in fig. 30):

1. Bovine embryo production and evaluation of cell numbers

2. mRNA gene expression analyses of the bovine IGF2, IGF2R and MASH2 genes

3. Sequencing of three fragments within the bovine IGF2 gene

4. Study of DNA methylation: Intragenic Differentially Methylated Regions (DMRs), methylation status of bovine preimplantation embryos from different origins

3.10.1 Bovine embryo production and evaluation of cell numbers

Bovine preimplantation embryos produced by in vitro fertilization and parthenogenesis, were employed for molecular analyses. The numbers of trophectoderm and inner cell mass cells were determined from a subset of blastocysts and expanded blastocysts. The cell numbers were used to calculate the relative amounts of transcriptional products on a per cell basis. Differences in the relative amounts of mRNAs were evaluated as cell specific gene expression i.e. in the trophectoderm or in the ICM, comparing in vitro fertilized and parthenogenetic bovine blastocysts. Size of blastocysts and expanded blastocysts at day 7 of development was determined. In addition, single trophectoderm and single plugs of inner cell mass cells were isolated from a few expanded blastocysts and were included in the gene expression analyses.

3.10.2 mRNA gene expression analyses of the bovine IGF2, IGF2R and MASH2 genes

The expression patterns of the bovine IGF2, IGF2R and MASH2 genes were analyzed by semi-quantitative RT-PCR. The three genes are known being imprinted in the mouse (DeCHIARA et al. 1991; BARLOW et al. 1991; GUILLEMOT et al.

1995). Mono- or bi-allelic gene expression should be resolved in this study by comparing in vitro fertilized and parthenogenetic blastocysts and isolated single trophectoderm and ICM cells derived thereof. In vivo collected expanded blastocysts served as controls. Due to the high number of embryos required to analyze bovine IGF2 gene expression, only blastocysts produced in vitro were analyzed.

The hypothesis of this study (fig. 29) was that maternally expressed genes such as the IGF2R and MASH2 genes (in the mouse) would show increased relative amounts of transcriptional products in parthenogenetic embryos (diploid maternal genome). Paternally expressed genes such as the IGF2 gene (in the mouse) would be expressed at a higher level in embryos derived from in vitro fertilization (paternal and maternal genome). If no differences were detected between the transcriptional products of in vitro fertilized and parthenogenetic embryos bi-allelic expression was assumed.

Figure 29: Hypothesis of mono-and bi-allelic gene expression

Maternally expressed genes would be expressed at a higher level in parthenogenetic embryos whereas paternally expressed genes would only be expressed in in vitro fertilized

Maternally expressed genes Paternally expressed genes

Single abundance

In vitroproduced embryos In vitroproduced

embryos

Parthenogenetic embryos Maternally expressed genes Paternally expressed genes

Single abundance

In vitroproduced embryos In vitroproduced

embryos

Parthenogenetic embryos

3.10.3 Sequencing of three fragments within the bovine IGF2 gene

The ovine IGF2 gene was analyzed for CpG islands to identify regions to sequence within the bovine IGF2 gene. This was necessary due to the lack of genomic sequence information for the bovine IGF2 gene in the GenBank database.

The mRNA sequence of the bovine IGF2 gene is available in the GenBank database (X53553). Fragments of the bovine IGF2 regions were obtained by PCR amplification first with ovine specific primers in bovine tissue and subsequently with bovine specific primers to sequence the complete fragment. CpG islands were verified within the bovine IGF2 fragments.

3.10.4 Study of DNA methylation: Intragenic Differentially Methylated Regions (DMRs), methylation status of bovine preimplantation embryos derived from different origins

Three DNA fragments within the bovine IGF2 gene were analyzed to identify intragenic DMRs by bisulfite sequencing. DMRs are established during primordial germ cell development and thought to be present in mature oocytes and sperm. DNA isolated from in vitro matured oocytes and frozen/thawed sperm was analyzed within the fragments of bovine introns 4, 5 and bovine exon 10.

The methylation status of the intragenic DMR was investigated in expanded day 7 blastocysts derived from different origins and in adult fibroblasts, which served as donor cells to generate nuclear transfer embryos. Adult fibroblasts were derived from a female and male slaughtered animal. These analyses were performed using bisulfite sequencing and served to determine methylation differences between embryos. Differences in the embryonic methylation status could indicate insufficient reprogramming and imprinting failure during in vitro production of bovine preimplantation embryos.

(A: androgenetic embryos; NT: nuclear transfer embryos; DC: donor cells of cloned embryos; Vivo: in vivo collected embryos) Bovine embryo production

and cell numbers of bovine embryos

derived from different origins

Gene expression analysis Sequencing of the bovine DNA methylation analyses

IGF2 gene

Determination of cell numbers by differential

staining

Collection and measurement of blastocysts (BL) and expanded blastocysts (eBL)

Isolation of trophectoderm (TE)

Isolation of inner cell mass (ICM)

Insulin-like growth factor 2 gene (IGF2)

Insulin-like growth factor 2 receptor gene (IGF2R)

Mammalian achaete-scute homolog 2 gene (MASH2)

PCR amplification of bovine tissue DNA

Bovine embryo production and cell numbers of bovine embryos