Analysis of imprinted gene expression in normal fertilized and uniparental preimplantation porcine embryos

Abstract

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos.

Figure 1. Relative expression levels of the eight imprinted genes in porcine MII oocytes and in diploid normal and uniparental embryos from the two-cell to the blastocyst stage.

The relative levels of mRNA were quantified using qRT-PCR and then calculated with the 2^{−ΔΔ Ct} method . Five replicate samples were examined for each class. The values from transcripts of the imprinted genes in PG and AG blastocysts, after normalization relative to the β ACTIN (internal control) gene, were compared to those of IVF counterparts which were taken as a standard (1). This data is presented as mean ± SEM. The relative abundance of eight imprinted genes among the different types of embryos at each stage are shown; A; the 2-cell (n = 25), B; the 4-cell (n = 25), C; the 8∼16-cell (n = 15), D; the morula (n = 10), and E; the blastocyst stage (n = 5) of porcine embryos.

Figure 2. Analysis of imprinted gene expression in in vivo derived and in vitro fertilized blastocysts.

Y-value is expressed as a relative fold change in mRNA levels in in vitro blastocysts compared with that of the in vivo ones defined as 1, (n = 10). This data is presented as mean ± SEM (n = 5).

Figure 3. Differential expression of XIST transcripts in individual in vivo blastocysts.

Each value derived from transcripts of the XIST gene in in vivo blastocysts, after normalization relative to β ACTIN (internal control), were compared with that of one of 10 in vivo blastocysts defined as 1. Of these, the labeled No. 3 and No. 4 samples were determined their sex by SRY gene; ^F and ^M indicate female and male embryos, respectively.

Figure 4. Imprinted gene expression of haploid and diploid PG blastocysts.

Haploid PG blastocysts were generated using the electrical activation method without cytochalasin D treatment. Zygotes possessing two polar bodies and a small pronucleus (presumed haploid) or with a polar body and a large pronucleus or two pronuclei (presumed diploid) were selected by Hoechst staining at 12 to 14 hr following parthenogenetic activation, respectively. Results for each sample were conducted in triplicate. Y-value is expressed as a relative fold change in mRNA levels in haploid PG blastocysts (n = 5) compared with that of the diploid ones (n = 5) defined as 1. The Data are presented as means ± SEM.

Figure 5. The methylation status of IGF2/H19 DMR3 in porcine haploid and diploid PG blastocysts.

The methylation patterns of DMR3 in porcine A; adult liver tissue (1×10⁵ cells), B; MII oocytes (n = 100), C; sperm (1×10⁶ sperm cells), D; IVF (n = 50), E; haploid PG (n = 100), and F; diploid PG (n = 50) blastocysts are shown. Individual circles indicate a CpG dinucleotide. Open and solid circles represent unmethylated and methylated CpGs, respectively. Each horizontal line represents one individual clone from three independently amplified PCR products.