Blastocyst morphology and gene expression analyses of the bovine IGF2, IGF2R and MASH2 genes

Semi-quantitative RT-PCR analyses revealed expression from both parental alleles of the IGF2 and the IGF2R genes whereas Mash2 was expressed mono-allelically, presumably from the paternal chromosome in bovine day 7 expanded blastocysts (fig. 32). The approach of this study employing in vitro fertilized (maternal and paternal genome) and parthenogenetic (two maternal genomes) embryos allows

transcriptional products in in vitro fertilized embryos compared to their parthenogenetic counterparts indicates paternal and/or bi-allelic expression. In case that the gene is paternally repressed and expressed only from the maternal allele, a higher relative amount of transcriptional products in parthenogenetic compared with in vitro fertilized embryos would indicate expression from only the two maternal alleles. This approach is only a first step in the characterization of a putative imprinting status. Beside, the ovine IGF2 gene was identified as imprinted by comparing the gene expression patterns of ovine parthenogenetic and IVF control fetuses (FEIL et al. 1998). In addition, no mRNA from paternally expressed genes was detected in parthenogenetic mice (WALSH et al. 1994; KANEKO-ISHINO et al.

1995; NISHITA et al. 1996). Although we could not discriminate the relative amounts of mRNA from the maternal or the paternal chromosome, the semi-quantitative RT-PCR assay used in this study can be a valuable tool to obtain first information on relative amounts of transcriptional products related to mono- or bi-allelic gene expression of putatively imprinted genes.

The semi-quantitative RT-PCR assay described in this study has been successfully employed in numerous studies in our laboratory and is highly sensitive and reproducible to determine relative amounts of individual mRNAs (WRENZYCKI et al. 2001; YASEEN et al. 2001; OROPEZA et al. 2004). Even tiny amounts of mRNA derived from single embryos were detected by this method (WRENZYCKI et al. 2001, 2002, 2003). Quantification of a specific mRNA is possible when a non-competitive agent such as a synthetic RNA molecule is added to the RT-PCR reaction (BUSTIN 2000). Rabbit globin mRNA has been added to the RT sample in our study and constitutes a suitable standard for quantification of transcriptional products derived from preimplantation embryos. In contrast to housekeeping genes frequently used as standards in endpoint and real time PCR assays (ROBERT et al.

2002; LONERGAN et al. 2003), the commercially available rabbit globin mRNA escapes environmental influences of the in vitro production protocol and has been proven as a reliable standard for semi-quantitative RT-PCR analyses. Previously, expression analyses of day 17 and day 60 bovine fetuses have revealed bi-allelic expression of the MASH2 gene with allele-specific RT-PCR. Thus, the bovine MASH2 gene cannot be considered as imprinted (ARNOLD et al. 2004). Parent-specific mRNA concentrations can be detected using allele-Parent-specific RT-PCR, but requires a polymorphism within the gene sequence. Identification of polymorphisms

within the IGF2, IGF2R and MASH2 sequences would have gone beyond the scope of this research project and therefore an initial characterization of imprinted gene expression was performed using semi-quantitative RT-PCR.

Mouse blastocysts express the Igf2 and the Igf2r genes from both parental alleles (FEIL et al. 1994). Our results demonstrate that bi-allelic expression of these two genes is conserved in mouse and cattle. However, the findings of a putative paternal expression of the bovine MASH2 gene in this study contradicts results from analyses in mouse blastocysts in which the paternal allele was turned off in early post-implantation embryos (TANAKA et al. 1999). Mash2 encodes a transcription factor critically involved in placental development in mouse embryos and is maternally expressed in trophectoderm cells (GUILLEMOT et al. 1995). Placentation in ruminants is non-invasive whereas human and rodents develop an invasive placenta and the fetus is in direct contact with the maternal circulation (RÜSSE and SINOWATZ 1991). It is conceivable that the MASH2 expression pattern has changed in ruminants during evolution. However, one has to bear in mind that the present experiments were performed with day 7 in vitro produced expanded blastocysts.

Recently, it was shown that bovine parthenotes possess fewer cells than bi-parental in vitro fertilized control embryos (99±29, 100±23 versus 185±60; LAGUTINA et al.

2004). The lower abundance of MASH2 mRNA in bovine parthenogenetic expanded blastocysts could also be attributed to a decreased number of trophectoderm cells.

Determination of effects derived from different cell numbers and/or blastocyst size required a more detailed analysis of the embryos employed in the semi-quantitative RT-PCR assay. Two types of blastocysts were discriminated: Blastocysts

<180 µm in size and expanded blastocysts >180 µm in size. In contrast to previous reports (VAN SOOM et al. 1997 a, b; LAGUTINA et al. 2004), we only observed subtle differences in the total cell number and allocation to inner cell mass and trophectoderm between in vitro fertilized and parthenogenetic blastocysts. The ratio between trophectoderm and inner cell mass was similar for all groups of blastocysts indicating that cell allocation was not different in the blastocysts used for the gene expression analyses.

Relative amounts of transcriptional products from the IGF2 gene were significantly increased in expanded in vitro fertilized blastocysts compared to

non-transcriptional products were often obtained despite rigorous optimization of PCR conditions. It is thought that in vitro culture systems and the relative amount of IGF2 mRNAs detected in bovine day 7 embryos are to some extent correlated (LIU et al.

1997; LONERGAN et al. 2003; GUTIERREZ-ADAN et al. 2004).

The IGF2R and MASH2 genes were both bi-allelically expressed in bovine in vitro fertilized and parthenogenetic blastocysts. Results obtained for the MASH2 gene are consistent with the bi-allelic MASH2 expression pattern reported by ARNOLD et al. (2004). Expanded blastocysts derived from in vitro fertilization and parthenogenesis contained relative concentrations of IGF2R and MASH2 mRNAs that were similar to those of in vivo derived expanded blastocysts. Thus, the expression pattern of both genes was not disturbed under in vitro culture conditions used in the present study. Nevertheless, PCR amplification of bovine MASH2 mRNA proved to be more prone compared to amplification of IGF2R mRNA. The relative amounts of transcriptional MASH2 products varied between single embryos similar to IGF2. Interestingly, the two genes (Mash2, Igf2) are located within the same cluster of imprinted genes in mouse and human (REIK and WALTER 2001b; VERONA et al.

2003). It could be possible that genes of this cluster are particularly susceptible to environmental conditions, an assumption that has yet to be proven.