2 IDENTIFICATION OF FIVE GENES EXPRESSED PREFERENTIALLY FROM THE PATERNAL X CHROMOSOME

Abstract

Imprinting mechanisms have been proposed to evolve from mechanisms to defend the genome against transposable elements. Compared with autosomal chromosomes, the X chromosome has generated a disproportionately high number of functional retroposed genes in mammalian species. However, few paternally expressed X-linked genes have been reported (http://igc.otago.ac.nz/home.html). In a previous microarray study that analyzed transcriptional differences between male and female bovine blastocysts, almost half of the upregulated genes in female embryos were present on the X-chromosome. The higher expression of X-linked genes in female compared with male embryos may be caused by preferential paternal allele expression or by double allele expression. In the first case, a lower expression for male and parthenogenetic embryos compared with their female counterparts is expected, as both lack a paternal X-chromosome; in the second case, a lower expression in male embryos compared with female and parthenogenetic embryos would be obtained, as they only have one X-chromosome. To discover putative imprinted genes on the X-chromosome, we analyzed the relative mRNA abundance of 8 X-linked transcripts (BEX1, CAPN6, FMR1NB, SAT1, BEX2, X24112, SRPX2, and UBE2A) in male, female, and parthenogenetic bovine blastocysts. Male and female embryos were produced by conventional IVF with unsorted semen, sexed by PCR, and pooled in groups of 10 to perform the reverse transcription (RT) reaction. Parthenogenetic embryos were obtained by activation in ionomycin + 6-DMAP. mRNA was extracted using the Dynabeads mRNA Direct Extraction Kit (Dynal Biotech, Oslo, Norway), RT was performed with Moloney murine leukemia virus reverse transcriptase, and relative abundance was analyzed by quantitative PCR using H2AFZ as a housekeeping gene. The expression level of 5 genes (BEX1, CAPN6, BEX2, SRPX2, and UBE2A) differed significantly between parthenogenetic and female blastocysts, suggesting that they are preferentially expressed from the paternal allele. These results suggest that an imprinting mechanism may play a role in preimplantation sexual dimorphism and provide a valuable tool to uncover X-linked paternally expressed imprinted genes.