Micro-Array Analysis Reveals That One Third of the Genes Actively Expressed Are Differentially Expressed Between Male and Female Bovine Blastocysts.

Abstract

In eutherian mammals, evidence has emerged that clearly demonstrates differences in growth rates and metabolism between male and female embryos which appear before sexual differentiation of the gonads and, therefore, could not be explained by sex-related hormonal differences. Though genetically identical for autosomal chromosomes, male and female embryos could display gender-specific transcriptional or translational regulation, leading to differential mRNAs or protein products for some genes. To illustrate the gender-specific differences in mRNA-level expression in bovine embryos produced in vitro, we compared gene expression patterns between male and female blastocyts using the GeneChip Bovine Genome Array (Affymetrix), which contains 24072 bovine gene probe sets. Bovine cumulus oocyte complexes (n=8858) were matured in vitro and fertilized with X- or Y-sorted sperm from 1 of 3 different bulls. The resulting zygotes were cultured in vitro to the blastocyst stage, and three pools of 60 male and 60 female blastocysts were used per bull (18 samples in total) for microarray analysis. RNeasy Micro kit (Qiagen) was used for RNA isolation and purification. Labelled cRNA was synthesized with a linear RNA amplification method (Message Amp Premier kit, Ambion) following the manufacurer's instructions and using 50 ng of total RNA as template for reverse transcription. After array hybridization, the arrays were scanned using a confocal scanner (GC3000_Affymetrix), and the image data were analysed by GeneChip Operating Software (GCOS 1.4, Affymetrix). For the Bioinformatic analysis dChip (www.dchip.org) and Affy/AffyPLM (Bioconductor) software were used. Statistical analysis was based on a regression model and ANOVA to analyze differences between groups and hierarchical supervised analysis was performed using Average linkage and Euclidean distance for classifying the samples. Bonferroni or FDR corrections were applied to reduce the total number of false positives. Approximately 3000 gene probes out of 9323 actively expressed exhibited statistically significant sex-linked expression; most differed by 2-fold or less. Distinct numbers of female upregulated and downregulated genes were identified according to the statistical correction used (1330 and 1591 respectively using FDR p<0.05; 290 and 92 at Bonferroni p<0.05). 10 genes were used for validation by qPCR. The majority of the genes showing more than a 2.4-fold difference in expression were uniquely present in the X- or Y-chromosome, suggesting potential roles in defining the gender-specific phenotypes. Interestingly, genes present in the X-chromosome that were upregulated in female blastocysts (at similar levels to XIST), are candidates to be imprinted (expressed predominantly from the paternally inherited X-chromosome). Furthermore, one copy of a gene present in the X-chromosome in mammals (ZRSR2) was found to be allocated in the nonrecombining portion of the Y-Chromosome. This region only contains genes responsible for large fitness effects which are either required for male function (e.g. genes involved in spermatogenesis) or for sex determination (e.g. the SRY gene on the Y chromosome of most mammals). This gene is an essential splicing factor only expressed in male embryos, which raises the possibility that splicing factors may govern sex determination, and that important steps in the mammalian sex determination process are likely to operate at the posttranscriptional level. (platform)