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Sex affects function of the developing mammalian embryo as early as the preimplantation period. There were two goals of the current objective. The first was to determine the degree and nature of differences in gene expression between female and male embryos in the cow at the morula stage of development. The second objective was to determine whether DKK1, a molecule known to alter differentiation of the blastocyst, would affect gene expression differently for female and male morulae. In Experiment 1, female and male embryos were treated with DKK1 at Day 5 after insemination. Morulae were harvested 24 h after treatment, pooled in groups of 20 for microarray analysis and RNA subjected to analysis of gene expression by microarray hybridization. There were 662 differentially expressed genes between females and males and 128 of these genes had a fold change ≥ 1.5 between the two sexes. Of the genes upregulated in females, 49.5% were located in the X chromosome. Functional analysis predicted that cell survival was greater in female embryos. Experiment 2 involved a similar design except that transcripts for 12 genes previously reported to be affected by sex, DKK1 or the interaction were quantified by quantitative polymerase chain reaction. Expression of all genes tested that were affected by sex in experiment 1 was affected in a similar manner in Experiment 2. In contrast, effects of DKK1 on gene expression were largely not repeatable in Experiment 2. The exception was for the Hippo signaling gene AMOT, which was inhibited by DKK1. In Experiment 3, embryos produced by fertilization with unsorted sperm were treated with DKK1 at Day 5 and abundance of transcripts for CDX2, GATA6, and NANOG determined at Days 5, 6 and 7 after insemination. There was no effect of DKK1 on expression of any of the three genes. In conclusion, female and male bovine embryos have a different pattern of gene expression as early as the morula stage, and this is due to a large extent to expression of genes in the X chromosomes in females. Differential gene expression between female and male embryos is likely the basis for increased resistance to cell death signals in female embryos and disparity in responses of female and male embryos to changes in the maternal environment.

Maturation of the oocyte involves nuclear and cytoplasmic changes that include post-translational processing of proteins. The objective was to investigate whether inhibition of proteasomes during maturation would alter competence of the bovine oocyte for fertilization and subsequent development. Cumulus-oocyte complexes were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0-6 h or 16-22 h after initiation of maturation. Treatment with MG132 early in maturation prevented progression to meiosis II and reduced fertilization rate and the proportion of oocytes and cleaved embryos that became blastocysts. Conversely, treatment with MG132 late in maturation improved the percentage of oocytes and cleaved embryos that became blastocysts without affecting nuclear maturation or fertilization rate. Optimal results with MG132 were achieved at a concentration of 10 µM -- effects were generally not observed at lower or higher concentrations. Using proteomic analysis, it was found that MG132 at the end of maturation increased relative expression of 6 proteins and decreased relative expression of 23. Among those increased by MG132 that are potentially important for oocyte competence are GAPDH, involved in glycolysis, TUBA1C, needed for organellar movement, and two proteins involved in protein folding (P4HB and HYOU1). MG132 decreased amounts of several proteins that exert antiapoptotic actions including ASNS, HSP90B1, PDIA3 and VCP. Another protein decreased by MG132, CDK5, can lead to apoptosis if aberrantly activated and one protein increased by MG132, P4HB, is anti-apoptotic. Finally, the pregnancy rate of cows receiving embryos produced from oocytes treated with MG132 from 16-22 h of maturation was similar to that for control embryos, suggesting that use of MG132 for production of embryos in vitro does not cause a substantial decrease in embryo quality. [ABSTRACT FROM AUTHOR]