Abstract

There is considerable evidence in the literature indicating that, depending on the culture environment, male pre-implantation embryos develop faster than females, with, for example, a higher proportion of early developing blastocysts being male. The aim of this study was to examine gender-related differences in gene expression in bovine blastocysts produced in vitro following IVF with sex-sorted, frozen–thawed semen. For blastocyst production, immature cumulus–oocyte complexes (COCs) recovered from the ovaries of slaughtered heifers were matured in vitro for 24 h. Matured COCs were randomly split into 2 groups and inseminated with frozen–thawed sperm sorted flow cytometrically for gender. Presumptive zygotes were cultured for 7 days in synthetic oviduct fluid medium. In a preliminary experiment, blastocysts (XX: n = 61; XY: n = 47) were recovered on Day 8 post-insemination and individually snap frozen in liquid nitrogen for verification of the sorting procedure. Sexing was performed with PCR using both male-specific (BRY4.a) and bovine satellite primers. The proportion of female and male blastocysts obtained with X- and Y-chromosome-bearing sperm was 87.2 and 80.3%, respectively. In a subsequent 5 replicates, Day 8 blastocysts were snap frozen in groups of 10 for analysis of mRNA relative abundance. These developmentally important genes were selected based on microarray analysis because they showed a high sensitivity to suboptimal in vitro culture conditions (data not shown). There was no difference in the relative abundance of desmocollin II (DcII), Na/K-ATPase alpha1 subunit (Na/K), ubiquitin-activating enzyme E2 (Ube2), fibroblast growth factor 4 (FGF4), and DNA methyltransferase 1 (Dnmt1) between male and female blastocysts. X-inactive specific transcript (Xist), interferon-tau (IFN), and a Sry-related HMG box transcriptoinal factor (Sox17) were significantly up-regulated in female blastocysts, whereas DNA methyltransferase 3a (Dnmt3a), DNA methyltransferase 3b (Dnmt3b), sarcosine oxidase (Sox), and the transcription factor Oct-4 were significantly up-regulated in males. In conclusion, differences in gene expression between male and female embryos exist and may be related to the well-described differences in the kinetics of development.