279 DIFFERENCES IN TRANSCRIPT ABUNDANCE BETWEEN MALE AND FEMALE BOVINE BLASTOCYSTS PRODUCED IN VITRO USING SEXED SEMEN

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.