181 TRANSCRIPTIONAL SEXUAL DIMORPHISM IN AUTOSOMAL GENES ON BOVINE DAY 14 EMBRYOS

Abstract

In bovine blastocysts, a large transcriptional sexual dimorphism affecting almost one-third of the genes is observed. At this early stage, sex chromosomes impose extensive transcriptional regulation on autosomal genes. However, little is known about the situation in further stages of embryo development. The aim of the present study was to determine the transcriptional sexual dimorphism on Day 14 bovine embryos for 10 candidate genes. In vitro-produced bovine blastocysts were transferred on Day 7 to the ipsilateral uterine horn of synchronized recipients. Seven days after embryo transfer, the recipients were slaughtered and Day 14 embryos were recovered by uterine flushing. Fourteen embryos of a similar size were selected for the analysis (8 males, 6 females). PolyA mRNA and cDNA synthesis and DNA extraction were performed individually. Embryo sexing was performed based on the expression of the Y-linked gene YZRSR2, and the result was confirmed on DNA by a PCR for BRY/Sat. The mRNA abundance relative to the housekeeping H2AZ was analysed for 5 genes known to display transcriptional sexual dimorphism at the blastocyst stage: the X-linked gene glucose-6-phosphate dehydrogenase (G6PD); the autosomal genes DNA methyltransferase 3 A (DNMT3A), gluthatione S-transferase mu 3 (GSTM3), laminin α 1 (LAMA1), progesterone receptor membrane component 1 (PGRMC1), and interferon tau (IFNT2); and 4 genes related to sex determination and differentiation: Wilms tumour 1 (WT1), GATA binding protein 4 (GATA4), zinc finger protein, multitype 2 (ZFPM2, also known as FOG2), and double-sex and mab-3-related transcription factor 1 (DMRT1). Statistical differences were assessed by ANOVA (P ≤ 0.05). Consistently with the situation observed in blastocysts, DNMT3A was significantly up-regulated in males (2.5 ± 0.5 v. 1 ± 0.2), and IFNT2 in females (1 ± 0.2 v. 1.8 ± 0.5), but G6PD, GSTM3, LAMA1, and PGRMC1 did not show significant differences. Among the genes linked with sex determination, WT1 was significantly up-regulated in males (1.7 ± 0.2 v. 1 ± 0.2), whereas the opposite situation was observed for GATA4 (1 ± 0.2 v. 1.5 ± 0.2). No differences between sexes were observed for ZFPM2 (1 ± 0.3 v. 1.1 ± 0.3) and DMRT1 (1.2 ± 0.2 v. 1 ± 0.1). In conclusion, transcriptional sexual dimorphism seems to be reduced in more advanced stages of embryo development, but 2 genes, related to DNA methylation (DNMT3A) and pregnancy recognition (IFNT2), maintained the sex-specific differences observed at the blastocyst stage. Furthermore, the expression of 3 potential activators of SRY (WT1, GATA4, and ZFPM2) and the regulator of male development DMRT1 was observed on Day 14 bovine conceptuses, and 2 of the genes (WT1 and GATA4) showed sex-specific transcriptional regulation.