221 TYPICAL HIF1-REGULATED GENES ARE UNALTERED BY OXYGEN IN BOVINE BLASTOCYSTS

Abstract

Oxygen-regulated gene expression in the bovine embryo contrasts markedly with that observed in the mouse. Under low (2%) post-compaction oxygen conditions moderate changes in gene expression are observed in the bovine blastocyst (Harvey et al. 2004 Biol. Reprod. 71, in press), compared with 3–4 fold increases in the mouse (Kind et al. 2004 Mol. Reprod. Dev., in press). Specifically, GLUT-1 (Harvey et al. 2004), myotrophin, and anaphase-promoting complex 1 (Harvey et al., unpublished) mRNAs are increased in bovine blastocysts following 2% oxygen culture, compared with those cultured under 20% oxygen. These oxygen-mediated differences in gene expression in the bovine are most likely regulated by hypoxia-inducible factor (HIF)2 transcription factor activity, as we have previously observed that HIF1α protein is not detectable in bovine embryos whereas HIF2α is readily detectable (Harvey et al. 2004). The aim of this study was to determine the effect of post-compaction oxygen concentration on the expression of typically HIF1-regulated and potential HIF2-regulated (suggested from a mouse knockout study; Scortegagna et al. 2003 Nat. Genet. 35, 371) genes in bovine blastocysts. In vitro-produced bovine embryos were generated using standard protocols. Compact morulae were randomly allocated to treatments under 2%, 7%, or 20% oxygen for 72 h from Day 5. Blastocyst RNA was isolated using TriReagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and samples were reverse-transcribed using Superscript II (Invitrogen, Melbourne, Australia). Amplification and analysis of cDNA was achieved by real-time PCR using specific primers and Sybr green PCR master mix (Applied BioSystems, Melbourne, Australia). Statistically significant differences in gene expression were analyzed by ANOVA, P < 0.05. Examination of expression of genes known to be regulated by HIF1 in somatic cells (reviewed by Semenza 2002 Biochem. Pharm. 64, 993) revealed no oxygen-mediated alteration in expression of aldose reductase, cyclooxygenase 2, or inducible nitric oxide synthase. However, the expression of lactate dehydrogenase A (LDHA) displayed a 4-fold increase under 2% oxygen, compared with 7% and 20% oxygen (P < 0.001). Expression of glutathione peroxidase, and CuZn- and Mn-superoxide dismutase (putative HIF2-regulated genes) was not influenced by oxygen concentration post-compaction. This study suggests that typical HIF1-regulated genes are not influenced by alterations in the external oxygen environment in the bovine embryo. These results complement previous observations that HIF1α protein is not detectable in blastocyst-stage bovine embryos, and suggest that LDHA may be an HIF2 target gene in the bovine embryo. As embryo development is influenced by oxygen concentration, levels of LDHA at the blastocyst stage may be used as a marker of oxygen responsiveness.