195 HEAT STRESS ALTERS THE TRANSCRIPTOME OF MATURING BOVINE OOCYTES

Abstract

Direct exposure of maturing oocytes to a physiologically relevant elevated temperature reduces embryo development after fertilisation and has been coincident with reduced de novo protein synthesis. Mechanisms responsible for heat-induced reductions in protein synthesis are unknown but may be related to alterations in the transcriptome of the maturing oocyte. To determine the extent to which this may occur, the impact of heat stress on the maternal pool of RNA in bovine oocytes was assessed using microarrays. After maturation for 24 h at 38.5°C (control) or 41°C (first 12 h only, 38.5°C thereafter; heat stress) oocytes were denuded from associated cumulus cells and lysed for RNA extraction or underwent IVF to assess developmental competence. Total RNA from oocytes was amplified by 3′-poly(A) priming or a combination of 3′-poly(A) and internal priming because oocyte transcripts may or may not have a polyadenylated tail. Amplified RNA was hybridised to GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA, USA; 8 oocyte pools per treatment were collected on 7 different occasions and amplified by 2 methods; n = 32 chips). Differential transcript abundance was determined using R and Bioconductor with only probes having a P < 0.01, a fold change of at least 1.3, and called present for at least half the arrays. Functional annotation of selected transcripts was performed using Gene Ontology and KEGG annotations (Bos taurus build 4.0) and DAVID (v 6.7) with significance level set at P < 0.10. Coincident with reduced blastocyst development (28.3 v. 15.2% for control v. heat stress, respectively; SEM = 3.6; P < 0.0003), heat stress altered the abundance of 159 transcripts (22 increased, 137 decreased); 130 of these were annotated. Use of DAVID demonstrated enrichment of genes important for mitochondrial function and RNA processing. Towards validating certain findings, the relative abundance of 3 mitochondrial transcripts (NDUFC2, COQ3, ATP5O) were assessed by quantitative PCR on non-amplified RNA from the oocyte samples used for the microarray study. Gene-specific primers were designed for 5′ and 3′ ends of transcripts when possible. Exposure to elevated temperature during the first 12 h of oocyte maturation reduced transcript levels of NDUFC2 at the 5′ and 3′ ends (P < 0.0001 and P = 0.003), COQ3 at the 3′ end (P = 0.02) and ATP5O at the 5′ end (P = 0.02). In conclusion, exposure of maturing cumulus-oocyte complexes to a physiologically-relevant elevated temperature altered the transcriptome in oocytes, especially certain transcripts important for mitochondrial function.

This research was supported in part by USDA National Institute of Food and Agriculture, Hatch Project No. 227701, the state of Tennessee through University of Tennessee AgResearch, Department of Animal Science, and East Tennessee Research and Education Center.