347 TRANSCRIPTIONAL REGULATION DURING IN VITRO BOVINE OOCYTE MATURATION: FACT OR ARTIFACT?

Abstract

Understanding the molecular dynamics of oocyte maturation may assist in improvements to maturation protocols and outcomes of in vitro embryo production. There are conflicting results on the occurrence of transcriptional changes during meiotic maturation. A previous microarray study from our group (Carter et al. 2007 Reprod. Fertil. Dev. 19 248) indicated upregulation of a number of transcripts during oocyte maturation in vitro. The aim of this study was to attempt to ascertain whether such changes are real or artifacts of methods used for analysis. COCs were aspirated from follicles of bovine ovaries collected at the local abattoir. Immature COC were immediately denuded and half of them were snap frozen in pools of 12 oocytes in liquid nitrogen. The remaining oocytes were matured in vitro for 24 h at 39°C under an atmosphere of 5% CO₂ in air with maximum humidity in TCM-199 supplemented with 10% (v/v) fetal calf serum and 10 ng mL^-1 epidermal growth factor. They were subsequently denuded and snap frozen in pools of 12 as above. Samples were stored at -80°C until analysis. Six different pools per stage were collected from four separate collections. Prior to RNA isolation, samples were spiked with luciferase mRNA and total RNA was isolated using RNeasy Micro Kit (Qiagen, Hilden, Germany). The RNA was then divided into two equal parts for priming either with Oligo (dT) or random primers during cDNA synthesis.The expression of eight genes (LUM, PLAT, SERPINE1, STC1, PLAU, ANXA1, TGFB2, and MAOA), previously reportedtobe up regulated (P < 0.05) during bovine oocyte maturation and luciferase was examined. Quantitative real-time PCR was used to compare transcript abundance and data were analyzed using the relative standard curve method. The expression level of these genes was normalized to the average luciferase levels and means were compared by the student t-test. With the exception of MAOA, the expression of all transcripts up regulated (P < 0.05) in the previous microarray study showed similar trend in this analysis. However, the relative expression levels varied with the type of primer used during cDNA synthesis. Oligo (dT) primed samples had higher relative expression ratios compared to samples with random primers, indicating the preferential amplification of polyadenylated transcripts by oligo primers in matured oocytes. Despite the variations in the ratio, the expression patterns remained the same, indicating the existence of transcription during the in vitro bovine oocyte maturation. Future studies will involve in vivo-matured oocytes and identify whether the observed transcription in the in vitro samples is a normal phenomenon or a consequence of suboptimal in vitro culture environment.

Supported by Science Foundation Ireland (07/SRC/B1156).