250 TRANSCRIPTIONAL PROFILING OF BOVINE OOCYTES DERIVED FROM THE GROWTH AND DOMINANCE PHASES OF THE FIRST FOLLICULAR WAVE

Abstract

Oocytes recovered at the growth phase have a higher blastocsyt rate (24.5%) than those recovered in the dominance phase (11.7%) (Machatkova et al. 2004 Theriogenology 61, 329–335). However, so far the intrinsic properties of these oocytes in relation to the gene expression patterns have not been investigated. Therefore, we aimed here to compare the transcriptional activity of bovine oocytes recovered at the growth and dominance phases of the first follicular wave. Sixty cyclic Simmental heifers (24 to 30 months old) were selected based on general clinical and ovarian normality, divided into 2 groups (30 heifers per group), and housed together. Estrous cycle was synchronized by 2 injections of PGF_2α with an 11-day interval, and onset of estrus was considered as Day 0. Ultrasonography-guided ovum pickup (OPU) was performed to collect oocytes from small follicles (3–5 mm) at the growth (Day 3) and dominance (Day 7) phases in 2 sessions per phase. Triplicate pools of oocytes (each with 20 oocytes) from each developmental phase were used for transcriptional analysis using custom bovine cDNA array with 2000 clones (Sirard et al. 2005 Reprod. Fertil. Dev. 17, 47–57). Array images were analyzed using GenePix^® Pro 4.0 (Axon Instruments, Inc., Union City, CA, USA), and then the data were LOESS normalized with GPRocessor 2.0a (http://bioinformatics.med.yale.edu/softwarelist.html). Microarray data were analyzed using Significance Analysis for Microarray (SAM) to obtain differentially regulated genes, followed by validation of 10 transcripts with real-time PCR. Data analysis revealed a total of 51 transcripts to be differentially regulated; out of these transcripts, 36 (70%) represented those with known function, 6 (12%) were those with unknown function, and 9 (18%) were novel transcripts. The quantitative real-time PCR validated the expression profile of 8 out of 10 to be in agreement with microarray results. Oocytes from the growth phase (Day 3) were found to be enriched with transcripts involved in protein biosynthesis (RPLP0, RPL8, RPL24, ARL6IP, RpS14, RpS15, RpS4x, and RPS3A), translation elongation (EF1A), ATP binding (ATP5A1), NADH dehydrogenase activity (FL396 and FL405), cytoskeleton (Actin, beta-Actin, H2AZ, and KRT8), calcium ion binding (S100A10 and ANXA2), signal transduction (G-beta like protein), and thiol-disulfide exchange intermediate (TXN). On the other hand, oocytes from the dominance phase (Day 7) were enriched with genes involved in cell cycle (CCNB1, CKS2, UBE2D3, and CDC31), transcription factors (MSX1, PTTG1, FANK1, and PWP1), aldehyde reductase activity (AKR1B1), nucleotide binding (TUBA6), growth factor (BMP15), and fertilization (ZP4). In conclusion, our results indicate that there are differences in transcriptional activity of oocytes derived from different stages of follicular development which may explain their reported differences in developmental competence. Transcripts identified in this study may be used as markers of oocyte competence.