352 EFFECTS OF FOLLICLE SIZE AND STAGE OF MATURATION ON mRNA EXPRESSION IN BOVINE IN VITRO-MATURED OOCYTES

Abstract

A well-orchestrated expression of genes is required to ensure mammalian oocyte progression from the first meiotic arrest to the metaphase II stage to achieve full developmental competence. DYNLL1 (cytoplasmic dynein light chain LC8), PMSB1 (proteasome beta subunit 1), and DYNC1I1 (cytoplasmic dynein 1 intermediate chain) are crucial genes for nuclear and cytoplasmic maturation. DYNLL1 and DYNC1I1 are constituents of the cytoplasmic dynein 1 complex, the main transport system of the cell. PMSB1 is a subunit of proteasome 20S that is the catalytic core of 26S proteasomes and belongs to the ubiquitin-dependent proteolytic system required for protein degradation. The goal of this study was to compare the relative abundance (RA) of the transcripts of the above genes in oocytes collected from different size follicles at different stages of IVM: germinal vesicle (GV), GV breakdown (GVBD), MI, and MII. Ovaries were collected at a local abattoir. Cumulus–oocyte complexes (COCs) were aspirated from follicles either <2 mm or 2–8 mm in size and were matured in M199, supplemented with 1% BSA-FAF, Suigonan (10 UI PMSG/5 UI HCG), and 100 µM cysteamin, at 39°C in 5% CO₂. First, meiotic progression and developmental competence were evaluated in both groups of oocytes by Hoechst staining and IVF/IVC (TALP medium/SOF-system), respectively. Denuded oocytes were frozen at -80°C for RNA analysis. Time points selected for freezing were 0 h for GV; 8 h for GVBD in oocytes from <2 mm follicles and 9 h for oocytes from 2-8 mm follicles; 15 h for MI; and 24 h for MII. The RA of the mRNAs were determined by semiquantitative RT-PCR. One-way and two-way analysis of variance was used for statistical analysis. Cleavage and blastocyst formation rates were significantly higher in the embryos from oocytes recovered from follicle size 2–8 mm; maturation rates were not statistically different between the two groups. The RA of DYNLL1 and DYNC1I1 were significantly higher in oocytes from follicle size 2–8 mm compared to oocytes from the small follicles. No significant difference in PMSB1 expression was found between the two groups. DYNLL1, DYNC1I1, and PMSB1 decreased significantly during IVM in oocytes from 2–8 mm follicles. Only DYNLL1 decreased significantly in the oocytes isolated from the small follicles. When comparing size and stages, DYNLL1 RA was significantly higher in GVBD and MI in the oocytes from follicle size 2–8 mm compared to the other group. A decreased expression of these genes is expected during maturation because the proteins are involved in transport into the cell and because of protein degradation. De novo synthesis of RNA would not be possible after GVBD. The higher mRNA expression of DYNLL1 and DYNC1I1 in oocytes recovered from the bigger follicles could be related to the higher proportion of blastocysts after IVF. The results provide insight into regulatory mechanisms of oocyte maturation and could serve as a marker to assess the developmental potency of bovine oocytes.