251 EVALUATION OF mRNA POLYADENYLATION STATUS OF CELL CYCLE-RELATED GENES IN BOVINE OOCYTES DURING MATURATION: PARTIAL RESULTS

Abstract

During oocyte growth, transcription activity results in the production of RNA and proteins, which are immediately used or stored. At this time, gene expression is, in part, controlled at the post-transcriptional level through deadenylation and polyadenylation processes. After fertilization, early embryonic cell cycles are mainly supported by maternal mRNA and proteins until the maternal embryonic transition. The extent of the poly A tail of mRNA is known as an important element to determine their stability and can be considered a key marker in embryonic development. Because cell cycle-related genes are implicated in the maturation process and early embryonic cell cycle, study of the poly A tail in oocytes during maturation and early embryonic development may be an useful marker of differential developmental competence. Thus, the aim of the present work was to determine the polyadenylation status of the cell cycle-related genes cdc2, CDK2, CDK4, and Cyclin A, B, D, and E during bovine oocyte in vitro maturation (IVM). A PCR-based technique previously developed by Salles and Strickland (1995 PCR Meth. Appl. 4, 317–321) was used, with some modifications. Cumulus–oocyte complexes obtained from 2- to 8-mm follicles were immediately frozen in liquid nitrogen or IVM for 24 h in TCM-199 supplemented with fetal bovine serum and hormones (LH, FSH, and estradiol). In both cases, cumulus cells were removed by pipetting after a 10-min 0.2% hyaluronidase incubation. The RNA was extracted from pools of 10 immature or IVM oocytes and incubated with phosphorylated oligo-dT and DNA ligase. Thereafter, an anchor adapter [poly(dT) anchor] was targeted to the 5 end of the oligo-dT and incubated with Superscript II (Invitrogen) for reverse transcription. Specific primers were designed at the end of a full known sequence of the genes described above, and PCR was conducted for each gene using a specific primer and the anchor primer. The PCR products were digested with restriction enzymes to verify their specificity and were submitted to polyacrylamide gel electrophoresis. All sequences were amplified in immature and matured oocytes from 2 replicates by using a very small sample. After the restriction enzyme treatment, the amplified products were digested as expected. Although differences in amplicon sizes could be observed between the immature and matured oocytes, these are preliminary results and a higher number of replicates are necessary to determine the changes in the polyadenylation status of these cell cycle-related genes and their biological function. In conclusion, the polyadenylation status of the cell cycle-related genes cdc2, CDK2, CDK4, and Cyclin A, B, D, and E could be effectively performed in small samples, and this may be a powerful tool for studying the differential developmental competence of bovine oocytes and early cleavage embryos.

FAPESP.