337 GENE EXPRESSION IN BOVINE OOCYTES AFTER MEIOSIS BLOCK WITH BUTYROLACTONE I

Abstract

The present study aimed to assess the effect of meiosis block on the expression of genes involved in apoptosis. Slaughterhouse bovine ovaries were collected soon after slaughter and transported to the laboratory in saline. Follicles with a diameter of 2–6 mm were aspirated, and oocytes were selected and distributed to the different treatments. For meiosis block (MB), oocytes were cultured for 24 h in TCM-199 supplemented with 100 µM butyrolactone I (BLI) and 3 mg mL^-1 BSA (B100) or with 10 µM BLI, but without BSA (B10). After the 24-h MB culture, the oocytes were subjected to IVM in TCM-199 supplemented with 10% fetal calf serum (FCS), 5.0 µg mL^-1 LH, 0.5 µg mL^-1 FSH, 0.2 mM pyruvate, and 10 µg mL^-1 gentamicin for 22 h. All cultures were at 38.5°C under an atmosphere of 5% CO₂ in air. As controls (C), a group of oocytes was collected immediately after aspiration (immature oocytes) and after IVM without prior meiosis block (mature oocytes). In the treated groups, oocytes were collected at the end of MB culture (immature oocytes) and at the end of IVM post-MB (mature oocytes). After removal of cumulus cells and zonae pelucidae, oocytes were frozen at -80°C in PBS with 0.1% PVA and 1 IU µL^-1 RNase inhibitor. Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen Brasil, Ltda., Sao Paulo, Brazil) according to the manufacturer's recommendations in an Applied Biosystems 7500 fast real-time PCR system (Applied Biosystems, Sao Paulo, Brazil). Efficiency of the reactions was assessed by the software LinRegPCR (Ramakers et al. 2003 Neurosci. Lett. 339, 62–66) and submitted to REST Beta 2005 V1.9.9 analysis (Relative Expression Software Tool). A level of significance of 5% was used. Relative expression of BAX and BCL2 genes did not differ (P > 0.05) when the expression of the constitutive gene GAPDH was used to normalize the data. However, without normalization, a significant reduction in expression was observed after IVM (P < 0.05). For immature oocytes, GAPDH, BAX, and BCL2 expression was 1.0% (for the three genes) for control; 0.26, 0.36, and 0.56% for B100; and 0.6, 0.44, and 0.26% for B10, respectively. After IVM, expressions were 0.06, 0.33, and 0.26% for control; 0.06, 0.19, and 0.25% for B100; and 0.02, 0.15, and 0.3% for B10, respectively. The results show a reduction in transcripts (GAPDH, BAX, and BCL2) after meiosis block, and this reduction was more pronounced in matured oocytes, irrespective of treatment. The reduction in transcripts during MB could influence oocyte competence, but the reduction of these transcripts during maturation seems to be inherent to the oocytes.

This work was supported by Fapesp, Brazil, grant # 03/01479-6.