247 CANDIDATE mRNA REGULATING MEIOTIC RESUMPTION IN BOVINE CUMULUS-OOCYTE COMPLEXES

When cumulus–oocyte complexes (COC) are cultured with follicle-stimulating hormone (FSH), germinal vesicle breakdown (GVBD) occurs and is transcriptionally dependent. In mice, expression of mRNA for nuclear receptor subfamily 4, member 1 (Nr4A1), and early growth response 1 (Egr1) is reduced when GVBD is blocked by the transcriptional inhibitor DRB. The objectives of this study were to define the period required for transcription initiation in bovine COC, determine the pattern of expression for Nr4A1 and Egr1 mRNAs in bovine COC, and reduce Nr4A1 mRNA expression using small interfering RNA (siRNA) to determine the effect on GVBD. In Experiment 1, pools of COC were randomly assigned to mature in TCM-199 supplemented with 10% estrous cow serum, 1 μg mL–1 estradiol, 200 nm pyruvate, 5 μg mL–1 FSH, and 50 μg mL–1 gentamicin. The DRB (120 μm) was added at 0, 30, 60, 90, 120, 150, or 180 min of culture (n = 27 ± 2 COC/treatment per replication; n = 5 replications). The COC were harvested at 20 h and assessed for meiotic stage. In Experiment 2, COC were randomly assigned to maturation medium with FSH and were harvested at 0, 30, 60, 90, and 180 min of culture. Semiquantitative RT-PCR was used to assess Nr4A1, Egr1, and GAPDH (housekeeper) mRNA levels (n = 39 ± 2 COC/treatment per replication; n = 10 replications). In Experiment 3a, COC were matured for 30 min with either FSH, FSH + DRB, or FSH + 25 nm, 50 nm, or 100 nm siRNA for bovine Nr4A1 (siNr4A1). Relative levels of Nr4A1 mRNA were assessed (58 ± 4 COC/treatment per replication; n = 4 replications). In Experiment 3b, COC were matured 9 h in medium containing either FSH, FSH + DRB, FSH + 50 nm siNr4A1, or FSH + 50 nm nonspecific siRNA (siNS). Oocytes were assessed for meiotic stage (n = 13 ± 1 COC/treatment per replication; n = 4 replications). Data were analyzed by one-way ANOVA and Duncan’s test. For Experiment 1, when DRB was added after the start of culture, progressively more oocytes underwent GVBD (least squares mean ± SEM; 99 ± 7%A, 6 ± 7%E, 33 ± 7%D, 64 ± 7%C, 77 ± 7%B,C, 91 ± 7%A,B, 87 ± 7%A,B, 90 ± 7%A,B for control, 0 + DRB, 30 + DRB, 60 + DRB, 90 + DRB, 120 + DRB, 150 + DRB, 180 + DRB; A,B,C,D,EP < 0.05). Thus, the gene transcription required for GVBD occurred between 0 and 60 min of culture. For Experiment 2, relative levels of Nr4A1 mRNA increased (P < 0.05) at 30 min of culture (100 ± 18% v. 192 ± 18%, for 0 v. 30 min). Relative levels of Egr1 mRNA did not change during culture. For Experiment 3a, the relative expression of Nr4A1 mRNA was decreased in cultures containing siNr4A1 (100 ± 6%A, 32 ± 6%D, 62 ± 6%C, 59 ± 6%C,D, 86 ± 6%B for FSH, FSH + DRB, FSH + 25 nm siNr4A1, FSH + 50 nm siNr4A1, FSH + 100 nm siNr4A1; A,B,C,DP < 0.05). For Experiment 3b, there were no differences in the proportions of COC undergoing GVBD at 9 h of culture in the FSH and FSH + siNS treatments. There was a decreased percentage of GVBD oocytes at 9 h of culture with either DRB or 50 nm siNr4A1 (95 ± 4%A, 4 ± 4%C, 65 ± 4%B, 97 ± 4%A for FSH, FSH + DRB, FSH + 50 nm siNr4A1, FSH + 50 nm siNS; A,B,CP < 0.05). These data are consistent with the hypothesis that Nr4A1 may play a role in regulating GVBD in bovine COC. This research was supported by USDA-NRI Grant 35205-12810, NIH Grant 1 R03 HD043875, and the North Carolina Agricultural Research Service.