Candidate mRNAs Regulating Meiotic Resumption in Bovine Cumulus-Oocyte-Complexes

Abstract

In bovine oocytes, the resumption of meiosis is characterized by the breakdown of the germinal vesicle (GVBD). When cumulus-oocyte complexes (COCs) are cultured in-vitro in the presence of gonadotropins, GVBD is characterized by an initial inhibitory phase, which is followed by an acceleration in the rate of GVBD. An initial transcriptional event is required for gonadotropin-induced in-vitro maturation. The objectives of this thesis were: 1) to define the time course required for transcriptional initiation in bovine cumulus oocyte complexes (COCs); 2) to determine the pattern of expression for Nr4A1 and Egr1 mRNAs in bovine COCs; and 3) to reduce the expression of Nr4A1 mRNA expression to determine its effect on oocyte maturation. Bovine COCs were cultured in the presence follicle stimulating hormone (FSH) alone or FSH with the transcriptional inhibitor, 5,6-dichloro-1-B-Dribofuranosylbenzamidazole (DRB), in order to refine the time course required for transcription initiation and to determine the pattern of expression for Nr4A1 and Egr1 mRNAs. All experiments contained a control group of COCs that were cultured for the entire duration in the presence of DRB. By adding DRB at 0, 30, 60, 90, 120, 150, or 180 minutes after the initiation of culture, it was determined that gene transcription required for GVBD occurs between 0 and 60 minutes after the start of culture. Analysis of COCs cultured for 0, 30, 60, 90 or 180 minutes demonstrated that Nr4A1 mRNA levels increased significantly (P<0.05) at 30 minutes after the start of culture, which is consistent with the time of transcription initiation required for GVBD. In contrast, Egr1 mRNA levels did not significantly differ throughout the culture period. Small interfering RNAs (siRNAs) designed from the sequence for Nr4A1 were used to reduce Nr4A1 mRNA expression and determine the effects of Nr4A1 mRNAs on GVBD in bovine COCs. Expression of Nr4A1 mRNA decreased in abundance in treatment groups containing siRNAs specific to Nr4A1 (siNr4A1) with the greatest decrease in expression occurring in the 25nM and 50nM siNr4A1 treatments. As expected, fewer COCs underwent GVBD when cultured in the presence of DRB at 9 and 20 hours as compared to COCs cultured in FSH alone. Additionally, no significant differences were observed between the FSH and nonspecific siRNA (siNS) treatment groups in the proportion of COCs undergoing GVBD at either 9 or 20 hours of culture. Fewer COCs cultured in the presence of 50nM siNr4A1 underwent GVBD by 9 hours of culture as compared to those cultured in FSH alone. The percentage of COCs that underwent GVBD did not differ between the siNr4A1 and FSH control treatments at 20 hours. The expression of Nr4A1 mRNA at 30 minutes after the start of culture did not differ with FSH, siNr4A1, or siNS treatments. In summary, gene transcription required for GVBD in bovine COCs occurs within 0 to 60 minutes of culture. Nr4A1 mRNAs are present in bovine COCs and these mRNA levels increase significantly after 30 minutes of culture. Furthermore, Egr1 mRNAs are present in bovine COCs, but Egr1 mRNA levels do not change throughout culture. Bovine COCs cultured with siNr4A1 showed a significant decrease in the percentage of oocytes undergoing GVBD after 9 hours of culture. In conclusion, it appears that Nr4A1 plays an active role in GVBD in bovine COCs.