Abstract
Objective1) further define the time course required for transcription initiation in bovine cumulus-oocyte-complexes (COC); 2) determine the pattern of expression for Nr4A1 and Egr1 mRNAs in bovine COC; and, 3) reduce Nr4A1 mRNA expression using small interfering RNAs (siRNA) to determine the effect on breakdown of the germinal vesicle (GVBD)MethodsA series of experiments were performed to define the time required for transcription initiation during FSH-induced maturation in bovine COCs, determine the pattern of expression for candidate mRNAs during GVBD, and use RNAi to determine their potential role in GVBD by examining whether candidate-specific siRNA can reduce mRNA expression in bovine COCs and affect the occurrence of GVBD.ResultsTranscription required for GVBD in bovine COC occurred as early as 30 min after culture initiation. Expression of Nr4A1 mRNA increased (P <0.05) at 30 min after culture initiation, consistent with the time of transcription initiation required for GVBD. Expression of Egr1 mRNA did not differ during culture. Expression of Nr4A1 mRNA was decreased (P <0.05) in COC cultured with 50nM siNr4A1 or with 120 µM of the transcriptional inhibitor DRB compared to controls. The proportion of COC undergoing GVBD at 9 hr of culture in FSH and non-specific siRNA (siNS) treatment groups did not differ. However, fewer (P <0.05) COC underwent GVBD at 9 hr of culture when in the presence of DRB or 50nM siNr4A1 compared to controls.Conclusionthese data support a role for Nr4A1 in regulating FSH-mediated and transcription-dependent GVBD in bovine COC cultured in vitro.
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