Candidate mRNAs Regulating Meiotic Resumptionin Bovine Cumulus-Oocyte-Complexes

COC;Egr1;Nr4a1;in vitro;oocyte maturation;cow;bovine;cumulus oocyte complex;oocyte

Hockney, Jessica Eileen ; Dr. William L. Miller, Committee Member,Dr. Charlotte E. Farin, Committee Chair,Dr. Robert M. Petters, Committee Member,Hockney, Jessica Eileen ; Dr. William L. Miller ; Committee Member ; Dr. Charlotte E. Farin ; Committee Chair ; Dr. Robert M. Petters ; Committee Member

University:North Carolina State University

关键词: COC;    Egr1;    Nr4a1;    in vitro;    oocyte maturation;    cow;    bovine;    cumulus oocyte complex;    oocyte;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/2490/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

In bovine oocytes, the resumption of meiosis is characterized by thebreakdown of the germinal vesicle (GVBD). When cumulus-oocyte complexes(COCs) are cultured in-vitro in the presence of gonadotropins, GVBD is characterizedby an initial inhibitory phase, which is followed by an acceleration in the rate ofGVBD. An initial transcriptional event is required for gonadotropin-induced in-vitromaturation. The objectives of this thesis were: 1) to define the time course requiredfor transcriptional initiation in bovine cumulus oocyte complexes (COCs); 2) todetermine 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 onoocyte 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 fortranscription initiation and to determine the pattern of expression for Nr4A1 and Egr1mRNAs. All experiments contained a control group of COCs that were cultured forthe 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 genetranscription required for GVBD occurs between 0 and 60 minutes after the start ofculture. Analysis of COCs cultured for 0, 30, 60, 90 or 180 minutes demonstratedthat Nr4A1 mRNA levels increased significantly (P<0.05) at 30 minutes after the startof culture, which is consistent with the time of transcription initiation required forGVBD. In contrast, Egr1 mRNA levels did not significantly differ throughout theculture period.Small interfering RNAs (siRNAs) designed from the sequence for Nr4A1were used to reduce Nr4A1 mRNA expression and determine the effects of Nr4A1mRNAs on GVBD in bovine COCs. Expression of Nr4A1 mRNA decreased inabundance in treatment groups containing siRNAs specific to Nr4A1 (siNr4A1) withthe greatest decrease in expression occurring in the 25nM and 50nM siNr4A1treatments. As expected, fewer COCs underwent GVBD when cultured in thepresence 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 nonspecificsiRNA (siNS) treatment groups in the proportion of COCs undergoingGVBD at either 9 or 20 hours of culture. Fewer COCs cultured in the presence of50nM siNr4A1 underwent GVBD by 9 hours of culture as compared to those culturedin FSH alone. The percentage of COCs that underwent GVBD did not differ betweenthe siNr4A1 and FSH control treatments at 20 hours. The expression of Nr4A1mRNA at 30 minutes after the start of culture did not differ with FSH, siNr4A1, orsiNS treatments.In summary, gene transcription required for GVBD in bovine COCs occurswithin 0 to 60 minutes of culture. Nr4A1 mRNAs are present in bovine COCs andthese mRNA levels increase significantly after 30 minutes of culture. Furthermore,Egr1 mRNAs are present in bovine COCs, but Egr1 mRNA levels do not changethroughout culture. Bovine COCs cultured with siNr4A1 showed a significantdecrease in the percentage of oocytes undergoing GVBD after 9 hours of culture. Inconclusion, it appears that Nr4A1 plays an active role in GVBD in bovine COCs.