Candidate mRNAs Regulating Meiotic Resumption in Bovine Cumulus-Oocyte-Complexes
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Date
2008-12-07
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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.
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Keywords
COC, Egr1, Nr4a1, in vitro, oocyte maturation, cow, bovine, cumulus oocyte complex, oocyte
Citation
Degree
MS
Discipline
Animal Science
