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Animal Science Departmental Report 2004-2005 Return to Beef Cattle articles
Functional Analysis
Using Short-Interfering (si)RNA of TRAM-6, a Novel Transcript associated with
Oocyte Maturation in Cattle C. E. Farin, J. R. Sommer, K. F. Rodriguez1, R. M. Petters and J. E. Alexander 1National Institutes of Environmental and Health Sciences, Research Triangle Park, NC AbstractMaturation of
cumulus-oocyte complexes (COC) with gonadotropins requires transcription of new
mRNAs. When COC are cultured with FSH and the transcriptional inhibitor DRB,
cumulus expansion and germinal vesicle breakdown (GVBD) are arrested. Using
differential mRNA display we identified a novel transcript associated with
maturation (TRAM-6) that is expressed during early maturation but not
when COC are inhibited by DRB. The objective of this study was to use siRNAs
targeted against TRAM-6 mRNA to assess its functional role in oocyte
maturation. Exp. 1: Pools of 50-80 bovine COC
(n=5/trt) were randomly assigned to culture in maturation medium consisting of
0.5ml TCM-199 with 2.5mg FSH, 0.5mg estradiol and 10% estrus cow serum with or without DRB (120mM) or with increasing doses of siTRAM-6 (25, 50
or 100nM). Exp. 2: COC pools (n=4/trt) were cultured in maturation medium with
one of the following treatments: control, 120mM DRB, 100nM non-specific siRNA (siNS) or 100nM
siTRAM-6. After 4 h of culture, COC pools were used to assess levels of mRNA
for TRAM-6, VEGF (vascular endothelial growth factor) and GAPD
(glyceraldehyde-3-phosphate dehydrogenase) by semi-quantitative RT-PCR. Exp. 3:
COC were cultured in maturation medium for either 8 h (n=9 pools/trt) or 20 h
(n=7 pools/trt) with one of the following treatments: control, 120mM DRB, 100nM siNS or 100nM siTRAM-6. Cumulus expansion was visually graded every
4 h. At the termination of culture, a subset of COC from each treatment were used
to assess oocyte meiotic stage. Remaining COC were used for assessment of TRAM-6,
VEGF and GAPD mRNA. Data
were analyzed using ANOVA and Duncan’s test.
At 4 h of culture, relative expression of TRAM-6:GAPD mRNA (least
squares means ± SEM) was decreased in the DRB and 100nM siTRAM-6 treatments but was
unaffected by 25nM TRAM-6, 50nM siTRAM-6 or 100nM siNS (Exp. 1: 100±13%a, 17±13%b, 101±13%a, 60±13%ab and 48±13%b for control, DRB, 25nM siTRAM-6,
50nM siTRAM-6 and 100nM siTRAM-6, abcP<.05; Exp. 2: 100±10%a, 14±10%b, 106±10%a and 68±10%c for control, DRB, 100nM siNS and
100nM siTRAM-6, abcP<.05).
Expression of GAPD and VEGF mRNAs, both present in COC at
the start of culture and unrelated to TRAM-6 mRNA, were unaffected by
any treatment. At 8 h, GVBD was
inhibited by treatment with DRB and siTRAM-6 (68±8%a, 3±8%b, 72±8%a and 30±8%c for control, DRB, siNS and
siTRAM-6, abcP<.05). At
20 h, the incidence of metaphase II (MII) oocytes was decreased in the DRB and
siTRAM-6 groups (96±8%a, 9±8%b, 94±8%a and 56±8%c for control, DRB, siNS and siTRAM-6, abcP<.05). Cumulus expansion was inhibited (P<.05)
by DRB but was not affected by any other treatment. In summary, TRAM-6 siRNA decreased the expression of TRAM-6
mRNA in bovine COC at 4 h of culture as well as the proportions of oocytes in
GVBD at 8 h and in MII at 20 h of culture but did not affect cumulus
expansion. In conclusion, these data
are consistent with the identification of a novel mRNA transcript, TRAM-6, that has a functional role in
regulating meiotic maturation in bovine COC.
Supported by USDA Grant #2002-35205-12810. |