324 FUNCTIONAL ANALYSIS USING SHORT-INTERFERING (si)RNA OF TRAM-6, A NOVEL TRANSCRIPT ASSOCIATED WITH OOCYTES MATURATION IN CATTLE

Abstract

Maturation of cumulus–oocyte complexes (COCs) with gonadotropins requires transcription of new mRNAs. When COCs are cultured with FSH and the transcriptional inhibitor DRB, cumulus expansion and germinal vesicle breakdown (GVBD) are arrested. Differential mRNA display was used to identify a novel transcript associated with maturation of COCs (TRAM-6) that is expressed during early maturation but not when maturation is 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 60–10 bovine COCs (n = 5/trt) were randomly assigned to culture in maturation medium consisting of 0.5 mL TCM-199 with 2.5 μg FSH, 0.5 μg estradiol, and 10% estrous cow serum with or without DRB (120 μM) or with increasing doses of siTRAM-6 (25, 50, or 100 nM). Exp. 2: COC pools (n = 4/trt) were cultured in maturation medium with one of the following treatments: control, 120 μM DRB, 100 nM non-specific siRNA (siNS), or 100 nM 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 semiquantitative RT-PCR. Exp. 3: COCs 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, 120 µM DRB, 100 nM siNS, or 100 nM siTRAM-6. Cumulus expansion was graded every 4 h. At the end of culture, a subset of COCs from each treatment was used to assess oocyte meiotic stage. Remaining COCs were used to assess 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 the 100 nM siTRAM-6 treatments but was unaffected by 25 nM TRAM-6, 50 nM siTRAM-6, or 100 nM siNS (Exp. 1: 100 ± 13%a, 17 ± 13%b, 101 ± 13%a, 60 ± 13%ab and 48 ± 13%b for control, DRB, 25 nM siTRAM-6, 50 nM siTRAM-6, and 100 nM siTRAM-6, respectively; abcP < 0.05; Exp. 2: 100 ± 10%a, 14 ± 10%b, 106 ± 10%a and 68 ± 10%c for control, DRB, 100 nM siNS, and 100 nM siTRAM-6, respectively; abcP < 0.05). There was no effect of treatment on expression of VEGF and GAPD mRNA, both of which are present in COCs at the start of culture and are unrelated to TRAM-6 mRNA. 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, siNSs and siTRAM-6, respectively; abcP < 0.05). 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, respectively; abcP < 0.05). Cumulus expansion was inhibited (P < 0.05) by DRB but was not affected by any other treatment. In summary, TRAM-6 siRNA decreased the expression of TRAM-6 mRNA in bovine COCs 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 COCs. This work was supported by USDA Grant #2002–35205–12810.