Maturation, Aging and Death in Reproductive Tissues
(M610) DIFFERENTIAL GENE EXPRESSION OF OOCYTES OBTAINED FROM GROWING AND PERSISTENT FOLLICLES.
Lingenfelter, Brandon1, Dailey, Robert1, Inskeep, E. Keith1, Vernon, Michael1, Poole, Daniel1, Rhinehart, Justin1, Yao, Jianbo1, 1 West Virginia University, Morgantown, WV
ABSTRACT- Eighty-six percent of embryos died by d 6 in inseminated cows that ovulated a persistent follicle, while donor embryos transferred into such cows on d 7 survived in normal percentages. Loss of embryos might be due to errors in RNA in the oocyte. The first objective was to determine if single bovine oocytes could be used to analyze specific genes expressed at low intensity. The second objective was to determine if embryonic loss, in cows that ovulate a persistent follicle, might be due to changes in RNA in the oocyte. Candidate genes chosen included: Poly (A) ribonuclease (PARN; RNA degradation), MSY2 (RNA masking), and YY1 (transcription). Cows were assigned at random to 4 groups (2 X 2 factorial) with growing follicles harvested on d 6 (G6) or d 8 (G8,) and persistent follicles harvested on d 13 (P13) or d 15 (P15) of the estrous cycle. Cows were super-stimulated with FSH at decreasing dosages on d 1 to d 4 (estrus = d 0) and cows in 3 of 4 groups received 25 mg PGF2 AM and PM on d 6; ovaries of cows in the 4th group (G6) were harvested on d 6. Cows in P groups received progesterone from CIDR-B devices on d 4 through 13 of the estrous cycle. Oocytes were aspirated immediately after ovariectomy via colpotomy, denuded of cumulus cells and stored at −80°C until isolation of RNA. Total RNA from individual oocytes was purified and subjected to T7 based linear amplification. Antisense RNA was purified and reverse transcription was performed to generate cDNA, which was used for real-time RT-PCR. Expression (p = 0.01) differed between G and P groups by Chi square analysis. Three oocytes from P13, 3 oocytes from P15 and 1 oocyte from G8 did not amplify when real-time RT-PCR was performed, but all oocytes amplified Histone H2a. Average numbers of cycles in 7 G oocytes were 26.6 ± 9.2 for MSY2, 37.5 ± 7.2 for YY1 and 36.3 ± 5.9 for PARN. Average cycle numbers, for 2 P oocytes for MSY2, YY1 and PARN were 30 ± 9.5, 36.9 ± 7.1 and 26.6 ± 6.1, respectively. Based on these data, RNA isolated from single bovine oocytes can be analyzed, and oocytes from growing and persistent follicles differed in expression of mRNA. Supported by project Hatch 27 (NE 1007) of the WV Agricultural and Forestry Experiment Station.
KEY WORDS: persistence, oocyte, Bos taurus