237 CHARACTERIZATION OF CYTOPLASMIC POLYADENYLATION SITES DURING PORCINE EMBRYOGENESIS

Abstract

During the development of a germinal vesicle (GV) stage porcine oocyte to the 4-cell stage embryo, there is a transcriptionally silent period during which the oocyte/embryo relies completely on the maternal contribution of mRNA. Regulation of transcription and degradation of the maternal mRNA is at least partially dependent on cytoplasmic polyadenylation elements (CPE) within the 3′ untranslated region of the mRNA. The goal of this study was to determine how single, double, and triple CPE sites affect the transcript levels of mRNA during embryogenesis. Expressed sequence tags from oocytes and 4-cell stage embryos were scanned for the presence of consensus CPE (TTTAAAA, TTAAAAA, ATAAAAA, TTAAAAT, ATTAAAA). We choose 19 different transcripts containing 1 to 3 CPE sites to examine their mRNA levels in GV, metaphase II (MII), and 2-cell and 4-cell stage embryos via real-time PCR. Ovaries were collected from a local slaughterhouse and aspirated, and those oocytes surrounded by cumulus cells were matured in vitro, fertilized, and cultured to the 2-or 4-cell stage. At each stage, 30 oocytes/embryos were pooled and flash frozen in liquid nitrogen, followed by storage at -80°C. RNA from oocyte/embryo pools was then extracted and reversed transcribed into cDNA using an oligo dT primer accompanied with purification using Tris. Real-time PCR was performed with primers designed for the corresponding transcripts using 3 biological and 2 technical replicates for each stage. Starting quantities were calculated based on the standard curve method and standardized to the housekeeping gene, YWHAG, and then MII, 2-cell, and 4-cell were compared to the GV stage. One-way ANOVA was done on the individual samples using proc GLM and proc power in Statistical Analysis System (SAS Institute, Inc., Cary, NC). Individual comparisons were made by a protected (P < 0.05) least squares means. The relative abundance of one of the two clones that contained the consensus TTAAAAA decreased after MII, whereas the other did not change. One of the two clones that contained the consensus TTTAAAA decreased (c-MOS) after MII, whereas the other did not change. One of the two clones with the consensus ATAAAAA decreased after the GV stage, whereas the other did not change. Of the two clones with the consensus TTAAAAT, one increased after the 2-cell stage, whereas the other decreased after GV The two clones that contained the ATTAAAA consensus did not change over the stages of embryos evaluated. Two of five clones with 2 CPE showed no change during these stages, whereas all four clones with 3 CPE decreased from GV through the 4-cell stage. Our results show that there was no significant trend when comparing 2 transcripts that contained the same type and number of CPE sites and that combinations of CPE sites can change the exact nature of when the transcript is available for translation. Thus, CPE might not be the only factors that regulate message stability.

Funding was received from Food for the 21st Century at the University of Missouri.