254 DIFFERENTIAL TRANSCRIPTION AND CYSTOPLASMIC POLYADENYLATION ELEMENTS IN PORCINE GERMINAL VESICLE AND METAPHASE II OOCYTES

Abstract

Identification of transcripts produced during porcine oocyte maturation is one of the first steps in defining pathways important to development. Practically, this information will aid in the refinement of in vitro culture processes, allowing for more efficient in vitro embryo production. To this end, mRNA was isolated from 50 in vitro-matured sow metaphase II stage oocytes (Bomed, Inc., Madison, WI, USA) (MIIs), 50 in vitro-matured gilt metaphase II-stage oocytes (MIIg), and 50 gilt germinal vesicle stage oocytes (GVO) by using the Dynabead^® system (Dynal, Inc., Lake Success, NY, USA) and amplified by using the SMART system (Clontech, Palo Alto, CA, USA). The PCR products were ligated into a pSport vector and transformed into electro-competent E. coli. Colonies were randomly picked and sequenced at the University of Missouri DNA Core. Sequences were clustered with similar sequences derived from a larger expressed sequence tag (EST) project (http://genome.rnet.missouri.edu/Swine/) by using the tlcluster program developed at the University of Iowa. Following clustering, individual clusters in the cDNA libraries were compared by using Fisher's exact test (P < 0.01) to determine if they were differentially represented. Two sets of comparisons were performed, one between the MIIs and MIIg libraries, and another between the GVO library and the combination of both metaphase II libraries (MII). The number of clusters per number of clones in the library was 966/1668 (GVO), 458/820 (MIIg), and 158/819 (MIIs). There were 15/419 clusters that were different between the MIIs and MIIg libraries, and 26/1269 that were different between the MII and GVO libraries. Potential cytoplasmic polyadenylation elements (CPEs) identified from the literature were found in the GVO and MII libraries by using a custom pattern-matching program. Of the clusters with differential expression, 4/15 (MIIs vs. MIIg) and 7/26 (MII vs. GVO) contained CPEs. Table 1 contains a partial list of differentially expressed genes and the sequence of their cytoplasmic polyadenylation elements. Many genes were found to be differentially expressed in both (MII vs. GVO and MIIs vs. MIIg) comparisons. Collectively, these findings will facilitate the elucidation of important developmental pathways in swine and other animals.

Table 1. Comparison of mRNA expression and cytoplasmic polyadenylation elements

This work was partially funded by a University of Missouri Life Sciences Mission Enhancement grant and Food for the 21st Century.