Submission Number: JAC-4-11-28
Abstract Number: 309
EFFECT OF SERUM ON mRNA PATTERN DURING IN VITRO MATURATION OF BOVINE OOCYTES.
S Jacek Rzucidlo and Steven L Stice*
Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA 1
A novel restriction fragment differential display (rfdd) RT-PCR has been used to examine differences in gene expression in bovine oocytes matured in vitro (Keskintepe and Brackett, Biol. Reprod. 1996, 55:333) in the presence (10%) or absence of fetal calf serum (FCS). At 21 h after the onset of in vitro maturation, cumulus cells were removed, and 100 of oocytes from each experimental group were transferred into microcentrifuge tube and immediately frozen in liquid nitrogen. Total RNA extracted from oocytes was processed using displayProfile kit (Display System Biotech). After synthesis from total RNA, cDNA was digested with TaqI and DNA linkers have been ligated to the ends of cDNA fragments. The cDNA template has been amplified in 64 PCRs using fluorescein labeled 5'-primer and one of the 64 specific 3'-primers, that anneal to the junction between the linker and cDNA fragment. Three bases, distinct for each 3'-primer, extend into the cDNA sequence, thus each PCR amplifies different subset of cDNA fragments. Products from single PCR are called "expression window" and they were separated on 6% polyacrylamide gel and analyzed using Long-Read Tower System (Visible Genetics). The analysis of mRNA profiles generated from each sample, revealed approximately 650 clearly identifiable products in size ranging from 90 to 500 bp. Proportion of oocytes reaching metaphase II was not significantly affected by the absence of serum; however, within the RNA sample originated from oocytes matured in the presence of FCS, 13 fragments have been found which were not represented in the other sample; and 19 fragments, not present in the other sample, have been found in RNA sample extracted from oocytes matured in the absence of FCS. These findings suggest that presence or absence of FCS during in vitro maturation can affect the pool of oocyte's mRNAs either directly or through the interactions with cumulus cells. Application of rfdd RT-PCR for analysis of embryonic RNA offers sensitive and relatively fast method of screening for differentially expressed transcripts.
Keywords: gene expression, rfdd RT-PCR, maturation in vitro, bovine oocytes
This abstract is being presented at: 8:00 AM in session:
Gene Regulation and Function I