|Back Topic Categories Search Previous Abstract Next Abstract|
HJ Do1 , JH Kim1 *, LR Abeydeera1 *, YM Han1 *, J Green2 , RM Roberts1 2 *, RL Matteri3 , BN Day1 *, RS Prather1 *
Department of Animal Science, University of Missouri, Columbia, MO 1
Department of Biochemistry, University of Missouri, Columbia, MO 2
Animal Physiology Research, Agricultural Research Service, USDA, Columbia, MO 3
Successful pregnancy requires multiple interactions between the conceptus and the mother. Pregnancy-associated glycoproteins (PAG), members of the aspartic proteinase gene family, may participate in these interactions. A fragment of PAG 2 has been identified as early as day 11 in pig embryos. There are no reports on the expression of PAG 1 and 2 genes in normal or parthenogenetic expanded blastocysts. The objective of this study was to determine whether PAG genes are expressed in expanded blastocysts. Day 7 expanded blastocysts produced in vitro or via electric activation of oocytes were analyzed by RT-PCR for the presence of mRNA specific for poPAG 1 and 2. Zonae pellucidae-free blastocysts were lysed by freezing-thawing and the whole lysate was used for RT-PCR. RT was performed on 30 embryos, and PCR was conducted on 10 embryo equivalents for PAG 1 and 2, and 5 embryo equivalents for -actin as an internal control. As a negative control, in vitro-derived embryos were subjected to RT without RTase prior to PCR to examine whether the products were derived from genomic DNA or contamination. PCR products were analyzed by eletrophoresis on agarose gels and subjected to Southern blotting with nonradioactive probes. Southern blot analysis revealed the presence of mRNA encoding poPAG 1 and 2 in fertilized (4 replicates) and parthenogenetic (1 replicate) blastocysts. Partial sequencing of PAG 1 and 2 in in vitro-derived blastocysts showed that they shared 99% and 100% identity, respectively, to poPAG genes identified in pig trophoblast at day 15. Our study indicates that poPAG 1 and 2 genes are expressed as early as day 7 in expanded blastocyst and that these genes are not maternally imprinted.
This abstract is being presented on Tuesday, August 3 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.