143 PRESENCE OF PROSTAGLANDIN F_2α RECEPTOR IN IN VITRO-DERIVED MORULA AND BLASTOCYST STAGE BOVINE EMBRYOS

Abstract

Culture of in vitro and in vivo-derived embryos in medium containing prostaglandin F_2± (PGF) decreased embryonic development to blastocyst stage and reduced hatching rates (Scenna et al. Prostaglandins 73, 215-226). Moreover, administration of an inhibitor of PGF synthesis at the time of embryo transfer in bovine recipients improved pregnancy rates (Schrick et al. 2001 Theriogenology 59, 335 abstr.). These findings indicate a direct negative effect of PGF on embryonic development. However, to our knowledge, no evidence of PGF receptor expression in morula or blastocyst stage bovine embryos is available in the literature. Therefore, the objective of the current study was to determine the presence of PGF receptor mRNA using real-time RT-PCR and protein expression by Western blotting in morula or blastocyst stage in vitro bovine embryos. Briefly, isolated total RNA from compact morula or blastocyst stage embryos and from bovine tongue epithelium (positive control for PGF receptor mRNA) were reverse-transcribed into cDNA. A volume from the RT reaction equivalent to 10 embryos per tube was utilized to determine transcripts for PGF receptor and Histone H2A (standard PCR control). Polymerase chain reaction was performed, and identity of PCR fragments was confirmed by ethidium-bromide-stained 2% agarose gel electrophoresis and by DNA sequencing. To determine protein expression, morula and blastocyst stage embryos were lysed in lysis buffer (10% SDS, 1 m Tris pH 7.5, 1 m NaF, 1 m DTT, 0.1 m EGTA with protease inhibitors) and stored at -20°C. Crude proteins isolated from bovine corpora lutea (positive control for PGF receptor protein), embryo samples, and prestained standards were separated by 12% SDS-PAGE under reducing conditions. Proteins were electrotransferred onto a PVDF membrane. Nonspecific binding sites in the PVDF membrane were blocked with 10% nonfat dry milk, and the blot was washed and incubated for 1 h at room temperature with a 1:1000 dilution of the primary antibody (rabbit polyclonal antibody against PGF receptor protein). Subsequently, the blot was washed and incubated for 1 h at room temperature with 1:1000 dilution of mouse anti-rabbit IgG conjugated with horseradish peroxidase. Finally, the blot was washed and revealed by chemiluminescence in a CCD camera. Results indicated that transcripts as well as the protein for PGF receptor were present in early stage bovine embryos. Identification of PGF receptor in morula and blastocyst stage bovine embryos may, in part, explain the increase in pregnancy rates after administration of a PGF synthesis inhibitor at the time of embryo transfer, which opens up the possibility to develop new strategies to prevent detrimental effects of PGF during early embryonic development.