Effect of purmorphamine on the mRNA expression of Sonic Hedgehog signaling downstream molecules in ovine embryo

Abstract

Abstract. Sonic Hedgehog (SHH) is a signaling pathway mediated through a receptor system which seems to have effects on oocyte maturation and embryonic development. Purmorphamine is an SHH agonist that performs a crucial role in the regulation of the activity of SHH receptors and downstream transcription factors. The aim of this study was to analyze the effect of purmorphamine on the mRNA expression of SHH signaling downstream molecules (Patched1, Glioma-Associated Oncogene1, Smoothened, Histone Deacetylase1, Histone Deacetylase2 and Histone Deacetylase3) in ovine two-cell embryo. Ovaries were obtained from a slaughterhouse, and cumulus-oocyte complexes were aspirated and cultured in maturation media containing 0, 250 or 500 ng mL −1 purmorphamine. Then, oocytes were fertilized and cultured in a CR1 culture medium and after 24 h, two-cell embryos were collected for RNA extraction. Gene expression was evaluated by real-time polymerase chain reaction (PCR). Results indicated that in 250 ng mL −1 purmorphamine, Smo, Ptch1 and Hdac3 expression reduced, Hdac1 expression increased, and Gli1 and Hdac2 expression levels did not change. In 500 ng mL −1 purmorphamine, Gli1 and Smo transcripts increased, while Ptch1, Hdac2 and Hdac3 transcripts decreased. Regarding to the presence of SHH signaling molecules in two-cell embryos and their response to purmorphamine, it can be suggested that SHH signaling is probably active before embryonic genome activation in ovine embryos.