Effects of a Sonic Hedgehog agonist on ovine oocyte maturation, epigenetic changes and development of parthenogenetic embryos

Abstract

Purmorphamine is a Sonic Hedgehog agonist that plays an important role in activity regulation of receptors and transcription factors. This study was carried out to assess the effects of Purmorphamine on nuclear and cytoplasmic maturation, epigenetic changes of in vitro matured oocytes and development of parthenogenetic ovine embryos. Ovine ovaries were randomly collected from industrial slaughterhouse, and cumulus-oocyte complexes were cultured in media containing 250 or 500ng/ml Purmorphamine or without Purmorphamine as a control group. Then, after in vitro maturation (IVM), Hoechst stain, cell tracker blue and histone H4K12 antibody were used, respectively to assess the nuclear and cytoplasmic maturation, and histone acetylation rate of matured oocytes. Matured oocytes were activated and cultured to the blastocysts stage in order to determine the parthenogenetic embryo cleavage rate. Expression of Histone Deacetylase 1, 2 and 3 (Hdac1, Hdac2 and Hdac3) genes were evaluated in matured oocytes by quantitative Real Time PCR. Results showed that although the concentration of 500ng/ml Purmorphamine had no significant influence on the oocyte nuclear maturation, it led to an increase in oocyte cytoplasmic maturation. Also, no significant difference in the rates of cleavage and blastocysts per cleavage was observed (P>0.05) in the groups treated with Purmorphamine compared to the control. Quantitative PCR analysis indicated that in 500ng/ml Purmorphamine treatment, Hdac 2 and 3 transcripts decreased and the H4K12 acetylation increased significantly (P<0.05). Consequently, this study showed that 500ng/ml Purmorphamine can induce the cytoplasmic maturation of oocytes, and may possibly be a good addative for improvement of ovine oocyte cytoplasmic maturation.