Sonic Hedgehog Supplementation Promotes Porcine Oocyte Maturation and Early Embryo Development.

Abstract

The Hedgehog signaling pathway plays an important role in cell proliferation in a variety of tissues of different species. However, its specific roles during mammalian oocyte maturation and early embryo development are unknown. This study aimed to investigate the effects of Sonic Hedgehog (Shh) protein supplement in the culture medium on oocyte maturation and their early development after parthenogenetic activation. Ovaries were colleted from the gilts at the slaughterhouse and oocytes-cumulus complexes (COCs) were aspirated from the follicles of 3 - 6 mm in diameter. The COCs were cultured in North Carolina State University-23 (NCSU-23) medium supplemented with different concentrations (0, 0.5, 1, or 2 μg/ml) of mouse recombinant Shh protein. Results showed that oocyte maturation rates were significantly increased in the 0.5-1 μg/ml (P < 0.05) groups compared to those in the control (92.4% and 90.4% vs. 81.9%) and the 2 μg/ml Shh supplementation (76.7%) groups. However, only oocytes with 0.5 μg/ml Shh supplementation had their blastocyst rate (46.1%) increased significantly (P < 0.05) as compared to the control (36.0%) and other treatment groups (37.5% in the 1 μg/ml group, 19.8% in the 2 μg/ml group). The total cell numbers per blastocyst were significantly greater in those with 0.5 μg/ml Shh supplementation as compared to those in all the other groups (P < 0.05). Furthermore, the MII oocytes matured in the 0.5 μg/ml Shh supplementation had higher cyclin B1 protein content than that in the control group (P < 0.05). To understand whether Shh protein exerts a direct effect on the oocyte, cumulus cell-free germinal vesicle (GV) stage oocytes were cultured with Shh (0.5 μg/ml ) for 44 h and a significantly greater (P < 0.05) maturation rate (28.2%) was observed compared to the control group (10.9%). Another evidence for Shh promoting oocyte maturation was also demonstrated by an elevation of intracellular calcium release induced by thimerosal (1.065 ± 0.101 and 0.942 ± 0.124 vs. 0.754 ± 0.064 in control, P < 0.05) treatment, when oocytes were matured with 0.5 μg/ml or 1 μg/ml of Shh supplement in the culture medium. In conclusion, our findings provide the first evidence that Hedgehog protein positively promotes porcine oocyte maturation and the subsequent development of the parthenogenetic embryos. However, the underlying mechanisms remain to be explored.