Sonic Hedgehog improves in vitro development of porcine parthenotes and handmade cloned embryos.
This study investigated the expression of Sonic Hedgehog (Shh) signaling pathway and its effect on porcine parthenogenetic (PA) embryo development. The Shh receptor Patched (Ptc1) and co-receptor Smoothened (Smo) were expressed at various stages of PA porcine embryos, at both mRNA and protein levels. Furthermore, the transcriptional activator Gli1 mRNA was first present in the 2-cell stage embryos, and was readily detected at the 4-cell stage and beyond. Culture medium supplemented with 0.5 μg/mL Shh optimized blastocyst rates (58.6 vs. 41.1%; P < 0.05) and the total number of cells per blastocyst (56.4 vs. 45.6 cells; P < 0.05); however, this response was prevented by simultaneous addition of 1 mM cyclopamine (an Shh inhibitor). Moreover, blastocysts that developed in medium containing 0.5 μg/mL Shh had lower apoptotic indices and reduced DNA damage (evaluated by TUNEL and comet assays, respectively). Based on Western-blot analysis, expression of phosphorylated Akt protein in Shh-treated blastocysts was higher than that of the control group (1.22- vs. 0.66-fold, P < 0.05), and less total PARP-1/2 protein was accumulated (0.7-fold, P < 0.05) in treated blastocysts compared to untreated controls. Furthermore, supplementation of Shh (1 μg/mL) also supported development of handmade cloned embryos (50.3 vs. 26.8%; P < 0.05) with reduced apoptotic rates (2.8 vs. 6.3%; P < 0.05). We inferred that the Shh signaling pathway existed in porcine PA embryos and we concluded that Shh supplementation improved the quality and developmental competence of early PA embryos, at least in part, by increasing cell proliferation and reducing apoptosis of the developing embryos.
Nguyen
Ngoc Tan
NT
Department of Animal Science, National Chung Hsing University, 250 Kuokuang Road, Taichung 402, Taiwan, ROC.
Lin
David Pei-Cheng
DP
Siriboon
Chawalit
C
Lo
Neng-Wen
NW
Ju
Jyh-Cherng
JC
D000818:Animals; D002459:Cell Fusion; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D004249:DNA Damage; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism*; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010312:Parthenogenesis; D015398:Signal Transduction; D013552:Swine / Q000196:embryology*; D014704:Veratrum Alkaloids / Q000494:pharmacology
D053823:Hedgehog Proteins; D014704:Veratrum Alkaloids; C000541:cyclopamine