Wang De-Chi, Huang Jan-Chi, Lo Neng-Wen, Chen Lih-Ren, Mermillod Pascal, Ma Wen-Lung, Chiang Hsin-I, Ju Jyh-Cherng
Department of Animal Science, National Chung Hsing University, Taichung 402, Taiwan, ROC; Hengchun Branch Institute, COA-LRI, Hengchun, Pingtung 946, Taiwan, ROC.
Hengchun Branch Institute, COA-LRI, Hengchun, Pingtung 946, Taiwan, ROC.
Theriogenology. 2017 Nov;103:52-58. doi: 10.1016/j.theriogenology.2017.07.029. Epub 2017 Jul 24.
The aim of this study was to investigate the effects of Shh (Sonic Hedgehog) protein on caprine oocyte maturation, early embryo development, and developmental competence after embryo transfer of vitrified-thawed in vitro-produced embryos. Cumulus-oocyte complexes (COCs) derived from abattoir were randomly allocated to the in vitro maturation (IVM) medium supplemented with 0 (Control), 0.125, 0.25, 0.5, or 1.0 μg mL recombinant mouse Shh protein. After IVM, COCs were fertilized with frozen-thawed semen and the presumptive zygotes were cultured on goat oviduct epithelial monolayers in M199 medium for 9 days. Our results showed that supplementation of Shh (0.25 or 0.5 μg mL) enhanced oocyte maturation as compared with the control group (92.4% and 95.0% vs. 86.2%, P < 0.05), yet the effect could be reversed by the simultaneous addition of cyclopamine (an inhibitor of Shh signaling by direct binding to the essential signal transducer Smo). Subsequently, an improved blastocyst rate (66.3 ± 10.9, P < 0.05) was observed for the embryos derived from the oocytes matured in the presence of 0.5 μg mL Shh compared with the control group (41.4 ± 12.9). Expressions of Shh, SMO and Gli1 were observed in the ovaries, granulosa cells, COCs, cumulus cells, oocytes and oviduct epithelia. Notably, Ptch1 was expressed in nearly all of the aforementioned tissues and cells except cumulus cells. The embryos exhibited a higher survival rates in the Shh-supplemented group (37.5%) compared to those without Shh supplementation (14.8%; P < 0.05) after embryo transfer. This study demonstrated the beneficial effects of Shh supplementation on oocyte maturation and subsequent embryo development both in vitro and in vivo, suggesting a functional existence of Shh signaling during the final stage of folliculogenesis and early embryogenesis in caprine.
本研究旨在探讨音猬因子（Shh）蛋白对山羊卵母细胞成熟、早期胚胎发育以及玻璃化冷冻-解冻体外生产胚胎移植后的发育能力的影响。将来自屠宰场的卵丘-卵母细胞复合体（COCs）随机分配到添加0（对照组）、0.125、0.25、0.5或1.0 μg/mL重组小鼠Shh蛋白的体外成熟（IVM）培养基中。IVM后，COCs用冷冻-解冻精液受精，假定受精卵在M199培养基中的山羊输卵管上皮单层上培养9天。我们的结果表明，与对照组相比，添加Shh（0.25或0.5 μg/mL）可提高卵母细胞成熟率（分别为92.4%和95.0%，而对照组为86.2%，P<0.05），但同时添加环杷明（一种通过直接结合关键信号转导蛋白Smo抑制Shh信号的抑制剂）可逆转该效应。随后，与对照组（41.4±12.9）相比，观察到来自在0.5 μg/mL Shh存在下成熟的卵母细胞的胚胎具有更高的囊胚率（66.3±10.9，P<0.05）。在卵巢、颗粒细胞、COCs、卵丘细胞、卵母细胞和输卵管上皮中观察到Shh、SMO和Gli1的表达。值得注意的是，Ptch1在除卵丘细胞外的几乎所有上述组织和细胞中均有表达。胚胎移植后，与未添加Shh的组（14.8%）相比，添加Shh的组胚胎存活率更高（37.5%；P<0.05）。本研究证明了添加Shh对体外和体内卵母细胞成熟及随后胚胎发育均有有益作用，表明Shh信号在山羊卵泡发生和早期胚胎发生的最后阶段存在功能性作用。
