College of Animal Science and Technology, Beijing University of Agriculture, Beijing, China.
Department of Agricultural, Forest and Food Sciences, University of Torino, Grugliasco, Italy.
Sci Rep. 2017 Jul 27;7(1):6705. doi: 10.1038/s41598-017-07066-2.
Embryonic cryopreservation has a relatively low survival rate because of cytoskeletal damage. However, molecular anti-freezing mechanisms have been largely unexplored. This study investigated the significance of RhoA, involved in embryonic development, and the Rho/RhoA-associated kinase (ROCK) signalling pathway in cryopreservation. The anti-freezing mechanism in murine dormant embryos, compared with normal blastocysts, was assessed by combining molecular, physiological and pharmacological approaches. Real-time PCR and western blotting experiments showed high RhoA expression in cryo-dormant and dormant embryos. RhoA GTPases were overexpressed on the surface of trophectoderm cells in dormant embryos. Treatment with Y-27632, a ROCK antagonist, decreased survival of both normal and dormant blastocysts, while recombinant RhoA protein remarkably increased survival, after freeze-thawing, of normal hatched blastocysts. Our findings elucidated the molecular mechanism of anti-freezing, involving RhoA phosphorylation, meditated by the Rho/ROCK signalling pathway, in hatched and diapaused murine blastocysts. In addition, evidence for a potentially protective additive suggests a new method for improving the anti-freezing potential of mammalian embryos, without protecting the zona pellucida.
胚胎冷冻保存的存活率相对较低，这是由于细胞骨架受损所致。然而，分子防冻机制在很大程度上尚未得到探索。本研究调查了胚胎发育过程中涉及的 RhoA 以及 Rho/RhoA 相关激酶 (ROCK) 信号通路在冷冻保存中的重要性。通过结合分子、生理和药理学方法，评估了与正常囊胚相比，小鼠休眠胚胎中的防冻机制。实时 PCR 和 Western blot 实验显示冷冻休眠和休眠胚胎中 RhoA 表达水平较高。RhoA GTPases 在休眠胚胎的滋养外胚层细胞表面过度表达。用 ROCK 拮抗剂 Y-27632 处理后，正常和休眠囊胚的存活率均降低，而重组 RhoA 蛋白显著提高了正常孵化囊胚冷冻解冻后的存活率。我们的研究结果阐明了涉及 RhoA 磷酸化的防冻分子机制，该机制由 Rho/ROCK 信号通路介导，存在于孵化和休眠的小鼠囊胚中。此外，有证据表明潜在的保护添加剂为提高哺乳动物胚胎的防冻潜力提供了一种新方法，而无需保护透明带。
