Al-Ghadi Muath Q, Alhimaidi Ahmad R, Iwamoto Daisaku, Al-Mutary Mohsen G, Ammari Aiman A, Saeki Kazuhiro O, Aleissa Mohammed S
King Saud University, College of Science, Zoology Dept. Riyadh, Saudi Arabia.
Kindai University Faculty of Biological -Oriented Sci. and Technology Dept. of Genetic Engineering. Wakayama, Japan.
Saudi J Biol Sci. 2020 Sep;27(9):2280-2286. doi: 10.1016/j.sjbs.2020.04.039. Epub 2020 Apr 29.
Although, it has been success in the generation of animal clones from somatic cells in various animal species, the information related to nuclear reprogramming of cloned embryos is found to be limited. This study aims to compares the effect of both Scriptaid (SCR) and Trichostatin (A) treatments in improving cloning efficiency, and embryos developmental rate of cloned sheep embryos . Three groups were formed, i.e., one SCR group, second TSA group, with both treatment concentrations of 5 nM, 50 nM, and 500 nM, respectively, and third were control group with 0 nM. : Ovaries of slaughtered sheep were collected and oocytes were recovered from antral follicles using aspiration method and maturation of oocytes were done. Then zona dissecting with micropipettes and oocyte enucleation were carried out under the micromanipulator. Later nuclear transfer, cell fusion and activation were done via cell fusion machine. Finally the embryo cultured in incubating chamber at the CO2 incubator up to 9 days. : In general the results showed that when the concentration increases the cleavage rate increased. The cleavage rates of the SCNT embryos treated with SCR at different concentrations are closely related to cleavage rate of embryos treated with TSA at same concentration; such as 39.47% for 500 nM TSA, 38.09% for 500 nM SCR; 18.6% for 50 nM TSA, 19.17% for 50 nM SCR, and 22.64% for 5 nM TSA, 17.18% for 5 nM SCR. As for the control group, the cleavage rate of the SCNT embryos cleavage ratewere27.47%., 30% and 30.85% respectively for bothtreatments. While there is a significant difference in TSA treatments at an eight-cell stage at the concentration (5 and 50 nM TSA) compared to the all other cleavage cell stages of (500 nM TSA and control). Also their were a differences between (50 nM of TSA) compared to the (50 nM SCR). Also there were a significant differences between the 16 cell stage at the (500 nM TSA) compared to other treatment (5 nM, 50 nM TSA and control). Regarding the SCR there were a significant difference at 8 cell stage between (5 nM SCR), compared to the other treatment (50 nM, 500 nM SCR and control). Also there were a significant difference at 16 cell stage between (50 nM, and 500 nM SCR), compared to the other treatment (5 nM SCR and control). While in the development of the embryos reach to blastocyst stage the SCR and the control group show a higher rate, in compered to TSA that did not show any development to blastocyst stage. The total SCR treatment showed (3/41 = 7.31%), and the total control showed (4/89 = 4.49%) blastula stage. It concludes that SCR improve the final development blastula stage compared to the TSA treatments that did not improved embryos reach to final developmental blastula stages may be due to spices differences or to the toxicity of TSA, especially at higher concentrations.
尽管在多种动物物种中已成功从体细胞生成动物克隆体,但发现与克隆胚胎核重编程相关的信息有限。本研究旨在比较司立通(SCR)和曲古抑菌素A(TSA)处理对提高克隆效率以及克隆绵羊胚胎发育率的影响。实验分为三组,即一组SCR组,二组TSA组,两种处理的浓度分别为5 nM、50 nM和500 nM,第三组为0 nM的对照组。采集屠宰绵羊的卵巢,采用抽吸法从卵泡中回收卵母细胞并使其成熟。然后用微吸管进行透明带切割并在显微操作器下进行卵母细胞去核。随后通过细胞融合仪进行核移植、细胞融合和激活。最后将胚胎在二氧化碳培养箱的培养室中培养9天。总体结果表明,随着浓度增加,卵裂率升高。不同浓度SCR处理的体细胞核移植(SCNT)胚胎的卵裂率与相同浓度TSA处理的胚胎卵裂率密切相关;例如,500 nM TSA的卵裂率为39.47%,500 nM SCR的卵裂率为38.09%;50 nM TSA的卵裂率为18.6%,50 nM SCR的卵裂率为19.17%,5 nM TSA的卵裂率为22.64%,5 nM SCR的卵裂率为17.18%。对于对照组,两种处理的SCNT胚胎卵裂率分别为27.47%、30%和30.85%。与所有其他卵裂细胞阶段(500 nM TSA和对照组)相比,在8细胞阶段,TSA处理浓度为(5和50 nM TSA)时有显著差异。此外,(50 nM TSA)与(50 nM SCR)之间也存在差异。与其他处理(5 nM、50 nM TSA和对照组)相比,在16细胞阶段,(500 nM TSA)也有显著差异。关于SCR,与其他处理(50 nM、500 nM SCR和对照组)相比,(5 nM SCR)在8细胞阶段有显著差异。此外,与其他处理(5 nM SCR和对照组)相比,(50 nM和500 nM SCR)在16细胞阶段有显著差异。在胚胎发育至囊胚阶段时,SCR组和对照组的比例较高,相比之下,TSA组未显示出任何发育至囊胚阶段的情况。SCR处理组总体显示(3/41 = 7.31%)处于囊胚期,对照组总体显示(4/89 = 4.49%)处于囊胚期。结论是,与TSA处理相比,SCR提高了最终发育至囊胚阶段的比例,TSA处理未能使胚胎达到最终发育囊胚阶段,可能是由于物种差异或TSA的毒性,尤其是在较高浓度时。