Elsheikh Adil Salim, Takahashi Yoshiyuki, Katagiri Seiji, Kanagawa Hiroshi
Department of Reproduction and Obstetrics, Faculty of Veterinary Medicine, University of Khartoum, Shambat, PO Box 32, Sudan.
Reprod Fertil Dev. 2006;18(6):697-701. doi: 10.1071/rd05159.
This study was carried out to test the ability of sucrose-exposed chemically enucleated mouse oocytes to support the development of reconstituted embryos in vitro. Cumulus-enclosed germinal-vesicle-stage mouse oocytes were matured in vitro to metaphase I stage and were chemically enucleated with 50 microg mL(-1) etoposide in tissue culture medium 199. The chemically enucleated oocytes were grouped into two groups. Group I was exposed to 0.75 M sucrose and group II was not exposed to sucrose. The zonae pellucidae of the chemically enucleated oocytes were removed with acid Tyrode's solution (pH 2.7). They were then aggregated into couplets with karyoplasts from pronuclear-stage embryos using phytohemagglutinin-P. The couplets were electrically fused to form reconstituted embryos. The reconstituted embryos were activated with 7% ethanol and cultured in vitro in simplex optimisation medium to test their developmental ability to the blastocyst stage. Some of the reconstituted embryos that developed to the blastocyst stage were used for chromosome counts to test their ploidy. The results of the present study showed that chemically enucleated oocytes exposed to sucrose supported the development of reconstituted embryos to the blastocyst stage (21.5%), whereas those not exposed to sucrose did not. The chromosome counts showed that the reconstituted embryos had normal ploidy (40 chromosomes). It is concluded that sucrose exposure improves the quality of chemically enucleated mouse oocytes. Thus they can be used as recipients for mouse embryo cloning and nucleocytoplasmic interaction studies.
本研究旨在测试经蔗糖处理的化学去核小鼠卵母细胞支持体外重构胚胎发育的能力。将卵丘包裹的生发泡期小鼠卵母细胞在体外培养至中期I期，并用50μg/mL依托泊苷在199培养基中进行化学去核。化学去核的卵母细胞分为两组。第一组暴露于0.75M蔗糖，第二组不暴露于蔗糖。用酸性台氏液（pH 2.7）去除化学去核卵母细胞的透明带。然后使用植物血凝素-P将它们与原核期胚胎的核体聚合成双联体细胞。双联体细胞经电融合形成重构胚胎。重构胚胎用7%乙醇激活，并在体外简单优化培养基中培养，以测试其发育至囊胚期的能力。一些发育至囊胚期的重构胚胎用于染色体计数以检测其倍性。本研究结果表明，暴露于蔗糖的化学去核卵母细胞支持重构胚胎发育至囊胚期（21.5%），而未暴露于蔗糖的则不能。染色体计数显示重构胚胎具有正常倍性（40条染色体）。结论是，蔗糖处理可提高化学去核小鼠卵母细胞的质量。因此，它们可作为小鼠胚胎克隆和核质相互作用研究的受体。
