Zakhartchenko V, Reichenbach H D, Riedl J, Palma G A, Wolf E, Brem G
Bayerisches Forschungszentrum für Fortpflanzungsbiologie, Oberschleissheim, Munich, Germany.
Mol Reprod Dev. 1996 Aug;44(4):493-8. doi: 10.1002/(SICI)1098-2795(199608)44:4<493::AID-MRD9>3.0.CO;2-G.
To determine the best developmental stage of donor embryos for yielding the highest number of clones per embryo, we compared the efficiencies of nuclear transfer when using blastomeres from morulae or morulae at cavitation, or when using inner-cell-mass cells of blastocysts as nuclear donors. This comparison was done both on in vivo-derived and in vitro-produced donor embryos. In experiment 1, with in vivo-derived donor embryos, nuclei from morulae at cavitation supported the development of nuclear transfer embryos to the blastocyst stage (36%) at a rate similar to that of nuclei from morulae (27%), blastomeres from morulae at cavitation being superior (P < 0.05) to inner-cell-mass cells from blastocysts (21%). The number of blastocysts per donor embryo was significantly (P < 0.05) higher when using nuclei from morulae at cavitation (15.7 +/- 4.1) rather than nuclei from morulae (9.8 +/- 5.5) or blastocysts (6.3 +/- 3.3). With in vitro-produced donor embryos (experiment 2), nuclei from morulae yielded slightly more blastocysts (32%) than nuclei from morulae at cavitation (29%), both stages being superior to nuclei from blastocysts (15% development to the blastocyst stage). Morulae at cavitation yielded a higher number of cloned blastocysts per donor embryo (11.5 +/- 5.9) than did morulae (9.3 +/- 3.2) and blastocysts (3.3 +/- 1.4). Transfer of cloned embryos originating from in vivo-derived morulae, morulae at cavitation, and blastocysts resulted in four pregnancies (10%), three pregnancies (7%), and one (17%) pregnancy on day 45. The corresponding numbers of calves born were 3 (4%), 3 (7%), and 0, respectively. After transfer of blastocysts derived from in vitro nuclear donor morulae (n = 16) and morulae at cavitation (n = 7), two (20%) and two (50%) recipients, respectively, were pregnant on day 45. However, transfer of seven cloned embryos from in vitro donor blastocysts to three recipients did not result in a pregnancy. Using in vitro-produced donor embryos, calves were only obtained from morula-stage donors (13%). Our results indicate that the developmental stage of donor embryos affects the efficiency of nuclear transfer, with morulae at cavitation yielding a high number of cloned blastocysts.
为了确定供体胚胎的最佳发育阶段，以便每个胚胎产生最多数量的克隆体，我们比较了使用桑葚胚或处于囊胚腔形成阶段的桑葚胚的卵裂球，或使用囊胚的内细胞团细胞作为核供体时的核移植效率。这种比较在体内来源和体外生产的供体胚胎上均进行。在实验1中，对于体内来源的供体胚胎，处于囊胚腔形成阶段的桑葚胚的细胞核支持核移植胚胎发育到囊胚阶段的比例（36%）与桑葚胚的细胞核（27%）相似，处于囊胚腔形成阶段的桑葚胚的卵裂球优于（P<0.05）囊胚的内细胞团细胞（21%）。当使用处于囊胚腔形成阶段的桑葚胚的细胞核（15.7±4.1）时，每个供体胚胎产生的囊胚数量显著（P<0.05）高于使用桑葚胚的细胞核（9.8±5.5）或囊胚的细胞核（6.3±3.3）时。对于体外生产的供体胚胎（实验2），桑葚胚的细胞核产生的囊胚（32%）略多于处于囊胚腔形成阶段的桑葚胚的细胞核（29%），这两个阶段均优于囊胚的细胞核（发育到囊胚阶段的比例为15%）。处于囊胚腔形成阶段的桑葚胚每个供体胚胎产生的克隆囊胚数量（11.5±5.9）高于桑葚胚（9.3±3.2）和囊胚（3.3±1.4）。移植源自体内来源的桑葚胚、处于囊胚腔形成阶段的桑葚胚和囊胚的克隆胚胎，在第45天时分别有4例妊娠（10%）、3例妊娠（7%）和1例妊娠（17%）成功。相应出生的犊牛数量分别为3头（4%）、3头（7%）和0头。在移植源自体外核供体桑葚胚（n = 16）和处于囊胚腔形成阶段的桑葚胚（n = 7）的囊胚后，分别有2例（20%）和2例（50%）受体在第45天时怀孕。然而，将7个源自体外供体囊胚的克隆胚胎移植到3个受体中未获得妊娠成功。使用体外生产的供体胚胎时，仅从桑葚胚阶段的供体获得了犊牛（13%）。我们的结果表明，供体胚胎的发育阶段会影响核移植效率，处于囊胚腔形成阶段的桑葚胚能产生大量克隆囊胚。
