Zakhartchenko V, Durcova-Hills G, Schernthaner W, Stojkovic M, Reichenbach H D, Mueller S, Steinborn R, Mueller M, Wenigerkind H, Prelle K, Wolf E, Brem G
Bavarian Research Center for Biology of Reproduction (BFZF), Oberschleissheim, Germany.
Mol Reprod Dev. 1999 Apr;52(4):421-6. doi: 10.1002/(SICI)1098-2795(199904)52:4<421::AID-MRD11>3.0.CO;2-P.
The developmental potential of bovine fetal germ cells was evaluated using nuclear transfer. Male and female germ cells at three stages of fetal development from 50- to 57-, 65- to 76- or 95- to 105-day-old fetuses were fused to enucleated oocytes 2 to 4 hr prior to activation with 7% ethanol (5 min) followed by 5 hr culture in 10 microg/ml cycloheximide and 5 microg/ml cytochalasin B. The in vitro development of nuclear transfer embryos derived from germ cells was compared with those derived from embryonic cells (blastomeres from day 5 or day 6 embryos). Blastocyst rate (38%) obtained with germ cells from 50- to 57-day-old fetuses tended to be higher than when using germ cells from 65- to 76- or 95- to 105-day-old fetuses (23% and 20%, respectively). Within each stage of fetal development, the proportion of blastocysts derived from male germ cells tended to be higher than that obtained with female germ cells, but due to the high variation between individual fetuses this difference was not significant. With the post activation procedure used in this study, germ cells from 50- to 57-day-old fetuses supported the development of nuclear transfer embryos to the blastocyst stage significantly (P<0.05) better than nuclei of embryonic cells (38% vs. 3%). After transfer of blastocysts derived from germ cells of 50-to 57- and 65- to 76-day fetuses, respectively, 45% (5/11) and 50% (3/6) recipients were pregnant on day 30. The corresponding pregnancy rates on day 90 were 36% (4/11) and 17%(1/6). One live male calf was delivered by cesarean section at day 277 of gestation. Our results show that nuclei of bovine fetal germ cells may successfully be reprogrammed to support full-term development of nuclear transfer embryos.
利用核移植技术评估了牛胎儿生殖细胞的发育潜能。分别采集50至57日龄、65至76日龄或95至105日龄这三个胎儿发育阶段的雄性和雌性生殖细胞，在使用7%乙醇（5分钟）激活前2至4小时，将其与去核卵母细胞融合，随后在含有10微克/毫升环己酰亚胺和5微克/毫升细胞松弛素B的培养液中培养5小时。将源自生殖细胞的核移植胚胎的体外发育情况与源自胚胎细胞（第5天或第6天胚胎的卵裂球）的核移植胚胎进行比较。来自50至57日龄胎儿的生殖细胞所获得的囊胚率（38%）往往高于使用65至76日龄或95至105日龄胎儿的生殖细胞时的囊胚率（分别为23%和20%）。在胎儿发育的每个阶段内，源自雄性生殖细胞的囊胚比例往往高于源自雌性生殖细胞的囊胚比例，但由于各个胎儿之间差异较大，这种差异并不显著。采用本研究中的激活后程序，50至57日龄胎儿的生殖细胞比胚胎细胞的细胞核更能有效地支持核移植胚胎发育至囊胚阶段（P<0.05）（38%对3%）。分别将源自50至57日龄和65至76日龄胎儿生殖细胞的囊胚移植后，在第30天，45%（5/11）和50%（3/6）的受体怀孕。在第90天，相应的怀孕率分别为36%（4/11）和17%（1/6）。在妊娠第277天通过剖宫产分娩出一头存活的雄性小牛。我们的结果表明，牛胎儿生殖细胞的细胞核可成功重编程，以支持核移植胚胎的足月发育。
