Nakamura Yoshiaki, Yamamoto Yasuhiro, Usui Fumitake, Atsumi Yusuke, Ito Yohei, Ono Tamao, Takeda Kumiko, Nirasawa Keijiro, Kagami Hiroshi, Tagami Takahiro
Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano, Japan.
Reprod Fertil Dev. 2008;20(8):900-7. doi: 10.1071/rd08138.
The aim of the present study was to improve the efficiency of endogenous primordial germ cell (PGC) depletion and to increase the ratio of donor PGCs in the gonads of recipient chicken embryos. A sustained-release emulsion was prepared by emulsifying equal amounts of Ca(2+)- and Mg(2+)-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 microg per 50 microL busulfan sustained-release emulsion was injected into the yolk. To determine the depletion and repopulation of PGCs in the gonads after 6 days incubation, whole-mount immunostaining was performed. The busulfan sustained-release emulsion significantly reduced the number of endogenous PGCs compared with control (P < 0.05). Moreover, the busulfan sustained-release emulsion significantly depleted endogenous PGCs compared with other previously reported busulfan delivery systems (P < 0.05), but with less variation, suggesting that the sustained-release emulsion delivered a consistent amount of busulfan to the developing chicken embryos. The PGC transfer study showed that the proportion of donor PGCs in the gonads of busulfan sustained-release emulsion-treated embryos after 6 days incubation increased 28-fold compared with control. In conclusion, the results demonstrate that exogenous PGCs are capable of migrating and settling in gonads from which endogenous PGCs have been removed using a busulfan sustained-release emulsion.
本研究的目的是提高内源性原始生殖细胞（PGC）清除效率，并提高受体鸡胚性腺中供体PGC的比例。通过使用过滤器将等量含10%白消安（溶解于N，N-二甲基甲酰胺）的无钙镁磷酸盐缓冲盐水与芝麻油乳化，制备了一种缓释乳剂。然后，将每50微升含75微克白消安的缓释乳剂注入卵黄。为了确定孵化6天后性腺中PGC的清除和重新填充情况，进行了全胚胎免疫染色。与对照组相比，白消安缓释乳剂显著减少了内源性PGC的数量（P<0.05）。此外，与其他先前报道的白消安给药系统相比，白消安缓释乳剂显著清除了内源性PGC（P<0.05），但变异较小，这表明缓释乳剂向发育中的鸡胚输送了一致量的白消安。PGC移植研究表明，孵化6天后，经白消安缓释乳剂处理的胚胎性腺中供体PGC的比例与对照组相比增加了28倍。总之，结果表明外源性PGC能够迁移并定居在使用白消安缓释乳剂清除了内源性PGC的性腺中。
