Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens
Yoshiaki Nakamura A B , Yasuhiro Yamamoto A , Fumitake Usui A , Yusuke Atsumi A , Yohei Ito A , Tamao Ono A , Kumiko Takeda B , Keijiro Nirasawa B , Hiroshi Kagami A and Takahiro Tagami B CA Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano 399-4598, Japan.
B National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan.
C Corresponding author. Email: tagami@affrc.go.jp
Reproduction, Fertility and Development 20(8) 900-907 https://doi.org/10.1071/RD08138
Submitted: 18 June 2008 Accepted: 27 July 2008 Published: 10 October 2008
Abstract
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 Ca2+- and Mg2+-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 μg per 50 μL 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.
Additional keyword: gonadal migration.
Acknowledgements
The authors thank the staff of the Poultry Management Section of the NILGS for taking care of the birds and providing the fertilised eggs. The authors also thank K. Hamano of the Faculty of Agriculture, Shinshu University for his helpful advice. The present study was supported by all members of the Animal Breeding and Reproduction Research Team, NILGS.
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