Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens

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 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.

Germline Replacement by Transfer of Primordial Germ Cells into Partially Sterilized Embryos in the Chicken1

“…A sustained-release emulsion [23] was used as the vehicle for busulfan delivery. Busulfan (Sigma-Aldrich, St. Louis, MO) was dissolved in N,Ndimethyl formamide (DMF; Nacalai Tesque, Kyoto, Japan), diluted 10-fold in Ca 2þ -and Mg 2þ -free PBS (PBS[À]).…”

“…After fixing in 4% PFA in PBS(À), gonads were rinsed three times with PBS-T for 15 min each. Fluorescent immunostaining was done as described elsewhere [23]. After blocking in 4.5% normal goat serum in PBS-T for 6 h, gonads were incubated overnight with rabbit anti-CVH antibody (1:10 000) and were then rinsed three times with PBS-T for 25 min each.…”

“…We have developed an efficient method for delivering busulfan (1,4-butanediol dimethanesulfonate), an alkylating agent with cytotoxic effect on chicken germ cells [21,22], to the chicken embryo [23]. This method enables an efficient depletion of the endogenous PGCs and repopulation with exogenous PGCs in early chicken embryos [24].…”

Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

“…However, in this study, germline transmission rate of the long-term cultured PGC-LCs were low (6%; 3/50). Thus, sterilization of the recipient should be essential for improvement of donorgermline transmission by use of irradiation (Carsience et al, 1993, Atsumi et al, 2009, Macdonald et al, 2010, Nakamura et al, 2012, busulfan treatment (Song et al, 2005, Nakamura et al, 2008. On the other hand, gametic differentiation ability of PGC-LCs might be decreased during long-term culture, this system should be progress so as to improve the ability of gametic differentiation in PGC-LCs.…”

X-irradiation Removes Endogenous Primordial Germ Cells (PGCs) and Increases Germline Transmission of Donor PGCs in Chimeric Chickens

“…In contrast, endogenous PGCs can be removed following administration of the alkylating agent busulfan (1,4-butanediol dimethanesulfonate) [11][12][13]. In regard to the latter, our group has developed an efficient method for delivering busulfan to developing chicken embryos using a sustained-release emulsion [14]. This method enables the efficient and stable depletion of endogenous PGCs.…”