Abstract
INTRODUCTION: Primordial germ cells (PGCs) are the major population of cells in the developing bilateral embryonic gonads. Little is known about the cellular responses of PGCs after treatment with toxic chemicals such as busulfan during embryo development. In this study, we investigated the elimination, restorative ability, and cell cycle status of endogenous chicken PGCs after busulfan treatment. METHODS: Busulfan was emulsified in sesame oil by a dispersion-emulsifying system and injected into the chick blastoderm (embryonic stage X). Subsequently, we conducted flow cytometry analysis to evaluate changes in the PGC population and cell cycle status, and immunohistochemistry to examine the germ cell proliferation. RESULTS: Results of flow cytometry and immunohistochemistry analyses after busulfan treatment showed that the proportion of male PGCs at embryonic day 9 and female PGCs at embryonic day 7 were increased by approximately 60% when compared with embryonic day 5.5. This result suggests the existence of a compensatory mechanism in PGCs in response to the cytotoxic effects of busulfan. Results of cell cycling analysis showed that the germ cells in the G0/G1 phase were significantly decreased, while S/G2/M-phase germ cells were significantly increased in the treatment group compared with the untreated control group in both 9-day-old male and female embryos. In addition, in the proliferation analysis with 5-ethynyl-2'-deoxyuridine (EdU) incorporation, we found that the proportion of EdU-positive cells among VASA homolog-positive cells in the 9-day embryonic gonads of the busulfan-treated group was significantly higher than in the control group. CONCLUSIONS: We conclude that PGCs enter a restoration pathway by promoting their cell cycle after experiencing a cytotoxic effect.