Transgenic zebrafish with fluorescent germ cell: a useful tool to visualize germ cell proliferation and juvenile hermaphroditism in vivo.

Juvenile zebrafish are hermaphroditic; undifferentiated gonads first develop into ovary-like tissues, which then either become ovaries and produce oocytes (female) or degenerate and develop into testes (male). In order to fully capture the dynamic processes of germ cells' proliferation and juvenile hermaphroditism in zebrafish, we established transgenic lines TG(beta-actin:EGFP), harboring an enhanced green fluorescent protein (EGFP) gene driven by a medaka beta-actin promoter. In TG(beta-actin:EGFP), proliferating germ cells and female gonads strongly expressed EGFP, but fluorescence was only dimly detected in male gonads. Based on the fluorescent (+) or nonfluorescent (-) appearance of germ cells seen in living animals, three distinct groups were evident among TG(beta-actin:EGFP). Transgenics in ++ group (44%) were females, had fluorescent germ cells as juveniles, and female gonads continuously fluoresced throughout sexual maturation. Transgenics in +- (23%) and -- (33%) groups were males. Fluorescent germ cells were transiently detected in +- transgenics from 14 to 34 days postfertilization (dpf), but were not detected in -- transgenics throughout their life span. Histological analyses showed that 26-dpf-old transgenics in ++, +-, and -- groups all developed ovary-like tissues: Germ cells in -- group juveniles arrested at the gonocyte stage and accumulated low quantities of EGFP, while those in ++ group juveniles highly proliferated into diplotene to perinucleolar stages and accumulated high quantities of EGFP. In +- group juveniles, degenerating oocytes, gonocytes, and spermatogonia were coexistent in transiently fluorescent gonads. Therefore, the fluorescent appearance of gonads in this study was synchronous with the differentiation of ovary-like tissues. Thus, TG(beta-actin:EGFP) can be used to visualize germ cells' proliferation and juvenile hermaphroditism in living zebrafish for the first time.