High accumulation of EGFP in female gonads of TG(β-actin:EGFP). (A) Comparison of EGFP accumulated in somatic tissues (black filled boxes) and female gonads (gray filled boxes) among seven distinct transgenic lines (β1, β3, β4, β5, β7, β9, and β11). EGFP content was evaluated with a fluorescence spectrophotometer and normalized against the same amount of a total soluble protein. Values are the means ± SE of five replicates. (B) SDS-PAGE (upper panel) and Western blot (lower panel) analyses of EGFP accumulated in gonads of β9 TG(β-actin:EGFP). S, soluble proteins extracted from somatic tissues; O, soluble proteins extracted from female gonads; NT, non-transgenics; TG, transgenics. Arrow indicates the positive signal of the 27-kDa EGFP protein. Protein markers are shown on the left; sizes are in kDa. (C) Sexually dimorphic expression of EGFP in the male (left) and female (right) gonads of β9 TG(β-actin:EGFP) at 60 dpf. TG(β-actin:EGFP) are viewed laterally; anterior to the left of the figure. (D) Transverse sections showing the relative intensity of fluorescent gonads in male (left) and female (right) TG(β-actin:EGFP). ms, muscle; ov, ovary; sk, skin; te, testis. Scale BAR = 250 μm in (C) and 200 μm in (D).

Subcellular distribution of EGFP and β-actin in female gonads of β9 TG(β-actin:EGFP) detected by cryosection or immunohistochemistry. (A–E) Paraffin sections of female gonads. Sections are stained with H&E. (F–J) Cryosections of female gonads. (K–O) Immunohistochemistry of female gonads with β-actin monoclonal antibody. (P–T) Immunohistochemistry of female gonads with troponin T monoclonal antibody. Overviews of gonadal sections at low magnification (A, F, K, P). High magnification of oocytes at the primary growth stage (B, G, L, Q), early (C, H, M, R), middle (D, I, N, S), and late (E, J, O, T) vitellogenic stages. gv, germinal vesicle; I, stage I (primary growth stage) oocyte; IIa, stage IIa (early vitellogenic stage) oocyte; IIb, stage IIB (middle vitellogenic stage) oocyte; III, stage III (late vitellogenic stage) oocyte. Scale BAR = 250 μm in (A, F, K, P), 100 μm in (E, J, O, T), 60 μm in (D, I, N, S), and 30 μm in (B, C, G, H, L, M, Q, R).

Visualization of juvenile hermaphroditism in living TG(β-actin:EGFP) by following the fluorescent appearance of gonads. (A–F) In -- group juveniles (33%), no fluorescent appearance of gonads could be detected from juveniles throughout sexual maturation. (G–L) In ++ group juveniles (44%), gonads were fluorescent from juveniles and continued to fluoresce throughout sexual maturation. (M–R) In +- group juveniles (23%), gonads transiently fluoresced at the juvenile stage but gradually lost their fluorescence in later development stages. Fish in all pictures are viewed laterally and the anterior is to the left. All pictures were recorded at the same exposure time (2 s). Development stages are indicated in each panel. dpf, day postfertilization; gc, germ cells. Scale BAR = 500 μm in (A, G, M) and 1 mm in all other pictures.

Gonadal histology of TG(β-actin:EGFP) in --, ++, and +- groups juveniles. (A–D) Transverse sections showing germ cell development in -- group juveniles. Germ cells of 14- to 26-dpf-old juveniles in -- group are solely composed of gonocytes and surrounded by somatic cells. (E–H) Transverse sections showing germ cell development in ++ group juveniles. Germ cells of 14- to 26-dpf-old juveniles in ++ group juveniles are solely composed of early diplotene oocytes. (I–L) Horizontal sections showing germ cell development in +- group juveniles. Degenerating diplotene oocytes, gonocytes, and spermatogonia coexist in the gonads of 26-dpf-old juveniles in the +- group. Developmental stages are indicated in each panel. dpf, day postfertilization; ed, early diplotene oocytes; go, gonocytes; po, perinucleolar oocytes; sc, somatic cells; sg, spermatogonia; so, spermatocytes; sp, spermatids. Scale BAR = 25 μm in (E–I) and 10 μm in all other pictures.

Acknowledgments
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Reprinted from Developmental Biology, 262, Hsiao, C.-D. and Tsai, H.-J., Transgenic zebrafish with fluorescent germ cell: a useful tool to visualize germ cell proliferation and juvenile hermaphroditism in vivo, 313-323, Copyright (2003) with permission from Elsevier. Full text @ Dev. Biol.