Generation of Transparent Zebrafish with Fluorescent Ovaries: A Living Visible Model for Reproductive Biology

Abstract

The transparent zebrafish enables researchers to study the morphology and distribution of cells and tissues in vivo. To capture the dynamic processes of germ cell proliferation and juvenile ovarian development in zebrafish in vivo, we established transgenic (TG) lines to allow us to monitor the changes in the ovaries of living fish. The original transgenic line with ovarian fluorescence was occasionally established. Although the cDNA integrated in the strain was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka β-actin promoter, expression of EGFP is restricted to the oocytes and gills in adult fish. Mutant strains with transparent bodies, roy and ruby, were isolated in zebrafish. In this study, we crossed the TG strain with fluorescent ovary with transparent strains and established the TG (β-actin:EGFP);ruby strain. The strain is highly transparent, and the oocytes are easily observed in living fish. We identified a fluorescent tissue that might contain the undifferentiated germ cells close to the cloaca in the strain. This strain can be used for analysis of ovarian development in vivo.

See-Thru-Gonad zebrafish line: developmental and functional validation

“…These traits provide a significant improvement over the transparent Tg(bactin:eGFP)-based line β-ruby, which does not allow identification of germ cells in fish younger than 2 wpf or in males (Hsiao & Tsai 2003, Akhter et al 2016. In addition, the Tg (vasa:vasa-eGFP) transgene is germline-specific (Krøvel & Olsen 2002), while GFP signal in β-ruby has been detected in the ovary, gills, as well as a small cluster of cells near the genital pore (Akhter et al 2016).…”

“…The Tg(bactin:eGFP) line was recently crossed with roy (−/−) and albino (alb; −/−) mutants to create β-ruby, a transparent and eGFP-expressing zebrafish line for use in reproductive biology (Akhter et al 2016). While the line provides improved transparency and visualization of the ovary it contains the same limitations as the parent Tg(bactin:eGFP) line; gonads cannot be identified until after 14 dpf, and differentiated testis do not express eGFP signal.…”

Next-generation and further transgenerational effects of bisphenol A on zebrafish reproductive tissues

“…To monitor the changes in the ovaries and testes of living fish during BPA administration, we used ovarian fluorescent and transparent transgenic zebrafish lines ( Fig. 4 A) ( Akhter et al., 2016 ; Takatsu et al., 2013 ). The fluorescence of oocytes in the studied strain was relatively strong in small oocytes at stages 1 and 2.…”

“…The resulting strain, TG ( β-actin: EGFP);roy , was highly transparent, and its oocytes were easily observed by fluorescence in living fish. We refer this strain as β-roy ( Akhter et al., 2016 ; Takatsu et al., 2013 ). The TG zebrafish were bred and maintained at 28.5 °C under a 14-h light/10-h dark cycle ( Westerfield, 1995 ).…”

“…Chemical powder of BPA was mixed with powdered fish food grains (Chroma, Kobe, Japan) by crashing with ceramic mortar and stored at room temperature in a dark box as previously described for sex-change experiment ( Akhter et al., 2016 ; Takatsu et al., 2013 ). Under these conditions, the BPA mixed in the food was stable for more than one year ( Fig.…”