Retinoid requirements in the reproduction of zebrafish.

This study examines whether retinoids are essential in the reproduction of zebrafish. Using RT-PCR, it was shown that the ovaries and testes express enzymes that synthesize and metabolize the hormone retinoic acid (RA) (raldh2 and cyp26a, respectively), and RA receptors (raraa, rarga, rxrba, rxrbb, rxrga but not rxrab). Three new isoforms of rxrba were also observed in a variety of tissues. In other experiments, zebrafish were exposed for 11 d to diethylaminobenzaldehyde (DEAB), an inhibitor of RA synthesis, or fed a retinoid deficient diet for 130 d in order to evaluate the functional requirements of retinoids in reproduction. DEAB altered cyp26a transcript numbers in the gonads, suggesting an impact on RA, and decreased the number of spawned eggs by 95%. The retinoid deficient diet decreased whole body retinoids (retinol and retinal) by 68% in females and 33% in males. Females fed the retinoid deficient diet also produced 73% fewer eggs that contained 78% less retinal than controls. Fertilization rates were not affected. These studies have shown that the RA receptors are expressed in zebrafish gonads, and RA is required for the spawning of eggs. Dietary retinoid content influences reproduction, while retinyl ester storage levels appear to be of little significance. Females were more susceptible to retinoid perturbation than males, likely due to the cost of retinal deposition in the eggs. Overall, these studies have shown retinoids play a fundamental role in the reproduction of zebrafish, and the lack of retinyl ester stores in controls that successfully spawned illustrates that we have only a limited understanding of the retinoid physiology and requirements of fish.

The effects of copper and benzo[a]pyrene on retinoids and reproduction in zebrafish.

This study examines whether a link exists between toxicant exposure, retinoids and reproduction in fish. Zebrafish were fed a control diet (8.1 microg Cu/g diet, 0 microg benzo[a]pyrene/g diet) or diets containing elevated copper (100 microg, 500 microg and 1000 microg Cu/g diet) or benzo[a]pyrene (B[a]P; 30 and 150 microg B[a]P/g diet) for 260 days. Toxicant-supplemented diets did not affect growth or mortality rates. While whole body retinoid levels in control zebrafish decreased during the experiment, females exposed to Cu or B[a]P for 200 days or more experienced additional losses of retinyl esters (45-100% depleted) and retinal (45% depleted in B[a]P-fed fish). Despite the reduced retinoids, Cu and B[a]P did not effect reproduction with respect to the number of eggs spawned, fertilization rates or egg retinal content (retinal was instead increased 55-65% in eggs from B[a]P-fed fish). There were no apparent deformities observed in 36 h post fertilization embryos from any treatment. It appears that although internal retinoid stores were depleted in adults, dietary retinoids were sufficient to meet the daily requirement for retinal deposition in the eggs and retinoic acid synthesis. This study has shown that retinoid levels in female zebrafish are sensitive to Cu and B[a]P, and are a good indicator of long-term exposure. It also brings to light the resiliency of the retinoid system in fish and the importance of the diet on the toxicological response. Specifically that dietary retinoids appear to support normal reproduction in the absence of internal retinoid stores.

Dietary retinoic acid induces hindlimb and eye deformities in Xenopus laevis.

This study investigated the effects of dietary retinoic acid (RA) on frog hindlimb development. Xenopus laevis (African clawed frog) tadpoles were fed a diet supplemented with 0, 1, 10, or 100 microg of RA/g of food for 2 or 5 d at different stages of metamorphosis. Hindlimb deformities were induced in the group fed 100 microg of RA/g of diet for 5 d. Exposures beginning at mid-hindlimb bud development induced bilaterally bent tibiafibula (bony triangles), while exposures later in hindlimb development induced deformities of the feet, including fusion of the 1st and 2nd clawed digits and reduced length of the 4th and 5th digits (due to reduced, missing, or misplaced phalanges). There were also cases of extra phalanges in the 5th digit. The eye was another target of RA exposure. In one experiment, 58% of the tadpoles fed 10 microg of RA/g had a smaller or absent right eye. Additionally, 11% of the tadpoles fed 100 microg of RA/g of diet developed a smaller or absent left eye. Waterborne heavy metals (Zn or Cu) modified RA effects on the hindlimb and eye. Co-exposure to metals and RA resulted in cases of unilateral bony triangles and reduced rates of smaller eyes. There were also cases of extra hindlimb digits in Zn-exposed animals. Dietary RA exposure in tadpoles can cause some deformities that differ from waterborne RA exposures in previous studies. RA also induced deformities that resemble those in affected wild frog populations (bony triangles), although the patterns of other deformities and missing segments (phalanges and metatarsals) are not similar to those documented in the wild.