Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.
The overall goal of this procedure is to isolate gonadal tissue from larval zebrafish and analyze the molecular properties of the isolated tissue This method that can help answer key questions in the zebrafish's sex development field. Such as the conservation of genes in regulating gonad development and the molecular underpinnings of the zebrafish sex development. The main advantage of this technique is that gonadal tissues from zebrafish larvae as early as 17 days post fertilization can be dissected for cellular and the molecular biological status.
Generally, individuals new to this method will struggle because of the small size and the dynamic remodel of the gonadal tissues in the larval zebrafish. To prepare for gondal disection, transfer two male and two female adult zebrafish to either side of a divided crossing tank. The following morning refresh the tank water and remove the barrier to initiate mating.
One to two hours after fertilization collect and transfer about forty eggs into a 100 millimeter plate with 40 milliliters of embryo medium, or EM.Incubate the embryos at 28.5 degrees Celsius for four days, refreshing the EM twice per day. Following the incubation transfer the larvae to one liter tanks. After five days post-fertilization, or DPF, feed live rotifers to the fish.
When the larvae reach ten DPF feed the fish live brine shrimp. Then, at 14 DPF, transfer the fish into a recirculating water system. Raise the larval zebra fish to 17 or 25 DPF.
Then measure the body length of the larvae at 17 and 25 DPF. Prepare two percent agar plates for dissection by adding four grams of agar to 200 milliliters of sterile water. Heat the mixture in a microwave until it turns transparent.
Cool the agar for 15 minutes, then pour it into 60 millimeter diameter petri dishes. Store the solidified agar plates at four degrees Celsius. To anesthetized 17 DPF larvae add crushed ice into the tank.
Transfer the anesthetized larvae to a 100 millimeter chilled petri dish with 30 milliliters of cold Ringer's solution. Incubate the fish in the chilled Ringer's solution for at least 15 minutes to fully anesthetize the larvae. With a plastic spoon transfer the larvae to a pre-cooled agar plate.
Submerge the entire fish body in ten milliliters of chilled Ringer's Solution and gently lay it on it's side. Under a stereomicroscope, at 25x magnification, use tweezers to clamp the fish trunk for stabilization. Then, with another pair of tweezers, rip the abdomen longitudinally from the anus to the heart.
Next, gently remove the skin and muscles on one side of the body to expose the internal organs. Then, carefully remove the massive organs ventral to the swim blader. Avoid damaging the gonad attached to the swim bladder.
Cut the connection between the swim bladder and interior body. Then, carefully pull out the entire swim bladder and the gonadal tissue. With tweezers, carefully separate the gondal tissue from the swim bladder.
And clean up the surrounding adipose tissue. Immediately transfer the isolated gonadal tissue to a pre-chilled, 1.5 milliliters centrifuge tube, containing 200 microliters of Ringer's Solution. Keep the tube on ice until all gonadal tissues are separated from the larvae.
To extract total RNA from larval gonadal tissue transfer the isolated gonads to a new RNase free 1.5 milliliter tube and remove the Ringer's Solution. Then, add 100 microliters of lysis solution. And vortex the tube until the tissue is completely lysed.
After performing a total RNA extraction, according to the manufacturer's instructions, add one tenth the volume of DNase I buffer, and one microliter of DNase I to the RNA solution, and gently mix the contents of the tube. Incubate the sample at 37 degrees Celsius for 20 minutes. Next, add one tenth the volume of DNase inactivation reagent to the RNA solution.
Incubate the sample at 70 degrees Celsius for ten minutes. Then, use a spectrophotometer to measure the concentration of the total RNA. To perform first strand cDNA synthesis use an oligo-dT linker primer and one to two micrograms of RNA to perform first strand cDNA synthesis, following the manufacturer's protocol.
Incubate the reaction at 45 degrees Celsius for 90 minutes. Then, terminate the reaction by heating the sample at 70 degrees Celsius for five minutes. Add one microliter on RNase H to the cDNA solution to remove the residual RNA.
Gently mix the contents of the tube and centerfuge the sample at approximately 13, 000 times G for ten seconds. Incubate the tube at 37 degrees Celsius for 20 minutes. Then, add 20 microliters of neucleous free water to the sample.
And store the cDNA at negative 70 degrees Celsius. Finally, use fluorescent dye to perform QCPR using the condition and primers listed in the text protocol. This figure shows typical gonadal tissue of lavaral zebrafish at 17 DPF.
The gonad is attached to the ventral side of the swim bladder, as seen here. At 17 DPF, the larvae contain the left and the right gonads, which are translucent. In most cases the gonads are surrounded by epithelial tissue and protonephridium.
At 25 DPF, the gonad is often wrapped by adipose tissue, and big or small gonads may be observed. Finally, to analyze the molecular properties of the isolated gonadal tissue, expression levels of the gonadal markers amh, cyp19a1a, nanos3, and vasa were examined by QPCR. The results show significant increases in marker genes in gonad tissue compared to controlled tissue.
Once mastered this technique can be done in an hour if it's performed properly. While attempting this procedure it's important to remember to provide consistent rearing conditions for larvae zebrafish, as it's critical to yield expected results. After it's development, this technique paved the way for researches in the field of sex development to explore sex determining mechanisms of zebra fish.
After watching this video you should have a good understanding of how to isolate gonadal tissues from larval zebra fish.
