Juvenile gonad staining was performed similarly to the adult protocol with some modifications:

Euthanized fish were decapitated and cut open along the ventral midline to expose the body cavity. Alternatively, an additional cut was made at the anal fin to expose the body cavity if the fish was too small to make a ventral cut. The fish were fixed in 4% PFA in PBT at 4° C for 16–18 hours with gentle rocking. The fish were placed into fresh tubes and washed in 0.2% PBT 3 times for a minimum of 5 minutes each. Gonads were dissected out in PBT and placed into a ceramic 12-well plate, 1 gonad per well.

Gonads were washed in an antibody block composed of 5% goat serum and 5% BSA in 0.2% PBT for 1 hour minimum on a 2D rocker at room temperature. Primary antibody chicken anti-Ddx4 [42] was added at 1:500 final dilution. Plate was left rocking gently overnight at 4° C.

The gonads were washed 3 times for a minimum of 30 minutes in PBT, then washed in antibody block as described above. Secondary antibody anti-chicken Alexa Fluor 488 was added at 1:300 final dilution. Plate was left rocking gently overnight at 4° C.

The gonads were washed 2 times for a minimum of 10 minutes each and dehydrated in a series of glycerol (Sigma-Aldrich, Catalog #: G5516-1L) washes for 1 hour minimum each: 30% glycerol with DAPI at 1:5000 dilution in PBT, 50% glycerol with DAPI at 1:5000 dilution in PBT, and 70% glycerol in PBT without DAPI. The gonads were mounted in 70% glycerol without DAPI on slides with vacuum grease applied to the four corners to hold the coverslip in place. Slides were stored at 4° C until imaging.

The mutant and control females were crossed with wild-type males the morning of fixation to reduce the overall numbers of eggs in the ovaries. Animals were euthanized and decapitated posterior to the pectoral fins and cut open the ventral side to the genital pore using scissors and placed in Bouin’s fixative overnight at room temperature with gentle shaking (10ML/fish). H&E staining of paraffin-embedded gonads was done according to [80]. Images were taken at 5X magnification on a Zeiss Axiophot microscope using a Leica DFC500 camera.

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