of the incubation during the six day incubation follicle cells proliferated significantly increasing the yield of cells for the experiments cells are washed and trypsinized at 28 u00b0 c for 15 minutes thereafter the cells are washed three times with medium m199 through centrifugation at 1000 rpm for 2 minutes and then subcultured in 24 well plates at a density of 1 u00d7 10 5 cells ml per well for 24 hours in complete m199 before hormone treatment the amount of cells was not enough for the experiment so we should start with 20 females to get enough material different concentrations of the supernatant will be used as a positive control hcg will be used at a 15 iu ml and carp pituitary extracts will also be included with the pituitary extract condition we can compare the amount of cells that should be used to observe an effect in the reproduction capacity of the female eel hcg was dissolved in 0 9 of nacl solution in a concentration of 20 iu ml each fish will receive 50 u03bcl of saline as a negative control hcg as a positive control and different concentrations of the supernatant or the purified fsh u03b2 and or lh u03b2 at 1 2 4 6 and 12 hours after injections fish are killed and ovaries removed for rna extraction at the end of the hormonal treatment total rna was isolated from zebrafish ovaries or follicular cells homogenized in liquid nitrogen and extracted using trizol reagent according to the manufacturer 39.