In Vivo Injection
Human CG (Sigma) was dissolved in 0.9% NaCl solution in a concentration of 20 IU/ml. Each fish received 50 ^l of saline (control) or hCG solution through i.m. injection. At 12 h, 24 h, and 30 h after injections, fish were killed, and ovaries were removed for protein extraction. The ovaries were homogenized in ice-cold 50 mM Tris-HCl (pH 7.2) containing 1 mM EDTA and 1 mM PMSF using a motor-driven polypropylene pestle. The homoge-nate was centrifuged at 10 000 X g, and the resultant supernatant was used for electrophoresis. Total protein from each of the control or treated samples was mixed with SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 0.05% bromophenol blue, 10% glycerol), and approximately 15-^g protein was loaded in each lane.
In Vitro Culture of Zebrafish Follicles
Ovaries were removed from zebrafish as described above and placed in a Petri dish in modified Cortland’s medium. Follicles were isolated through manual dissection and separated into groups according to size. For each experiment, follicles collected from 4-5 fish were pooled, and 15-25 follicles were placed into each well of a 24-well culture plate in 1 ml of medium. They were then incubated with either Cortland’s medium alone (control) or hormones at room temperature (25°C). At 18 and 24 h after incubation, the rate of maturation was scored. Germinal vesicle breakdown (GVBD) has been used as an index for maturation, and it can be observed under a dissecting microscope equipped with transmitted light after follicles are placed in a clearing solution (5% acetic acid in Cortland’s medium) for 10 min. Follicles that have undergone GVBD can also be identified because they become translucent. Both methods were used to score percentage of GVBD. There was no difference in percentage of GVBD scored between two different methods used. Recombinant human (rh) activin A, rh-inhibin A, and rh-FS-288 were kindly provided by Dr. A.F. Parlow (NHPP).
All values are expressed as the mean ± SEM of pooled data from 2-4 experiments. Multiple group comparisons were performed by one-way ANOVA, followed by Scheffe’s multiple-comparison procedure, using the Statistical Analysis System (SAS) program (Cary, NC). Unpaired Student’s £-tests were used if only two groups were to be compared. P < 0.05 was considered significant.