To confirm the identities of PCR products, DNA fragments were recovered from gels using Gene Clean II kit (Bio 101, Vista, CA) and were sequenced using PCR primers. All sequencing runs were performed using a DNA sequencer (Applied Biosystems, Inc., Foster City, CA) at the York University (Toronto, ON, Canada) Core Facility for Molecular Biology. Sequencing data were analyzed using the Blast program (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD; http:Wwww.ncbi.nlm.nih.gov/).
Protein Extraction and Western Blot Analysis
Fish were anesthetized using MS-222 (Sigma) and decapitated. Ovaries were subsequently removed and washed in Cortland’s balanced saline. Concentrations of solubilized proteins were determined using a Bio-Rad Protein Assay Kit (Bio-Rad Labs., Richmond, CA). Proteins were separated in a 12.5% SDS-polyacrylamide gel under nonreducing conditions using a Bio-Rad mini-gel apparatus. After electrophoresis, proteins were transferred to a polyvinyli-dene difluoride (PVDF) membrane (Amersham-Pharmacia). After incubation with the blocking solution (Tris-buffered saline containing 0.1% Tween-20 and 5% skim milk) overnight at 4°C, the membrane was incubated with a monoclonal mouse anti-human activin (3A antibody (1:200 dilution; Cedarlane Laboratories Ltd., Hornby, ON, Canada) or mouse anti-human FS antibody (1:5000 dilution, obtained from Dr A.F. Parlow, National Hormone and Pituitary Program [NHPP], Rockville, MD), overnight at 4°C. Subsequently, the membranes were washed and then incubated for 1 h at room temperature with a horseradish peroxidase-conjugated (anti-mouse IgG diluted 1:3000 for (3A or antirabbit IgG diluted 1:50 000 for FS; Amersham-Pharmacia). Immunoreactive signals were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham-Phar-macia).