Estradiol (0.01 or 1.0 nM) was added to the cells for the final 24 h of culture. After treatment, medium was collected from all cultures and stored at -20°C until hormone assays were performed.
To immunoneutralize activin, monoclonal antibody to activin B (anti-activin, 25 ^g/ml, kindly supplied by Ralph Schwall, Genentech, Inc., San Francisco, CA) was added to individual wells (n = 5) of 24-well plates for the final 24 h of culture. At the end of treatment medium was collected and stored at – 20°C until hormone assays were performed.
Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was prepared from pituitary cells using the single-step method of Chomczynski and Sacchi. One hundred nanograms of total RNA were reverse transcribed to cDNA using reverse transcription-polymerase chain reaction (RT-PCR) Ready-to-Go beads (Amersham Pharmacia, Piscataway, NJ). Briefly, total RNA and 0.5 ^g oli-go(dT) were incubated for 75 min at 42°C with beads, then for 10 min at 95°C. For the amplification step, [a32P]dCTP (0.5 fxCi/^l), primers and target-specific primers were added to each tube. Samples were withdrawn every two cycles (ranging from 14 to 34 cycles) to determine amplification kinetics of the reaction. Products were separated on 6% TBE polyacrylamide gels. Gels were dried and reaction products were visualized on a PhosphorImager Cassette (Molecular Dynamics, Sunnyvale, CA) for 24-48 h at room temperature and optical densitometry was performed using ImageQuant Software (Molecular Dynamics). The oligonucleotide primers used for the amplification of cDNAs specific to FSHp, activin pB, follistatin, and p-actin are shown in Table 1. Conditions were validated for all three transcripts and for p-actin (used as the internal standard) as follows: denaturation at 95°C for 5 min (1 cycle), 30 sec at 95°C, 30 sec at 56°C, and 1 min at 72°C (30 cycles) and 10 min at 72°C for extension. To reduce variability the same master mix of reagents (including p-actin primers) was used for each transcript (FSHp, activin pB, and follistatin). For each PCR reaction, samples were run in duplicate.