Studies of the intrinsic antiuterotropic activity of murine alpha-fetoprotein.

We previously reported that uterine growth in immature mice given estradiol (E2) is strongly curtailed by co-administration of trace amounts of alpha-fetoprotein (AFP). We used a semiquantitative bioassay for this antiuterotropic activity to evaluate AFP purification and storage methods. AFP was isolated from Nya:NYLAR mouse amniotic fluid (MAF) by polyacrylamide gel electrophoresis plus Blue Sepharose affinity chromatography (PAGE/BS), and by affinity chromatographies employing immobilized E2 and rabbit anti-AFP. E2 (0.5 micrograms) and purified AFP (0.1-50 micrograms) were incubated in 0.10 ml PBS for 1 hr at 22 degrees C and given intraperitoneally to female pups, and their uterine weights determined 23 hr later. The antiuterotrophic action increased with AFP dose up to 1 microgram (AFP/E2 molar ratio = 0.008) and declined at higher doses. Greatest inhibition by 1.0 microgram AFP occurred in 15-18 day old NYLAR pups weighing 5-8 g, but the magnitude (approximately 3 mg) was insufficient for generating a dose/response standard curve. We therefore express the activity of a preparation as the % inhibition that 1.0 microgram of its immunoreactive AFP content imposes on the uterine growth that 0.5 micrograms E2 would elicit. NYLAR AFP was more effective in NYLAR pups than in Swiss or Colony 22 pups suggesting strain specificity. High activity (54-88% inhibition) was found in all 9 batches of PAGE/BS AFP prepared over a 2 year period. The anti-AFP affinity product had similar activity, while the immobilized E2 product showed less. AFP in MAF was inactive but treatment with Norit or dextran/Norit generated partial activity. Acid/Norit treatment to remove fatty acids produced fully active AFP but in low yield. Purified AFP in storage retained activity for 1-2 weeks at 4 degrees C, up to 6 weeks at -20 degrees C, and up to 4 months in dithiothreitol-supplemented buffer. The activity is thus inhibited by other MAF constituents and decays by a temperature sensitive process that is slowed by an antioxidant.