Abstract
We tested the hypothesis that cyclic AMP plays a significant role in modulating the growth of embryonic chick cartilage by determining whether cyclic AMP levels change in growing embryonic cartilage and whether cyclic AMP could stimulate embryonic cartilage growth in a long term in vitro organ culture. Cyclic AMP levels were low (0.1 pmol/mg wet wt) in 8-d chick embryo pelvic cartilage, and increased progressively through the 11th d of embryonic development at which time they reached a maximum (1.8 pmol/mg wet weight) and thereafter remained constant. We developed an in vitro organ culture system to determine whether cyclic AMP, a factor known to stimulate radiolabeled precursor incorporation into macromolecules in short-term studies does, in fact, stimulate growth of cartilage. Individual pelvic cartilages were isolated from 9-d chick embryos, placed in serum-free medium (BGJb-FJ modification) and incubated for 3 to 5 d during which time they increased in size (39 and 60% in length, respectively), wet weight (90 and 141%, respectively), and content of total soluble protein (30 and 48%, respectively). N6-monobutyryl cyclic AMP (BtcAMP) added to the medium caused a dose-dependent (0.05 to 1.0 mM) stimulation of growth. After 3 d of incubation, 1.0 mM BtcAMP increased wet weight (125%), [14C]leucine incorporation into protein (75%), and [3H]thymidine incorporation into DNA (48%) compared with control cartilages incubated in medium alone. 1-methyl-3-isobutyl xanthine, a phosphodiesterase inhibitor, also increased cartilage growth above control while sodium butyrate, AMP, and ATP had no effect. Histological examination of cartilage grown in medium was similar to that of cartilage developing in ovo, whereas, cartilage grown in medium containing BtcAMP showed marked hypercellularity with many immature chondrocytes. Our observations are compatible with the hypothesis that cyclic AMP can significantly modulate the growth of embryonic cartilage.