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UBC Theses and Dissertations

A comparative study of collagen synthesis during avian and mammalian secondary palate development : effects of 5-fluorouracil Benkhaial, Gheith S.


A study was undertaken to examine whether collagen synthesis is critical for shelf reorientation. In the initial experiments in quail, a dose of 100μg 5-fluorouracil (5-FU) administered on day 4 of incubation was determined to be the best dose-time regimen to induce cleft palate. Pregnant hamsters were given 81mg/kg 5-FU intramuscularly or 1m1 saline on day 11 of gestation. Control and treated embryonic palates dissected from hamsters between days 11 and 13 of gestation, and from quail between days 5 and 10 of incubation, were incubated in a growth medium supplemented with 14C-proline. The samples were used for either: 1. Collagen digestion assay to determine the rate of collagen synthesis; 2.Total protein determination; 3. High performance liquid chromatography (HPLC)to determine hydroxyproline (HYP) levels; or 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine different collagenisotypes. In addition, embryos from both hamster and quail were processed for light microscopy (LM). The LM results showed that, in hamster 5-FU induces cleft palate by delaying the reorientation of palatal shelves, while in quail the drug widened the gap between the palatal shelves. The data on collagen synthesis showed that in control hamster a spurt in the collagen synthesis was seen in palate between days 12:00 (12 day: 0 hour) and 12:04 of gestation, which is the period of shelf reorientation. In 5-FU exposed hamster palates, the rate of collagen synthesis was lower than controls until day 12:04 of gestation followed by a spurt on day 12:12 of gestation. In 5-FU-treated embryos palatal shelf reorientation took place between days 12:16 and 13:00 of gestation. In the developing secondary palate of both the control and 5-FU- treated quail, the rate of collagen synthesis peaked on day 8 of incubation. The collagen synthesis, however, was lower in 5-FU-treated than in the control palates. HYP levels in both control and 5-FU-treated hamster palate indicated that although an equal amount of new collagen was synthesized in both groups, the shelf reorientation was delayed in the drug-treated embryos. The HYP data from control and 5-FU-treated quail indicated that, in addition to new collagen a considerable amount of non-collagenous protein may also have been synthesized during quail palate morphogenesis. SDS-PAGE showed that only type I collagen was synthesized during palate development in both the control and 5-FU-treated hamster and quail. It was suggested that since (1) in birds, a spurt in collagen synthesis occurs in the absence of shelf reorientation, (2) an equal amount of new collagen was synthesized in both the control and 5-FU-treated hamster embryos during the period of normal reorientation, and (3) in 5-FU-treated hamster embryos, are covery in collagen synthesis occurs prior to, and a reduction at the time of initiation of delayed shelf reorientation, collagen synthesis may not cause shelf reorientation in mammals.

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