HORMONAL EFFECTS ON CALCIFICATION AND BONE DEVELOPMENT IN THE CHICK EMBRYO

The ability of parathyroid hormone (PTH), calcitonin (CT) and the vitamin D metabolites 25-hydroxycholecalciferol (25 D-3) and 1,25-dihydroxycholecalciferol (1,25D-3) to stimulate calcium release from embryonic chick bone was assessed in vitro. Limb bones from embryos of twelve days of incubation were chosen as targets for the hormones because this is the start of the increased calcium movement from the shell into the embryonic circulation. The calcium is then used by the embryo for skeleton formation.

Homogenates of parathyroid glands from adult turtles, Chrysems picta and C. scripta were capable of stimulating the release of previously incorporated calcium-45 from the bones after 8, 24 and 72 hours of incubation in vitro. At 8 hours, all doses increased calcium release 90%. At 24 and 72 hours, the responses to the homogenate were dose-dependent. An estimate of 1.25 IU/gland of turtle PTH was made based on a comparison of the stimulatory capacities of turtle PTH and bovine PTH. These two hormones produced parallel dose-response curves in this system.

Addition of bovine PTH to prelabeled bone cultures resulted in a significant increase in the release of calcium-45 into the culture medium after 24, 48 and 72 hours incubation. Maximal resorption occurred at 48 hours incubation. Low doses of salmon CT decreased the calcium-45 release from bone but higher doses of salmon CT significantly increased calcium-45 release. When bones were incubated with both bovine PTH and salmon CT, the amount of calcium-45 release was consistently less than that caused by the bovine PTH alone. Escape from salmon CT inhibition of PTH-mediated resorption may have occurred at 72 hours of incubation.

When 25D-3 and 1,25D-3 were assessed for their ability to stimulate calcium release in the assay, it was found that both metabolites produced significant calcium-45 releases into the medium after 24 and 48 hours incubation. The response to the metabolites was always greater at 48 hours than the response seen at 24 hours and a clear dose-dependency was evident at the end of 48 hours of incubation. 1,25D-3 was shown to be the more potent resorption-inducing agent at all concentrations of the metabolites that were tested. These concentrations were within the circulating range of vitamin D metabolites in chick embryo plasma.

Parathyroid glands from chick embryos of 12-20 days of incubation were homogenized and assayed for calcium-mobilizing ability in the chick bone culture system. At all ages tested, the glands were shown to contain a calcium-mobilizing substance. The potency of the gland homogenates increased between days 12-16 of incubation, dropped on day 17, remained low on days 18 and 19, and returned to the day 16 level by the 20th day. This reduction in calcium-mobilizing ability may correspond with the release of CT from the embryonic chick parathyroid glands which occurs during the later stages of incubation. The calcium-mobilizing substance present in the homogenates was not subject to structural analysis so it is not possible to state with certainty that it is PTH. Gland homogenates produced dose-response curves parallel to that of the response to bovine PTH and this is a good indication that PTH was being measured in the assay.

These experiments suggest that embryonic bone is responsive to hormonal stimulation as early as day 12 incubation. The time course of limb development in the embryo can be correlated with the presence of PTH, CT and vitamin D in the embryo. This fact, taken together with the responses of the embryo to the hormones in other putative targets suggests that hormones are involved in bone formation in the avian embryo.