Frederick C. Leung

Purification and properties of chicken growth hormone and the development of a homologous radioimmunoassay

Highly purified growth hormone (GH) has been isolated from pituitary glands of chicken (Gallus domesticus), and a specific homologous radioimmunoassay (RIA) has also been developed. The purified chicken GH was active in the rat tibia bioassay and it gave a dose-dependent response which paralleled that of the bovine GH standard. High pressure liquid chromatography revealed that the purified chicken GH was homogenous. Chicken GH had an Rf value of 0.2 in disc electrophoresis, and a MW of 26,000 from sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was estimated to be 7.6 by gel isoelectric focusing. The amino acid composition of chicken GH was found to be similar to that of mammalian GH, and the NH2-terminal amino acid was threonine. Partial sequencing (114 amino acids) of the chicken GH showed 79% homology with bovine GH. An antiserum was developed to the purified chicken GH in a rabbit, and it was used to develop a homologous RIA using 125I-labeled chicken GH as the ligand. The purified chicken GH was iodinated via the lactoperoxidase method to a specific activity of approximately 100 microCi/micrograms. Plasma from chickens, medium from incubation of pituitary glands, and homogenates of pituitary glands gave parallel dilution-response curves with the chicken GH standard. Mammalian GH, prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) showed no cross-reaction with the 125I-labeled chicken GH. Purified turkey GH showed parallel dose response with the chicken GH, but purified turkey PRL did not cross-react. Chicken FSH and LH also showed no inhibition of binding. The minimum detectable concentration of the assay was 0.93 ng/tube, and the intraassay and interassay coefficients of variation were 9 and 16%, respectively. The specific binding of 125I-labeled chicken GH to a microsomal fraction isolated from chicken liver was identified, and the specific binding was generally low (1-4%). Turkey PRL, and chicken LH and FSH showed no inhibition of the 125I-labeled chicken GH hepatic binding and the ontogeny of the hepatic GH receptor binding sites in male and female chickens was examined.

Diminished Hepatic Growth Hormone Receptor Binding in Sex-Linked Dwarf Broiler and Leghorn Chickens:

Abstract Hepatic growth hormone (GH) receptor binding was compared in normal and sex-linked dwarfs (SLD) from both Hubbard and Cornell strain chickens. At 6, 8, and 20 weeks of age, hepatic GH receptor binding in the Hubbard SLD chickens was significantly lower than that of normal fast-growing birds. At 20 weeks of age, only 2 of 22 SLD chickens in the Hubbard broiler strain showed positive binding at a high enough level to allow for Scatchard analysis. The affinity constants and binding capacities of these two SLD chickens were numerically (but not significantly) lower than those of the normal fast-growing birds. We further examined hepatic GH receptor binding in two closely related White Leghorn strains of chickens that have been maintained as closed breeding populations for many years. We observed no detectable hepatic GH binding in the Cornell SLD chickens (N = 20), as compared to the normal-growing control strain (K strain). In both SLD strains, pretreatment with 4 M MgCl2 did not enhance GH binding, suggesting that there was no endogenous GH binding to the receptor. Based on these data, we suggest that the lack, or greatly reduced number, of GH receptors may be a major contributing factor to the dwarfism observed in these strains.

Effects of dietary thyroid hormones on growth, plasma T3 and T4, and growth hormone in normal and hypothyroid chickens

Cockerels and pullets fed with T3 or T4 for 2 weeks showed a decrease in both body weight gain and feed efficiency. The reduction in body weight gain and feed efficiency was dose related in cockerels where T3 or T4 were fed at 0.1, 1.0, and 10.0 ppm levels. T3 and T4 at 0.1 and 1.0 ppm had no significant effects on growth or feed efficiency in pullets, but the 10.0-ppm level of T3 and T4 caused a reduction of -55.24 and -28.18%, respectively, in body weight gain as compared with control birds. T3 was more active than T4 in reducing growth and was toxic when fed at 10.0 ppm both in cockerels and pullets. Both propylthiouracil (PTU)- and methimazole-treated cockerels showed a decrease in rates of gain. T3 and T4 at a dietary level of 0.1 ppm were equipotent in promoting growth in these PTU- and methimazole-treated cockerels, but 10.0 ppm caused a further reduction in body weight gain. Plasma T3 levels were found to be significantly higher in birds that were fed either T3 or T4. Plasma T4 levels were higher in T4-fed birds, but significantly lower in T3-fed birds as compared with controls. Both PTU- and methimazole-treated cockerels had significantly lower plasma T3 and T4 concentrations, but elevated plasma GH concentrations. Dietary T3 and T4 at 1.0 and 10.0 ppm significantly lowered plasma GH concentrations. In summary, these results indicated that T3 was more active than T4 in reducing body weight gain in intact normal birds, but that they were equally potent in promoting growth in PTU- and methimazole-treated hypothyroid birds.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogenesis of immunocytochemically demonstrable somatotrophs in the adenohypophyseal pars distalis of the developing chick embryo

Growth hormone (GH)-containing cells in the hypophyseal pars distalis of the chick embryo were identified immunohistochemically using anti-chicken GH sera. GH cells are first demonstrable in Rathkes Pouch as early as Day 4.5 of incubation. By Day 9.5, when the two lobes (rostral and caudal) of the pars distalis are easily recognized, GH cells are confined exclusively to the caudal lobe. Although the number of GH cells increases gradually during embryonic development, there is a statistically significant difference between Days 10.5 and 12.5 in both the cellular density and the percentage of somatotrophs. GH cells, which contain coarse granules evenly distributed throughout the cytoplasm, varied in diameter from 9.3 micron on Day 4.5 to 11.8 micron on Day 20.5, while the nuclear diameter of these cells increased from 2.8 micron on Day 4.5 to 4.9 micron on Day 20.5. There was a statistically significant difference in the nuclear/cytoplasmic ratio between Days 4.5 and 13.5 and between Days 13.5 and 20.5 of incubation. Aspects of the regulation of growth hormone synthesis and secretion in the chick embryo are discussed.

Effects of Dietary Thyroid Hormones on Growth and Serum T3, T4, and Growth Hormone in Sex-Linked Dwarf Chickens

Abstract Dwarf pullets fed with either T3 or T4 at 0.1, 1.0, and 10.0 ppm for 2 weeks showed no improvement in their body weight gain as compared with birds that were fed a control diet. Birds fed T3 or T4 at 10.0 ppm showed poorer growth, body weight gain, and feed efficiency than control birds. Pullets fed 1.0 ppm of T3 showed significantly better feed efficiency than control birds. Serum T3 concentrations were significantly higher when birds were fed 1.0 ppm of T3 or 10.0 ppm of T3 or T4. Plasma T4 concentrations were also higher in T4 fed birds (1.0 ppm and 10.0 ppm), but were significantly lower in T3 fed birds (1.0 ppm and 10.0 ppm) than in control birds. In birds fed T3 or T4 at 1.0-ppm and 10.0-ppm levels serum growth hormone concentrations were significantly lower as compared with control birds. In conclusion, exogenous T3 and T4 did not correct the sex-linked dwarfism of dwarf chickens. Such dwarfism is characterized by low circulating levels of T3 and T4.

Interaction of Human Pancreatic Growth Hormone-Releasing Factor, Thyrotropin-Releasing Hormone, and Somatostatin on Growth Hormone Release in Chickens

Abstract The effects of synthetic somatostatin (SRIF) on serum growth hormone (GH) concentrations stimulated by exogenous administration of synthetic thyrotropin-releasing hormone (TRH) and/or human pancreatic GH-releasing factor (hpGRF) were investigated in 4-week-old cockerels. In addition, the additive effects of TRH and hpGRF on serum GH were examined. TRH and hpGRF, when given in combination intravenously, produced an additive effect on serum GH concentration that peaked 10 min after the injection. The somatostatin did not significantly affect basal GH concentrations when given alone, but did significantly decrease the magnitude of the GH response to hpGRF. In contrast, SRIF did not significantly decrease the stimulatory effects of TRH on GH release. These results suggest that TRH and hpGRF are potent GH releasers in vivo and that their stimulating effects on GH release are additive, suggesting different mechanisms for their stimulation. The results obtained from the combination studies suggest that the main site of the stimulatory action of hpGRF is at the pituitary, and that SRIF significantly inhibited the rise in serum GH induced by a synthetic hpGRF, but not that induced by TRH.

Expression of the Bovine Growth Hormone Gene in Cultured Rodent Cells

Expression of recombinant DNA has been achieved in both prokaryotic and eukaryotic cells. This chapter deals with expression of bovine growth hormone recombinant DNA in cultured mammalian cells.