SUMMARY

An assay system, using the characteristic migration of follicle-stimulating hormone (FSH) on polyacrylamide gels, was developed to evaluate the effects of hypothalamic factors on amino acid incorporation into FSH in the rat adenohypophysis incubated in vitro. Follicle-stimulating hormone extracted from rat pituitary secretory granules was found to migrate on 7·5% polyacrylamide gels, pH 9·5, as a protein with an R _F of 0·614. The characteristic R _F was used to measure [14C]amino acid incorporation into FSH in rat adenohypophyses in vitro. Follicle-stimulating hormone synthesis increased after addition of crude hypothalamic extract to the incubation medium. Hormone synthesis was also increased three times after the addition of dibutyryl cyclic AMP.

SUMMARY

DNA polymerase activity was found in the cytoplasmic fraction and in isolated nuclei from anterior pituitary glands of male rats. The enzyme activity was assayed by measuring the incorporation of [³H]dTTP into DNA in a medium containing Tris-HCl buffer (pH 8·5), the four deoxyribonucleoside triphosphates, Mg^{2 +}, ATP and activated calf thymus DNA. The DNA polymerase activity decreased with age in glands from animals aged 25 days to over a year but increased after oestrone treatment in vivo. These changes in activity, more pronounced in the cytoplasmic fraction than in the isolated nuclei, were similar to changes in DNA synthesis measured in anterior pituitary glands under the same physiological conditions.

Isolated nuclei also retained endogenous DNA synthetic activity in the absence of added template. Addition of a cytoplasmic fraction to the reaction medium stimulated activity by as much as 1·9-fold but the degree of stimulation was the same whether the cytoplasm was from young, old or oestrone-treated animals.

ABSTRACT

The average concentration of GH in blood is high at birth and declines during the period of sexual maturation in bulls. The objectives of these studies were (1) to define age-related changes in vivo in the pulsatile secretion of GH from birth to puberty, (2) to determine whether pituitary cell content of GH and characteristics of the secretion of GH in vitro reflect age-related changes in vivo, and (3) to examine whether responsiveness to GH-releasing hormone (GHRH) and somatostatin (SRIF) in vitro changed with age in Holstein bull calves.

In experiment 1, calves were bled every 15 min for 12 h at <1, 12 and 42 weeks of age (n= 5/group), these being representative of infantile, juvenile and pubertal stages of development. Calves were killed 3 to 5 days later and the pars distalis of the anterior pituitary gland was enzymatically dispersed into a suspension of single cells. Aliquots of cells were extracted with 0·01 mol NaHCO₃/l to determine the content of GH and cultured for 18 and 72 h. As expected, the average concentration of GH in plasma decreased with age (P<0·001). The initial decrease in GH was caused by a reduction in the baseline concentration between birth and 12 weeks of age. There was a marked decrease in GH pulse amplitude between 12 and 42 weeks of age and a further reduction in the baseline concentration. In contrast, the pulse frequency of GH increased (P<0·05) from <1 week to 12 weeks of age and remained constant thereafter. The initial intracellular content of GH and basal release of GH into media were similar at birth and 12 weeks of age. However, intracellular content of GH and basal release were decreased (P<0·01) after 18 and 72 h of culture from cells obtained from 42-week-old calves when compared with cells obtained from calves <1 and 12 weeks of age. At each age, the total amount of GH present in the cultures at 18 and 72 h was greater than intracellular GH, indicating net synthesis in culture.

In experiment 2, pituitary responsiveness to GHRH and SRIF were evaluated in vitro using cells obtained from calves at <1, 12 and 25 weeks of age. GHRH stimulated and SRIF inhibited the release of GH in a dose-dependent manner. The dose required to achieve 50% of the maximum response for GHRH and SRIF was 46·9 pmol/l and 4·76 nmol/l respectively and these were not influenced by age. However, the maximum response to GHRH was greatest at 25 weeks of age and this was correlated with the initial cell content.

In summary, the decrease in the average concentration of GH in plasma in bull calves was caused initially by a reduction in the baseline concentration of GH and then by a decline in GH pulse amplitude. The decrease in GH levels between birth and 12 weeks of age was not accompanied by changes in the initial GH content of the pituitary, the ability of somatotrophs to secrete GH in vitro, or changes in pituitary sensitivity to GHRH or SRIF. Thus, the changes in the secretion of hypothalamic factors may account for the initial decrease in GH. However, the low circulating level of GH at 42 weeks of age was associated with a reduced somatotroph content of GH and a decrease in the ability to secrete GH in culture, indicating a fundamental change in somatotroph function in older animals that is retained in vitro.

Journal of Endocrinology (1993) 139, 307–315