Isolation and characterization of gonadotropin-rich secretory granules from rat: depletion by GnRH and by a high affinity agonist.
Gonadotropin secretory granules were purified from rat pituitary tissue by differential centrifugation followed by a continuous metrizamide density gradient separation procedure. Fractions were characterized by radioimmunoassay, electron microscopy and polyacrylamide gel electrophoresis. The LH granule peak distribution across the gradient was biphasic with peaks at densities 1.287 and 1.275 (g/ml). Samples taken from the leading and trailing regions of the LH peak remigrated to similar positions in a second gradient indicating that the pattern was not an artifact caused by reversible aggregation during the isolation procedure. Reproducible separation of the LH granule peak distribution enabled us to assess the effect of secretagogues. GnRH (2 micrograms/rat) and a metabolically stable GnRH agonist (buserelin; 1 microgram/rat) both caused a 6- to 8-fold increase in serum LH and a corresponding reduction (23-25%) in the amount of LH present in the granules. In both cases the reduction was more pronounced on the granules at the higher (1.287 g/ml) density and the bimodal LH distribution was shifted to lower densities (1.280 and 1.265 g/ml). Granules containing growth hormone and prolactin migrated in the denser (1.322-1.318 g/ml) region of the gradient whereas granules containing thyroid stimulating hormone and follicle stimulating hormone were present close to the LH peak. Electron microscopic inspection of ultrathin (60 nm) sections verified that the fractions enriched in immunoassayable gonadotropins contained predominantly granules. In the less dense (1.20 g/ml) regions, mitochondria and membranous debris were present. Fifteen days after ovariectomy, total granule LH content increased 6- to 7-fold and estrogen supplementation at 5 micrograms/rat, but not 1 microgram/rat, reversed this effect to that of sham-operated animals.