要旨
We have developed RIAs using antisera directed against the cryptic peptide of the GnRH precursor (termed GnRH−associated peptide, GAP) and have used these together with a GnRH assay to characterize proGnRH−derived peptides in rat hypothalamic extracts. On Sephadex chromatography we have identified three molecular forms of GAP−like immunoreactivity (GAP−LI), in addition to the GnRH decapeptide. The largest of these forms is an 8.0−kilodalton (kD) GAP−LI which appears to be the complete proGnRH peptide. The second is a 6.5−kD GAP−LI, and is similar to the complete cryptic peptide (i.e. proGnRH14−69 or GAP1.56). The third peptide is a 2.5 kD C−terminal fragment of the cryptic peptide, representing a processed form of GAP. In whole hypothalamic extracts from normal rats the 8.0−kD form was the major form, comprising 60−70% of the total GAP−LI. All three forms were present in three distinct areas of the rat hypothalamus, namely median eminence (ME), anterior and mid−hypothalamus. However in the ME the proportion of 8.0−kD GAP−LI was significantly reduced and the proportion of 6.5−kD GAP−LI significantly increased compared to anterior and mid−hypothalamic samples (p less than 0.05). In whole hypothalamic extracts from pregnant and lactating rats the total content of proGnRH−derived peptides was reduced but the relative proportions of these peptides were not significantly changed from normal female rats. However, in postlactating rats, 2 weeks after removal of pups, the total levels of GAP−LI were unchanged compared to normals, but the percentage of 8.0−kD GAP−LI was significantly decreased and the percentage of 2.5−kD GAP−LI significantly increased compared to normals (p less than 0.05), suggesting that proGnRH may undergo additional processing dependent on physiological condition. In fetal and neonatal rats the proportion of the 6.5−kD peptide was increased and that of the 8.0−kD peptide decreased compared to adults, and this change became less significant with increasing age. In ovariectomized rats the proportion of 6.5−kD GAP−LI was increased and that of 8.0−kD GAP−LI decreased; this change was partially reversed with steroid treatment. Both the 6.5 and 2.5−kD forms were released by high K+ stimulation of neonatal hypothalamic cells in culture. These results indicate that there is differential processing of the proGnRH precursor within the hypothalamus and in altered physiological states
