Characterization of LH-RH immunoreactivity in mammalian pituitary neural lobe by HPLC

High performance liquid chromatography was used to characterize luteinizing hormone-releasing hormone (LH-RH) immunoreactivity that was previously identified immunocytochemically in the pituitary neural lobes of bats, ferrets and humans. Extracts of bat posterior lobe and hypothalamus, ferret posterior lobe and hypothalamus and human neurohypophysis were partially purified with C-18 Bond-Elut cartridges. Samples were chromatographed using a C-18 reverse phase HPLC column, and LH-RH-immunoreactive moieties were separated by gradient elution (TFA/acetonitrile solvent system). For bats and ferrets, the major peak of neural lobe LH-RH immunoreactivity eluted with a retention time identical to that of hypothalamic LH-RH. Synthetic mammalian standard added to bat and ferret hypothalamic extracts coeluted as a single peak with the predominant form of LH-RH immunoreactivity present in those tissues. In humans, the peak of LH-RH immunoreactivity in neural lobe extracts coeluted with synthetic standard. These results provide strong evidence that the LH-RH-immunoreactive fibers which terminate within the neural lobe contain authentic LH-RH. Additional minor peaks of LH-RH immunoreactivity were observed in posterior lobe and hypothalamic extracts of both bats and ferrets. Comparisons of posterior lobe content of LH-RH immunoreactivity across species verify that the neural lobe projection is a major component of the LH-RH system in bats, whereas it is represented only minimally in the laboratory rat.