Detection of Immunoreacting LH-RF Neurons in Rodents

A highly specific antibody to LH-RF was generated in female rabbits by frequent immunization with synthetic LH-RF-BSA conjugate.In vitro incubation gave 35-40% binding of 125I-LH-RF and a linear dose-response relationship against cold LH-RF (from 3-2500 pg) using 1: 1500 dilutions of the antiserum. This antiserum showed no marked immunocross-reactivity with rat LH and FSH, Substance P, oxytocin, lysine and arginine vasopressin or synthetic TRF compared with synthetic LH-RF. These substances were less than 0.03% displacement of LH-RF under these conditions. The precipitin line disappeared after absorption by BSA, though the antiserum reacted with the LH-RF-BSA conjugate on the Ouchterlony agar plate test. Intravenous injections of 0.8 ml of this antiserum to 4-day cycling female rats on the day of proestrus blocked ovulation on the following day. In these rats, LH surge on the evening of proestrus was blocked, but LH was maintained at basal levels. Two to five ng of LH-RF was detected in adult mature rat hypothalamus. Subcellular fractions were radioimmunoasayed for LH-RF The synaptosomal fraction contained 5% LH-RF as compared. with whole hypothalamic tissue (47.6ng in 17 rats). For immunohistochemical demonstrations, the antibody was applied to rats and mice hypothalami by new and direct immunoenzyme techniques. Adult Wistar-Imamichi rats and ICR mice were decapitated and the hypothalami were prefixed with 2% paraformaldehyde and freeze-dried paraffin or cryostat sections were postfixed with Zamboni's or PLP solutions (found to be the most satisfactory). LH-RF was detected in the cytoplasm of some cell bodies close to the ependymal lining in the lower part of the arcuate nucleus. Other specific LH-RF neurons were observed in the medial preoptic, prechiasmatic, anterior hypothalamic, ventromedial hypothalamic and mammilloinfundibular (or ventrolateral premammillary)nuclei. LH-RF was also localized in the organum vas culosum of the lamina terminalis (OVLT). Ependymal tanycytes, however, were never immunoreactive upon absorption of the antiserum with rat or mouse liver powder prior to the immunohistochemical incubation on the rat or mouse brain tissue, respectively, (indirect method). The other penventricular organs were not studied.