Studies of proteins in the posterior lobe of the pituitary gland

Abstract

Ovine neurophysin was extracted from fresh-frozen whole pituitary glands using hydrochloric acid at pH 1.2-1.6. After salt precipitation neurophysin was separated from other extracted protein by gel-exclusion chromatography on Sephadex G.75. Ion exchange chromatography on CM-sephadex C.50 using acetate buffer in a pH gradient was employed to resolve the neurophysin fraction into three components. Contamination of the preparations by non-neurohypophysial hormones was investigated. Antibodies which were raised in rabbits against ovine and porcine neurophysin were observed to have reactivity with neurophysins of the same and different species as the source of the antigen. Soluble proteins in the neural lobes of a number of mammals were separated by electrophoresis on starch-gel and tested, by immunodiffusion, for immunological reactivity with an anti-porcine neurophysin-II serum. A number of proteins, sometimes more than three per animal, reacted positively with the antiserum. Immunofluorescence histochemistry was used to demonstrate neurophysin-like antigen in tissue slices. Initially tissue was frozen unfixed and sectioned in a cryostat prior to fixation in 95% (v/v) aqueous ethanol. subsequently an improved method was developed in which tissues were fixed in saline-formalin and embedded in paraffin and immunohistochemistry carried out on deparaffinised sections. Immunofluorescence on tissue fixed in saline formalin was more intense and cells were resolved more sharply than on slices of fresh-frozen tissue. Neurophysin antigen was observed in the magnocellular nuclei of adult rats, guinea-pigs and rabbits and also in accessory cell groups in the hypothalamus. Material reactive against anti-neurophysin sera was also present in the neurohypophysis. Furthermore, neurophysin-like material was demonstrated in axons of the hypothalamo-neurohypophysial fibre tracts, especially in guinea-pig tissue slices. Immunoreactive material in axons was more abundant in tissue from dehydrated guinea-pigs. Neurophysin-like antigen, as detected by immunodiffusion and immunofluorescence was depleted in the neural lobes of dehydrated rate. Staining by aldehyde fuchsin showed that dehydration also caused a reduction in neurosecretory material in rat neurohypophyses. In the guinea-pig neurohypophysis, neurosecretory material was observed to have a lobular distribution whereas immunofluorescence was lobular on frozen sections but of even intensity on paraffin sections. Dehydration caused a depletion of histochemically-detected material in the guinea-pig neural lobe. Immunofluorescence histochemistry enabled detection of neurophysin antigen in rat hypothalami at 17 days gestational age. Fetal calves of 130 days and rabbits of 29 days gestation were also seen to have immunoreactive material in the hypothalamus and neural lobe. Evidence for modification of neurophysin-like material in the perikaryon or in proximal parts of the axona of the guinea-pig hypothalamo-neurohypophysial system was obtained by observing the effects of different treatments of HNS tissue blocks and slices on histological stains. Elements of the hypothalamo-neurohypophysial system which were stained by immunofluorescence were not always stained by aldehyde fuchsin dye.