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B. T. PICKERING

SUMMARY

Purification of cobra neurohypophysial extracts has led to the isolation of two fractions with uterotonic activity. One of these was eluted from carboxymethylcellulose columns at the same place as oxytocin. Its amino acid composition and pharmacological properties are compatible with it being a mixture of oxytocin and 8-isoleucine oxytocin (mesotocin). The second fraction had the chromatographic properties of 8-arginine oxytocin (arginine vasotocin) and had the amino acid composition of this compound, but the degree of potentiation of its oxytocic activity by magnesium ions may imply the presence of yet another active peptide in this fraction.

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B. T. PICKERING and SUSAN McWATTERS

Neurohypophysial hormones have been investigated in both the Australian lungfish (Follett & Heller, 1963, 1964) and the African lungfish (Follett & Heller, 1962, 1963, 1964; Sawyer & van Dyke, 1963). The African lungfishes (Protopterus aethiopicus) used by Follett & Heller (1964) had been caught in Lake Victoria, Uganda, and their glands contained two hormones with the properties of arginine vasotocin (AVT) and 8-isoleucine oxytocin as did the pituitaries of the Australian lungfish (Neoceratodus forsteri) examined at the same time. Sawyer & van Dyke (1963) used Protopterus aethiopicus which came from a different lake (Lake George, Uganda) and glands from these fishes contained AVT and oxytocin, although in another batch of glands Sawyer (personal communication) found evidence for the presence of 8-isoleucine oxytocin. Both groups of workers, however, suggested the possibility of a mixture of both the oxytocin-like peptides in the glands they had analysed. The present work was undertaken to

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B. T. PICKERING and H. HELLER

SUMMARY

Two peptides with uterotonic activity have been isolated from the pituitary gland of a holocephalian elasmobranch fish: Hydrolagus collei. One of them had an amino acid composition compatible with that of oxytocin itself, and also had the pharmacological properties of this hormone. The other peptide which was present in much smaller amounts was basic by chromatography and had the pharmacological characteristics of [8-arginine]-oxytocin. It was not completely purified because of the small amount available, but its amino acid composition was in accord with that of vasotocin. The implications of the presence of oxytocin in such a primitive fish on the phylogeny, and hence probably the evolution, of neurohypophysial hormones are discussed.

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JEAN H. LACEY and B. T. PICKERING

The contents and membranes of the secretory granules in the rat neurohypophysis were labelled in vivo with [35S]cysteine and [3H]choline respectively. Density-gradient centrifugation of the labelled granules showed the membrane label to be distributed largely between two peaks: one associated with intact granules and one with the characteristics of empty granule envelopes. Stimulation of hormone release in vitro led to the movement of membrane label from the intact granule fraction to the other one, consistent with the recapture of membrane as large vesicles. Freezing and thawing the crude granule fraction, ostensibly to aid osmotic fracture, produced a single membrane component with a density intermediate between the two original fractions.

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B. T. PICKERING and C. W. JONES

SUMMARY

A method is described for the preparation of isotopically pure [3H]oxytocin and [3H]vasopressin from the pooled posterior pituitary glands of groups of four or five rats after the intracisternal injection of [3H]tyrosine. Hormones were separated from deproteinized neurohypophysial extracts by chromatography on Amberlite CG-50, and further purified by chromatography on carboxymethylcellulose. In this way, samples of the hormones were obtained with a very high degree of isotopic purity. The method is suitable for studying the effects of stimuli to the hypothalamo—neurohypophysial system on the biosynthesis and transport of the hormones.

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R. W. SWANN and B. T. PICKERING

SUMMARY

Rat neural lobes have been separated into subcellular fractions by differential centrifugation at various times after an intracisternal injection of [35S]cysteine or [3H]choline. Both isotopes led to a rise and fall in the radioactivity of neurosecretory granules (NSG) which paralleled that found previously for the neurohypophysial hormones and the neurophysins. While the radioactivity of the NSGs resulting from [35S]cysteine injection was predominantly associated with granular contents, [3H]choline injections led to a preferential labelling of the granular membrane. There was no indication of a sequential movement of radioactivity from the NSG-membrane fraction into the microsomal fraction (containing the so-called small vesicles) which might be expected if granular membrane were recaptured as small vesicles after release of secretory product by exocytosis. When release was stimulated in injected animals by giving them 2% NaCl solution to drink, 35S disappeared from the gland as expected, but 3H was retained and, moreover, appeared in the NSG-membrane fraction; results compatible with membrane conservation occurring by recapture of large vesicles. There was an indication that some of the neurophysin in the NSG was membrane-bound and that this too was retained after release of the granular contents.

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MARGARET A. MCPHERSON and B. T. PICKERING

A number of antisera have been raised against individual rat neurophysins. One of these proved suitable for the specific radioimmunoassay of rat vasopressin–neurophysin and data validating such a use are presented. Use of this antiserum showed that vasopressin–neurophysin is present in rat neurohypophyses in equimolar amounts to vasopressin, while in the hypothalamus the assay detected twice as much neurophysin-like material as hormone. A number of partially specific antisera for rat oxytocin–neurophysin have been obtained and the best of these was raised against the minor neurophysin.

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H. D. Nicholson, R. T. S. Worley, H. M. Charlton and B. T. Pickering

ABSTRACT

Immunoreactive oxytocin is present in the testis and it has been shown that this hormone increases the contractility of seminiferous tubules. We have investigated the relationship between testicular oxytocin, tubular movements and the effects of LH and testosterone using, as a model, the hypogonadal (hpg/hpg) mouse, which is deficient in hypothalamic LH-releasing hormone (LHRH). Whilst both testicular oxytocin and seminiferous tubule movements, resembling those seen in the rat, can be found in normal adult mice, neither can be found in hypogonadal mice. After 2 weeks of treatment with LH (200 ng to 100 μg daily) low levels of testicular oxytocin and tubular movements were observed. Treatment with large doses of testosterone for 2–12 weeks led to higher concentrations of testicular oxytocin and tubular movements resembling those seen in the normal adult mouse. The results support the evidence that testicular oxytocin modulates seminiferous tubule movements. We suggest that testosterone may play a part in the accumulation of oxytocin in the testis.

J. Endocr. (1986) 110, 159–167

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H. D. Nicholson, R. T. S. Worley, S. E. F. Guldenaar and B. T. Pickering

ABSTRACT

An oxytocin-like peptide is present in the interstitial cells of the testis, and testicular concentrations of oxytocin have been shown to increase seminiferous tubule movements in vitro. We have used the drug ethan-1,2-dimethanesulphonate (EDS), which depletes the Leydig cell population of the adult rat testis, to examine further the relationships between the Leydig cell, testicular oxytocin and tubular movements. Adult rats were injected i.p. with a single dose of EDS (75 mg/kg) or of vehicle (25% dimethyl sulphoxide). Histological study 3 and 10 days after treatment with EDS showed a reduction in the number of interstitial cells, and levels of oxytocin immunoreactivity were undetectable by radioimmunoassay. Immunostaining revealed very few oxytocin-reactive cells. Spontaneous contractile activity of the seminiferous tubules in vitro was also dramatically reduced, but could be restored by the addition of oxytocin to the medium. Four weeks after EDS treatment, the interstitial cells were similar to those in the control animals both in number and in immunostaining; immunoassayable oxytocin was present and tubular movements were normal. The EDS effect, seen at 3 and 10 days, was not altered by daily treatment with testosterone. However, repopulation of the testes with oxytocin-immunoreactive cells was not seen until 6 weeks in the testosterone-treated animals.

We suggest that the Leydig cells are the main source of oxytocin immunoreactivity in the testis and that this oxytocin is involved in modulating seminiferous tubule movements and the resultant sperm transport. The results also imply that testosterone does not play a major role in controlling tubular activity in the mature rat.

J. Endocr. (1987) 112, 311–316

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H. D. Nicholson, A. J. Smith, S. D. Birkett, P. A. Denning-Kendall and B. T. Pickering

ABSTRACT

Vasopressin (VP)-like immunoreactivity (IR) has been located in the testes of several species of mammal. There is evidence that most of this IR in the rat does not represent authentic arginine vasopressin (AVP) and that a second AVP-like peptide may exist. We have studied testis samples from the pig, which produces lysine vasopressin (LVP) in its pituitary, and have found both LVP- and AVP-like IR.

High-performance liquid chromatography (HPLC) of testis extracts showed two peaks of VP-IR. The first peak co-eluted with authentic LVP and was recognized only by antisera which cross-reacted with LVP. The second peak co-eluted with authentic AVP and was recognized by antisera raised against AVP. Both VP-like peptides bound to a neurophysin affinity column and the HPLC elution profiles of the bound peptides were similar to those of the authentic hormones. When the LVP-like material was oxidized with performic acid, a peak of IR running in the same position as oxidized authentic LVP on HPLC was produced. Similarly, the performic acid-oxidized AVP-like material co-eluted with oxidized authentic AVP.

The presence of both LVP- and AVP-like peptides in the pig testis may mean that more than one gene is involved. A second VP-like gene could also explain the anomalies of VP-IR in other species.

J. Endocr. (1988) 117, 441–446