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Jonathan D Johnston and Debra J Skene

Gland (Harris 1955), which is often considered to have established neuroendocrinology as a discipline and is celebrated in this special issue of the journal. Lerner was seeking to identify the molecule(s) in bovine pineal glands known to cause blanching

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There is evidence that substances possessing higher molecular weights than the pineal indole derivatives may be responsible for the antigonadotrophic activity of pineal extracts (Cheesman & Fariss, 1970; Moszkowska, Kordon & Ebels, 1971; Benson, Matthews & Rodin, 1972). This possibility is suggested first by the fact that pineal extracts are more active than the melatonin which they contain (Benson, Matthews & Rodin, 1971), and secondly by the discovery that the activity of pineal extracts can be separated from the melatonin using Sephadex G-10 and G-25 (Ebels, 1967; Benson et al. 1972). The non-melatonin pineal activity, referred to as pineal antigonadotrophic factor (PAG), was retained in the 500–1000 molecular weight fraction after ultrafiltration on Diaflo membranes and was found in a ninhydrinpositive peak when chromatographed on Sephadex G-25 (Benson et al. 1972). Considering that these studies were performed with defatted bovine pineal powder, one would suspect that the active substance is

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Pinealocytes constitute 90% of the cell population in the pineal organ (Quay, 1965) and are thought to have an endocrine function (Kelly, 1962; Thieblot & Blaise, 1963; Zweens, 1965). Removal of the pineal in prepubertal rats induces gonadal hypertrophy (Kitay, 1954) and Gittes & Chu (1965) reported that the effects of pinealectomy on the oestrous cycle of female rats could be consistently reversed by transplantation of isogeneic pineal tissue. It seems, therefore, that a pineal—gonadal interrelationship exists. This investigation was undertaken to study the cellular changes in the rat pineal gland after gonadectomy.

The pineal organ of each member of four groups of rats was studied: (a) A prepubertal group of 24 males and 24 females gonadectomized at 1–2 weeks of age and killed 10–12 weeks later; (b) Twelve males and 12 females were used as controls for group a. They were sham-operated at 10–12 days of age and killed

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Neurophysin-like proteins have been extracted and purified from bovine pineal glands and were found to share the biochemical and immunological properties of neurophysin I and neurophysin II respectively, and to be associated with at least one biologically and immunologically active neurohormone, probably vasotocin.

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Unforeseen difficulties were encountered in attempts to use the 24 hr. spontaneous activity rate of rats in revolving cages, as a basis for the standardization of inhibitors. Even when litter-mates were used the range of activity of the individual rats was extremely wide. The differences between the average counts of revolutions reached up to ± 600%. However, the activity rate of individual animals over a period of 1–4 weeks showed a variation of up to ± 20 % only. This agrees with the findings of Irwin (1961). Since Reiss, Davis, Sideman & Plichta (1963) have found that pineal extracts influence the spontaneous activity, the pineal and other endocrine glands of fast-running and slow-running animals were investigated.

Male Sprague-Dawley rats were used. Running activity was measured in vertically revolving activity drums located on three racks of one stand. The rats were rotated from cage to cage every 2 or 3 days throughout

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D. J. Skene, I. Smith and J. Arendt


A sensitive, specific, reproducible and practical radioimmunoassay for the determination of 5-methoxytryptophol (ML) in pineal glands of different species has been developed. High-affinity specific antisera were produced by immunization of sheep with ML–bovine serum albumin. Iodinated ML, used as the radiolabel, was synthesized by direct iodination of ML using 1,3,4,6,-tetrachloro-3,6-diphenylglycouril as the oxidant. Sensitivity of the assay was 0·005 pmol/tube. The validity of the assay was checked using classical techniques. Cross-reactivity with other indoles was negligible. Parallel inhibition curves were obtained for rat, hamster, sheep and tortoise pineal homogenates. Using thin-layer chromatography, tortoise pineal immunoreactivity also co-chromatographed with standard ML. Samples (n = 5) with ML concentrations of 0·013, 0·052 and 0·209 pmol/tube had intra-assay coefficients of variation of 9·8, 5·7 and 7·6% respectively. Their respective interassay coefficients of variation were 17·7 16·5 and 11·4% (n = 8). The pineal concentration of ML was found to be species dependent. Afternoon ML levels were 0·052± 0·002 (s.e.m.) pmol/gland in the rat (n = 16), 0·539 ±0·089 pmol/gland in the hamster (n = 16), 1·73±0·225 nmol/g in the sheep (n = 10) and 7·15± 0·465 pmol/gland in the tortoise (n = 4). The ratio of ML:melatonin content in the pineal gland also showed a large interspecies variation with values of 0·02 in the rat, 0·22 in the sheep, 2·7 in the hamster and 17 in the tortoise.

J. Endocr. (1986) 110, 177–184

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Institute of Endocrinology, Bucharest, Romania

(Received 20 September 1977)

We have previously reported that the pineal gland of the mammalian foetus, which comprises mainly specialized secretory ependymal cells (Anderson, 1965), synthesizes the nonapeptide arginine-vasotocin (AVT; Pavel, Dorcescu, Petrescu-Holban & Ghinea, 1973; Pavel, Goldstein, Ghinea & Calb, 1977). However, in contrast with the foetal pineal gland, the predominant cell type in the pineal gland of the adult mammal is the parenchymal cell or pinealocyte (Quay, 1965); there is a marked reduction in the number of ependymal cells and they are restricted to the pineal recess (Anderson, 1965), which shows the same secretory characteristics as the subcommissural organ (Palkovits, 1965). Since we have postulated the ependymal origin of mammalian AVT (Pavel, 1971; Pavel et al. 1973; Pavel, 1975; Pavel et al. 1977), it seemed worthwhile to investigate the synthesis of AVT in the adult pineal gland in order to elucidate, if only

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Ovine pineal gland extracts were examined throughout the year for content and molecular form of immunoreactive (IMR) LH releasing hormone (LH-RH). The content of IMR LH-RH in pineal glands collected in spring and summer was 160–2230 pg/gland, while the content in pineal glands collected in autumn and winter was lower (31–39 pg/gland). Pineal gland IMR LH-RH, purified by affinity and Sephadex G-25 chromatography, yielded displacement curves parallel to those of hypothalamic IMR LH-RH and synthetic LH-RH in radioimmunoassays employing four antisera which require different regions of the decapeptide for effective binding, suggesting considerable similarities in the structure of the molecule. The majority of pineal gland IMR LH-RH behaved identically to hypothalamic IMR LH-RH and synthetic LH-RH on gel filtration, cation exchange chromatography, high-pressure liquid chromatography and cellulose thin-layer chromatography. However, a small amount of a less positively charged LH-RH species was also present in all pineal gland extracts. Our findings indicate that hypothalamic decapeptide LH-RH occurs in the ovine pineal gland.

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University of Liège, Department of Internal Medicine, Radioimmunoassay Laboratory, Boulevard Piercot 23, B-4000 Liège, Belgium

(Received 20 October 1975)

The human foetal pineal contains (Pavel, Dumitru, Klepsh & Dorcescu, 1973 b) and synthesizes (Pavel, Dorcescu, Petrescu-Holban & Ghinea, 1973 a) arginine-vasotocin (AVT) which could be the physiological antigonadotrophic factor (Pavel et al. 1973 b). Neurophysins, the carriers of vasopressin (ADH) and oxytocin along the hypothalamo-neurohypophysial tract have been isolated from bovine pineal glands (Reinharz, Czernichow & Vallotton, 1974) and identified in one human pineal tumour (Legros, Louis, Grotschel-Stewart & Franchimont, 1975). In the latter study we showed that AVT was present in 'free form' unbound to neurophysins. Here we confirm the presence of immunoreactive neurophysins and AVT in the normal foetal pineal.

Five glands were removed as rapidly as possible after therapeutic abortion, immediately put in cold acetone and later homogenized in 1ml NaCl (0·9%) by ultrasonication. After centrifugation (4,200g,

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Department of Physiology and Biophysics, Colorado State University, Fort Collins, Colorado 80523, U.S.A.

(Received 13 September 1977)

Mammalian pineal gland activity is controlled by environmental lighting schedules. Light exerts its influence via a neuronal pathway originating in the retina (Moore & Klein, 1974) and as a consequence of this photoperiodic control, the concentration of melatonin in the plasma is raised during periods of darkness and depressed during periods of light (Rollag & Niswender, 1976). The response of the pineal gland to photostimulation is surprisingly rapid. Within 5 min of a darkness to light transition, there is a precipitous decline in pineal N-acetyltransferase activity in the rat (Deguchi & Axelrod, 1972; Klein & Weller, 1972). In sheep, peripheral concentrations of melatonin decline within 5–10 min of a darkness to light transition (Rollag, O'Callaghan & Niswender, 1978). A circadian rhythm of blood flow to the pineal gland analogous to the rhythm of melatonin