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F. R. BURNET
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P. C. B. MACKINNON
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SUMMARY

The rate of [35S]methionine incorporation into protein in discrete cerebral areas was measured before and after the administration of oestradiol benzoate (OB) to chronically ovariectomized rats. The circadian rhythm of incorporation which is normally seen in the intact cyclic female rat was deleted by ovariectomy. A daily rhythm of incorporation reappeared, however, in all the brain areas studied 30 h after a single injection of OB (20 μg), and was still present 12 days later.

The release of luteinizing hormone (LH) after administration of 20 μg OB was measured in chronically ovariectomized animals and was found to be biphasic. High levels of LH after ovariectomy were initially reduced by negative feedback, but this phase was followed 52 h later by a facilitation of LH release between 15.00 and 18.00 h. The facilitation of LH release at this time of day was still detectable 12 days after the initial injection.

The evidence for a functional link between the rhythm of neural activity which is reflected by [35S]methionine incorporation, and the ability to 'time' the facilitation of LH release is discussed.

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A. M. L. van DELFT
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J. KAPLANSKI
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P. G. SMELIK
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SUMMARY

Two days after a single or repeated administration of p-chlorophenylalanine (p-CPA; 316 mg/kg i.p.) in the rat, a marked diminution to 20% of control levels of brain 5-hydroxytryptamine, partial but not complete abolition of the circadian pituitary—adrenal rhythm and a profound decrease in adrenal responsiveness to ether stress or exogenous adrenocorticotrophin were found. Pituitary—adrenal activity in unstressed or stimulated animals was assessed by the level of corticosterone in plasma and the corticosteroid production by adrenal tissue in vitro. The effect of p-CPA administration on changes in pituitary—adrenal periodicity were concomitant with a reduction in food intake and body growth and a pronounced increase in adrenal weight. In the light of the marked functional alterations after p-CPA treatment, the proposed relationship between decreased brain 5-hydroxytryptamine content and pituitary—adrenal periodicity is questioned.

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N. D. HORSEMAN
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A. H. MEIER
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In the teleost, Fundulus grandis, injections of prolactin early in the light phase cause an immediate 50% depression in the rate of hepatic lipogenesis ([14C]acetate incorporation); 10 h later, that rate has returned to levels not different from controls. Injections of prolactin late in the light phase cause an even more dramatic immediate depression of lipogenesis (79%) followed by a gradual increase in lipogenic rate which is 2·6 times higher than the control rate after 24 h. The stimulation of lipogenesis by prolactin is blocked by simultaneous treatment with indomethacin, an inhibitor of prostaglandin synthesis. These circadian phase-dependent effects of prolactin on hepatic lipogenesis are discussed with reference to possible mechanisms of action exerted by endogenous prolactin rhythms.

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P. Boulanger
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M. Desaulniers
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G. M. Dupuy
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G. Bleau
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K. D. Roberts
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A. Chapdelaine
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Radioimmunoassays were used for the measurement of several androgens in canine plasma and in the liquid of the vas deferens. Large variations in the plasma concentrations of androstenedione, testosterone and 5α-dihydrotestosterone occurred during a period of 24 h, but there was no evidence of a circadian rhythm. The ratios of the androgen concentration in the liquid of the vas deferens compared with that in the peripheral plasma were: androstenedione, 4·6; testosterone, 1·9; 5α-dihydrotestosterone, 13·6; 5α-androstane-3α,17β-diol, 17·0; 5α-androstane-3β,17β-diol, 22·4. These high levels of androgens in the liquid of the vas deferens could play a role in the development of prostatic hypertrophy.

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M. A. F. MURRAY
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C. S. CORKER
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Recent studies of the circadian rhythm of steroidal and gonadotrophic hormones (Hellman, Nakada, Curti, Weitzman, Kream, Roffwarg, Ellman, Fukushima & Gallagher, 1970; Katongole, Naftolin & Short, 1971; Sederberg, Binder & Kehlet, 1971; Naftolin, Yen & Tsai, 1972) have shown that it is far from being the smooth diurnal variation usually reported. Instead rapid fluctuations in the levels of the hormones occur throughout the day. The present study was undertaken to confirm the findings of Naftolin et al. (1972) regarding luteinizing hormone (LH) and to attempt to correlate the plasma levels of LH with changes in plasma testosterone. Blood samples (5 ml) were obtained at 10-min intervals over an 8-h period, between 09.00 and 17.00 h, from three normal men. The men (aged 25–30 years) were recumbent throughout the experiment, except when urinating, and were awake and allowed to read or converse. Frequent sampling was facilitated by the use of an

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G. MOLINO
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L. PEROTTI
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M. MARINO
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R. DARDANELLI
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It is well-known that adrenocortical secretory activity shows a characteristic oscillating course with a 24-h period. This is promoted by a corresponding circadian rhythm of corticotrophin (ACTH) secretion, normally related to the alteration of sleep with the waking state. In the urine, this phenomenon is expressed by a urinary steroid flow which rises during the morning hours reaching a maximum between 08.00 and 12.00 h and subsequently drops to its lowest level at about 24.00 h. It is thus possible to assess the effects on the adrenal cortex of a strictly physiological corticotrophic stimulus such as that to which the adrenal cortex is subjected during the early morning hours (Ceresa, Angeli, Boccuzzi et al. 1969, 1970). In fact, any qualitative change induced by endogenous ACTH in adrenal steroid secretory activity may be detected at the urinary level by variations of the metabolic pattern.

The present investigation was done in 16

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C. W. COEN
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P. C. B. MacKINNON
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Ovariectomized rats in which <7% of the suprachiasmatic nuclei had been spared by bilateral radiofrequency lesions were distinguishable from those with >40% of the nuclei by their consistent failure to show the oestrogen-induced daily surge of LH, either with or without pharmacological manipulations of serotonin (5-HT), and also by their loss of the normal rhythmicity of drinking. Minor damage to structures adjacent to the suprachiasmatic nuclei was similar in both groups. The identical facility with which electrical stimulation of the preoptic area induced LH release in the two groups of animals suggested that they were not characterized by different degrees of damage to the preopticotuberal pathway. These results are considered in relation to evidence indicating that the suprachiasmatic nuclei represent the densest concentration of 5-HT terminals in the forebrain and also the site of a mechanism involved in the generation of circadian rhythms.

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W. J. FULKERSON
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Department of Animal Science and Production, University of Western Australia, Nedlands, Western Australia 6009, Australia

(Received 13 March 1978)

The activity of the adrenal gland is believed to be governed by the secretion of corticotrophin (ACTH) in a positive stimulus/negative feedback equilibrium. There is increasing evidence that in man, the secretion of corticosteroids never actually reaches a steady-state condition and that the circadian rhythm displayed by these hormones in the circulation is therefore the result of a number of secretory episodes over a 24 h period (Hellman, Nakada, Curti, Weitzman, Kream, Roffwarg, Ellman, Fukushima & Gallagher, 1970; Weitzman, Fukushima, Nogeire, Roffwarg, Gallagher & Hellman, 1971). Data presented by McNatty, Cashmore & Young (1972) also raise the possibility that a similar pattern of hormone release may exist in the sheep. However, McNatty et al. (1972) collected samples relatively infrequently and it is hard to define peaks in cortisol concentration. With more

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GA Lincoln
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H Andersson
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A Loudon
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Melatonin-based photoperiod time-measurement and circannual rhythm generation are long-term time-keeping systems used to regulate seasonal cycles in physiology and behaviour in a wide range of mammals including man. We summarise recent evidence that temporal, melatonin-controlled expression of clock genes in specific calendar cells may provide a molecular mechanism for long-term timing. The agranular secretory cells of the pars tuberalis (PT) of the pituitary gland provide a model cell-type because they express a high density of melatonin (mt1) receptors and are implicated in photoperiod/circannual regulation of prolactin secretion and the associated seasonal biological responses. Studies of seasonal breeding hamsters and sheep indicate that circadian clock gene expression in the PT is modulated by photoperiod via the melatonin signal. In the Syrian and Siberian hamster PT, the high amplitude Per1 rhythm associated with dawn is suppressed under short photoperiods, an effect that is mimicked by melatonin treatment. More extensive studies in sheep show that many clock genes (e.g. Bmal1, Clock, Per1, Per2, Cry1 and Cry2) are expressed in the PT, and their expression oscillates through the 24-h light/darkness cycle in a temporal sequence distinct from that in the hypothalamic suprachiasmatic nucleus (central circadian pacemaker). Activation of Per1 occurs in the early light phase (dawn), while activation of Cry1 occurs in the dark phase (dusk), thus photoperiod-induced changes in the relative phase of Per and Cry gene expression acting through PER/CRY protein/protein interaction provide a potential mechanism for decoding the melatonin signal and generating a long-term photoperiodic response. The current challenge is to identify other calendar cells in the central nervous system regulating long-term cycles in reproduction, body weight and other seasonal characteristics and to establish whether clock genes provide a conserved molecular mechanism for long-term timekeeping.

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J. GUILLEMANT
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S. GUILLEMANT
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Adrenocortical cyclic AMP (both total and protein-bound), and adrenal and plasma concentrations of corticosterone were measured in male rats killed at selected intervals throughout the day. The animals had previously been synchronized for 3 weeks in natural lighting. Adrenal and plasma levels of corticosterone showed similar circadian fluctuations and the onset of their ascending phases started at 13.00 h, maximum concentrations being reached at 21.00 h. On the other hand, a time-lag between the circadian variations of total cyclic AMP and protein-bound cyclic AMP could be seen in adrenocortical tissue. The onset of an increase in adrenocortical protein-bound cyclic AMP was apparent at 15.00 h and the peak occurred at 21.00 h, while total adrenocortical cyclic AMP did not begin to rise before 19.00 h and was maximal at 04.00 h. No direct link between total cyclic AMP and protein-bound cyclic AMP could be seen during the dark phase, suggesting a functional compartmentalization of cyclic AMP in the adrenal gland of the rat. While the ascending phase of the rhythm in steroidogenesis preceded the rise in total adrenocortical cyclic AMP by about 4 h an excellent synchrony between the respective patterns of corticosterone concentration and protein-bound cyclic AMP was noticed.

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