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I. plevrakis, C. Clamagirand and G. Pontonnier

ABSTRACT

Human granulosa cells were collected from preovulatory follicles during follicular puncture for in-vitro fertilization. They were cultured in serum-free medium supplemented with ascorbic acid. Using a combination of high-performance liquid chromatography and radioimmunoassay, the oxytocin material present in the cell extracts and secreted into the medium was identified. When cells were deprived of ascorbate, intermediary forms resulting of the post-translational processing of pro-oxytocin/neurophysin were detected. These data demonstrate that oxytocin biosynthesis occurs in human granulosa cells.

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T. CHARD

The first radioimmunoassay was described by Yalow & Berson in 1960, since when the technique has been widely used in physiological and patho-physiological studies of the protein hormones. The success in this field has led to a number of attempts to develop similar assays for the small peptide hormones (molecular weight less than 3000). However, these compounds present certain difficulties which have considerably retarded progress. The purpose of the present review is to discuss the problems of the radioimmunoassay of the neurohypophysial peptides, with particular reference to oxytocin and vasopressin.

The problems of the radioimmunoassay of small peptide hormones

There are two major problems. (1) Being of low molecular weight (about 1000) they are poor immunogens. As a result, the preparation of high-affinity antisera is considerably more exacting than in the case of larger molecules. (2) Their levels in the circulation are, in molar terms, considerably lower than those of

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T. Higuchi, K. Honda, T. Fukuoka, H. Negoro and K. Wakabayashi

ABSTRACT

A highly sensitive and specific radioimmunoassay (RIA) for oxytocin was developed and used to measure oxytocin concentrations during both suckling and parturition in individual rats. In urethane-anaesthetized rats, the suckling stimuli, provided by ten pups, induced intermittent increases in intramammary pressure of about 10 mmHg. This was associated with a significant (P < 0·01) increase in serum oxytocin levels from 19·5 ± 4·5 (s.e.m., n = 9) to 49·1 ± 7·4 pmol/l (n = 9) in the samples taken within 30 s from the time of the peak in the pressure. These rises in serum oxytocin returned rapidly to the basal levels as expected from the short half-life (1·46 min) of oxytocin in general circulation.

On day 22 or 23 of gestation, serum oxytocin levels remained stable until 0–0·5 h before the first fetus was expelled. They then increased significantly (P < 0·01) from 27·6± 4·6 pmol/l (n = 19) in samples taken 0–0·5 h before to 45·1 ± 5·6 pmol/l in samples taken after the expulsion of the first fetus and gradually increased until the last fetus was expelled. Serum oxytocin concentrations then declined but remained higher than those observed before the first fetus had been born until at least 1–1·5 h after the expulsion of the last fetus. Thus, this oxytocin RIA revealed increased concentrations of the hormone in blood during both suckling and parturition in the rat.

J. Endocr. (1985) 105, 339–346

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P. Melin, J. Trojnar, B. Johansson, H. Vilhardt and M. Åkerlund

ABSTRACT

With the aim of developing inhibitors of vasopressin-and oxytocin-induced uterine activity, 17 analogues of 1-deamino-oxytocin were synthesized by the solid-phase method. Modifications were made at positions 2, O-methyltyrosine (Tyr(OMe)) and O-ethyltyrosine (Tyr(OEt)),d-Tyr,d-Tyr(OEt),d-Trp; 4, Val,Thr and 8, Orn,Cit,Arg,d-Arg.

The analogues were tested for antiuterotonic activity in vitro and in vivo in the rat and in vitro on myometrial strips from non-pregnant women and pregnant women at term. Their selectivity was also investigated in blood pressure and antidiuretic bioassays in rats. Results were compared with those from an original antiuterotonic analogue 1-deamino-2-Tyr(OEt)-oxytocin (d(OEt)-oxytocin). In the rat in vitro and in vivo all analogues possessed higher antiuterotonic activity than d(OEt)-oxytocin. The negative logarithm of the molar concentration of the antagonist which reduced the effect of a dose of agonist to that of half the dose (pA2) was between 7·6 and 8·9 for all the new inhibitors compared with 7·2 for d(OEt)-oxytocin. The highest pA2 value was found for 1-deamino-2-Tyr(OMe)-8-Orn-oxytocin (8·9 ± 0·2, s.e.m.) and 1-deamino-2-Tyr(OEt)-4-Thr-8-Orn-oxytocin (8·9 ± 0·6). In myometrium from non-pregnant women the most potent peptide was 1-deamino-2-d-Tyr(OEt)-4-Th r-8-Orn-oxytocin (17·2 ± 2·0 times more potent that d(OEt)-oxytocin). In myometrium from pregnant women the inhibitory effects of the majority of the analogues were less pronounced. In the rat in vivo the most potent analogue 1-deamino-2-d-Trp-4-Val-8-Orn-oxytocin was 19·9 ± 2·5 times more active than d(OEt)-oxytocin. Exchanging l-tyrosine for the d form generally increased inhibitory activity as well as specificity of the analogues. Alkylation of the d-tyrosine residue did not appear to be necessary for inhibition. Substitution with d-tryptophan at position 2 gave analogues with high inhibitory potency in the rat in vitro and in vivo, but which exhibited weak effects in women in vitro. There was no correlation between the inhibitory effects on myometrium from non-pregnant and pregnant women nor between rat and human data. The high antiuterotonic activity of 1-deamino-2-d-Tyr(OEt)-4-Val-8-Orn-oxytocin and 1-deamino-2-d-Tyr(OEt)-4-Thr-8-Orn-oxytocin combined with low blood pressure and antidiuretic effects make these two analogues interesting for clinical studies.

J. Endocr. (1986) 111, 125–131

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B. A. EDWARDS

Radioactivity accumulates in the posterior pituitary after intravenous injections of [3H]oxytocin (Aroskar, Chan, Stouffer, Schneider, Murti & du Vigneaud, 1964) or tritiated lysine vasopressin ([3H]LVP) (Willumsen & Bie, 1969). These results allow for the possibility that the neurohypophysial hormones may be taken up by the pituitary after the release of endogenous, or the administration of exogenous, oxytocin and vasopressin. The experiments in vitro of Pliška, Thorn & Vilhardt (1971) and Edwards (1971) showed that uptake of radioactivity occurred after incubation of neural lobes with labelled vasopressin but that much of the activity was not associated with hormone. The present communication describes a study on the uptake of [3H]oxytocin by neural lobes of rats incubated in vitro.

Halved neural lobes were incubated in a bicarbonate buffer (McIlwain & Rodnight, 1962) containing [3H]oxytocin (prepared in the Departement de Biologie, Commissariat à l'Energie Atomique, France) and non-radioactive purified oxytocin

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M. D. Stoneham, B. J. Everitt, S. Hansen, S. L. Lightman and K. Todd

ABSTRACT

In male New Zealand white rabbits, it was shown that oxytocin but not vasopressin concentrations in plasma were markedly raised after ejaculation. In male Wistar rats, oxytocin infused into the internal carotid artery reduced the number of intromissions made before ejaculation but had no other significant effect. Infusion of oxytocin into the third ventricle increased the latencies to the first mount and intromission and lengthened post-ejaculatory refractory periods. It is suggested that oxytocin released into the periphery during coitus, while not essentially involved in ejaculation, may exert effects on the genital periphery. Behavioural effects of centrally administered oxytocin suggest that it may play a role in the neural mechanisms underlying post-ejaculatory refractoriness.

J. Endocr. (1985) 107, 97–106

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Akiko Katoh, Hiroaki Fujihara, Toyoaki Ohbuchi, Tatsushi Onaka, W Scott Young III, Govindan Dayanithi, Yuka Yamasaki, Mitsuhiro Kawata, Hitoshi Suzuki, Hiroki Otsubo, Hideaki Suzuki, David Murphy and Yoichi Ueta

Introduction The neurohypophyseal hormones arginine vasopressin (AVP) and oxytocin (OXT) are mainly synthesised in discrete groups of magnocellular neurosecretory cells (MNCs) that are located in the hypothalamus. The gene expression, synthesis and

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Yoko Fujiwara, Masami Hiroyama, Atsushi Sanbe, Junji Yamauchi, Gozoh Tsujimoto and Akito Tanoue

Introduction Arginine-vasopressin (AVP) and oxytocin (OT) are neurohypophysial hormones synthesized in the paraventricular nucleus and supraoptic nucleus of the hypothalamus. AVP acts in many organs and plays a variety of

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R. R. CHAUDHURY and J. M. WALKER

SUMMARY

Oxytocin injected intravenously into anaesthetized rabbits disappears rapidly from the blood stream, and its disappearance is retarded in animals whose liver or kidneys have been excluded from the circulation. The rate of disappearance of injected vasopressin is similar to that of oxytocin in intact rabbits. Homogenates of rabbit liver and kidney inactivate oxytocin rapidly in vitro; the site of inactivation by the kidney is in the tubules and not in the glomeruli.

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T. Higuchi, K. Uchide, K. Honda and H. Negoro

ABSTRACT

Blood levels of oxytocin during parturition in pelvic-neurectomized rats were determined by radioimmuno-assay. Four out of 29 pelvic-neurectomized rats completed parturition within 23 days of pregnancy. These rats exhibited an increase in blood levels of oxytocin during parturition similar to those of sham-operated control rats, but delivery took longer and there was a higher percentage of still-births. The rise in blood levels of oxytocin was smaller in the 16 rats in which the first fetus was expelled but where delivery did not end within day 23 of gestation than that in sham-operated controls. Levels did not increase in the other nine rats which failed to deliver the first fetus within 23 days of pregnancy. They did, however, show signs indicating delivery, such as stretch movements, vaginal bleeding and/or excretion of mucus within 23 days of gestation. Oxytocin infusion (2 mu./min) for 2–4 h increased uterine contractions in the pelvic-neurectomized rats but failed to reduce the percentage of still-births or the duration of delivery. Immuno-neutralization of circulating oxytocin by anti-oxytocin serum in intact pregnant rats resulted in a significant but much smaller prolongation of the duration of delivery compared with that observed in pelvic-neurectomized rats. The rise in blood levels of oxytocin during pregnancy may be induced, at least in part, by the Ferguson reflex via the pelvic nerve and may thus facilitate the process of delivery. A shortage of oxytocin secretion may not, however, be the main cause of the dystocia in pelvic-neurectomized rats.

J. Endocr. (1986) 109,149–154