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T. Higuchi
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K. Honda
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H. Negoro
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ABSTRACT

The influence of oestrogen on LH and oxytocin responses to immobilization stress, and the involvement of noradrenergic afferent neurones in these responses, was examined in ovariectomized rats with or without pretreatment with oestrogen or after noradrenergic transmitter blockade. Immobilization of the rats on a board in a supine position for 1 h brought about a rapid decrease in LH levels and an increase in oxytocin levels in the blood of ovariectomized rats. Oestradiol benzoate (20 μg) injected s.c. the day before immobilization, decreased basal LH levels but had no effect on basal oxytocin levels. Immobilization stress applied to oestrogen-treated rats induced a small but significant increase in LH concentrations and a rise in oxytocin concentrations similar to that in rats without oestrogen pretreatment. A dopamine-β-hydroxylase inhibitor or phenoxybenzamine (α-adrenoceptor blocker) injected into ovariectomized rats reduced basal LH levels and increased basal oxytocin levels in the blood. Immobilization stress induced an increase in LH levels in rats treated with dopamine-β-hydroxylase inhibitor, but had no effect in rats treated with phenoxybenzamine. Stress induced a larger increase in blood oxytocin levels in rats treated with either drug compared with that in control rats injected with vehicle. On the other hand, propranolol (β-adrenoceptor blocker) had no effect on basal or stress-induced changes in LH or oxytocin levels in the blood. These results indicate that the LH response to stress, which might be mediated through α-adrenergic neurones, depends on the circulating oestrogen or LH levels before the stress. In contrast, the oxytocin response to stress may not be mediated by noradrenergic neurones and may not be influenced by oestrogen.

J. Endocr. (1986) 110, 245–250

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T. Higuchi
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H. Negoro
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J. Arita
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ABSTRACT

Prolactin, GH, TSH, adrenaline and noradrenaline responses to the stress of immobilization were compared between lactating and non-lactating dioestrous rats. The concentrations of GH in plasma were reduced to a similar degree by the immobilization of lactating and non-lactating rats, and TSH levels were unchanged in both groups. The increases in plasma concentrations of adrenaline and noradrenaline induced by stress were significantly smaller in lactating than in non-lactating rats. Immobilization caused a marked increase in prolactin levels in the plasma of non-lactating rats but no increase in lactating rats. These changes may help to save energy and maintain milk production during the period of lactation.

Journal of Endocrinology (1989) 122, 495–498

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T. Higuchi
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K. Honda
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S. Takano
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H. Negoro
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ABSTRACT

The release of oxytocin in response to an osmotic stimulus and immobilization stress was compared in lactating rats 8–12 days after delivery and in non-lactating rats. Intravenous injection of hypertonic saline or immobilization stress induced an increase in blood oxytocin levels in both lactating and non-lactating rats, but the increment in the former was significantly lower than that in the latter. The lower responsiveness of oxytocin release to stress in lactating rats was not altered by ovariectomy 2 days after parturition. Oxytocin release induced by electrical stimulation of the anteroventral third ventricle (an osmoreceptive area), paraventricular nucleus and neurohypophysis was significantly lower, to a similar extent, in lactating rats compared with non-lactating rats. These findings indicate that the structural reorganization reported in the hypothalamo-neurohypophysial system may not function to facilitate release of oxytocin in response to stress and osmotic stimulus in lactating rats. The reduced responsiveness of the release of oxytocin is independent of the influence of ovarian hormones, and may be due to the low ability of the oxytocin neurone itself to release oxytocin, and/or due to the activated inhibitory influence on the oxytocin neurone in the lactating rat.

J. Endocr. (1988) 116, 225–230

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T. Higuchi
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K. Uchide
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K. Honda
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H. Negoro
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ABSTRACT

Developmental changes in levels of oxytocin in the blood and the pituitary gland and in oxytocin responses to oxytocin-releasing stimuli were investigated in the rat from the fetus close to term to the 40-day-old young adult. The oxytocin content of the pituitary gland rose gradually from fetuses of 21 days of gestation to 40-day-old rats. Pituitary oxytocin levels expressed in terms of body weight also increased up to day 25 after birth and declined slightly thereafter. In contrast, serum concentrations of oxytocin increased from day 21 of pregnancy up to day 5 after birth but were stable thereafter. Oxytocin levels in both blood and the pituitary gland were equal in 23-day-old fetuses and 1-day-old infants born on day 22 of pregnancy. There was no difference in serum and pituitary oxytocin levels in newborn pups and unborn littermates of day 22 or 23 of gestation. The i.p. injection of hypertonic saline induced a significant increase in serum oxytocin levels on day 5 and later, but no effect in the fetus on day 22 of gestation and in the 1-day-old infant. The responsiveness to the osmotic stimuli increased after 5 days of age. The i.p. injection of diethyl-dithiocarbamate, a noradrenaline synthesis inhibitor, or phenobarbitone was effective in raising blood oxytocin levels only in rats older than 10 and 20 days of age respectively. These findings, that a gradual increase in oxytocin levels in both blood and the pituitary gland without an apparent increase in its release and the absence of a pituitary response to oxytocin-releasing stimuli during the perinatal period, do not support a role for fetal oxytocin in the initiation of labour in the rat.

J. Endocr. (1985) 106, 311–316

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T. Higuchi
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Y. Tadokoro
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K. Honda
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H. Negoro
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ABSTRACT

A detailed secretory profile of oxytocin during suckling and parturition was determined in unanaesthetized freely moving rats. Ten pups were reunited with their mothers after 12–15 h of separation. Unless the milk-ejection reflex occurred, there was no difference in serum oxytocin levels before separation and during the suckling of either four or five, or nine or ten pups. Serum oxytocin levels increased abruptly by 50·1 ± 4·2 (s.e.m.) pmol/l (n = 9) at milk ejection, and declined rapidly with a half-life of about 1·5 min. The peak concentration of blood oxytocin at each milk ejection was independent of the previous suckling period; values from the first three milk-ejection reflexes following the introduction of the pups and those observed 3–5 h after introduction were similar. The process of parturition was monitored by recording intra-uterine pressure with a balloon implanted in the uterus. On day 22 or 23 of pregnancy, continuous and rhythmical contractions of the uterus occurred (onset of parturition), but serum levels of oxytocin (21·1 ± 1·9 pmol/l; n = 13) did not alter until the expulsive phase. During the expulsive phase, fetuses were delivered after fetus-expulsion reflexes which were recorded as sudden large increases in intra-uterine pressure. Basal levels of oxytocin in the blood increased during this phase (32·5 ± 4·4 pmol/l; n = 13) and, in addition, rose by about 15 pmol/l and declined slowly after fetus-expulsion reflexes. The increase, however, was quite different from that seen at milk-ejection reflexes.

J. Endocr. (1986) 110, 251–256

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

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T. Higuchi
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K. Honda
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T. Fukuoka
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H. Negoro
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K. Wakabayashi
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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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C-X Liu
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S Takahashi
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T Murata
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K Hashimoto
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T Agatsuma
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S Matsukawa
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T Higuchi
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Abstract

Large changes in the responsiveness of target organs to oxytocin are thought to originate from alteration of the number of oxytocin receptors (OTR). To elucidate the molecular mechanisms regulating the synthesis of the OTR, we developed a competitive reverse transcription-PCR protocol to measure OTR mRNA. We synthesized cRNA comprising a small stuffer introduced into the target mRNA. Using this cRNA as an internal standard, we made a quantitative estimation of OTR mRNA. Application of this method to the rat uterus revealed that the mean levels of OTR mRNA remained unchanged until 1030–1100 h on day 21 of pregnancy, increased significantly after 2200–2230 h on the same day and declined rapidly after parturition. A similar rapid increase in uterine OTR mRNA content was observed in rats given prostaglandin on day 18, inducing premature delivery on day 19 of pregnancy. All parturient rats had higher OTR mRNA levels regardless of whether parturition was spontaneous or prostaglandin induced. However, in a few rats, OTR mRNA remained as low as that observed during mid pregnancy even on day 22 of gestation, the expected day of parturition in about 70% of the rats in our colony. A similar increase in uterine OTR mRNA content to that observed at parturition was induced by oestrogen treatment for 3 days in ovariectomized virgin rats, but concomitant injection of progesterone did not influence the effect of oestrogen. The present results revealed that the large increase of uterine OTR at the peripartum period is accompanied by an increase in OTR mRNA content that may be brought about, at least in part, by increased oestrogen secretion following luteolysis.

Journal of Endocrinology (1996) 150, 479–486

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T Higuchi
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C-X Liu
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H Saito
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H Negoro
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S Matsukawa
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Abstract

Oxytocin (OT) is a neurohypophysial hormone with potent stimulating activity of the pregnant uterus, but its physiological role in parturition is still unclear. Recently, OT was found to be synthesized in the pregnant uterus, indicating that OT originating from the uterus, not from the posterior pituitary gland, may trigger the onset of labour. In order to define the factors responsible for the induction of uterine OT, the effect of ovarian steroid hormones and conceptus on the induction of OT mRNA in the rat uterus was examined by Northern and dot blot hybridization analysis. OT mRNA in the uterus started to increase on day 14 of pregnancy and showed very high levels at the time of parturition. Uterine OT mRNA was not altered by any steroid treatment, oestradiol-17β (0·2 μg), progesterone (4 mg) or both in combination, for 6 days. The gravid horn of the uterus had 3·6-fold as much OT mRNA as the non-gravid horn on day 21 of pregnancy in hemipregnant rats with one ligated oviduct. The ovarian steroid hormones could not induce accumulation of OT mRNA in the uterus of ovariectomized rats, at least under the conditions used, but the presence of a conceptus may be critical for the very high levels of OT mRNA.

Journal of Endocrinology (1995) 146, 81–85

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T Murata
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E Murata
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CX Liu
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K Narita
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K Honda
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T Higuchi
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The present study was designed to investigate a possible role for ovarian steroids in the regulation of rat uterine oxytocin receptor (OTR) mRNA expression before labour. By using a competitive RT-PCR system, we have previously reported that parturition was associated with high levels of uterine OTR mRNA in all the animals examined. On the other hand, near term, some rats showed high OTR mRNA levels while others did not. We therefore examined the changes in OTR mRNA expression before and during prostaglandin F(2)(alpha) (PGF(2)(alpha))-induced parturition; a paradigm adopted to reduce the variation in the onset of parturition. Injection of PGF(2)(alpha) on day 18 of pregnancy significantly increased OTR mRNA expression in all the rats within 24 h of treatment, suggesting that the variation in OTR mRNA levels during spontaneous parturition may be due to the difference in the timing of the onset of parturition. The increase in OTR mRNA was significantly abolished by injection of the anti-oestrogen compound, tamoxifen. The stimulatory action of oestrogen on OTR mRNA expression was then examined in the presence or absence of ovarian factors. Pregnant rats were ovariectomized (OVX) or sham-operated on day 18 of pregnancy and either oestrogen or vehicle was administered 6 h after the surgical operation. Oestrogen increased OTR mRNA significantly in OVX rats 18 h after administration compared with sham-operated animals. Moreover, ovariectomy alone on day 18 of pregnancy increased OTR mRNA expression to a level which reached statistical significance 24 h after the operation. In addition, oestrogen treatment increased OTR mRNA levels in OVX virgin rats in which progesterone tubes were implanted for 1 week and removed 6 h before oestrogen injection. The stimulatory effect of oestrogen was not observed in rats in which the progesterone tubes were implanted for 1 week and not removed. These results suggest that the decline of progesterone is necessary for the expression of the stimulatory effects of oestrogen on uterine OTR mRNA.

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