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Recordings of intramammary pressure have been used in recent experiments to demonstrate the intermittent pattern of milk ejection (ME) in the rat (Wakerley & Lincoln, 1971; Lincoln, Hill & Wakerley, 1973). This technique provides 'on-line' but indirect evidence of oxytocin release. Other naturally occurring substances (bradykinin, 5-hydroxytryptamine, acetylcholine and vasopressin) can initiate contractions of the mammary gland and thus mimic the effect of oxytocin (Bisset, Clark, Haldar, Harris, Lewis & Rocha e Silva, 1967). The following experiments were designed to identify oxytocin as the active milk-ejecting principle released during suckling.
Bisset, Clark & Haldar (1970) described an in-vivo method of identifying oxytocin using an antagonist, N-carbamyl-O-methyl-oxytocin, which acts as a competitor for oxytocin receptors. The effects of this antagonist on the contractions of the mammary gland during natural ME were examined. Lactating rats were anaesthetized with tribromoethanol (Lincoln et al. 1973) and two mammary glands in the
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ABSTRACT
Gonadotrophin-releasing activity of oxytocin has previously been demonstrated in vitro and in vivo. This study investigated whether oxytocin is also able to induce LH accumulation in pituitary cells. Following trypsin digestion and mechanical dispersion, pituitary cells from female rats were incubated with oxytocin (100 nmol/l) for 24 h. LH release stimulated by oxytocin increased (P < 0·001) progressively during the incubation indicating a different secretory pattern from the more rapid but less sustained secretion stimulated by gonadotrophin-releasing hormone. Oxytocin also enhanced (P < 0·01) total LH accumulation in the incubation system (released plus cell contents) which was apparent after 7–11 h of stimulation. The release of LH stimulated by oxytocin was reduced by the protein synthesis inhibitor cycloheximide (10 μmol/l). However, cycloheximide did not completely block oxytocin-stimulated LH release; there remained some LH release above that seen in non-stimulated controls (P < 0·01) revealing the presence of a cycloheximide-resistant component in the release mechanism. Furthermore, accumulation of total LH in 24 h incubations was suppressed (P < 0·01) by cycloheximide.
The advancement in LH release which oxytocin has been shown to induce in vivo in pro-oestrous rats was accompanied by an early reduction of pituitary LH stores. However, the fall normally observed in LH content during the surge was markedly attenuated by the oxytocin treatment. Thus, loss of pituitary LH stores was less in oxytocin-treated rats than in saline-treated controls, even though net LH release into plasma was increased. Therefore, oxytocin stimulated the replenishment of LH stores.
Although the mechanism(s) remains to be defined and the relationships between in-vitro and in-vivo results are as yet uncharacterized, the present study demonstrates that oxytocin treatment stimulates LH production in both dispersed cells and intact pituitaries in situ.
Journal of Endocrinology (1992) 132, 277–283
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SUMMARY
A method for the bioassay of oxytocin in the lactating rat mammary gland in vitro is described. The changes in transparency induced by oxytocin in a flat segment of the gland are recorded by means of a CdS photoresistor and a simple Wheatstone bridge. The sensitivity of the assay in routine use is about 0·5–10 μu. oxytocin, the index of precision being about 0·1.
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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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ABSTRACT
The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234·4 ± 32·8 pmol/g per h (n = 24) during 60-min incubations.Activators of protein kinase C: phorbol 12,13-dibutyrate (n = 8), phorbol 12-myristate,13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0·2 μmol/l). Phospholipase C (PLC; 50–250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4).
These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum.
Journal of Endocrinology (1990) 124, 225–232
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ABSTRACT
The distribution of [3H]vasopressin- and [3H]oxytocin-binding sites was examined, using an autoradiographical technique, in the kidney of Long–Evans and Brattleboro rats. Two types of binding sites with affinities in the nanomolar range were detected: one, located on glomeruli, bound both vasopressin and oxytocin; the other, on collecting ducts, bound vasopressin selectively. In the presence of 10 μmol oxytocin/1, [3H]vasopressin labelling was abolished in glomeruli, but only reduced in collecting ducts; [3H]oxytocin labelling was completely abolished by 10 μmol vasopressin/1. These observations are discussed in relation to known effects of neurohypophysial hormones on renal physiology.
J. Endocr. (1987) 113, 179–182
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ABSTRACT
The effect of relaxin on electrically evoked release of oxytocin from the posterior pituitary was examined by monitoring changes in intramammary pressure in the anaesthetized lactating rat.
The amount of oxytocin released by electrical stimulation of the neurohypophysis in vivo was dramatically reduced following i.v. injection of highly purified porcine relaxin (2·5–10 μg/rat). Relaxin inhibited oxytocin release in a dose-dependent manner and the onset of inhibition occurred within 6–10 min and lasted for 10–60 min. No effect on the sensitivity of the mammary gland to exogenous oxytocin was observed after relaxin treatment. During the period of inhibition, i.v. injection of the opioid antagonist naloxone chloride (1 mg/kg) completely and immediately restored electrically evoked oxytocin release. The neurohypophysis is known to contain endogenous opioid peptides, therefore the effect of relaxin on electrically stimulated release of oxytocin from the rat isolated neural lobe in vitro was examined. Relaxin (500–2000 ng/ml) failed to inhibit oxytocin release in vitro.
The results suggest that relaxin can inhibit the release of oxytocin from terminals in the neurohypophysis, but by an indirect mechanism. This action appears to be mediated through endogenous opioid peptides whose source is not clear. They are unlikely to be of neurohypophysial origin and may probably come from the adrenal medulla, since acute adrenalectomy negated the inhibitory effect of relaxin on oxytocin release.
J. Endocr. (1986) 109, 393–397
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SUMMARY
Ovariectomized, oestrogen-primed rabbits were injected intravenously with varying doses of oxytocin immediately after insemination with a standardized inseminate. The does were killed 30 min after insemination and the number of spermatozoa in different segments of the genital tract was determined. The most significant finding was that when 80 mu. oxytocin were given, mass transport of semen into the uterus occurred. This suggests a coordinated response of the genital tract to a critical oxytocin stimulus which facilitates mass transcervical passage of fluids. However, when 80 mu. oxytocin were given to intact oestrous rabbits, no significant effect on sperm transport was detected.
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SUMMARY
A simple and rapid method is described for labelling oxytocin with 131I at a high specific activity. This method is compared with those of previous workers. A satisfactory antiserum has been raised by direct intra-lymph node injection of oxytocin adsorbed to carbon microparticles. A number of methods for separating antibody-bound from free oxytocin are described, and reasons given for preferring a procedure using ammonium sulphate precipitation. These data form the basis for developing a radioimmunoassay intended for the determination of oxytocin in human plasma.
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ABSTRACT
The presence of oxytocin receptors in ovine oviduct has been investigated. High-affinity binding sites for [3H]oxytocin were detected in crude membrane fractions prepared from the oviducts of ewes killed during the oestrous period. The dissociation constant calculated for these sites in competition studies was 1·7 nmol/l. Similar dissociation constants were calculated for [Arg8]-vasopressin and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4, Gly7]-oxytocin, indicating that these sites represent oxytocin receptors. At least one additional site of lower affinity and undetermined identity was present.
The relative concentration of oxytocin-binding sites in preparations of oviduct membranes were estimated in ewes killed at different stages of the oestrous cycle using a single concentration of [3H]oxytocin. Binding was low during the luteal phase of the cycle but increased to a maximum at oestrus (77·7 fmol/mg protein). Binding fell after ovulation, reaching what appeared to be basal concentrations by the early luteal stage of the cycle. Binding to oviductal membranes from prepubertal, anoestrous and pregnant ewes was also low, but in anoestrous animals which had been treated with progesterone and oestrogen it was similar to values measured in ewes at oestrus. These results are consistent with the existence of oviductal oxytocin receptors which are regulated by ovarian steroids.
We conclude that oxytocin receptors are present in the oviduct of the ewe around the time of ovulation. The significance of oxytocin to events taking place in the oviduct at this time remains to be determined.
Journal of Endocrinology (1990) 124, 353–359