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E L Matthews
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V J Ayad
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Abstract

The purpose of the present study was to investigate the presence of high-affinity oxytocin-binding sites (putative oxytocin receptors) in the cervix of the non-pregnant ewe. [3H]Oxytocin binding to the peripheral layers of cervical tissue (comprising the serosal layer and the least dense collagenous and muscular layers) and the remaining dense collagenous cervical tissue were studied separately. [3H] Oxytocin-binding sites were detected in membrane fractions prepared from both of these regions, but binding to the peripheral cervix was variable and binding site concentrations were low, hence these were not characterized further. A high-affinity oxytocin-binding site, having a dissociation constant of 1·8 nmol/l, was characterized in the dense collagenous regions of the cervix of ewes killed during the oestrous period. Similar dissociation constants were determined for [Arg8]-vasopressin and the oxytocin-specific agonist [Thr4, Gly7]-oxytocin in competition studies.

[3H] Oxytocin binding to peripheral cervical tissue and to the dense collagenous cervix was generally low or undetectable during the luteal phase, but increased in both tissues around the time of luteolysis. Although specific binding to both tissues was variable during the oestrous period, it was higher at this time than during the luteal phase. [3H] Oxytocin-binding site concentrations were also found to be higher within the inner dense collagenous cervix of oestrous ewes than of pregnant, ovariectomized or anoestrous animals. During the oestrous cycle, oxytocin-binding site concentrations reached a maximum in the dense collagenous cervical tissue on the day of oestrus (141·8 ±44 (s.e.m.) fmol/mg protein), showing a general decline during the following days back to luteal phase values. This compared with concentrations of 513·3 ±132·1 and 216·1 ± 13·9 fmol/mg protein, measured for comparative purposes in endometrial and myometrial membrane preparations, respectively, on the day of oestrus in the same group of ewes. However, in membrane preparations of peripheral cervical tissue higher concentrations were measured on day 14 than on the following 2 days and maximal concentrations were not reached until the day after oestrus (52·7 ± 4·2 fmol/mg protein). Concentrations were maintained at similar values during the subsequent 2 days and significant specific binding was still measurable in both regions of the cervix on day 5.

The localization of oxytocin-binding sites within dense collagenous cervical tissue was investigated autoradiographically using the 125I-labelled specific oxytocin receptor antagonist [1(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2, Thr4, Orn8, Tyr9 -NH2]-vasotocin. The only clear specific labelling was localized to the luminal epithelium of the uterine section of the cervix of oestrous ewes, with labelling in ewes in the luteal phase clearly reduced or absent.

The results demonstrate the presence of a high-affinity oxytocin-binding site within the cervix of the oestrous ewe which is associated with secretory cells and which undergoes similar changes in concentration during the oestrous cycle to uterine oxytocin receptor sites. The significance of this novel putative site of oxytocin action remains to be established.

Journal of Endocrinology (1994) 142, 397–405

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V. J. Ayad
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T. J. Parkinson
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E. L. Matthews
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M. L. Wild
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ABSTRACT

Previous autoradiographic studies have suggested that the regulation of oxytocin receptors differs between endometrial cell types during the ovine oestrous cycle, and that those present on luminal epithelial cells are of particular importance to the regulation of prostaglandin F release during luteal regression. The present autoradiographic study compares the distribution of the endometrial oxytocin receptor in day-15 non-pregnant and pregnant ewes. The distribution of the endometrial oxytocin receptor in day-15 non-pregnant ewes infused with systemic or intrauterine oxytocin has also been investigated.

Continuous, s.c. infusion of oxytocin (150 mmol/24 h) into ewes (n = 6) between days 10 and 15 of the oestrous cycle significantly increased plasma oxytocin concentrations (to approximately 100 pmol/l). There was no similar increase in systemic oxytocin concentrations in ewes receiving intrauterine (i.u.) oxytocin infusions (10 nmol/24 h) between days 10 and 15 of the oestrous cycle (n = 6). Luteolysis was inhibited in all six animals infused with oxytocin (s.c.) and endometrial oxytocin receptor concentrations were significantly lower on day 15 in these animals (12·8 ± 6·5 (s.e.m.) fmol/mg protein; P<0·001) and in pregnant ewes (18·4 ± 15·4 fmol/mg protein; P <0·001; n = 8) than in ewes infused with saline (248·6±67·1 fmol/mg protein; n = 6). While the 125I-labelled oxytocin receptor antagonist, [1-(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2, Thr4, Orn8, Tyr9-NH2]-vasotocin (125I-labelled OTA) clearly labelled glandular epithelia, luminal epithelium and caruncular stromal cells specifically on day 15 in saline (s.c.)-infused ewes, such specific labelling appeared to be reduced or absent from pregnant animals and those infused with oxytocin (s.c.). A significant reduction in the density of labelling of caruncular stroma (P < 0·05) and luminal epithelium (P < 0·001) was confirmed using quantitative densitometric analysis. The reduction in the labelling of endometrium in oxytocin-infused ewes was not caused by the binding of exogenous oxytocin to endometrial binding sites. Oxytocin infusion (i.u.) did not inhibit luteolysis, nor was there any significant difference in the endometrial oxytocin receptor concentration in this group of ewes on day 15 compared with those infused with saline (i.u.). There was also clear specific labelling of luminal epithelial cells with 125I-labelled OTA in ewes receiving oxytocin infused i.u. and quantification of autoradiograms failed to differentiate between these animals and those infused with saline (i.u.).

It was concluded that systemic oxytocin infusion and the early establishment of pregnancy led to a clear reduction in the endometrial oxytocin receptor concentration on luminal epithelial cells, glandular epithelial cells and caruncular stromal cells, but that i.u. oxytocin infusions did not affect any of these receptor populations and notably not the luminal epithelial oxytocin receptor. The results support the contention that the luminal epithelial oxytocin receptor is involved in the luteolytic process.

Journal of Endocrinology (1993) 137, 423–431

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V. J. Ayad
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E. L. Matthews
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D. C. Wathes
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T. J. Parkinson
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M. L. Wild
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ABSTRACT

The present study was designed to determine the localization of the endometrial oxytocin receptor during the ovine oestrous cycle, particularly on day 14, the time of initiation of luteal regression in the ewe. Samples were obtained from 29 ewes at different stages of the oestrous cycle (several during the luteal phase and on every day between day 14 (− 2) and day + 3 of the oestrous period).

Oxytocin receptors were localized autoradiographically in sections of uterine tissue, using the 125I-labelled oxytocin receptor antagonist [1-(β-mercapto-β,β-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2,Thr4,Orn8,Tyr9-NH2]-vasotocin (125I-labelled OTA). There was some variation in the pattern of 125I-labelled OTA labelling between different uterine tissue samples from the same ewe and also between samples obtained from different ewes thought to be at the same stage of the oestrous cycle. A clear overall pattern did, however, emerge with 125I-labelled OTA-binding sites distributed between luminal epithelial cells, glandular epithelial cells and caruncular stromal cells to varying extents on different days of the cycle.

During the luteal phase (days 5–12) clear specific labelling of endometrial tissue was generally absent. On day 14 labelling was evident on the luminal epithelium, but only in nine tissue samples out of a total of 18 studied, indicating that the entire luminal surface did not contain oxytocin receptors at this time. Between the day before oestrus and day 3 of the oestrous cycle the luminal epithelium was consistently labelled. The most extensive labelling of the remaining endometrial tissue was observed on the day of oestrus, with 125I-labelled OTA-binding sites clearly present on the stromal cells within caruncles and on a large proportion of secretory epithelia. This contrasted with the day before and the day after oestrus when labelling of glandular tissue was confined to the superficial endometrium, and labelling of caruncular stromal cells, although sometimes evident, was never as intense as on day 0. On days 2 and 3 labelling varied between being similar to that found on day 1 and being confined to the luminal epithelium and very few superficial secretory glands.

The results of this study lead us to conclude that the oxytocin receptor shows a differential distribution between stromal cells, epithelial cells lining secretory glands and luminal epithelial cells during the oestrous cycle; that the steroidal regulation of the oxytocin receptor differs between endometrial cell types; and that control of the luminal epithelial oxytocin receptors is probably of particular importance to the regulation of prostaglandin F release at luteal regression and during the maternal recognition of pregnancy.

Journal of Endocrinology (1991) 130, 199–206

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D. C. Wathes
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V. J. Ayad
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M. Kumari
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E. L. Matthews
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C. M. Wathes
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ABSTRACT

Blood samples were collected twice daily (09.30 and 17.00 h) via indwelling jugular-vein cannulae from five ewes throughout an entire oestrous cycle. Additional frequent samples were taken at 30-min intervals for 8 h on each of days 3 (early luteal phase), 9 (mid-luteal phase), 12 (late luteal phase) and 0 (day of oestrus). Plasma concentrations of arginine vasopressin and oxytocin were measured in all samples by radioimmunoassay and progesterone was measured in the twice-daily samples only.

Both oxytocin and progesterone showed the expected pattern of plasma concentrations, increasing during the early luteal phase, reaching a plateau and declining either preceding (oxytocin) or at (progesterone) luteolysis. Vasopressin concentrations showed a significant dependence on the day of cycle (P<0·05, analysis of variance) with concentrations lowest at oestrus and minor peaks on days 4 and 8–9. There was no correlation between the concentrations of vasopressin and progesterone. Vasopressin values were significantly higher in the morning than in the afternoon samples (1·3 cf. 0·9 pmol/l; P<0·001). Analysis of the frequent samples showed a significant (P<0·001) dependence on the time of day for vasopressin but not oxytocin. Values were high throughout the morning, declined to a trough at 15.00 h and rose again by 17.00 h.

We conclude that there is a minor variation in the vasopressin concentration during the oestrous cycle, which is not related to the circulating progesterone concentration but could be regulated by oestradiol. We also provide evidence for a diurnal rhythm in the release of vasopressin into the plasma in the ewe. The concentrations of vasopressin measured are unlikely to be sufficient to influence the activity of the reproductive tract in vivo.

Journal of Endocrinology (1992) 134, 107–113

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