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C. Sernia, R. T. Gemmell, and W. G. Thomas

ABSTRACT

There is inconclusive evidence that oxytocin acts directly on the corpus luteum and affects steroidogenesis. Since any such action would probably be mediated by oxytocin receptors, these should be present in luteal tissue. In this study, homogenates of corpora lutea from both pregnant and non-pregnant ewes were examined for oxytocin receptors by radio-receptor assay. Specific oxytocin binding was not observed in luteal tissue during the oestrous cycle. However specific binding was found in the corpora lutea of pregnant ewes; appearing at a fetal head length of approximately 0·65 cm (about 30 days of pregnancy) and persisting to a head size of 11 cm, the largest size examined in this study. The affinity (K d) of the receptor was calculated as 2·9 ± 0·3 nmol/l (s.e.m.; n = 9), a value similar to that obtained for the uterus. The receptor number ranged from a low of 8·7± 3·2 fmol/mg protein (n = 6) at a head size of <0·65 cm, to a maximum of 40·1 ± 6·5 fmol/mg protein (n = 25) at a head size of 2·5–3·75 cm. These values were lower than our estimate of 588 ± 39 fmol/mg protein (n = 5) for the uterus. It is concluded that a direct action of oxytocin on the corpus luteum is possible but only after the first month of pregnancy and not in the corpus luteum of the oestrous cycle.

Journal of Endocrinology (1989) 121, 117–123

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

. Akerlund M , Bossmar T, Brouard R, Kostrzewska A, Laudanski T, Lemancewicz A, Serradeil-Le Gal C & Steinwall M 1999 Receptor binding of oxytocin and vasopressin antagonists and inhibitory effects on isolated myometrium from preterm and term pregnant

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X H Zhang, S Filippi, L Vignozzi, A Morelli, R Mancina, M Luconi, S Donati, M Marini, G B Vannelli, G Forti, and M Maggi

oxytocin receptor (OTR) gene and protein in rabbit and human cavernous tissue in a similar concentration to that found in other portions of the male genital tract ( Vignozzi et al. 2004 ), classically considered the main male target of oxytocin (OT), such

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E. L. Sheldrick and A. P. F. Flint

ABSTRACT

Specific binding of [3H]oxytocin to high affinity sites (hormone receptors) in membrane preparations from uterine tissues of the ewe has been determined at varying stages of the oestrous cycle and in pregnancy. Mean receptor concentrations in caruncular and inter-caruncular endometrium and in myometrium were 14·2, 1·9 and 13·0 fmol/mg protein respectively between days 10 and 13 of the cycle. By the day of oestrus these values had increased to 749, 1085 and 179 fmol/mg protein. These increases in receptor concentrations coincided with luteolysis and falling plasma progesterone levels and followed the preovulatory decline in peripheral oxytocin and rise in ovarian venous oestradiol-17β. Receptor concentrations were low in all uterine tissues from pregnant animals between days 14 and 19 after oestrus. Analysis of binding parameters by Scatchard plot suggested a single population of receptor molecules in each of the tissues studied with apparent dissociation constants in the range 1·9–2·2 nmol/l. A number of naturally occurring neurohypophysial peptides inhibited binding of [3H]oxytocin to the receptor from ewes at oestrus; the cross-reactions of arginine vasopressin and vasotocin exceeded that of oxytocin.

Use of a receptor binding assay to measure oxytocin in extracts of corpora lutea on days 4 and 10 after oestrus gave values similar to those obtained by radioimmunoassay, suggesting the absence of other receptor-active peptides in the corpus luteum.

It is concluded that the oxytocin receptor is present in both components of the endometrium, as well as in the myometrium and that changes in uterine receptor concentrations before oestrus are consistent with receptor activation by steroid hormones.

J. Endocr. (1985) 106, 249–258

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R J Windle, J M Judah, and M L Forsling

Abstract

The effect of three oxytocin receptor antagonists on the renal actions of oxytocin and vasopressin was investigated in conscious male rats infused with hypotonic saline. Infusion of oxytocin at 100 pg/min produced plasma concentrations of 12·7 ± 3·3 pmol/l and led to significant increases in sodium excretion, urine flow and glomerular filtration rate (GFR). The increase in sodium excretion of 42 ± 9% during oxytocin infusion was significantly decreased by all three antagonists to 15 ± 5% (10 ng [mercapto-proprionic acid1,d-Tyr(Et)2, Thr4,Orn8]-oxytocin/min), 13 ± 5% (5 ng desGly9[d-Trp2,Thr4,Orn8]-dC6oxytocin/min) and 4 ± 5% (1 ng d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr(NH2)9]-vasotocin/min). Similarly, the increase in urine production of 22 ± 5% associated with oxytocin infusion was significantly decreased to 4 ± 3% (5 ng desGly9[d-Trp2,d-Thr4,Orn8]-dC6oxytocin/min) and 1 ± 4% (1 ng d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr(NH2)9]-vasotocin/min). All three antagonists blocked the oxytocin-induced increase in GFR when infused at 10 ng/min. Infusion of vasopressin at 160 pg/min produced plasma concentrations of 10·1 ± 2·1 pmol/l and this led to a significant increase in sodium excretion and a significant decrease in urine flow rate. None of the antagonists had any effect on the natriuretic or antidiuretic actions of vasopressin suggesting that different receptors are involved in these renal actions of the two peptides.

Journal of Endocrinology (1997) 152, 257–264

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R A D Bathgate and C Sernia

Abstract

In this study arginine vasopressin (AVP) and oxytocin (OT) receptors have been characterized in the brushtail possum. AVP receptors were characterized using [3H]AVP and the radioiodinated AVP V1a receptor antagonist 125I-labelled [(C6H5-CH2CO)-O-methyl-d-Tyr-Phe-Gln-Asn-Arg-Pro-Arg-Tyr- NH2] while OT receptors were characterized using the radioiodinated OT receptor antagonist 125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Orn8, Tyr-NH2 9]-vasotocin. The receptor affinities and densities have been compared with the rat AVP and OT receptors. Low densities of OT receptors were present in the possum ovary and kidney. High densities of AVP-binding sites were found in the possum adrenal, testis, mesenteric artery, ovary and renal medulla and lower densities in the possum liver. The AVP-binding sites showed marked differences in ligand-binding characteristics from the rat AVP V1a and V2 receptors. Receptor affinities were similar between tissues, except for a distinctly lower value in the renal medulla. It is concluded that the brushtail possum expresses AVP receptors with distinct ligand specificities from those of the rat AVP V1a and V2 receptors.

Journal of Endocrinology (1995) 144, 19–29

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R A D Bathgate and C Sernia

Abstract

In this study oxytocin (OT) receptors have been characterized and localized in the testis of the rat using the radioiodinated OT receptor antagonist 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr9-NH2]-vasotocin (OTA). Receptor density and localization have been compared with the rat testis arginine vasopressin (AVP) receptor using the radioiodinated AVP V1a receptor antagonist 125I-labelled d(CH2)5Sar7-AVP and the radioiodinated linear AVP V1a antagonist 125I-labelled [(C6H5-CH2CO)-O-methyl-d-Tyr-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2]. 125I-labelled OTA bound with high affinity to membrane fractions of the rat testis (K a = 13·8 ± 1·25 litres/nmol), mammary tissue (K a=20·3± 4·36 litres/nmol) and uterus (K a=27·8±0·74 litres/nmol). Competition studies with various OT and AVP receptor agonists and antagonists confirmed that the binding was to OT receptors. AVP receptors in the testis were found to be identical to AVP V1a receptors in the liver. The AVP receptor density in the testis was much higher than the OT receptor density (109 ±12·3 vs 5·2 ±0·79 (mean ± s.e.m.) fmol/mg protein). Autoradiographical localization showed that both OT and AVP receptors were present in the interstitial spaces in the testis consistent with binding to Leydig cells. AVP receptors were also localized on the epithelial surfaces of the seminiferous tubules and on testicular blood vessels. This study has, for the first time, found OT receptors in the testis of the rat which have similar ligand-binding characteristics to mammary and uterine OT receptors. The receptor localizations are consistent with binding to Leydig cells.

Journal of Endocrinology (1994) 141, 343–352

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S Phaneuf, G Asbóth, M P Carrasco, G N Europe-Finner, F Saji, T Kimura, A Harris, and A López Bernal

Abstract

We have recently provided evidence for the desensitization of oxytocin receptors in human myometrial cells. In the present study, we have investigated the possible mechanisms by which oxytocin (OT) might regulate OT receptor density. The steady state level of OT binding in cultured myometrial cells was 220 × 103 binding sites/cell, but this was time-dependently reduced to 27 × 103 sites/cell by exposure to OT for up to 20 h. Similarly, OT exposure decreased the binding of OT to cell membranes. In contrast, Western blotting data showed that the total amount of OT receptor protein was not affected by OT treatment for up to 48 h. Flow cytometry experiments demonstrated that OT receptors are not internalized during prolonged exposure of the cells to OT. However, RNase protection assays and Northern analysis showed that OT receptor mRNA was reduced by OT treatment to reach a new low steady state level with a time course similar to that of the disappearance of cell surface OT binding sites. Possible mechanisms involved in mRNA down-regulation include transcriptional suppression and destabilization of mRNA by RNA binding proteins.

Journal of Endocrinology (1997) 154, 7–18

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V. J. Ayad and D. C. Wathes

ABSTRACT

Oxytocin-binding sites in the endometrium and myometrium of the non-pregnant ewe were characterized. [3H]Oxytocin bound to a single site in both tissues with high affinity; dissociation constants were determined to be 1·96 nmol/l in endometrium and 2·12 nmol/l in myometrium. Oxytocin binding was enhanced by divalent cations with a similar order of potency in both tissues: Co2+> Mn2+> Ni2+> Mg2+ > Zn2+> Ca2+. The endometrial and myometrial binding sites showed the same specificity for oxytocin analogues and related peptides, having high affinity for oxytocin, [Arg8]-vasopressin, [Lys8]-vasopressin, and the oxytocin-specific agonists [Gly7]-oxytocin and [Thr4,Gly7]-oxytocin. The results suggest that oxytocin receptors present in the endometrium and myometrium of the ewe are similar both to each other and to classical oxytocin receptors.

Journal of Endocrinology (1989) 123, 11–18

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D C Wathes, G E Mann, J H Payne, P R Riley, K R Stevenson, and G E Lamming

Abstract

The regulation of oxytocin, oestradiol and progesterone receptors in different uterine cell types was studied in ovariectomized ewes. Animals were pretreated with a progestogen sponge for 10 days followed by 2 days of high-dose oestradiol to simulate oestrus. They then received either low-dose oestradiol (Group E), low-dose oestradiol plus progesterone (Group P) or low-dose oestradiol, progesterone and oxytocin (via osmotic minipump; Group OT). Animals (three to six per time-point) were killed following ovariectomy (Group OVX), at oestrus (Group O) or following 8, 10, 12 or 14 days of E, P or OT treatment. In a final group, oxytocin was withdrawn on day 12 and ewes were killed on day 14 (Group OTW). Oxytocin receptor concentrations and localization in the endometrium and myometrium were measured by radioreceptor assay, in situ hybridization and autoradiography with the iodinated oxytocin receptor antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2 9]-vasotocin. Oestradiol and progesterone receptors were localized by immunocytochemistry.

Oxytocin receptors were present in the luminal epithelium and superficial glands of ovariectomized ewes. In Group O, endometrial oxytocin receptor concentrations were high (1346 ± 379 fmol [3H]oxytocin bound mg protein−1) and receptors were also located in the deep glands and caruncular stroma in a pattern resembling that found at natural oestrus. Continuing low-dose oestradiol was unable to sustain high endometrial oxytocin receptor concentrations with values decreasing significantly to 140 ± 20 fmol mg protein−1 (P<0·01), localized to the luminal epithelium and caruncular stroma but not the glands. Progesterone treatment initially abolished all oxytocin receptors with none present on days 8 or 10. They reappeared in the luminal epithelium only between days 12 and 14 to give an overall concentration of 306 ± 50 fmol mg protein−1. Oxytocin treatment caused a small increase in oxytocin receptor concentration in the luminal epithelium on days 8 and 10 (20 ± 4 in Group P and 107 ± 35 fmol mg protein−1 in Group OT, P<0·01) but the rise on day 14 was not affected (267 ± 82 in Group OT and 411 ± 120 fmol mg protein−1 in Group OTW). In contrast, oestradiol treatment was able to sustain myometrial oxytocin receptors (635 ± 277 fmol mg protein−1 in Group O and 255 ± 36 in Group E) and there was no increase over time in Groups P, OT and OTW with values of 61 ± 18, 88 ± 53 and 114 ± 76 fmol mg protein−1 respectively (combined values for days 8–14). Oestradiol receptor concentrations were high in all uterine regions in Group O. This pattern and concentration was maintained in Group E. In all progesterone-treated ewes, oestradiol receptor concentrations were lower in all regions at all time-points. The only time-related change occurred in the luminal epithelium in which oestradiol receptors were undetectable on day 8 but developed by day 10 of progesterone treatment. Progesterone receptors were present at moderate concentrations in the deep glands, caruncular stroma, deep stroma and myometrium in Group O. Oestradiol increased progesterone receptors in the luminal epithelium, superficial glands, deep stroma and myometrium. Progesterone caused the loss of its own receptor from the luminal epithelium and superficial glands and decreased its receptor concentration in the deep stroma and myometrium at all time-points. There was a time-related loss of progesterone receptors from the deep glands of progesterone-treated ewes between days 8 and 14.

These results show differences in the regulation of receptors between uterine regions. In particular, loss of the negative inhibition by progesterone on the oxytocin receptor by day 14 occurred only in the luminal epithelium, but is unlikely to be a direct effect of progesterone as no progesterone receptors were present on luminal epithelial cells between days 8 and 14. The presence of oxytocin receptors in the luminal epithelium of ovariectomized ewes suggests that oestradiol is not essential for oxytocin receptor synthesis at this site. Oestradiol was able to sustain its own receptor at all sites, but high circulating progesterone was always inhibitory to oestradiol receptors. In general, oestradiol stimulated progesterone receptors in epithelial cells whereas progesterone abolished its own receptor from epithelial cells over a period of time, but had a lesser effect on stromal cells. The concentration of all three receptors is therefore differentially regulated between different uterine cell types, suggesting the importance of paracrine effects which remain to be elucidated.

Journal of Endocrinology (1996) 151, 375–393