Internalization and desensitization of the oxytocin receptor is inhibited by Dynamin and clathrin mutants in human embryonic kidney 293 cells.

Oxytocin (OT) has long been used as an uterotonic during labor management in women, and yet responses to OT infusion remain variable and unpredictable among patients. The investigation of oxytocin receptor (OTR) regulation will benefit labor management, because the clinical practice of continuous iv infusion of OT is not optimal. As with other G protein-coupled receptors, it is likely that the OTR internalizes and/or desensitizes upon continuous agonist exposure. The mechanisms by which this might occur, however, are unclear. Here we explore OTR internalization and desensitization in human embryonic kidney cells by utilizing inhibitors of heterologous second messenger systems and recently available mutant cDNA constructs. We report rapid and extensive internalization and desensitization of the OTR upon agonist exposure. Internalization was unaffected by inhibitors of protein kinase C or Ca(2+) calmodulin-dependant kinase II but was significantly reduced after transfection with dominant-negative mutant cDNAs of G protein-coupled receptor kinase 2, beta-Arrestin2, Dynamin, and Eps15 (a component of clathrin-coated pits). Moreover, desensitization of the OTR, measured by a calcium mobilization assay, was also inhibited by the aforementioned cDNA constructs. Thus, our data demonstrate, for the first time, the importance of the classical clathrin-mediated pathway during agonist-induced OTR internalization and desensitization.

Coincident increases in oxytocin receptor expression and EMG responsiveness to oxytocin in the ovine cervix at oestrus.

This study has localised oxytocin receptor (OTR) mRNA expression within the cervix of non-pregnant ewes and related this to changes in the sensitivity of the cervical musculature to administered oxytocin (OT) during the oestrous cycle by recording electromyographic (EMG) activity. Cervices were collected from 34 ewes at specified time points throughout the cycle. OTR mRNA expression was localised by in situ hybridisation and results were expressed as optical density measurements from autoradiographs in each of four different cervical regions. EMG recordings were made for up to 8 h per day from four non-pregnant ewes undergoing seasonal oestrous cycles between Days -3 and +3 relative to oestrus (Day 0). The highest concentrations of OTR mRNA were detectable within the luminal epithelium (LE) of the cervix, with values increasing from Day 15 of the cycle, peaking during the follicular phase (P<0.001, compared to the mid-luteal phase) and returning to basal by Day 2. There was a small but significant increase in OTR mRNA hybridisation (above basal/luteal phase values) within the stromal cells (STR) adjacent to the lumen (P<0.05) during the same time period, but no differences from basal values were detectable in the dense collagenous annular ring or in tissue superficial to this. Analysis of pooled EMG activity recorded daily from the cervix indicated that endogenous contractile activity was higher on Day 0 than on the Days +1 (P<0.05), -2, +2 and +3 (P<0.001). The response to bolus intravenous (i.v.) injections of 25 mU OT (25 mU) varied with day of the cycle. This dose produced a measurable and significant response on Days 0 (P<0.001) and +1 (P<0.001), but not on any of the other days, indicating that the sensitivity of the cervical musculature to OT peaked on these days. These data show that the cervix is highly responsive to OT at oestrus. This coincides with an increase in OTR mRNA expression in the luminal epithelial cells, suggesting the likely production of an intermediary messenger between the epithelial and smooth muscle cells.

Characterization and localization of a putative oxytocin receptor in the cervix of the oestrous ewe.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of oxytocin and vasopressin on oestrogen-induced electromyographic activity recorded from the uterus and oviduct of anoestrous ewes.

Oxytocin and the related peptide [Arg8]vasopressin (AVP) have previously been shown to bind with equally high affinity to oxytocin binding-sites (presumed oxytocin receptors) present within the uterus and oviduct of oestrous ewes. There is a possibility, therefore, that AVP mediates oxytocic actions through these binding sites. For the present study, ewes in seasonal anoestrus were treated with oestradiol-17 beta (50 micrograms subcutaneously, daily for 2-4 days). It was shown initially that this treatment stimulated the development of high-affinity oxytocin binding-sites (Kd 4.4 +/- 0.8 nmol L-1) which had similar affinity for AVP (Kd 4.2 +/- 0.9 nmol L-1) in the myometrium. The efficacy of oxytocin and AVP in vivo were compared by recording electromyographic (EMG) activity from the ampullary-isthmic junction of the left oviduct and the left uterine horn of four conscious ewes. Before oestradiol treatment there was no EMG response to oxytocin even at supraphysiological (1000 mU) doses. During oestradiol treatment, EMG activity was consistently increased in response to injections of 25 mU and 100 mU oxytocin via the jugular vein, but not to saline or 100 mU AVP. Higher doses of AVP were not investigated because of the possibility of cardiovascular side effects. A subsequent blood sampling experiment showed that maximal concentrations of oxytocin and AVP (achieved in peripheral plasma during the first 2 min following injection into the jugular vein) were of a similar order of magnitude after injection of equivalent doses of the two peptides. It is concluded that AVP probably does not mediate biological activity through the oxytocin receptor in non-pregnant ewes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of pregnancy and systemic or intrauterine oxytocin infusion on the distribution of endometrial oxytocin receptors in the ewe: an autoradiographic study.

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 F2 alpha 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).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of oxytocin infusion on secretion of progesterone and luteinizing hormone and the concentration of uterine oxytocin receptors during the periovulatory period in cloprostenol-treated ewes.

Oxytocin infusions were initiated on day 10 of the oestrous cycle in ewes, and luteal regression was induced by injection of 100 micrograms cloprostenol on day 12. Blood samples were collected at frequent intervals via an indwelling jugular vein cannula to measure concentrations of progesterone and luteinizing hormone (LH) during the luteal and follicular phases in saline (n = 6) and oxytocin (n = 5) infused animals. The oxytocin infusion maintained peripheral plasma concentrations of 53 +/- 3.2 pg oxytocin ml-1 (mean +/- SEM) compared with values of about 1 pg ml-1 during oestrus in control ewes. Oxytocin infusion had no effect on luteal phase progesterone concentrations, the timing of luteolysis, basal luteinizing hormone (LH) secretion, LH pulse frequency, or the timing or height of the LH surge. Treated ewes came into oestrus significantly earlier than controls (P < 0.05) but ovulated normally. Uterine samples collected 96 h after cloprostenol injection (approximately day 2 of the cycle) showed that oxytocin receptor concentrations were significantly higher in the endometrium in ewes that had been given a 5 day oxytocin infusion than in control animals (556 and 262 fmol mg-1 protein, respectively: geometric means from ANOVA, P < 0.001), whereas myometrial receptor concentrations were not affected (113 and 162 fmol mg-1 protein, respectively). We conclude that the previously reported delay in luteal development caused by oxytocin infusion (Wathes et al., 1991) is not due to the inhibition or delay of ovulation, but must instead occur via a direct influence on the developing corpus luteum.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of plasma vasopressin and oxytocin concentrations during the oestrous cycle of the ewe.

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 (midluteal 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 less than 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 less than 0.001). Analysis of the frequent samples showed a significant (P less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic localization of oxytocin receptors in the endometrium during the oestrous cycle of the ewe.

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-(beta-mercapto-beta, beta-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 labeling 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of oxytocin infusion during oestrus and the early luteal phase on progesterone secretion and the establishment of pregnancy in ewes.

In Experiment 1, an osmotic minipump containing oxytocin was implanted s.c. in ewes for 12 days beginning on Day 10 of the oestrous cycle, producing approximately 100 pg oxytocin/ml in the plasma. Two days after the start of infusion, all ewes were injected with 100 micrograms cloprostenol and placed with a fertile ram. At slaughter 22 days later, 9 (75%) of the 12 control (saline-infused) ewes were pregnant compared with 1 (11%) of the 9 ewes infused with oxytocin. In the control group, midcycle plasma concentrations of oxytocin were significantly higher in nonpregnant than in pregnant ewes. In Experiment 2, an infertile ram was used throughout to avoid any possible effects of pregnancy and oxytocin infusions were given at different stages of the oestrous cycle. Otherwise the protocol was similar to that in Exp. 1. Oxytocin infusion during luteolysis and the early follicular phase had no effect on the subsequent progesterone secretion pattern, but infusions beginning the day before cloprostenol-induced luteolysis and lasting for 7 or 12 days and infusions beginning on the day of oestrus for 4 days all delayed the subsequent rise in plasma progesterone by approximately 3-4 days. In these animals, the cycle tended to be longer. It was concluded that an appropriate oxytocin secretion pattern may be necessary for the establishment of pregnancy in ewes and that a high circulating oxytocin concentration during the early luteal phase delays the development of the young corpus luteum.

Characterization and localization of oxytocin receptors in the uterus and oviduct of the non-pregnant ewe using an iodinated receptor antagonist.

Some of the binding characteristics of a novel oxytocin receptor ligand 125I-labelled [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2,Thr4,Orn8,Tyr9-NH2]-vasotocin ([125I]OTA) have been determined in the sheep uterus. The compound was subsequently used for the autoradiographic localization of oxytocin receptors in the uterus and oviduct of the ewe. Specific binding of [125I]OTA to crude membrane fractions of ovine endometrium was time-dependent and was unaffected by the addition of cations to incubation media. Endometrial membranes contained a single population of saturable, high-affinity binding sites for the iodinated ligand (dissociation constant (Kd) 0.23 +/- 0.08 nmol/l) and unlabelled oxytocin competed with [125I]OTA for binding sites with high affinity (Kd 1.29 +/- 0.4 nmol/l) in the presence of Mg2+ In contrast, unlabelled OTA was able to compete with high affinity (Kd 1.13 +/- 0.16 nmol/l) in the absence of cation. Competition studies with a number of oxytocin analogues and related peptides and the tissue distribution of [125I]OTA binding sites also indicated that [125I]OTA bound to the ovine oxytocin receptor. This was further validated by autoradiographic studies which showed specific labelling with [125I]OTA to be greater to uterus and oviduct obtained from ewes which had been killed within 2 days of oestrus than to similar tissue from ewes killed during the luteal phase. In both the ampullary and isthmic regions of the oviduct and the myometrium, [125I]OTA binding sites were confined to smooth muscle. Endometrial binding sites for [125I]OTA were consistently located on the luminal epithelium and epithelial cells lining secretory glands.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxytocin receptors in the oviduct during the oestrous cycle of the ewe.

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.

Characterization of endometrial and myometrial oxytocin receptors in the non-pregnant ewe.

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+ greater than Mn2+ greater than Ni2+ greater than Mg2+ greater than Zn2+ greater than 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.

Effect of active immunization against oxytocin on gonadotrophin secretion and the establishment of pregnancy in the ewe.

A group of 14 ewes was actively immunized against oxytocin coupled to carrier protein, and comparisons of the reproductive status of these animals were made against ewes immunized against carrier protein only (N = 5) and untreated controls (N = 6). The last two groups were indistinguishable and were therefore combined as a single control group for analysis of the results. Oestrous cycle lengths were significantly extended in oxytocin-immunized ewes (P less than 0.005) with 42% of cycles lasting greater than 18 days. Cloprostenol treatment in the mid-luteal phase resulted in apparently normal luteal regression and re-ovulation, but luteal phase FSH concentrations and follicular phase LH concentrations were elevated in the immunized ewes, although surge levels of both hormones were unaffected. Measurements of free oxytocin concentrations in the blood suggested that these were significantly raised in treated animals. Progesterone concentrations in peripheral plasma were not altered by treatment. Mating resulted in a conception rate of 91% in control ewes compared with only 28% in oxytocin-immunized animals (P less than 0.01). There was no evidence of any conceptus material in the uteri of non-pregnant immunized ewes 25 days after service. Some had re-ovulated, whereas the ovaries of others contained mature corpora lutea which had been maintained. Ovarian histology appeared normal. We conclude that active immunization against oxytocin influences gonadotrophin secretion and reduces fertility. The site(s) of action for both of these effects needs to be determined.

Role of prostaglandin F-2 alpha and oxytocin in the regression of GnRH-induced abnormal corpora lutea in anoestrous ewes.

Anoestrous Romney Marsh ewes with (+P) and without (-P) progesterone pretreatment were induced to ovulate by multiple low-dose injection of GnRH followed by a bolus injection of GnRH. Luteal function was assessed by twice daily measurement of plasma progesterone. Animals were slaughtered on Days 3 or 5 after the end of GnRH treatment and CL and endometrium were recovered. In all Day-5 ewes, blood samples were collected at 30-min intervals for 8 h on Days 3 and 5 for measurement of PGFM and oxytocin. At slaughter 92% of the Group +P ewes had ovulated compared with 54% of the Group -P ewes. The ovaries of some of the Group -P ewes only contained luteinized cysts either alone or in association with CL. In the ewes that ovulated, progesterone profiles were normal in all Group +P ewes, whereas Group -P ewes had 'normal' or 'abnormal' profiles in which plasma progesterone was declining prematurely. All of the CL from ewes with abnormal progesterone profiles were associated with follicular cysts, and were significantly smaller and with a lower progesterone content on Day 5. PGFM levels decreased (P less than 0.05) between Days 3 and 5 in ewes in Groups +P and -P with 'normal' CL but increased (P less than 0.01) in Group -P ewes with 'abnormal' CL. Oxytocin levels were lower in Group -P ewes with 'abnormal' CL on Day 5, than in 'normal' ewes in Groups -P (P less than 0.01) or +P (P less than 0.05). In 3/5 Day-5 ewes with 'abnormal' CL there was a clear association between a major peak of oxytocin and a rise in PGFM during the frequent sampling period on Day 3 or Day 5, and endometrial oxytocin binding sites were present at slaughter. This suggests that the premature regression of 'abnormal' CL occurs via the normal luteolytic mechanism. Although ewes in Groups +P and -P with 'normal' CL had similar progesterone profiles, plasma oxytocin was significantly higher (P less than 0.05) in the Group -P ewes and oxytocin binding sites were present only in this group, suggesting that progesterone pretreatment can influence the production of both oxytocin and its receptor.