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Hormonal regulation of oxytocin-induced prostaglandin F2α secretion by the bovine and ovine uterus in vivo
Domestic Animal Endocrinology 21 (2001) 127–141
Hormonal regulation of oxytocin-induced prostaglandin F2␣ secretion by the bovine and ovine uterus in vivo G.E. Mann*, J.H. Payne, G.E. Lamming University of Nottingham, School of Biosciences, Division of Animal Physiology, Sutton Bonington, Loughborough, LE12 5RD, UK Received 10 April 2001; accepted 20 June 2001
Abstract In long-term ovariectomized ewes and cows, endometrial oxytocin receptors rest at relatively high levels but oxytocin is unable to induce prostaglandin F2␣ release. A series of studies were carried out to investigate the roles of physiological levels of progesterone and estradiol in “activating” these receptors in terms of permitting oxytocin-induced prostaglandin F2␣ release. In long-term ovariectomized cows, treatment with progesterone, but not estradiol, resulted in the induction of responsiveness to oxytocin. This responsiveness appeared within 2 d of progesterone treatment, reached a maximum by 6 d and was maintained to Day 18. In ovariectomized ewes, while estradiol treatment did induce temporary responsiveness to oxytocin after 3 d of treatment, treatment with progesterone was required to induce sustained responsiveness that appeared by Day 9 of treatment and was maintained to Day 12. Measurement of endometrial receptors for oxytocin revealed a significant decline in oxytocin receptors by Day 6 of progesterone treatment when responsiveness to oxytocin was maximal, demonstrating that receptor concentrations were not a limiting factor. The most likely mechanism by which progesterone treatment induces responsiveness to oxytocin may be through the up regulation of post receptor signaling pathways and/or enzymes involved in prostaglandin synthesis. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction It is now well established that the two ovarian hormones progesterone and estradiol play a major role in the control of the development of oxytocin receptors and prostaglandin F2␣(PGF2␣) release in both cattle and sheep [for reviews see 1, 2, 3, 4]. Numerous studies * Corresponding author. Tel.: ⫹01159 516326; fax: ⫹01159 516326. E-mail address: [email protected] (G.E. Mann). 0739-7240/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 0 1 ) 0 0 1 0 5 - 9
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have used steroid-treated ovariectomized ewe and cow models to further elucidate the roles of these two hormones. These studies have shown that by pre treating ovariectomized animals with progesterone and estradiol, to mimic a previous cycle, and then replacing progesterone and estradiol at physiological levels a pattern of oxytocin receptor development and PGF2␣ release can be recreated that closely matches that seen in intact cyclic animals (ewe: 5, 6, 7; cow: 8). The advantage of such models is that precise concentrations of progesterone and estradiol can be achieved without the complication of disruption to the gonadotrophic feedback control of endogenous ovarian hormone secretion. While the endometrial oxytocin receptors appears to be controlled by progesterone and estradiol, deprivation of endogenous ovarian hormone secretion results in high levels of the receptor in long term ovariectomized ewes (734 ⫾ 139 fmol/mg protein, 9) and cows (325 fmol/mg protein, 8). These receptor concentrations are considerably higher than those seen at the initiation of luteolysis, but despite high levels of receptor in these long-term ovariectomized animals, treatment with oxytocin cannot induce PGF2␣ release. Thus, these oxytocin receptors are “inactive” in terms of their ability to stimulate the release of PGF2␣ in response to an exogenous oxytocin challenge. Treatment with progesterone alone is sufficient to induce responsiveness to oxytocin in terms of PGF2␣ in both cattle [8,10] and sheep [5,6]. A number of known actions of progesterone on the uterus may account for this. These include increases in phospholipid stores [11] and the induction of regulatory enzymes such as prostaglandin synthetase [12,13] and phospholipase C [14]. It is known that in the cow, treatment with progesterone alone is sufficient to induce responsiveness to oxytocin (PGF2␣ release) within 1 wk, with responsiveness rising to a maximum by 2 wk [10]. However, the effects of shorter progesterone treatment on PGF2␣ and the effect of progesterone treatment on oxytocin receptor populations in long-term ovariectomized animals are not known. While studies have examined the short-term effects of estradiol treatment and the modulating effect of estradiol on progesterone treatment, little is known about the effects of longer-term estradiol treatment. While in the long-term ovariectomized ewe a number of studies have examined the effects of estradiol and progesterone on PGF2␣ secretion [15–17], little work has focussed on the effects of progesterone and estradiol on oxytocin-induced PGF2␣ release. Short-term treatment of anestrous ewes with high levels of estradiol (1 mg over 24h) has, however, been shown to induce responsiveness to oxytocin [18]. In this paper, we describe a series of studies in which we have examined the role of physiological concentrations of progesterone and estradiol in the “activation” of endometrial responsiveness to oxytocin, in terms of allowing oxytocin to induce PGF2␣ release, in long term ovariectomized ewes and cows. The main aims were to further elucidate the mechanisms controlling PGF2␣ release and to examine any differences in responses between these two closely related species. 2. Materials and methods 2.1. Experimental animals The cows used in these studies were all mature Galloway x Shorthorn cows ovariectomized several years previously and not treated with progesterone or estradiol for at least one
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month before each study. The ewes were all mature Dorset ewes ovariectomized one month prior to the initiation of studies. All animals were maintained indoors on a diet of hay and concentrate pellets throughout the duration of the experiments. All experiments were carried out under the control of an appropriate animal experimentation project license. 2.2. Blood sampling and oxytocin challenges Prior to the start of treatment a jugular vein of each animal was cannulated under local anesthesia (lignocaine s.c. as Lignovet 2% w/v, C-Vet, Bury St Edmunds, UK) with a 30 cm (cow) or 20 cm (ewe) indwelling jugular catheter (Secalon universal tubing, BOC Health Care, Swindon, UK) using a 12 gauge needle and guide wire. Cannulae were then maintained for the duration of the experiment and used for the collection of all blood samples. To monitor endometrial responsiveness to oxytocin, animals were injected with a single i.v. bolus of oxytocin (cow 50iu in 5 ml saline; ewe 5iu in 0.5 ml saline; Hoechst UK Ltd, Milton Keynes, UK). The oxytocin was administered via the jugular cannula, which was then flushed with a further 5 ml saline to ensure animals received the full amount of oxytocin. Plasma concentrations of 13, 14 dihydro-15-keto PGF2␣ (PGFM), the principle metabolite of PGF2␣, were measured in blood samples collected at 20 min intervals for 1 hr before the injection of oxytocin and then at 10 min intervals for 1h after the challenge. All samples were collected into heparinized tubes, centrifuged at 1500 g for 10 min and the plasma stored at ⫺20°C until assayed. 2.3. Experimental design 2.3.1. Study 1 The aims of this study were to determine the time course by which progesterone can induce responsiveness to oxytocin in long term ovariectomized ewes and to determine whether treatment with physiological levels of estradiol can induce responsiveness to oxytocin. Progesterone and estradiol were administered at levels designed to produce luteal phase concentrations of the two hormones. Treatment was administered for 12 d as it is known that this is sufficient time for progesterone to induce responsiveness to oxytocin. Oxytocin challenges were administered at 3-d intervals over this period to determine the time at which responsiveness was first observed. Two groups of 3 ovariectomized ewes were treated with either progesterone or estradiol for 12 d. Progesterone treatment was by twice daily im injection of 15 mg progesterone (Sigma Chemical Co., Poole, Dorset) in 0.5 ml corn oil. Estradiol was administered via a subcutaneous (sc) silastic implant (1 cm tube containing estradiol 17; 19). Daily blood samples were collected throughout treatment to monitor plasma hormone concentrations. Ewes were challenges with 5iu oxytocin on Days 0, 3, 6, 9, and 12 of hormone treatment. 2.3.2. Study 2 The aims of this study were to determine the time course by which progesterone can induce responsiveness to oxytocin this long term ovariectomized cow model system and to determine whether treatment with physiological levels of estradiol can induce responsiveness
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to oxytocin. Progesterone and estradiol were administered at levels designed to produce luteal phase concentrations of the two hormones. Treatment was continued for 18 d to ensure sufficient time to induce maximum responsiveness to oxytocin. Oxytocin challenges were administered at 6-d intervals to determine the time course over which maximum responsiveness developed. Two groups of 4 long-term ovariectomized cows were treated for 18 d with either progesterone or estradiol. Estradiol was administered via a sc silastic implant (2.5 cm tube containing estradiol-17; 20). Progesterone was administered via a CIDR-B intravaginal device (InterAG, New Zealand). Daily blood samples were collected throughout treatment to monitor plasma hormone concentrations. On Days 0, 6, 12 & 18 animals were challenged with a single i.v. bolus of 50iu oxytocin. 2.3.3. Study 3 The aim of this study was to expand on Study 2 to more precisely determine the time at which progesterone can induce responsiveness to oxytocin in long term ovariectomized cows. A group of 4 ovariectomized cows were treated with progesterone via an intravaginal CIDR-B intravaginal device, as in Study 2. However, in this study cows were administered oxytocin challenges on Days 0, 2, 4 and 6 of progesterone treatment. Daily blood samples were collected throughout treatment to monitor plasma hormone concentrations. 2.3.4. Study 4 The aim of this study was to determine the effects of progesterone treatment on endometrial oxytocin receptor concentrations. A group of 3 long-term ovariectomized cows were treated for 18 d with progesterone via a CIDR-B intravaginal device as in Study 2. Daily blood samples were collected throughout treatment to monitor plasma hormone concentrations and on Days 0, 6, 12 & 18 endomtrial biopsies were collected to determine endometrial oxytocin binding capacity. Endometrial biopsies were collected via a trans-cervical technique [21]. During biopsy collection animals were first sedated by an i.m. injection of 20 mg xylazine (Rompun; Bayer U.K. Ltd, Cambridge, UK) and the biopsy forceps were guided through the cervix by trans-rectal manipulation. Once in the uterus a single sample of 300 – 600 mg endometrial tissue was collected. Animals were then administered prophylactic antibiotic (Duplocillin LA; Mycofarm UK, Cambridge, UK). Samples were collected from left and right lower and middle sections of the uterine horns to avoid repeated sampling of the same site. We have previously demonstrated that oxytocin receptor concentrations do not differ between horns or between regions of the horn or between caruncular and intercaruncular endometrium in the cow [21]. Immediately following collection samples were snap frozen in liquid nitrogen and then stored in liquid nitrogen until processed. 2.3.5. Study 5 The aim of this study was to determine the effects of progesterone treatment on uterine PGF2␣ secretion directly, rather than through measurement of PGFM in the peripheral circulation. Groups of 3 ovariectomized ewes were either left untreated (n ⫽ 3) or were treated with progesterone by twice daily im injection of 15 mg progesterone (Sigma Chemical Co., Poole, Dorset, UK) in 0.5 ml corn oil. Daily blood samples were collected
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throughout treatment to monitor plasma hormone concentrations. On Day 10 of treatment, ewes underwent surgical mid-line laparotomy under general anesthesia. The reproductive tract was exteriorized and the ovarian artery and uterine veins were cannulated. Ewes were sampled at 10 min intervals from the uterine vein and jugular vein for 30 mins. 2.3.6. Study 6 The aim of this study was to determine the difference in the pattern of change of plasma PGFM concentration following oxytocin challenge between ovariectomized ewes and cows. This was not a specific study in itself, but a comparison of data from Studies 1 and 2. The data from these 2 studies were re-evaluated to provide a comparison of the pre and post oxytocin concentrations of PGFM found in cows and ewes in the two individual studies. Data from challenges in which a maximum response was observed were combined to produce a composite response profile. In ovariectomized ewes (Study 1), data were combined from oxytocin challenges carried out on Days 9 and 12. In ovariectomized cows (Study 2), data were combined from oxytocin challenges carried out on Days 12 and 18. This approach does not provide an experimentally controlled comparison between the two species but does give valuable insight into the differences that exist. 2.4. Assays Plasma samples were assayed for PGFM after extraction with acidified diethyl ether by radioimmunoassay [22], using antibody supplied by Dr H. Dobson. The sensitivity of the assay was 15 pg/ml, and the intra- and inter-assay coefficients of variation were 14.6% and 16.2% respectively. Progesterone was measured in plasma samples after extraction with petroleum ether by radioimmunoassay [23] with antiserum obtained from Dr B.J.A. Furr. The sensitivity of the assay was 0.2 ng/ml, and the intra- and inter-assay coefficients of variation were 8.6% and 11.3% respectively. Estradiol was measured in plasma using a modified radioimmunoassay kit (Serono Diagnostics Ltd, Woking, Surrey, UK) [24]. The sensitivity of the assay was 0.5 pg/ml, and the intra- and inter-assay coefficients of variation were 6.3% and 9.5% respectively. PGF2␣ was measured by radioimmunoassay [25]. Sensitivity was 0.4 ng/ml and intra and inter assay coefficients of variation 8.9 and 12.6% respectively. Oxytocin binding by the membrane preparations was then determined by binding assay [26] modified for use in bovine samples [27]. Results were expressed as fmol oxytocin bound/mg total protein [28]. The sensitivity of the assay was 20 fmol oxytocin bound/mg total protein. 2.5. Statistical analysis Differences in plasma concentrations of PGFM and PGF2␣ following oxytocin challenge were analyzed by repeated sample analysis of variance with day of challenge and time as main factors. Results on endometrial oxytocin binding in Study 4 were analyzed by analysis of variance with day as the main factor. In Study 6, plasma concentrations of PGFM following oxytocin challenge were analyzed by repeated sample analysis of variance with species and time as main factors. All data were analyzed without transformation.
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3. Results 3.1. Study 1 In Study 1, in ovariectomized ewes treated with progesterone, the mean peripheral plasma progesterone concentration was 2.1 ⫾ 0.1 ng/ml while the mean peripheral plasma estradiol concentration was 0.7 ⫾ 0.1 pg/ml. In the ovariectomized ewes treated with estradiol, the mean peripheral plasma estradiol concentration was 2.7 ⫾ 0.7 pg/ml while the plasma progesterone concentration remained below the sensitivity of the assay (⬍0.2 ng/ml). In ewes treated with progesterone, no response to oxytocin was seen on Day 0, 3 or 6 of treatment. However, by Day 9 a large increase in plasma PGFM was observed following oxytocin (P ⬍ 0.01) that was maintained at a similar level until Day 12 (Fig. 1). Treatment with estradiol resulted in a response to oxytocin on Day 3 (P ⬍ 0.01). This response was, lost by Day 6 and remained absent for the remaining treatment period (Fig. 1). 3.2. Study 2 In Study 2, in ovariectomized cows treated with progesterone, the mean peripheral plasma progesterone concentration was 7.3 ⫾ 0.1 ng/ml while the mean peripheral plasma estradiol concentration was 0.6 ⫾ 0.1 pg/ml. In the ovariectomized cows treated with estradiol, the mean peripheral plasma estradiol concentration was 1.9 ⫾ 0.6 pg/ml, while the plasma progesterone concentration remained below the sensitivity of the assay (⬍0.2 ng/ml). Treatment with progesterone resulted in the initiation of a large response to oxytocin by Day 6 (P ⬍ 0.001), which was maintained at a similar level until Day 18 (Fig. 2). Treatment with estradiol did not induce any responsiveness to oxytocin at any of the time points investigated. 3.3. Study 3 In this study, in ovariectomized cows treated with progesterone, the mean peripheral plasma progesterone concentration was 6.1 ⫾ 0.6 ng/ml while plasma concentrations of estradiol were low (0.5 ⫾ 0.2 pg/ml). Compared with Day 0, a significant increase in plasma PGFM following oxytocin challenge was seen on Day 2 (P ⬍ 0.05), Day 4 (P ⬍ 0.05) and Day 6 (P ⬍ 0.001) (Fig. 3). The increase on Day 6 was larger than that seen on Day 2 or Day 4 (P ⬍ 0.01). 3.4. Study 4 In this study, in ovariectomized cows treated with progesterone, the mean peripheral plasma progesterone concentration was 6.8 ⫾ 0.1 ng/ml. Compared with Day 0, there was a significant decline in oxytocin binding by Day 6 (P ⬍ 0.01) as well as Days 12 and 18 (P ⬍ 0.05) (Fig. 4). In addition, the level of oxytocin binding on Day 6 was lower than on Days 12 and 18 (P ⬍ 0.05).
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Fig. 1. Mean ⫾ sem plasma concentration of PGFM before and after oxytocin challenge (5iu oxytocin iv) in long term ovariectomized ewes treated with either progesterone (n ⫽ 3) or estradiol (n ⫽ 3) and challenged on Days 0, 3, 6, 9 and 12.
3.5. Study 5 In ovariectomized ewes treated with progesterone, the mean plasma concentration of progesterone was 2.6 ⫾ 0.2 ng/ml while in the untreated group it was below the limit of assay
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Fig. 2. Mean ⫾ sem plasma concentration of PGFM before and after oxytocin challenge (50iu oxytocin iv) in long term ovariectomized cows treated with either progesterone (n ⫽ 4) or estradiol (n ⫽ 4) and challenged on Days 0, 6, 12 and 18.
detection (⬍0.2 ng/ml). The PGF2␣ concentration in uterine venous plasma of ewes treated with progesterone was considerably higher than in untreated ewes (6.9 ⫾ 1.6 compared with 0.6 ⫾ 0.2 ng/ml; P ⬍ 0.01). This increased secretion of PGF2␣ was reflected in higher concentrations of PGFM in the peripheral circulation (57.7 ⫾ 4.3 compared with 42.3 ⫾ 2.6 pg/ml; P ⬍ 0.05). 3.6. Study 6 Mean plasma concentration of PGFM before and after oxytocin challenge in cows and ewes are shown in Fig. 5. Prior to oxytocin challenge, plasma concentrations of PGFM were
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Fig. 3. Mean ⫾ sem plasma concentration of PGFM before and after oxytocin challenge (50iu oxytocin iv) in ovariectomized cows treated with progesterone and challenged on Days 0, 2, 4 and 6 (a-d).
similar in ovariectomized ewes and cows. However, following oxytocin administrations PGFM concentration rose much faster in ewes than in cows such that plasma concentration of PGFM was higher in ewes at 10 (P ⬍ 0.01) and 20 (P ⬍ 0.05) mins following oxytocin administration. Furthermore, by 50 mins PGFM concentrations in ewes had begun to decline while in cows concentrations were still increasing. 4. Discussion In long term ovariectomized cows treatment with oxytocin is unable to induce any increase in PGF2␣ release from the uterus despite the presence of oxytocin receptors on the
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Fig. 4. Mean ⫾ sem oxytocin binding in endometrial biopsies collected from long term ovariectomized cows treated with progesterone and biopsied on Days 0, 6, 12 and 18 of treatment. (Different letters differ significantly; ab, bc P ⬍ 0.05; ac P ⬍ 0.01).
endometrium. In this study, following treatment with progesterone but not estradiol, oxytocin was able to induce uterine PGF2␣ release, measures as an increase in the plasma concentration of PGFM, the principle metabolite of PGF2␣. In ovariectomized ewes, while estradiol treatment did induce temporary responsiveness to oxytocin, treatment with progesterone was required to induce sustained responsiveness to oxytocin. In ovariectomized ewes, responsiveness to oxytocin did not develop until Day 9 of progesterone treatment and was completely absent on Days 3 and 6. Conversely, in the cow, responsiveness was present as early as Day 2 and was maximal by Day 6 of progesterone treatment. Thus in the cow it would appear that progesterone acts much faster than in the ewe, despite the longer cycle length in the cow. The reason for this difference is not known but presumably results from differences in either the degree to which the deficient post receptor mechanisms are suppressed between the two species or the rate at which they can be up regulated. Differences may also exist in progesterone receptor activity between the two species, though this was not investigated in this study. In both ewes and cows, the principal “trigger” for the initiation of luteolysis is the appearance of the oxytocin receptors on the uterine endometrium, binding of oxytocin to these newly developed receptors generating the release of luteolytic PGF2␣ episodes. This increase in oxytocin receptor is thought to result from the removal of the inhibitory action of progesterone that results from progesterone’s inhibition of its own receptor at this time
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Fig. 5. Composite responses to oxytocin challenge (cows 50i.u; ewes 5i.u.) in long term ovariectomized cows (squares) and ewes (circles) treated with progesterone. Graphs are based on days when a maximum response to oxytocin was observed (cows Day 12 and 18, n ⫽ 3; ewes Days 9 and 12, n ⫽ 3). ** P ⬍ 0.01; * P ⬍ 0.05.
[2,29,30]. In the ewe, it is thought that the loss of progesterone action on the uterus allows oestrogen receptor to up regulate with oestrogen then stimulating the development of oxytocin receptors [29,31]. However, in cattle, oxytocin receptor development on the luminal epithelium coupled with the initiation of luteolytic PGF2␣ release has been demonstrated in the absence of any change in oestrogen receptor [32]. In the present study, the time taken for progesterone to “activate” the uterus to respond to oxytocin, in the long term ovariectomized ewe was similar to the time of exposure of the uterus to progesterone during the luteal phase, prior to the onset of luteolytic PGF 127 127 release in the cyclic ewe [33]. This may suggest a possible role of the period of exposure to progesterone in controlling the timing of luteolysis in the ewe. However, in the ovariectomized cows in Study 2, it took only 6 d for maximum responsiveness to oxytocin to develop suggesting that, in the cyclic cow, the uterus has received sufficient progesterone “priming” to allow maximum responsiveness to oxytocin several days before the onset of luteolysis. In both ewes [34] and cows [35] it has been demonstrated that if endometrium from long term ovariectomized animals is placed in explant culture responsiveness to oxytocin develops “spontaneously” in the absence of any hormone treatment. This is in contrast to the results of the present in vivo study where, in the absence of appropriate steroid hormone treatment, no responsiveness to oxytocin had developed. In the absence of any direct influence of the culture system on tissue responsiveness, this would suggest that by placing
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tissue in culture it might be removed from the influence of a yet unidentified inhibitory mechanism operating in vivo. The levels of oxytocin receptor found in the uteri of long-term ovariectomized ewes and cows are well in excess of the concentrations required for luteolytic PGF2␣ release to occur. Oxytocin receptor concentrations of around 120 fmolmg⫺1 protein have been demonstrated at luteolysis and 560 fmolmg⫺1 protein at estrus compared to levels of 250 fmolmg⫺1 protein in the present study [21]. Furthermore, a marked increase in oxytocin induced PGF2␣ production has been shown in heifers between Days 13 and 16 despite only a slight increase in endometrial oxytocin receptor concentration, a large increase in oxytocin receptor concentration not occurring till Day 19 [36]. Thus in intact cows, luteolytic PGF2␣ release requires only a modest rise in uterine oxytocin receptors. Thus, the lack of responsiveness to oxytocin in long-term ovariectomized animals is not due to inadequate oxytocin receptor concentrations. One important point to note is that the binding assay used in the present study does not identify the pattern of oxytocin receptor localization within the endometrium. In intact ewes [2] and cows [32], the development of responsiveness to oxytocin is preceded by a large increase in oxytocin receptor mRNA expression in the luminal epithelium of the uterus. Receptors only appear in the deeper uterine tissues once luteolysis has begun [2]. The importance of oxytocin receptor concentrations in the luminal epithelium is not surprising as the majority of PGF 127 127 release occurs from epithelium and not stromal cells [37]. Thus in long term ovariectomized animals the location of the oxytocin receptor may be an important factor in determining responsiveness to oxytocin. However, a study in long-term ovariectomized ewes clearly demonstrated high levels of both oxytocin receptor mRNA expression and oxytocin receptor protein localized in the luminal epithelium an superficial glands [38]. Thus in long term ovariectomized animals it does not appear that inappropriate localization of oxytocin receptors is a contributing factor to the lack of responsiveness to oxytocin. In the cow, measurement of endometrial oxytocin receptor concentrations revealed a significant reduction in receptor concentrations after 6 d of progesterone treatment, at a time when responsiveness to oxytocin was developing. This finding of dissociation between oxytocin responsiveness and oxytocin receptor concentrations during progesterone treatment of ovariectomized animals has also been reported in the ewe [9]. The presence of oxytocin receptors in the absence of responsiveness to oxytocin coupled with the finding that as responsiveness develops oxytocin receptor levels fall demonstrates that the ability to release PGF2␣ in response to oxytocin may be controlled by receptor “activation” or post receptor function and not by receptor concentrations. In Study 5, treatment of ovariectomized ewes with progesterone for 10 d raised uterine vein PGF2␣ concentrations from low levels (0.6 ng/ml) to levels comparable to those seen in intact ewes (progesterone treated ovariectomized 6.9 ⫾ 1.1 ng/ml compared with intact ewes 3.0 ⫾ 0.4 ng/ml; 39). Thus, it would appear that in the absence of progesterone PGF2␣ secretion is inhibited while following progesterone treatment PGF2␣ is secreted at the normal level. It has previously been shown that treatment of short-term (3– 4 d) ovariectomized ewes with progesterone increases both uterine tissue content of PGF2␣ and PGF2␣ production by tissue during incubation [17] and has been shown to increase PGF2␣ secretion [15]. It is not
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surprising that progesterone is required for PGF2␣ synthesis as progesterone is known to induce a variety of important regulatory enzymes such as prostaglandin synthetase [12,13] and phospholipase C [14]. In Study 6, there were considerable differences in the pattern of increase in plasma PGFM concentration following oxytocin between progesterone- treated ovariectomized ewes and cows. The faster increase in PGFM may reflect either a faster release of PGF2␣ or a higher rate of metabolism of PGF2␣ to PGFM. The earlier decline in PGFM concentration may also result from a faster metabolism in the ewe although differences in the pattern of PGF2␣ release may also be a causal factor. In conclusion, these results demonstrate a clear temporal difference in the up regulation of endometrial responsiveness to oxytocin between ewes and cows. This difference highlights an unexpected lack of similarity in this aspect of the control of the luteolytic mechanism between these two closely related species. Further work is required to identify the post receptor signaling mechanisms and/or components of the PGF2␣ synthetic pathways that are deficient in long term ovariectomized animals. Aknowledgements The authors are grateful to SJ Mann, M Hunter and DV Scholey for technical assistance and to the University of Nottingham animal care staff. The work was supported by the Ministry of Agriculture Fisheries and Food, The Milk Development Council and the BBSRC. References [1] Silvia WJ, Lewis GS, McCracken JA, Thatcher WW, Wilson L. Hormonal regulation of uterine secretion of prostaglandin F2a during luteolysis in ruminants. Biol Reprod 1991;45:655– 63. [2] Wathes DC, Lamming GE. The oxytocin receptor, luteolysis, and the maintenance of pregnancy. J Reprod Fert 1995;Suppl 49:53– 67. [3] McCracken JA, Custer EE, Lamsa JC. Luteolysis: a neuroendocrine-mediated event. Physiol Rev 1999;79: 263–323. [4] Mann GE, Lamming GE, Robinson RS, Wathes DC. The regulation of interferon- production, and uterine hormone receptors during early pregnancy. J Reprod Fert 1999;Suppl 54:317–28. [5] Homanics GE, Silvia WJ. Effects of progesterone, and estradiol-17 on uterine secretion of prostaglandin F2␣ in response to oxytocin in ovariectomized ewes. Biol Reprod 1988;38:804 –11. [6] Vallet JL, Lamming GE, Batten M. Control of endometrial oxytocin receptor, and uterine response to oxytocin by progesterone, and oestradiol. J. Reprod. Fert 1990;90:625–34. [7] Beard AP, Hunter MG, Lamming GE. Quantitative control of oxytocin-induced PGF2␣ release by progesterone and oestradiol in ewes. J Reprod Fert 1994;100:143–50. [8] Lamming GE, Mann GE. Control of endometrial oxytocin receptors, and prostaglandin F2␣ production in the cow by progesterone and estradiol. J Reprod Fert 1995;103:69 –73. [9] Payne JH, Mann GE, Lamming GE. Progesterone action in ovariectomized ewes passively immunized against estradiol. J Reprod Fert 1994;Abstr Series 14:70. [10] Lafrance M, Goff AK. Effects of progesterone, and estradiol-17 on oxytocin-induced release of prostaglandin F-2␣ in heifers. J Reprod Fert 1988;82:429 –36. [11] Vallet JL, Bazer FW. Effect of ovine trophoblast protein–1, oestrogen, and progesterone on oxytocininduced phosphatidylinositol turnover in endometrium of sheep. J Reprod Fert 1989;87:755– 61.
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[12] Raw RE, Curry TE, Silvia WJ. Effect of progesterone and estradiol on the concentration, and activity of cyclooxygenase in the ovine uterus. Biol Reprod 1988; Suppl 38:104 (abstract). [13] Salamonson LA, Hampton AL, Clements JA, Findlay JK. Regulation of gene expression, and cellular localization of prostaglandin synthase by oestrogen, and progesterone in the ovine uterus. J Reprod Fert 1991;92:393– 406. [14] Raw RE, Silvia WJ. Activity of phospholipase C, and release of prostaglandin F2␣ by endometrial tissue from ovariectomized ewes receiving progesterone and estradiol. Biol Reprod 1991;44:404 –14. [15] Scaramuzzi RJ, Baird DT, Boyle HP, Land RB, Wheeler AJ. The secretion of prostaglandin F from the autotransplanted uterus of the ewe. J Reprod Fert 1974;49:157. [16] Ford SP, Weems CW, Pitts RE, Pexton JE, Butcher RL, Inskeep EK. Effects of estradiol 17, and progesterone on prostaglandin F in sheep uteri and uterine venous plasma. J Anim Sci 1975;41:1407–13. [17] Louis TM, Parry DM, Robinson JS, Thorburn GD, Challis JRG. Effects of exogenous progesterone, and oestradiol on prostaglandin F and 13, 14-dihydro-15-oxo prostaglandin F2␣ concentrations in uteri, and plasma of ovariectomized ewes. J Endocrin 1977;73:427–39. [18] Sharma SC, Fitzpatrick RJ. Effect of oestradiol-17b, and oxytocin treatment on prostaglandin F alpha release in the anoestrous ewe. Prostaglandins 1974;6:97–105. [19] Mann GE, Campbell BK, McNeilly AS, Baird DT. The role of inhibin, and estradiol in the control of gonadotrophin secretion in the ewe. J Endocrin 1992;133:381–91. [20] Mann GE, Lamming GE. Effect of the level of oestradiol on oxytocin-induced prostaglandin F2␣ release in the cow. J Endocrin 1995;145:175– 80. [21] Mann GE, Lamming GE. Use of repeated biopsies to monitor endometrial oxytocin receptors in cows. Vet Rec 1994;135:403–5. [22] Kaker ML, Murray RD, Dobson H. Plasma hormone changes in cows during induced or spontaneous calvings, and the early post partum period. Vet Rec 1984;115:378 – 82. [23] Haresign W, Foster JP, Haynes NB, Crighton DB, Lamming GE. Progesterone levels following treatment of seasonally anoestrous ewes with synthetic LH-releasing hormone. J Reprod Fert 1975; 43:269 –79. [24] Mann, GE, Lamming GE, Fray MD. Plasma estradiol and progesterone during early pregnancy, and the effects of treatment with buserelin. Anim Reprod Sci 1995;37:121–31. [25] Kelly RW, Deam S, Cameron MJ, Seamark RF. Measurement of prostaglandins as their methyl oximes. Prost Leuk Med 1986;24:1–14. [26] Sheldrick EL, Flint APF. Endocrine control of uterine oxytocin receptors in the ewe. J Endocrin 1985;106: 249 –58. [27] Jenner LJ, Parkinson TJ, Lamming GE. Uterine oxytocin receptors in cyclic, and pregnant cows. J Reprod Fert 1989;91:49 –58. [28] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75. [29] McCracken JA, Schramm W, Okulicz WC. Hormone receptor control of pulsatile secretion of PGF2␣ from the uterus and its abrogation in early pregnancy. Animal Reproduction Science 1984;7:31–55. [30] Spencer TE, Bazer FW. Temporal and spatial alterations in uterine estrogen receptor, and progesterone receptor gene expression during the estrous cycle and early pregnancy in the ewe. Biology of Reproduction 1995;53:1527– 43. [31] Spencer TE, Ott TL, Bazer FW. ␣-interferon: pregnancy recognition signal in ruminants Proceedings of Experimental Biology and Medicine 1996;213:215–29. [32] Robinson RS, Mann GE, Lamming GE, Wathes DC. The effect of pregnancy on the expression of uterine oxytocin, oestrogen and progesterone receptors during early pregnancy in the cow. J Endocrin 1999;160: 21–33. [33] Fairclough RJ, Moore LG, Peterson AJ, Watkins WB. Effect of oxytocin on plasma concentrations of 13, 14-dihydro-15-keto prostaglandin F and oxytocin-associated neurophysin during the estrous cycle and early pregnancy in the ewe. Biol Reprod 1984;31:36 – 43.
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[34] Sheldrick EL, Flick-Smith HC, Bendall DE, Flint APF. Absence of the oxytocin-induced prostaglandin F2␣ secretory response in uterus from ovariectomized ewes and the activation of the response in vitro. J Endocrin 1995;145:299 –305. [35] Mann GE. Hormone control of prostaglandin F2␣ production and oxytocin receptor concentrations in bovine endometrium in explant culture. Domest Anim Endocrinol 2001;20:217–226. [36] Mirando MA, Willard CB, Whiteaker SS. Relationships among endometrial oxytocin receptors, oxytocin stimulated phosphinosotide hydrolysis and prostaglandin F2␣ secretion in vitro and plasma concentrations of ovarian steroids before, and during corpus luteum regression in cyclic heifers. Biol Reprod 1993;48: 874 – 82. [37] Fortier MA, Guilbault LA, Grasso F. Specific properties of epithelial and stromal cells from endometrium of cows. J Reprod Fert 1988;83:239 – 48. [38] Wathes DC, Mann GE, Payne JH, Stevenson KR, Riley PR, Lamming GE. Regulation of oxytocin estradiol and progesterone receptor concentration in different uterine regions by estradiol progesterone and oxytocin in ovariectomized ewes. J Endocrin 1996;151:375–93. [39] Payne JH, Lamming GE. The direct influence of the embryo on uterine PGF2␣, and PGE2 production in sheep. J Reprod Fert 1994;101:737– 41.
Hormonal regulation of oxytocin-induced prostaglandin F2␣ secretion by the bovine and ovine uterus in vivo G.E. Mann*, J.H. Payne, G.E. Lamming University of Nottingham, School of Biosciences, Division of Animal Physiology, Sutton Bonington, Loughborough, LE12 5RD, UK Received 10 April 2001; accepted 20 June 2001
Abstract In long-term ovariectomized ewes and cows, endometrial oxytocin receptors rest at relatively high levels but oxytocin is unable to induce prostaglandin F2␣ release. A series of studies were carried out to investigate the roles of physiological levels of progesterone and estradiol in “activating” these receptors in terms of permitting oxytocin-induced prostaglandin F2␣ release. In long-term ovariectomized cows, treatment with progesterone, but not estradiol, resulted in the induction of responsiveness to oxytocin. This responsiveness appeared within 2 d of progesterone treatment, reached a maximum by 6 d and was maintained to Day 18. In ovariectomized ewes, while estradiol treatment did induce temporary responsiveness to oxytocin after 3 d of treatment, treatment with progesterone was required to induce sustained responsiveness that appeared by Day 9 of treatment and was maintained to Day 12. Measurement of endometrial receptors for oxytocin revealed a significant decline in oxytocin receptors by Day 6 of progesterone treatment when responsiveness to oxytocin was maximal, demonstrating that receptor concentrations were not a limiting factor. The most likely mechanism by which progesterone treatment induces responsiveness to oxytocin may be through the up regulation of post receptor signaling pathways and/or enzymes involved in prostaglandin synthesis. © 2001 Elsevier Science Inc. All rights reserved.
1. Introduction It is now well established that the two ovarian hormones progesterone and estradiol play a major role in the control of the development of oxytocin receptors and prostaglandin F2␣(PGF2␣) release in both cattle and sheep [for reviews see 1, 2, 3, 4]. Numerous studies * Corresponding author. Tel.: ⫹01159 516326; fax: ⫹01159 516326. E-mail address: [email protected] (G.E. Mann). 0739-7240/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 0 1 ) 0 0 1 0 5 - 9
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have used steroid-treated ovariectomized ewe and cow models to further elucidate the roles of these two hormones. These studies have shown that by pre treating ovariectomized animals with progesterone and estradiol, to mimic a previous cycle, and then replacing progesterone and estradiol at physiological levels a pattern of oxytocin receptor development and PGF2␣ release can be recreated that closely matches that seen in intact cyclic animals (ewe: 5, 6, 7; cow: 8). The advantage of such models is that precise concentrations of progesterone and estradiol can be achieved without the complication of disruption to the gonadotrophic feedback control of endogenous ovarian hormone secretion. While the endometrial oxytocin receptors appears to be controlled by progesterone and estradiol, deprivation of endogenous ovarian hormone secretion results in high levels of the receptor in long term ovariectomized ewes (734 ⫾ 139 fmol/mg protein, 9) and cows (325 fmol/mg protein, 8). These receptor concentrations are considerably higher than those seen at the initiation of luteolysis, but despite high levels of receptor in these long-term ovariectomized animals, treatment with oxytocin cannot induce PGF2␣ release. Thus, these oxytocin receptors are “inactive” in terms of their ability to stimulate the release of PGF2␣ in response to an exogenous oxytocin challenge. Treatment with progesterone alone is sufficient to induce responsiveness to oxytocin in terms of PGF2␣ in both cattle [8,10] and sheep [5,6]. A number of known actions of progesterone on the uterus may account for this. These include increases in phospholipid stores [11] and the induction of regulatory enzymes such as prostaglandin synthetase [12,13] and phospholipase C [14]. It is known that in the cow, treatment with progesterone alone is sufficient to induce responsiveness to oxytocin (PGF2␣ release) within 1 wk, with responsiveness rising to a maximum by 2 wk [10]. However, the effects of shorter progesterone treatment on PGF2␣ and the effect of progesterone treatment on oxytocin receptor populations in long-term ovariectomized animals are not known. While studies have examined the short-term effects of estradiol treatment and the modulating effect of estradiol on progesterone treatment, little is known about the effects of longer-term estradiol treatment. While in the long-term ovariectomized ewe a number of studies have examined the effects of estradiol and progesterone on PGF2␣ secretion [15–17], little work has focussed on the effects of progesterone and estradiol on oxytocin-induced PGF2␣ release. Short-term treatment of anestrous ewes with high levels of estradiol (1 mg over 24h) has, however, been shown to induce responsiveness to oxytocin [18]. In this paper, we describe a series of studies in which we have examined the role of physiological concentrations of progesterone and estradiol in the “activation” of endometrial responsiveness to oxytocin, in terms of allowing oxytocin to induce PGF2␣ release, in long term ovariectomized ewes and cows. The main aims were to further elucidate the mechanisms controlling PGF2␣ release and to examine any differences in responses between these two closely related species. 2. Materials and methods 2.1. Experimental animals The cows used in these studies were all mature Galloway x Shorthorn cows ovariectomized several years previously and not treated with progesterone or estradiol for at least one
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month before each study. The ewes were all mature Dorset ewes ovariectomized one month prior to the initiation of studies. All animals were maintained indoors on a diet of hay and concentrate pellets throughout the duration of the experiments. All experiments were carried out under the control of an appropriate animal experimentation project license. 2.2. Blood sampling and oxytocin challenges Prior to the start of treatment a jugular vein of each animal was cannulated under local anesthesia (lignocaine s.c. as Lignovet 2% w/v, C-Vet, Bury St Edmunds, UK) with a 30 cm (cow) or 20 cm (ewe) indwelling jugular catheter (Secalon universal tubing, BOC Health Care, Swindon, UK) using a 12 gauge needle and guide wire. Cannulae were then maintained for the duration of the experiment and used for the collection of all blood samples. To monitor endometrial responsiveness to oxytocin, animals were injected with a single i.v. bolus of oxytocin (cow 50iu in 5 ml saline; ewe 5iu in 0.5 ml saline; Hoechst UK Ltd, Milton Keynes, UK). The oxytocin was administered via the jugular cannula, which was then flushed with a further 5 ml saline to ensure animals received the full amount of oxytocin. Plasma concentrations of 13, 14 dihydro-15-keto PGF2␣ (PGFM), the principle metabolite of PGF2␣, were measured in blood samples collected at 20 min intervals for 1 hr before the injection of oxytocin and then at 10 min intervals for 1h after the challenge. All samples were collected into heparinized tubes, centrifuged at 1500 g for 10 min and the plasma stored at ⫺20°C until assayed. 2.3. Experimental design 2.3.1. Study 1 The aims of this study were to determine the time course by which progesterone can induce responsiveness to oxytocin in long term ovariectomized ewes and to determine whether treatment with physiological levels of estradiol can induce responsiveness to oxytocin. Progesterone and estradiol were administered at levels designed to produce luteal phase concentrations of the two hormones. Treatment was administered for 12 d as it is known that this is sufficient time for progesterone to induce responsiveness to oxytocin. Oxytocin challenges were administered at 3-d intervals over this period to determine the time at which responsiveness was first observed. Two groups of 3 ovariectomized ewes were treated with either progesterone or estradiol for 12 d. Progesterone treatment was by twice daily im injection of 15 mg progesterone (Sigma Chemical Co., Poole, Dorset) in 0.5 ml corn oil. Estradiol was administered via a subcutaneous (sc) silastic implant (1 cm tube containing estradiol 17; 19). Daily blood samples were collected throughout treatment to monitor plasma hormone concentrations. Ewes were challenges with 5iu oxytocin on Days 0, 3, 6, 9, and 12 of hormone treatment. 2.3.2. Study 2 The aims of this study were to determine the time course by which progesterone can induce responsiveness to oxytocin this long term ovariectomized cow model system and to determine whether treatment with physiological levels of estradiol can induce responsiveness
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to oxytocin. Progesterone and estradiol were administered at levels designed to produce luteal phase concentrations of the two hormones. Treatment was continued for 18 d to ensure sufficient time to induce maximum responsiveness to oxytocin. Oxytocin challenges were administered at 6-d intervals to determine the time course over which maximum responsiveness developed. Two groups of 4 long-term ovariectomized cows were treated for 18 d with either progesterone or estradiol. Estradiol was administered via a sc silastic implant (2.5 cm tube containing estradiol-17; 20). Progesterone was administered via a CIDR-B intravaginal device (InterAG, New Zealand). Daily blood samples were collected throughout treatment to monitor plasma hormone concentrations. On Days 0, 6, 12 & 18 animals were challenged with a single i.v. bolus of 50iu oxytocin. 2.3.3. Study 3 The aim of this study was to expand on Study 2 to more precisely determine the time at which progesterone can induce responsiveness to oxytocin in long term ovariectomized cows. A group of 4 ovariectomized cows were treated with progesterone via an intravaginal CIDR-B intravaginal device, as in Study 2. However, in this study cows were administered oxytocin challenges on Days 0, 2, 4 and 6 of progesterone treatment. Daily blood samples were collected throughout treatment to monitor plasma hormone concentrations. 2.3.4. Study 4 The aim of this study was to determine the effects of progesterone treatment on endometrial oxytocin receptor concentrations. A group of 3 long-term ovariectomized cows were treated for 18 d with progesterone via a CIDR-B intravaginal device as in Study 2. Daily blood samples were collected throughout treatment to monitor plasma hormone concentrations and on Days 0, 6, 12 & 18 endomtrial biopsies were collected to determine endometrial oxytocin binding capacity. Endometrial biopsies were collected via a trans-cervical technique [21]. During biopsy collection animals were first sedated by an i.m. injection of 20 mg xylazine (Rompun; Bayer U.K. Ltd, Cambridge, UK) and the biopsy forceps were guided through the cervix by trans-rectal manipulation. Once in the uterus a single sample of 300 – 600 mg endometrial tissue was collected. Animals were then administered prophylactic antibiotic (Duplocillin LA; Mycofarm UK, Cambridge, UK). Samples were collected from left and right lower and middle sections of the uterine horns to avoid repeated sampling of the same site. We have previously demonstrated that oxytocin receptor concentrations do not differ between horns or between regions of the horn or between caruncular and intercaruncular endometrium in the cow [21]. Immediately following collection samples were snap frozen in liquid nitrogen and then stored in liquid nitrogen until processed. 2.3.5. Study 5 The aim of this study was to determine the effects of progesterone treatment on uterine PGF2␣ secretion directly, rather than through measurement of PGFM in the peripheral circulation. Groups of 3 ovariectomized ewes were either left untreated (n ⫽ 3) or were treated with progesterone by twice daily im injection of 15 mg progesterone (Sigma Chemical Co., Poole, Dorset, UK) in 0.5 ml corn oil. Daily blood samples were collected
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throughout treatment to monitor plasma hormone concentrations. On Day 10 of treatment, ewes underwent surgical mid-line laparotomy under general anesthesia. The reproductive tract was exteriorized and the ovarian artery and uterine veins were cannulated. Ewes were sampled at 10 min intervals from the uterine vein and jugular vein for 30 mins. 2.3.6. Study 6 The aim of this study was to determine the difference in the pattern of change of plasma PGFM concentration following oxytocin challenge between ovariectomized ewes and cows. This was not a specific study in itself, but a comparison of data from Studies 1 and 2. The data from these 2 studies were re-evaluated to provide a comparison of the pre and post oxytocin concentrations of PGFM found in cows and ewes in the two individual studies. Data from challenges in which a maximum response was observed were combined to produce a composite response profile. In ovariectomized ewes (Study 1), data were combined from oxytocin challenges carried out on Days 9 and 12. In ovariectomized cows (Study 2), data were combined from oxytocin challenges carried out on Days 12 and 18. This approach does not provide an experimentally controlled comparison between the two species but does give valuable insight into the differences that exist. 2.4. Assays Plasma samples were assayed for PGFM after extraction with acidified diethyl ether by radioimmunoassay [22], using antibody supplied by Dr H. Dobson. The sensitivity of the assay was 15 pg/ml, and the intra- and inter-assay coefficients of variation were 14.6% and 16.2% respectively. Progesterone was measured in plasma samples after extraction with petroleum ether by radioimmunoassay [23] with antiserum obtained from Dr B.J.A. Furr. The sensitivity of the assay was 0.2 ng/ml, and the intra- and inter-assay coefficients of variation were 8.6% and 11.3% respectively. Estradiol was measured in plasma using a modified radioimmunoassay kit (Serono Diagnostics Ltd, Woking, Surrey, UK) [24]. The sensitivity of the assay was 0.5 pg/ml, and the intra- and inter-assay coefficients of variation were 6.3% and 9.5% respectively. PGF2␣ was measured by radioimmunoassay [25]. Sensitivity was 0.4 ng/ml and intra and inter assay coefficients of variation 8.9 and 12.6% respectively. Oxytocin binding by the membrane preparations was then determined by binding assay [26] modified for use in bovine samples [27]. Results were expressed as fmol oxytocin bound/mg total protein [28]. The sensitivity of the assay was 20 fmol oxytocin bound/mg total protein. 2.5. Statistical analysis Differences in plasma concentrations of PGFM and PGF2␣ following oxytocin challenge were analyzed by repeated sample analysis of variance with day of challenge and time as main factors. Results on endometrial oxytocin binding in Study 4 were analyzed by analysis of variance with day as the main factor. In Study 6, plasma concentrations of PGFM following oxytocin challenge were analyzed by repeated sample analysis of variance with species and time as main factors. All data were analyzed without transformation.
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3. Results 3.1. Study 1 In Study 1, in ovariectomized ewes treated with progesterone, the mean peripheral plasma progesterone concentration was 2.1 ⫾ 0.1 ng/ml while the mean peripheral plasma estradiol concentration was 0.7 ⫾ 0.1 pg/ml. In the ovariectomized ewes treated with estradiol, the mean peripheral plasma estradiol concentration was 2.7 ⫾ 0.7 pg/ml while the plasma progesterone concentration remained below the sensitivity of the assay (⬍0.2 ng/ml). In ewes treated with progesterone, no response to oxytocin was seen on Day 0, 3 or 6 of treatment. However, by Day 9 a large increase in plasma PGFM was observed following oxytocin (P ⬍ 0.01) that was maintained at a similar level until Day 12 (Fig. 1). Treatment with estradiol resulted in a response to oxytocin on Day 3 (P ⬍ 0.01). This response was, lost by Day 6 and remained absent for the remaining treatment period (Fig. 1). 3.2. Study 2 In Study 2, in ovariectomized cows treated with progesterone, the mean peripheral plasma progesterone concentration was 7.3 ⫾ 0.1 ng/ml while the mean peripheral plasma estradiol concentration was 0.6 ⫾ 0.1 pg/ml. In the ovariectomized cows treated with estradiol, the mean peripheral plasma estradiol concentration was 1.9 ⫾ 0.6 pg/ml, while the plasma progesterone concentration remained below the sensitivity of the assay (⬍0.2 ng/ml). Treatment with progesterone resulted in the initiation of a large response to oxytocin by Day 6 (P ⬍ 0.001), which was maintained at a similar level until Day 18 (Fig. 2). Treatment with estradiol did not induce any responsiveness to oxytocin at any of the time points investigated. 3.3. Study 3 In this study, in ovariectomized cows treated with progesterone, the mean peripheral plasma progesterone concentration was 6.1 ⫾ 0.6 ng/ml while plasma concentrations of estradiol were low (0.5 ⫾ 0.2 pg/ml). Compared with Day 0, a significant increase in plasma PGFM following oxytocin challenge was seen on Day 2 (P ⬍ 0.05), Day 4 (P ⬍ 0.05) and Day 6 (P ⬍ 0.001) (Fig. 3). The increase on Day 6 was larger than that seen on Day 2 or Day 4 (P ⬍ 0.01). 3.4. Study 4 In this study, in ovariectomized cows treated with progesterone, the mean peripheral plasma progesterone concentration was 6.8 ⫾ 0.1 ng/ml. Compared with Day 0, there was a significant decline in oxytocin binding by Day 6 (P ⬍ 0.01) as well as Days 12 and 18 (P ⬍ 0.05) (Fig. 4). In addition, the level of oxytocin binding on Day 6 was lower than on Days 12 and 18 (P ⬍ 0.05).
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Fig. 1. Mean ⫾ sem plasma concentration of PGFM before and after oxytocin challenge (5iu oxytocin iv) in long term ovariectomized ewes treated with either progesterone (n ⫽ 3) or estradiol (n ⫽ 3) and challenged on Days 0, 3, 6, 9 and 12.
3.5. Study 5 In ovariectomized ewes treated with progesterone, the mean plasma concentration of progesterone was 2.6 ⫾ 0.2 ng/ml while in the untreated group it was below the limit of assay
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Fig. 2. Mean ⫾ sem plasma concentration of PGFM before and after oxytocin challenge (50iu oxytocin iv) in long term ovariectomized cows treated with either progesterone (n ⫽ 4) or estradiol (n ⫽ 4) and challenged on Days 0, 6, 12 and 18.
detection (⬍0.2 ng/ml). The PGF2␣ concentration in uterine venous plasma of ewes treated with progesterone was considerably higher than in untreated ewes (6.9 ⫾ 1.6 compared with 0.6 ⫾ 0.2 ng/ml; P ⬍ 0.01). This increased secretion of PGF2␣ was reflected in higher concentrations of PGFM in the peripheral circulation (57.7 ⫾ 4.3 compared with 42.3 ⫾ 2.6 pg/ml; P ⬍ 0.05). 3.6. Study 6 Mean plasma concentration of PGFM before and after oxytocin challenge in cows and ewes are shown in Fig. 5. Prior to oxytocin challenge, plasma concentrations of PGFM were
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Fig. 3. Mean ⫾ sem plasma concentration of PGFM before and after oxytocin challenge (50iu oxytocin iv) in ovariectomized cows treated with progesterone and challenged on Days 0, 2, 4 and 6 (a-d).
similar in ovariectomized ewes and cows. However, following oxytocin administrations PGFM concentration rose much faster in ewes than in cows such that plasma concentration of PGFM was higher in ewes at 10 (P ⬍ 0.01) and 20 (P ⬍ 0.05) mins following oxytocin administration. Furthermore, by 50 mins PGFM concentrations in ewes had begun to decline while in cows concentrations were still increasing. 4. Discussion In long term ovariectomized cows treatment with oxytocin is unable to induce any increase in PGF2␣ release from the uterus despite the presence of oxytocin receptors on the
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Fig. 4. Mean ⫾ sem oxytocin binding in endometrial biopsies collected from long term ovariectomized cows treated with progesterone and biopsied on Days 0, 6, 12 and 18 of treatment. (Different letters differ significantly; ab, bc P ⬍ 0.05; ac P ⬍ 0.01).
endometrium. In this study, following treatment with progesterone but not estradiol, oxytocin was able to induce uterine PGF2␣ release, measures as an increase in the plasma concentration of PGFM, the principle metabolite of PGF2␣. In ovariectomized ewes, while estradiol treatment did induce temporary responsiveness to oxytocin, treatment with progesterone was required to induce sustained responsiveness to oxytocin. In ovariectomized ewes, responsiveness to oxytocin did not develop until Day 9 of progesterone treatment and was completely absent on Days 3 and 6. Conversely, in the cow, responsiveness was present as early as Day 2 and was maximal by Day 6 of progesterone treatment. Thus in the cow it would appear that progesterone acts much faster than in the ewe, despite the longer cycle length in the cow. The reason for this difference is not known but presumably results from differences in either the degree to which the deficient post receptor mechanisms are suppressed between the two species or the rate at which they can be up regulated. Differences may also exist in progesterone receptor activity between the two species, though this was not investigated in this study. In both ewes and cows, the principal “trigger” for the initiation of luteolysis is the appearance of the oxytocin receptors on the uterine endometrium, binding of oxytocin to these newly developed receptors generating the release of luteolytic PGF2␣ episodes. This increase in oxytocin receptor is thought to result from the removal of the inhibitory action of progesterone that results from progesterone’s inhibition of its own receptor at this time
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Fig. 5. Composite responses to oxytocin challenge (cows 50i.u; ewes 5i.u.) in long term ovariectomized cows (squares) and ewes (circles) treated with progesterone. Graphs are based on days when a maximum response to oxytocin was observed (cows Day 12 and 18, n ⫽ 3; ewes Days 9 and 12, n ⫽ 3). ** P ⬍ 0.01; * P ⬍ 0.05.
[2,29,30]. In the ewe, it is thought that the loss of progesterone action on the uterus allows oestrogen receptor to up regulate with oestrogen then stimulating the development of oxytocin receptors [29,31]. However, in cattle, oxytocin receptor development on the luminal epithelium coupled with the initiation of luteolytic PGF2␣ release has been demonstrated in the absence of any change in oestrogen receptor [32]. In the present study, the time taken for progesterone to “activate” the uterus to respond to oxytocin, in the long term ovariectomized ewe was similar to the time of exposure of the uterus to progesterone during the luteal phase, prior to the onset of luteolytic PGF 127 127 release in the cyclic ewe [33]. This may suggest a possible role of the period of exposure to progesterone in controlling the timing of luteolysis in the ewe. However, in the ovariectomized cows in Study 2, it took only 6 d for maximum responsiveness to oxytocin to develop suggesting that, in the cyclic cow, the uterus has received sufficient progesterone “priming” to allow maximum responsiveness to oxytocin several days before the onset of luteolysis. In both ewes [34] and cows [35] it has been demonstrated that if endometrium from long term ovariectomized animals is placed in explant culture responsiveness to oxytocin develops “spontaneously” in the absence of any hormone treatment. This is in contrast to the results of the present in vivo study where, in the absence of appropriate steroid hormone treatment, no responsiveness to oxytocin had developed. In the absence of any direct influence of the culture system on tissue responsiveness, this would suggest that by placing
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tissue in culture it might be removed from the influence of a yet unidentified inhibitory mechanism operating in vivo. The levels of oxytocin receptor found in the uteri of long-term ovariectomized ewes and cows are well in excess of the concentrations required for luteolytic PGF2␣ release to occur. Oxytocin receptor concentrations of around 120 fmolmg⫺1 protein have been demonstrated at luteolysis and 560 fmolmg⫺1 protein at estrus compared to levels of 250 fmolmg⫺1 protein in the present study [21]. Furthermore, a marked increase in oxytocin induced PGF2␣ production has been shown in heifers between Days 13 and 16 despite only a slight increase in endometrial oxytocin receptor concentration, a large increase in oxytocin receptor concentration not occurring till Day 19 [36]. Thus in intact cows, luteolytic PGF2␣ release requires only a modest rise in uterine oxytocin receptors. Thus, the lack of responsiveness to oxytocin in long-term ovariectomized animals is not due to inadequate oxytocin receptor concentrations. One important point to note is that the binding assay used in the present study does not identify the pattern of oxytocin receptor localization within the endometrium. In intact ewes [2] and cows [32], the development of responsiveness to oxytocin is preceded by a large increase in oxytocin receptor mRNA expression in the luminal epithelium of the uterus. Receptors only appear in the deeper uterine tissues once luteolysis has begun [2]. The importance of oxytocin receptor concentrations in the luminal epithelium is not surprising as the majority of PGF 127 127 release occurs from epithelium and not stromal cells [37]. Thus in long term ovariectomized animals the location of the oxytocin receptor may be an important factor in determining responsiveness to oxytocin. However, a study in long-term ovariectomized ewes clearly demonstrated high levels of both oxytocin receptor mRNA expression and oxytocin receptor protein localized in the luminal epithelium an superficial glands [38]. Thus in long term ovariectomized animals it does not appear that inappropriate localization of oxytocin receptors is a contributing factor to the lack of responsiveness to oxytocin. In the cow, measurement of endometrial oxytocin receptor concentrations revealed a significant reduction in receptor concentrations after 6 d of progesterone treatment, at a time when responsiveness to oxytocin was developing. This finding of dissociation between oxytocin responsiveness and oxytocin receptor concentrations during progesterone treatment of ovariectomized animals has also been reported in the ewe [9]. The presence of oxytocin receptors in the absence of responsiveness to oxytocin coupled with the finding that as responsiveness develops oxytocin receptor levels fall demonstrates that the ability to release PGF2␣ in response to oxytocin may be controlled by receptor “activation” or post receptor function and not by receptor concentrations. In Study 5, treatment of ovariectomized ewes with progesterone for 10 d raised uterine vein PGF2␣ concentrations from low levels (0.6 ng/ml) to levels comparable to those seen in intact ewes (progesterone treated ovariectomized 6.9 ⫾ 1.1 ng/ml compared with intact ewes 3.0 ⫾ 0.4 ng/ml; 39). Thus, it would appear that in the absence of progesterone PGF2␣ secretion is inhibited while following progesterone treatment PGF2␣ is secreted at the normal level. It has previously been shown that treatment of short-term (3– 4 d) ovariectomized ewes with progesterone increases both uterine tissue content of PGF2␣ and PGF2␣ production by tissue during incubation [17] and has been shown to increase PGF2␣ secretion [15]. It is not
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surprising that progesterone is required for PGF2␣ synthesis as progesterone is known to induce a variety of important regulatory enzymes such as prostaglandin synthetase [12,13] and phospholipase C [14]. In Study 6, there were considerable differences in the pattern of increase in plasma PGFM concentration following oxytocin between progesterone- treated ovariectomized ewes and cows. The faster increase in PGFM may reflect either a faster release of PGF2␣ or a higher rate of metabolism of PGF2␣ to PGFM. The earlier decline in PGFM concentration may also result from a faster metabolism in the ewe although differences in the pattern of PGF2␣ release may also be a causal factor. In conclusion, these results demonstrate a clear temporal difference in the up regulation of endometrial responsiveness to oxytocin between ewes and cows. This difference highlights an unexpected lack of similarity in this aspect of the control of the luteolytic mechanism between these two closely related species. Further work is required to identify the post receptor signaling mechanisms and/or components of the PGF2␣ synthetic pathways that are deficient in long term ovariectomized animals. Aknowledgements The authors are grateful to SJ Mann, M Hunter and DV Scholey for technical assistance and to the University of Nottingham animal care staff. The work was supported by the Ministry of Agriculture Fisheries and Food, The Milk Development Council and the BBSRC. References [1] Silvia WJ, Lewis GS, McCracken JA, Thatcher WW, Wilson L. Hormonal regulation of uterine secretion of prostaglandin F2a during luteolysis in ruminants. Biol Reprod 1991;45:655– 63. [2] Wathes DC, Lamming GE. The oxytocin receptor, luteolysis, and the maintenance of pregnancy. J Reprod Fert 1995;Suppl 49:53– 67. [3] McCracken JA, Custer EE, Lamsa JC. Luteolysis: a neuroendocrine-mediated event. Physiol Rev 1999;79: 263–323. [4] Mann GE, Lamming GE, Robinson RS, Wathes DC. The regulation of interferon- production, and uterine hormone receptors during early pregnancy. J Reprod Fert 1999;Suppl 54:317–28. [5] Homanics GE, Silvia WJ. Effects of progesterone, and estradiol-17 on uterine secretion of prostaglandin F2␣ in response to oxytocin in ovariectomized ewes. Biol Reprod 1988;38:804 –11. [6] Vallet JL, Lamming GE, Batten M. Control of endometrial oxytocin receptor, and uterine response to oxytocin by progesterone, and oestradiol. J. Reprod. Fert 1990;90:625–34. [7] Beard AP, Hunter MG, Lamming GE. Quantitative control of oxytocin-induced PGF2␣ release by progesterone and oestradiol in ewes. J Reprod Fert 1994;100:143–50. [8] Lamming GE, Mann GE. Control of endometrial oxytocin receptors, and prostaglandin F2␣ production in the cow by progesterone and estradiol. J Reprod Fert 1995;103:69 –73. [9] Payne JH, Mann GE, Lamming GE. Progesterone action in ovariectomized ewes passively immunized against estradiol. J Reprod Fert 1994;Abstr Series 14:70. [10] Lafrance M, Goff AK. Effects of progesterone, and estradiol-17 on oxytocin-induced release of prostaglandin F-2␣ in heifers. J Reprod Fert 1988;82:429 –36. [11] Vallet JL, Bazer FW. Effect of ovine trophoblast protein–1, oestrogen, and progesterone on oxytocininduced phosphatidylinositol turnover in endometrium of sheep. J Reprod Fert 1989;87:755– 61.
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