Fenton, Stoke-on-Trent), streptomycin sulphate (50 µg mlâ1), ..... Zhang J, Weston PG and Hixon JE (1992) Role of progesterone and oestradiol in the regulation ...
Journal of Reproduction and Fertility (2000) 119, 287–292
Oestradiol regulation of oxytocin receptor expression in cyclic bovine endometrium S. T. Leung and D. C. Wathes* Department of Veterinary Basic Sciences, The Royal Veterinary College, Boltons Park, Hawkshead Road, Potters Bar, Hertfordshire EN6 1NB, UK
Oestradiol treatment can increase uterine oxytocin receptor expression in vivo. The actions of oestrogen are usually mediated via its receptor, but it also has direct nongenomic effects in some cells. This study investigated the effect of oestradiol and the role of the oestradiol receptor in regulating endometrial oxytocin receptor expression in the bovine uterus. Explant cultures (in triplicate) from late luteal phase non-pregnant endometrium received the following treatments: control (serum-free medium), oestradiol (0.1 and 0.01 µmol l–1), oestradiol (0.1 µmol l–1) with the oestradiol receptor antagonist ICI 182780 (0.5 µmol l–1), and ICI 182780 (0.5 µmol l–1) alone. Explants were collected 12, 24 and 48 h after the start of culture. Oxytocin receptor mRNA expression in the explants was measured by in situ hybridization and oxytocin protein concentrations were measured by autoradiography with the iodinated oxytocin receptor antagonist d(CH2)5 [Tyr (Me)2 Thr4 Tyr NH29]-vasotocin (125I-labelled oxytocin receptor antagonist). Oxytocin receptor mRNA and protein expression were initially low but spontaneous upregulation occurred in the luminal epithelium between 24 and 48 h (P < 0.01). Oestradiol increased oxytocin receptor mRNA upregulation in the first 24 h (P < 0.05) but the effect on 125I-labelled oxytocin receptor antagonist binding was not significant. ICI 182780 inhibited the oestrogenic effect but had no significant effect on oxytocin receptor mRNA expression when given alone. In conclusion, the results showed that oestradiol exerts its effect via the oestradiol receptor. Oestradiol facilitates oxytocin receptor gene transcription by increasing it more rapidly, but spontaneous upregulation of endometrial oxytocin receptor still occurs in the absence of oestradiol.
Introduction In ruminants, oxytocin stimulates the pulsatile release of PGF2α via the endometrial oxytocin receptor, resulting in regression of the corpus luteum. Therefore, the regulation of oxytocin receptor expression plays a crucial role in determining the timing of the onset of luteolysis (Flint et al., 1994; Wathes and Lamming, 1995; McCracken et al., 1999). The concentration of oxytocin receptor, both mRNA and protein, is low during the luteal phase and is significantly upregulated during the follicular phase, peaking at oestrus, in both bovine and ovine uterine tissues (Fuchs et al., 1990; Jenner et al., 1991; Stevenson et al., 1994). Upregulation of oxytocin receptor expression in the ovine uterus precedes that of the oestradiol receptor in the endometrial luminal epithelium (Wathes and Hamon, 1993) and in the placentome during mid-pregnancy (Leung et al., 1998), indicating that oestradiol receptor is not involved in initiating the upregulation of oxytocin receptor expression. However, oestrogen may also initiate non-genomic reactions *Correspondence. Received 10 December 1999.
in the absence of the oestradiol receptor (Ignar-Trowbridge et al., 1996). Therefore, regulation of the expression of endometrial oxytocin receptor may be sensitive to the variation of oestrogen directly, without the involvement of the oestradiol receptor. During early pregnancy in ruminants, upregulation of oxytocin receptors is prevented by the production of interferon τ (IFN-τ) by the embryonic trophoblast (Roberts et al., 1992). In ewes, there is evidence that the inhibitory effect of IFN-τ on oxytocin receptor expression may be mediated by an initial inhibition in the concentration of uterine oestradiol receptors (Lamming et al., 1995; Spencer et al., 1995). However, in the early pregnant cow, the embryo is able to suppress the development of oxytocin receptor on day 16 despite the presence of oestradiol receptors in the luminal epithelium at this time (Robinson et al., 1999). Therefore, there may be a species difference between cattle and sheep in the regulation of uterine oxytocin receptor expression. Several studies in ewes have shown that oestradiol administration in vivo can stimulate endometrial oxytocin receptor expression (Hixon and Flint, 1987; Beard and Lamming, 1994; Spencer et al., 1995). In contrast, studies in
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vitro have demonstrated that a spontaneous upregulation of oxytocin receptors is observed in cultured bovine and ovine endometrial epithelium in the absence of oestrogen once the tissues are removed from the uterus (Sheldrick et al., 1993; Horn et al., 1998; Leung and Wathes, 1999). This finding indicates that endometrial oxytocin receptor expression is normally regulated by an inhibitory mechanism. However, oestradiol can facilitate oxytocin receptor expression in vitro in late pregnant ovine endometrium, resulting in a more rapid increase in the oxytocin receptor concentration at both the transcriptional and post-transcriptional levels (Leung and Wathes, 1999). Taken together, these results indicate that oxytocin receptor expression is modulated by both positive and negative regulators and that oestradiol cannot be the primary regulator of oxytocin receptor gene expression. Therefore, the aims of this study were to examine the effect of oestradiol on oxytocin receptor expression in bovine endometrium and to determine the role of oestradiol receptor in mediating the oestradiol regulatory effect in this species using the oestradiol receptor-specific antagonist ICI 182780. The samples collected from the explant cultures were analysed for oxytocin receptor mRNA by in situ hybridization and for oxytocin binding using [I125]-oxytocin antagonist.
Materials and Methods Reagents Chemicals were purchased from Sigma Chemical Co. (Poole) or Merck/BDH (Poole) unless otherwise specified.
Tissue collection and culture Holstein–Friesian cows were killed by captive bolt on days 15–16 of the oestrous cycle, which is shortly before the normal upregulation of expression of oxytocin receptor in the endometrium of cyclic cows (Robinson et al., 1999). This status was confirmed by measurement of the initial endometrial oxytocin receptor mRNA concentration in each cow used in the study (see Results section). The intact uterus was collected within 15 min after the animals were killed, placed on ice for transfer to the laboratory and opened in a laminar flow hood within 2 h. Complete cross-sections of uterine horn (5 cm in length) were frozen in isopentane in liquid nitrogen and stored at –80⬚C to determine the oxytocin receptor mRNA expression at the time of tissue collection. Strips of intercotyledonary endometrium were dissected and transferred to defined basic medium. All the collected tissues were chopped into cubes of 1 mm3 using a mechanical tissue chopper (McIlwain Laboratory Engineering Ltd, Guildford). The endometrial pieces were blotted with a sterile lens cleaning tissue and weighed to provide 0.15 g per culture dish. The tissue pieces were placed on a metal grid (stainless steel, 30 mm ⫻ 30 mm ⫻ 0.5 mm) cushioned with a layer of lens cleaning tissue in dishes (50 mm ⫻ 15 mm, single dent; BDH) containing 6 ml treatment medium (see below), and cultured at 37⬚C and 5% CO2 for up to 48 h in a humidified incubator.
Medium preparation The basic medium used was Dulbecco’s minimum essential medium–nutrient F12 mix (1:1) with 15 mmol Hepes l–1 (GibcoBRL, GibcoBRL Life Technologies Ltd, Paisley) containing penicillin sodium (50 iu ml–1; NVS, Fenton, Stoke-on-Trent), streptomycin sulphate (50 µg ml–1), sodium hydrogen carbonate (1.125 g l–1) and BSA (1.125 g l–1). The medium was sterilized by filtration (0.22 mm filter; Millipore, Watford) and 1 ml insulin– transferrin–selenium mixture (ITS) was added to 1 l filtered medium to provide final concentrations of 5 µg l–1, 5 µg l–1 and 5 ng l–1, respectively. The medium was stored at 4⬚C until used. Stock solutions of oestradiol and an oestradiol receptor antagonist (ICI 182780, kindly provided by Dr A. E. Wakeling, Zeneca Pharmaceuticals, Macclesfield, Cheshire, UK) were prepared in absolute ethanol. The stock solutions were diluted to the working concentrations in basic medium through a serial dilution. All treatment media were prepared within 72 h before the experiment and stored at 4⬚C.
Experimental treatments All treatments were tested in triplicate on tissue collected from at least three separate cows. The treatments included the following: (i) control (basic medium); (ii) oestradiol (0.1 and 0.01 µmol l–1); (iii) ICI 182780 (0.5 µmol l–1); (iv) oestradiol (0.1 µmol l–1) + ICI 182780 (0.5 µmol l–1); and (v) ICI 182780 (0.5 µmol l–1) alone. The medium was changed at 24 h. Endometrial explants were collected at 0, 12, 24 and 48 h for analysis. Tissue pieces were mounted in cryo-M-bed (Bright, Huntingdon), frozen in dry ice and stored at –80⬚C. The viability of the tissue after 48 h in culture was checked by examining the histology of the explants by haematoxylin and eosin staining.
In situ hybridization Sections (10 µm thick) of endometrial explants frozen in blocks of Cryo-M-bed were cut and thaw mounted on slides coated with poly-L-lysine (0.1 mg ml–1). The sections were fixed in 4% (w/v) paraformaldehyde in PBS (0.13 mol NaCl l–1, 0.007 mol Na2HPO4 l–1) for 5 min, washed in PBS for 2 min (⫻3) and dehydrated in 75% followed by 95% absolute ethanol each for 5 min. The sections were stored in 95% absolute ethanol at 4⬚C until used. The in situ hybridization procedures were applied as described by Wathes et al. (1996) and Leung and Wathes (1999). Briefly, the oxytocin receptor DNA probe (antisense; 45mer synthetic oligonucleotide), end-labelled with dATP35 S (Amersham International plc, Amersham; 100 k c.p.m. per 100 µl hybridization buffer per slide) was added to the sections, covered with a parafilm coverslip, and incubated at 43⬚C overnight. After incubation, the sections were washed at room temperature for 30 min followed by 1 h at 55⬚C in 1 ⫻ sodium saline citrate (SSC; 15 mmol sodium chloride l–1, 15 mmol sodium citrate l–1, pH 7.0) containing 0.2% (w/v) sodium thiosulphate-5 hydrate. The slides were dehydrated
Oestradiol regulation of oxytocin receptor expression (a)
(b)
(c)
(d)
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Fig. 1. Localization of oxytocin receptor in explant cultures of bovine intercotyledonary endometrium obtained on day 15 of the oestrous cycle and cultured for 48 h in vitro. Autoradiographs showing the localization of oxytocin receptor binding sites on sections treated with the 125I-labelled oxytocin receptor antagonist (a) alone (total counts) or (b) with excess unlabelled oxytocin (non-specific binding control). Emulsion-coated slides treated with either antisense (c) or sense (d, control) oligonucleotide probes showing that oxytocin receptor mRNA expression was confined to the luminal epithelium (LE). Scale bars represent (a,b) 3 mm and (c,d) 10 µm.
in a gradient of ethanol, air-dried and exposed to hyperfilmβmax (Amersham International plc) for 2 weeks. A labelled oligonucleotide of the sense sequence was used as the negative control for each sample. Sections from a bovine uterus collected at oestrus were used as the positive control in each batch of sections processed.
Autoradiography The method was based on that described by Ayad et al. (1991). Sections (20 µm thick) were cut from frozen explant samples, pre-washed in ice-cold phosphate buffer (0.1 mol l–1, pH 7.4, 0.1% BSA) without magnesium chloride (MgCl2) followed by incubation with 300 µl iodinated oxytocin receptor antagonist d(CH2)5 [Tyr (Me)2 Thr4 Tyr NH29]vasotocin (125I-labelled oxytocin receptor antagonist; 1250 c.p.m. ml–1 in buffer containing MgCl2 (2.04 g l–1)). This gave the maximum binding (total counts). The non-specific binding solution containing excess unlabelled oxytocin (10 mg ml–1; Bachem UK Ltd, Saffron Walden) was added to the control slides. The slides were then incubated at room temperature for 1 h. After incubation, the slides were washed vigorously in fresh ice-cold phosphate buffer containing MgCl2 for 1 min (⫻3), ice-cold distilled water (1 min) and air-
dried at room temperature. The slides were exposed to hyperfilm-βmax (Amersham International plc) for 48 h.
Photographic emulsions The procedures were similar to the instructions provided by the manufacturing company (LM-1, Amersham International plc). Completely dried slides were dipped into the emulsion vertically for 5 s at 43⬚C and allowed to dry horizontally at room temperature and then on a metal plate pre-cooled with dry ice for 10 min each. The slides were placed into a light-tight box with anhydrous silica gel, sealed and incubated at 4⬚C for 3 weeks. After incubation, the slides were dipped into developer (Phenisol; Ilford Limited, Ilford) for 5 min, stop bath (0.5% (v/v) acetic acid) for 1 min, fixative (47% (w/v) sodium thiosulphate pentahydrate) for 10 min and distilled water for at least 10 min before counterstaining with Harris’ haematoxylin and eosin to identify the types of cell.
Data analysis Images of the films from both the in situ hybridization and the 125I-labelled oxytocin receptor antagonist autoradiography
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experiments were quantified by measuring the absorbance of specific areas (identified using haematoxylin and eosin staining) using an image analysis system (Seescan plc, Cambridge) as described by Leung and Wathes (1999). The results were expressed as arbitrary absorbance units on a linear scale from 0.01 to 2.1. Measurements were made of four slides per sample for each method used (two antisense or total count and two sense or non-specific binding). As expression of oxytocin receptor was confined to the luminal epithelium, measurements were only taken from this type of cell and three representative regions were chosen per section (Fig. 1). The sense and non-specific binding values were subtracted from the antisense and total counts to produce a mean value of specific hybridization or binding for each sample. The inter-batch coefficient of variation (calculated from the control sections from a cow in oestrus) was 12%. All the samples from the same tissue culture experiment were processed for in situ hybridization at the same time to remove any inter-batch variation in the analysis of treatment effects. Data from each treatment were analysed by a three-way ANOVA with the incubation time (hour), the treatment effect (compared with the control at each time point) and the animal from which the tissue was obtained as factors. When the result from the initial ANOVA showed a significant treatment effect (P < 0.05), the data at each specific time point were subsequently analysed by Newman–Keul’s test to confirm where the differences lay.
Results Expression of oxytocin receptor in the endometrium The concentrations of endometrial oxytocin receptor mRNA in intact uterus before chopping and at 0 h after tissue chopping were 0.02 ⫾ 0.002 and 0.04 ⫾ 0.018 absorbance units, respectively (n = 6). These low values confirmed that tissue was collected before initiation of the luteolytic upregulation in oxytocin receptor, which occurs in the late luteal phase. After culture, the expression of oxytocin receptor mRNA was confined to the luminal epithelium (Fig. 1). Expression was spontaneously and significantly upregulated in the endometrial luminal epithelium in the basic medium between 24 and 48 h after the start of culture (Table 1; P < 0.01). 125I-labelled oxytocin receptor antagonist binding also increased significantly between 24 and 48 h (Table 1; P < 0.01).
Effect of oestradiol on oxytocin receptor expression in the endometrial luminal epithelium Oestradiol upregulated oxytocin receptor mRNA expression in the endometrial luminal epithelium (Table 1). For the 0.1 µmol l–1 dose, the effect was significant at both at 12 and 24 h (P < 0.05), whereas for 0.01 µmol l–1 the difference was only significant at 24 h. Although there was a tendency towards a slightly higher oxytocin receptor mRNA concentration in oestradiol-treated medium at 48 h, this
value was no longer significantly different from the control. The 125I-labelled oxytocin receptor antagonist binding was higher in both the oestradiol-treated groups at 24 h compared with the controls, but this difference was too small to achieve statistical significance as the low values were close to the detection limit of the technique, so the variances were large in relation to the means. An attempt was also made to measure the protein concentrations at 12 h using a radioreceptor assay with [3H]oxytocin (Jenner et al., 1991), but the low receptor concentration made it impossible to obtain accurate measurements at this time (data not shown).
Effect of oestradiol receptor antagonist on oxytocin receptor expression in the endometrial luminal epithelium The oestradiol receptor antagonist ICI 182780 alone had no significant effect on oxytocin receptor expression in the endometrium during the incubation period, measured as either mRNA or 125I-labelled oxytocin receptor antagonist binding (Table 1). However, addition of ICI 182780 to the oestradiol treatment abolished the stimulatory effect of oestradiol on oxytocin receptor mRNA expression (Table 1). A similar, although non-significant, trend was seen with the 125 I-labelled oxytocin receptor antagonist binding.
Discussion The results of the present study in cows confirm observations in sheep that in ovine endometrium collected during the luteal phase (Sheldrick et al., 1993) or late gestation (Leung and Wathes, 1999) oxytocin receptor expression, both mRNA and protein, is spontaneously upregulated in vitro in the absence of exogenous hormonal treatments. A similar observation was noted in cultures of luminal epithelium derived from non-pregnant bovine endometrium (Horn et al., 1998). This upregulation still occurred in the presence of an oestradiol receptor inhibitor, further confirming that oestradiol is not required. These data indicate that the expression of endometrial oxytocin receptor is normally regulated by an inhibitory mechanism. Alternatively, oxytocin receptor expression may be stimulated by an intermediary transcription factor which may be expressed in response to either oestradiol receptor activation or to another factor that is induced by the culture conditions. However, oestradiol treatment in vivo induces an initial upregulation of endometrial oxytocin receptor expression in ewes (Vallet et al., 1990; Beard and Lamming, 1994; Spencer et al., 1995). If oestradiol treatment is continued over several days, it is unable to maintain the increased oxytocin receptor expression and oxytocin receptor concentrations decrease again (Zhang et al., 1992; Lamming and Mann, 1995; Wathes et al., 1996). Experiments in ewes have also shown that withdrawal of progesterone alone is sufficient to initiate endometrial oxytocin receptor expression but that oestradiol administration at the time of progesterone withdrawal can facilitate the upregulation of oxytocin receptor gene expression (Leavitt et al., 1985; Vallet et al., 1990; Zhang et al., 1992). Studies in vitro support this facilitatory effect, since
Oestradiol regulation of oxytocin receptor expression
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Table 1. The effect of oestradiol and the oestradiol receptor antagonist ICI 182780 on oxytocin receptor mRNA expression and 125 I-labelled oxytocin receptor antagonist binding in the luminal epithelium of bovine endometrial explants Time in culture Treatment
n
12 h
24 h
48 h
Oxytocin receptor mRNA Control Oestradiol (0.1 µmol l–1) Oestradiol (0.01 µmol l–1) ICI 182780 (0.5 µmol l–1) Oestradiol (0.1 µmol l–1) + ICI (0.5 µmol l–1)
7 7 3 6 6
0.05 ⫾ 0.018a 0.09 ⫾ 0.025b 0.05 ⫾ 0.020 0.05 ⫾ 0.018 0.06 ⫾ 0.057
0.04 ⫾ 0.013ac 0.11 ⫾ 0.038d 0.09 ⫾ 0.017d 0.05 ⫾ 0.024 0.04 ⫾ 0.023
0.14 ⫾ 0.044e 0.16 ⫾ 0.045 0.18 ⫾ 0.054 0.15 ⫾ 0.056 0.12 ⫾ 0.047
3 3 3 3 3
0.02 ⫾ 0.004a 0.01 ⫾ 0.002 0.02 ⫾ 0.003 0.02 ⫾ 0.001 0.02 ⫾ 0.002
0.02 ⫾ 0.003a 0.03 ⫾ 0.007 0.03 ⫾ 0.007 0.02 ⫾ 0.005 0.01 ⫾ 0.003
0.06 ⫾ 0.021e 0.05 ⫾ 0.011 0.07 ⫾ 0.007 0.04 ⫾ 0.003 0.03 ⫾ 0.006
125
I-labelled oxytocin receptor antagonist binding Control Oestradiol (0.1 µmol l–1) Oestradiol (0.01 µmol l–1) ICI 182780 (0.5 µmol l–1) Oestradiol (0.1 µmol l–1) + ICI (0.5 µmol l–1)
Each treatment was tested in triplicate on tissues from at least three separate cows. Endometrium was collected on days 15–16 in the late luteal phase. Results are expressed as absorbance units (mean ⫾ SEM) and are analysed in comparison with tissues from the corresponding controls from the same time point (b > a, P < 0.05; d > c, P < 0.05). Control samples were also compared between time points (e > a, P < 0.01).
oestradiol speeded up the spontaneous increase in oxytocin receptor mRNA expression in late pregnant ovine endometrial explant culture but did not increase the final concentration of oxytocin receptor mRNA (Leung and Wathes, 1999). In contrast, Horn et al. (1998) demonstrated that oestradiol had no direct effect on oxytocin receptor expression in bovine endometrial epithelial cells, although oestradiol receptor mRNA was detected in the cells using RT–PCR. Similarly, Sheldrick and Flick-Smith (1993) found that long-term (96 h) treatment of ovine endometrial explants with oestradiol in vitro in the range 1 pmol l–1 to 10 µmol l–1 had no effect, whereas a higher dose of 100 µmol oestradiol l–1 significantly reduced oxytocin receptor protein concentrations. In the present study, oestradiol facilitated endometrial oxytocin receptor expression initially (12–24 h), but had no effect once oxytocin receptor expression reached a plateau after 48 h. These results are similar to those in late pregnant ovine endometrial explant culture (Leung and Wathes, 1999). The discrepancy between the present results and those of Horn et al. (1998) and Sheldrick and Flick-Smith (1993) is probably due to the sampling time points, as in both of the last two studies measurements were made after at least 72 h when, on the basis of our previous work, oxytocin receptor expression would have reached the plateau. Another possibly important difference between these experiments is that local factors originating from other cells in the endometrium would be absent in the epithelial cell culture but may play a role in regulating oxytocin receptor expression. Taken together, the results of the work both in vivo and in vitro indicate that oestradiol can cause a shortterm upregulation in oxytocin receptor expression, but that the effect cannot be maintained for more than 1–2 days. The mechanism leading to the subsequent downregulation of the stimulatory effect has not been investigated, but is unlikely to be caused only by oestradiol receptor downregulation, as
oestradiol receptors were present in the studies reported by Horn et al. (1998). It is likely that the initial stimulation of oxytocin receptor expression by oestradiol is mediated via oestradiol receptors, which are present in the bovine luminal epithelium during the luteal phase of the oestrous cycle (Robinson et al., 1999). The role of oestradiol receptors in enhancing oxytocin receptor transcription is supported by the results obtained using the oestradiol receptor specific antagonist ICI 182780, which abolished the facilitatory effect of oestradiol on oxytocin receptor expression although ICI 182780 alone had no effect. However, the site of action of the oestradiol receptor is uncertain. The promoter region of the oxytocin receptor gene contains three half oestradiol response elements (Inoue et al., 1994), indicating that oestradiol may modulate the expression of oxytocin receptor directly. However, these oestradiol response element sites appear to be inactive as the oestradiol receptor does not bind to the oestradiol response element sites immediately upstream to the start site of the transcription of bovine oxytocin receptor (R. Ivell, personal communication) and oestradiol does not activate transcription from this promoter region in reporter gene assays (A. P. F. Flint, personal communication). These results do not contradict the present findings directly, since oestradiol may function through an indirect mechanism involving a second intermediate gene. However, the data are consistent with the hypothesis that local factors from the endometrium are required to regulate oxytocin receptor expression in the endometrial epithelium via interaction with the oestradiol receptor. In conclusion, spontaneous upregulation of oxytocin receptor expression in the bovine endometrial luminal epithelium occurred when the uterine tissues were removed from the animal. Oestradiol speeds up the spontaneous upregulation of oxytocin receptor expression via the oestradiol receptor but it is not essential for this process.
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The work was supported by the BBSRC. The authors thank Sarah Mann, Robert Robinson and George Mann for their assistance with tissue collection and Barbara Wilsmore for photography. The ICI 182780 was kindly supplied by A. E. Wakeling, Zeneca Pharmaceuticals.
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