Oxytocin Receptor Development in Ovine Uterus and Cervix Throughout Pregnancy and at Parturition As Determined by in Situ Hybridization Analysis D

Oxytocin receptor development in ovine uterus and cervix throughout pregnancy and at parturition as determined by in situ hybridization analysis D. C. Wathes, H. Flick Smith, S. T. Leung,, K. R. Stevenson, S. Meier and G. Jenkin Babraham Institute, Babraham, Cambridge CB2 4AT, UK; ^Department of Farm Animal and Equine Medicine and Surgery, Royal Veterinary College, Hawkshead Road, Potters Bar, Herts EN6 1NB, UK; and Department of Physiology, Monash University, Clayton, Victoria 3168, Australia

The development of uterine oxytocin receptors is an important regulatory step in the initiation of labour. Paracrine production of oxytocin by uterine and placental tissues may also be involved in some species. Placentome, intercotyledonary endometrium, myometrium and fetal membranes were collected from 3\p=n-\5ewes each, at regular intervals throughout pregnancy and from eight ewes during labour. Localization of mRNA encoding oxytocin and its receptor was by in situ hybridization; oxytocin peptide concentrations were measured by radioimmunoassay and oxytocin receptor concentrations were measured by autoradiography and radioreceptor assay. In the intercotyledonary endometrium, mRNA encoding the oxytocin receptor was located in the luminal epithelium only. Both the epithelial and myometrial receptors were detected at low concentrations from the fourth week of gestation onwards, with a major increase associated with the onset of labour. In the placentomes, oxytocin receptors were localized to a stromal capsule surrounding the placental villi. Expression in this region was maximal in mid-gestation, declining in the second half of pregnancy and remaining low during labour. Cervical oxytocin receptors were detected at low concentrations in the epithelium and the muscular/connective tissue layers from day 22 of pregnancy onwards. There was no evidence for the local uterine production of oxytocin in the ewe; mRNA encoding oxytocin was undetectable and oxytocin concentrations were always < 23 pg g \m=-\1wet mass of tissue. These results suggest that regulation of the timing of oxytocin receptor development varies between the different tissue types, despite a similar steroidal background. The receptors in the luminal epithelium are probably associated with the ability of exogenous oxytocin to induce the release of PGF2\g=a\throughout most of pregnancy. The increase in receptors in both the intercotyledonary endometrium and myometrium at term suggest an involvement in labour, whereas their role in caruncular stroma in mid-pregnancy is unknown.

Introduction whereas vaginocervical stimulation caused by passage of the lamb at birth leads to a more pronounced rise (Mitchell el al, an role Oxytocin plays important in parturition, acting as a 1982; Kendrick et al, 1991). It is, however, uncertain whether potent myometrial stimulant at picomolar concentrations increased oxytocin release from the neurohypophysis triggers (Fuchs, 1985). In addition, there is evidence from a variety of the onset of labour. In this respect, the marked increase in the species, including humans (Fuchs et al, 1982) and sheep concentration of oxytocin receptors in both endometrium and et that it can increase the and (Mitchell al, 1975), synthesis myometrium at this time may be more important (Fuchs, 1985). release of via an action on in the prostaglandin F2a receptors The first evidence for an extra-hypothalamic source of endometrium. and immunoreactiv¬ Oxytocin gene expression oxytocin synthesis came from studies on the ovine corpus ity in the paraventricular and supraoptic nuclei of the ewe rise luteum (Ott and Scott, 1910; Wathes and Swann, 1982). In late with further in gestation a increase at term (Broad et al, ruminants, luteal oxytocin synthesis is initiated at the time of 1993). There is a modest increase in the plasma concentration ovulation, and concentrations of mRNA and peptide peak in of associated with the onset of uterine oxytocin contractions, the early and mid-luteal phases, respectively (for review, see *Address for correspondence. Wathes and Denning-Kendall, 1992), but decline and remain Received 5 July 1995. low during gestation (Sheldrick and Flint, 1983). More recent Downloaded from Bioscientifica.com at 09/23/2021 08:38:09PM via free access 24 ti — —H·—'-~"-.imi .-*.»m" * ii n »in -

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Fig. 1. Representative trace of uterine electromyographic activity recorded from a ewe on day 148—149 of gestation before and during the onset of labour. Tissue was collected after the development of high frequency, high amplitude contractions. studies in other species have shown that oxytocin can also be oestrus (day 0). The reproductive tracts were removed after synthesized by both the endometrium and placenta of rats and administration of an overdose of pentobarbitone sodium humans, with concentrations increasing at term (Lefebvre et al, (Lethobarb: Duphar Veterinary Ltd, Southampton) at the fol¬ 1992; Chibbar et al, 1993). This suggests that there is a lowing time points during pregnancy: days 13—14 (n = 6), day paracrine loop between peptide and receptor within the par¬ 22 (»1 = 5), days 30-35 (rt = 4), days 45-55 (n = 4), day 70 turient uterus and may obviate the need for oxytocin of (n = 3), day 105 (n = 3), days 138-140 (n = 4), during labour on neurohypophyseal origin to act as an initiating stimulus for day 148.8 ± 0.5 (n = 8), 1-2 h post partum (n = 4) and day 2 parturition. post partum (n = 1). In the uteri and oviducts of nonpregnant ewes, oxytocin receptor concentrations increase at luteolysis to reach peak concentrations at oestrus, before declining again in the early Electromyographic recordings luteal (Roberts et al, 1976; Sheldrick and Flint, 1985; phase The time of the onset of labour was established by fitting el al, At oestrus, are in Ayad 1990). oxytocin receptors present ewes with uterine EMG electrodes between the luminal caruncular eight recording epithelium, glands, stroma and day 117 and day 121 of gestation. Electrodes made of myometrium (Ayad et al, 1991; Wathes and Hamon, 1993; multistranded stainless steel Teflon-coated wire (1.2 m long; Stevenson et al, 1994). Administration of exogenous oxytocin Cooner Wire Co., Chatsworth, CA), covered with a vinyl can stimulate release of the metabolite PGF2ct 13,14,dihydro- catheter (Durai Plastics and Engineering, Durai, NSW) were 15-keto (PGFM) from the endometrium into prostaglandin F2a sutured to the body of the uterus in a triangular pattern 5 mm the circulation of ewes at luteolysis and peripheral nonpregnant apart. Signals derived from these electrodes were amplified on from about 17 of onwards (Meier et al, 1995). day gestation a Grass 7P3 amplifier (Grass Instruments, Quincey, MA) with Oxytocin has also been in the control of myometrial implicated high pass filtering at 0.3 Hz. These signals were recorded on a contractions at oestrus (Gilbert et al, 1992) and during preg¬ two or four channel Grass Polygraph (Model 7D). Electromyo¬ (Nathanielsz, 1973; et al, 1984). for nancy Lye Receptors graphic activity was monitored throughout the later stages of are also in the cervix of ewes oxytocin present nonpregnant and the animals were characterized as in and and pregnancy being (Matthews Ayad, 1994), changes in the electromyo¬ labour when the contractile activity recorded showed continu¬ (EMG) of the cervix have been recorded at graphic activity ous contractions with a low of It high frequency, high amplitude different stages gestation (Garcia-Villar et al, 1984). is duration (Harding et al, 1982; Jenkin and Nathanielsz, 1994). therefore that oxytocin could modulate the of possible activity Tissue collection was performed when this type of EMG the tract reproductive during pregnancy. activity was observed with visual observations of The the together purpose of present study was to determine the cervical opening (Fig. 1). localization and concentration of oxytocin receptors in the ovine uterus, placenta and cervix throughout gestation, and to investigate whether the uterus or placenta are sources of local Tissue collection oxytocin synthesis in this species. The reproductive tract was removed immediately after slaughter. In early pregnancy (days 13-30), whole transverse Materials and Methods sections of uterine horn and cervix approximately 1.5 cm long were cut, wrapped in aluminium foil, frozen in isopentane cooled in liquid N2 and stored at 80°C. At later stages, Animals - whole placentomes, half placentomes, sections of uterine wall Ewes of various breeds with an average duration of ges¬ (comprising both intercotyledonary endometrium and myo¬ tation of 147 ± 2.5 days (mean ± sd) were mated at natural metrium) and cervix were frozen separately. Additional pieces Downloaded from Bioscientifica.com at 09/23/2021 08:38:09PM via free access of placentome, intercotyledonary endometrium, myometrium Membranes (50 pg protein per tube) were incubated with and allantochorion were also collected for determination of [3,5-3H-Tyr]oxytocin (5 nmol I"1, 1224 GBq mmol; NEN oxytocin binding capacity and peptide extraction. Research Products) in 0.1ml Tris-HCl buffer (25 mmol 1_1 pH 7.6, containing 0.1% (w/v) BSA). Nonspecific binding was

determined in the of 10 1 . presence pmol oxytocin ~ Initial Reagents experiments were performed to determine the optimum incu¬ bation time and cation concentration. All data here Chemicals were purchased from Chemical Co. (Poole, reported Sigma were obtained after incubation for 30 min at 25°C in the Dorset) or Merck (Poole, Dorset) unless otherwise stated. presence of 1 mmol MnCl2 I-1. After incubation, tubes were placed in an ice bath and 2 ml chilled Tris—HC1 buffer were added In situ hybridization to stop the reaction. Separation of free from receptor- bound oxytocin was by filtration under vacuum through glass Localization of mRNAs encoding oxytocin receptor (OTR microfibre filters (GF/F; Whatman International Ltd, Maidstone, mRNA) and oxytocin peptide (OT mRNA) used 35S-Iabelled Kent) in a sampling manifold (Millipore UK Ltd, Watford). 45 base ovine-specific oligodeoxynucleotide probes as Inter- and intra-assay coefficients of variation were 19.0 and described by Broad et al (1993) and Stevenson et al (1994). 14.1%, respectively. The limit of detection, calculated as 2 sd ' The oxytocin receptor probe was complementary to bases above NSB, was 6.1 fmol [3H]oxytocin bound mg~ protein. 877-921 of the receptor cDNA (Stewart et al, 1993) and the oxytocin probe was complementary to bases 771—816 of the oxytocin—neurophysin 1 cDNA (Ivell et al, 1990; Jones and Oxytocin extraction Flint, 1990). In brief, 18 µ cryostat sections were thaw- mounted onto poly-L-lysine coated slides, fixed in paraformal- Tissue samples were extracted as described by Stevenson dehyde and dehydrated in ethanol. The probes were end- et al (1991). Approximately 1 g of tissue was partially thawed, labelled at the 3' end with 35S-labelled dATP (NEN Research weighed, chopped and homogenized in 10 ml ice cold Niall's Products, Stevenage, Herts). After overnight hybridization, the solution (15% (v/v) trifluoroacetic acid (TFA), 5% (v/v) formic slides were subjected to low (30 min at room temperature) and acid, 1% (w/v) sodium chloride in 1 mol HC1 1_I). The high (1 h at 55°C) stringency washes in 1 SCC, 0.2% (w/v) homogenate was centrifuged at 20 000 g tor 20 min at 4°C. sodium thiosulfate pentahydrate solution, pH 7.0 (where The resulting supernatant was filtered through Whatman No. 1 1 SCC is 15 mmol sodium chloride 1~ , 15 mmol sodium filter paper and a Millipore 0.22 pm filter, then applied to a citrate 1 ~:), dehydrated and exposed to Hyperfilm ß-max Sep-Pak C18 column (Waters Chromatography Division, (Amersham International pic, Amersham, Bucks) for 14 days. Milford, MA) equilibrated with 3 ml acetonitrile (ACN) in 0.1% The intensity of labelling on each section was measured from (v/v) TFA and then 10 ml distilled water. After addition of the the autoradiographs as absorbance measurements using image sample, the column was washed with 10 ml 10% (v/v) ACN in analysis (Seescan pic, Cambridge, Cambs) as described by 0.1% (v/v) TFA and the oxytocin eluted with 3 ml 80% (v/v) Stevenson et al. (1994). ACN in 0.1% (v/v) TFA. The sample volume was reduced by rotary film evaporation at 35°C for 4 h and the remaining extract was freeze dried. Autoradiography Oxytocin receptor binding sites were visualized by auto¬ radiography using the iodinated oxytocin receptor antagonist Oxytocin radioimmunoassay d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]-vasotocin (I25I-labelled OTA), Freeze dried extracts were reconstituted in 0.5 ml phosphate kindly supplied by M. Manning (Medical of Ohio, ~ College buffer (10 mmol 1 ), 7.2, mixed allowed to Toledo, OH) as described by Wathes and Hamon (1993). pH thoroughly, stand for 30 min at room temperature, mixed and Sections for total binding (TB) were incubated with 300 µ centrifuged at 2000 g for 10 min. The was assayed as described 5I-labelled OTA alone (0.1—0.4 nmol 1~ ), whereas for non¬ supernatant ~: by Stevenson et al (1991). The detection limit of the was specific binding (NSB), 10 pmol unlabelled oxytocin I assay 0.25 pg per tube and the intra-assay coefficient of variation was (Bachern UK Ltd, Saffron Waiden, Essex) was also included. 10.9%. A corpus luteum from a nonpregnant ewe was extracted and assayed at the same time as the uterine samples, as a control. Oxytocin binding measurements positive Microsomal membrane preparations for oxytocin receptor binding studies were prepared using the method of Sheldrick Statistical analyses and Flint (1985). Protein concentrations were determined by the method of Lowry et al (1951), with volumes reduced Data were analysed by analysis of variance (ANOVA) with for use in 96-well microtitre plates (No 611 F96; Sterilin, time of sample collection taken as the treatment. Log transfor¬ Hounslow), which were read at 720 nm (Argos 400 Microplate mations were performed where necessary to ensure homogen¬ reader, Canberra Packard, Pangbourne). Concentrations were eity of variance. When anova revealed a significant treatment calculated with reference to a BSA standard line (0.2-1.0 mg effect (P < 0.05), the individual time points were subsequently ml- ) assayed with the samples. compared by Student's t tests. Downloaded from Bioscientifica.com at 09/23/2021 08:38:09PM via free access Fig. 2. ¡ situ hybridization showing expression of mRNA encoding the oxytocin receptor (OTR mRNA) in ovine uterus during pregnancy. Serial sections were probed with antisense or sense (control) oligodeoxynucleotide probes. The majority of sense sections were blank and are not illustrated. The scale bar represents 2 mm in each case, (a) A complete cross-section through the end of a uterine horn at day 45, showing OTR mRNA in the luminal epithelium (LE), (b) A section through a placentome at day 46, showing intense OTR mRNA expression in the plate of caruncular stroma (CS) below the villous region (V). (c) Cross-section of a placentome collected during labour, showing OTR mRNA expression in the surrounding luminal epithelium; expression in the caruncular stroma capsule is, however, low at this stage, (d) Myometrium (M) collected during labour with intense OTR mRNA expression, (e) Section of uterine wall obtained 1 h post partum, with OTR mRNA still present in both the luminal epithelium and myometrium; although the intercotyledonary endometrium (ICE) in between is not labelled, (f) Adjacent section to that shown in (e), showing the lack of specific labelling when treated with the sense probe.

Results In the intercotyledonary region, it was localized to the luminal epithelium and myometrium (Fig. 2). Concentrations in both Oxytocin receptor (OTR) in situ hybridization regions remained low throughout most of pregnancy from days 22—140, although there was a small but significant No OTR mRNA could be detected in the uterus on days increase at day 70 (Fig. 3). Tissue collected from animals during 13—14 of pregnancy (absorbance < 0.01), but it was apparent labour showed the same localization but there was a significant on day 22 and then remained throughout the rest of gestation. increase in intensity of expression. Concentrations had declined Downloaded from Bioscientifica.com at 09/23/2021 08:38:09PM via free access from this peak by 1-2 h post partum but were still high at this stage in comparison to the rest of pregnancy. They had decreased further by day 2 post partum in the one animal examined at this time point. In the placentomes, OTR mRNA developed in the caruncu¬ lar stroma at the base, forming a plate below the region penetrated by the villi (Fig. 2). There was intense expression in this region between day 35 and day 70 (absorbance 0.29 ± 0.071, mean ± SEM; = 6). As pregnancy progressed, the width of this layer decreased and it formed a capsule-like structure surrounding the placentome. Expression of OTR mRNA in this region was significantly lower during labour than earlier in gestation (absorbance 0.04 ± 0.015; = 6; < 0.01). The precise timing of the decline in intensity of expression could not, however, be determined, as most tissue samples collected during the second half of pregnancy had been cut in an inappropriate orientation. In the luminal epi¬ . thelium, which extended from the intercotyledonary region o up the sides of the placentome OTR mRNA was also present (Fig. 2). In the cervix, OTR mRNA was not detectable in early ce pregnancy, but appeared in the surface epithelium and muscular o and connective tissue layers by day 22 and then remained Q. CD detectable at low concentrations throughout gestation, with (b) absorbance values of 0.03 ± 0.006 and 0.02 ± 0.005 in epithe¬ 0.20-1 lium and muscular/connective tissue, = In o respectively (n 18). % seven of eight animals from which tissue was collected during labour, no cervical oxytocin receptor mRNA could be detected; although in the remaining one animal, judged to be in 0.15- mid-labour from the EMG recordings, concentrations were raised (Abs 0.11 in epithelium and 0.06 in muscular/connective tissue).

0.10- U5l-labelled OTA binding The autoradiographical studies showed the same pattern of localization as the OTR mRNA, with receptors present in the 0.05 luminal epithelium, myometrium and caruncular capsules of the placentomes from the third week of gestation onwards (Fig. 4). In these regions, binding was displaced by the addition of cold oxytocin. In addition, there was a high degree of nonspecific binding of 125I-labelled OTA to the villous region of the placentomes which could not be displaced by cold oxytocin (Fig. 4).

Day of pregnancy Oxytocin radioreceptor assay for in the Fig. 3. Expression of mRNA encoding the oxytocin receptor (OTR Binding site concentrations [3H]oxytocin intercoty¬ mRNA) in the ovine uterus during pregnancy measured as absorbance ledonary endometrium and myometrium followed a similar units on an arbitrary scale from autoradiographs in (a) luminal trend to that observed in the in situ hybridization analysis (Fig. epithelium and (b) myometrium. Values are means ± sem of 3—5 5). In both these regions, binding site concentrations remained animals at each time point, except during labour (n = 7) and at 2 days relatively low throughout pregnancy with a small increase at = Concentrations on 14 were at or below post partum (pp; 1). day 70 days and a second larger rise during labour. Binding sites the detection limit (indicated arrow on the axis). There was a by (which were displaced by cold oxytocin) were also detected in effect of time on OTR mRNA expression (anova; significant the placentomes. Their concentration followed a different P< 0.001). Significance increases are shown in relation to the concen¬ trend, at day 70 (P<0.01), but with no rise during tration measured at day 22 as follows: *P<0.05, **P<0.01, peaking labour These results were similar to those ***P< 0.001. There was a decrease between labour and (Fig. 5). again significant which showed 1-2 h post partum (P<0.01). The day 2 post partum values had obtained by in situ hybridization analysis, high concentrations OTR mRNA in stroma apparently declined further but were not included in the analysis as of the caruncular capsule only a single sample was examined. in the first half of pregnancy, but low values at parturition. Downloaded from Bioscientifica.com at 09/23/2021 08:38:09PM via free access Fig. 4. Oxytocin receptor localization in the ovine uterus during pregnancy, demonstrated by binding of I25I-labelled OTA. Serial sections were treated in the absence (total binding, TB) and presence (non-specific binding, NSB) of an excess of unlabelled oxytocin. The scale bars represent 2 mm. (a) TB and (b) NSB cross-sections of placentome at day 70 of gestation, illustrating localization of oxytocin receptor binding sites in the caruncular stroma capsule (CS). The NSB section was treated with 10 µg oxytocin ml- '. (c) TB and (d) NSB of endometrium collected 1 h post partum showing that the binding in the luminal epithelium (LE) is displaced by cold oxytocin, whereas that in the placental villi (V) is not, the concentration of cold was increased to 100 ml '. This that the although oxytocin µg ~ finding suggests only labelling in the LE represents oxytocin binding sites, and correlates with the lack of OTR mRNA in the villous region detected by in situ hybridization (Fig. 2b, c). The intercotyledonary endometrium (ICE) is not labelled.

Oxytocin mRNA in situ hybridization the early luteal phase (Roberts et al, 1976; Sheldrick and Flint, 1985). In nonpregnant animals, oxytocin receptors first re¬ There was no evidence for oxytocin gene in expression appear in the luminal on day 14 of the cycle (Ayad either the uterus or or epithelium (endometrium, placentome myometrium) et al, 1991; Wathes and Hamon, 1993; Stevenson et al, 1994). allantochorion at any stage of pregnancy (data not shown). Their presence at this time is essential to generate the pulses of PGF2a release from the endometrium that initiate luteolysis Oxytocin measurements in tissue extracts (Flint et al, 1992; Wathes et al, 1993; Wathes and Lamming, 1995). Luteal regression is prevented in pregnant animals by were on extracts of Oxytocin assays performed placentome, the of the secretory IFN- endometrium, and allantocho¬ production conceptus protein intercotyledonary myometrium between day 10 and 22, which acts on the rion at 105 and 140 of labour and day directly days pregnancy, during endometrium to inhibit oxytocin receptor formation (Roberts 1-2 h Concentrations were at or below the post partum. et al, 1992; Stewart et al, 1992). The data presented here sensitivity of the assay in all samples, with no value in excess that ~ suggest IFN- delays oxytocin receptor development in of 23 pg g wet mass of tissue. In contrast, the oxytocin the luminal epithelium by about one week, with synthesis concentration of the corpus luteum from a ewe nonpregnant starting as soon as IFN- concentrations decline. used as a control was 600 positive ng g ~ were in the uterus . Oxytocin receptors subsequently present throughout pregnancy, although there were major differences Discussion in the timing of OTR mRNA expression between the luminal epithelium and myometrium and the caruncular stroma capsule. Oxytocin receptor concentrations in the uteri of cyclic ewes are In both the luminal epithelium and myometrium, receptor maximal at oestrus, declining to undetectable amounts during concentrations remained relatively stable during most of Downloaded from Bioscientifica.com at 09/23/2021 08:38:09PM via free access (a) pregnancy, with a small rise at 10 weeks and a five- to ten-fold 2000- increase at term. In pregnant cows, oxytocin receptor concen¬ trations in both the intercotyledonary endometrium and myo¬ metrium also rise from about day 20 onwards, although the pattern differs in that there is a steady increase throughout 1500- pregnancy in both tissues, with no further rise at the onset of labour (Fuchs et al, 1992a). In contrast, the concentration of oxytocin receptors in ovine placentomes was highest in the first half of gestation and did not increase during labour. No mRNA encoding the oxytocin receptor could be detected at the feto—maternal interface, although it was present in both the caruncular stroma capsule and the surrounding epithelium. These results again differ from those reported for the cow, in which there is a significant rise in the concentration of oxytocin binding sites in both fetal and maternal components of the placentomes at parturition (Fuchs et al, 1992b). Numerous studies in ewes have indicated that oxytocin receptor concentrations are regulated by both oestradiol and progesterone (for review see Wathes and Lamming, 1995). Our previous data from nonpregnant ewes suggest, however, that epithelial and myometrial oxytocin receptors are controlled in a different way. The initial appearance of oxytocin receptors in 1500 the luminal epithelium on day 14 of the oestrous cycle was related to the length of exposure to progesterone and was not dependent on changes in circulating concentrations of either oestradiol or progesterone, or to changes in uterine progester¬ one receptor concentration or localization (Wathes and Hamon, 1993; Wathes and Lamming, 1995; Lamming et al, 1995). In contrast, the subsequent development of oxytocin receptors in the myometrium between day 15 and oestrus occurred as progesterone concentrations fell, circulating oestradiol concen¬ trations rose and oestradiol receptors appeared in the myo¬ metrium, suggesting dependence on oestradiol (Wathes and Hamon, 1993). The maternal oestradiol and progesterone O, at are similar to those observed I profiles parturition during luteolysis (Heap el al, 1977), but it is not yet known whether the increase in epithelial oxytocin receptors at term also precedes that of the myometrial receptors. Although there is clear evidence for upregulation of oxy¬ tocin receptors by oestradiol in many species, recent work has failed to locate classic palindromic oestrogen response elements on the promoter region of the oxytocin receptor gene, although are (Inoue et al, 1994; Rozen 1000- half-palindromes present et al, 1995). It therefore remains to be determined whether the action of oestradiol on gene transcription is direct. Consensus sequences that may be influenced by cytokines have been identified in the oxytocin receptor gene (Inoue et al, 1994; Rozen et al, 1995), but their possible role in the control of oxytocin receptor expression also needs to be established.

500- Fig. 5. Oxytocin receptor concentrations in different regions of the ovine uterus during pregnancy, measured by radioreceptor assay using ['HJoxytocin in (a) intercotyledonary endometrium, (b) myo¬ metrium and (c) placentome; pp: post partum. Values are means ± sem of 2—5 animals at each time point. There was a significant effect of h time on oxytocin receptor concentration in each region (anova; intercotyledon < 0.001, myometrium and placentome < 0.01). Bars with different letters were significantly different as follows: b > a, < 0.001; Ob, < 0.001; e > d, < 0.05; f > d, < 0.001; g > d, e and f, < 0.001; j > h, < 0.001.

Downloaded from Bioscientifica.com at 09/23/2021 08:38:09PM via free access Day of pregnancy Further work is required to determine the basis for the neurohypophysis is maintained during pregnancy in cows variations in OTR mRNA expression between different sites in (Fuchs, 1985). The further increase in oxytocin receptor con¬ the uterus. It is possible that the local concentrations of steroid centration at term would significantly enhance the myometrial hormones within the placentomes may vary owing to such sensitivity to oxytocin at this time. factors as the local concentrations of steroid receptors, the rate Oxytocin can also increase the EMG activity of the cervix and extent of steroid metabolism or the lipid solubilities of the (Garcia-Villar et al, 1982) and influence cervical connective hormones within the different areas (Challis et al, 1975). tissue metabolism (Wiqvist et al, 1984). Low concentrations of With the exception of 125I-labelled OTA binding to the villi, OTR mRNA were present on both the epithelium and muscu¬ measurements in endometrium and myometrium of OTR lature of the cervix during pregnancy and could therefore mRNA from the in situ hybridization experiments were in mediate the changes in cervical EMG activity noted during agreement with those of receptor protein estimated by both gestation (Garcia-Villar et al, 1984). Oxytocin receptors have 125I-labelled OTA autoradiography and radioreceptor assay previously been localized to the cervical epithelium of non¬ using [3H]oxytocin. This indicates that control, including the pregnant ewes by autoradiography with 1Z5I-labeIIed OTA upregulation at parturition, is chiefly at the level of transcrip¬ (Matthews and Ayad, 1994). We have confirmed these findings tion rather than translation. A similar conclusion was reached in using in situ hybridization (S. T. Leung and D. G Wathes, studies on the human uterus (Takemura et al, 1994). The reason unpublished observations) but at no stage of pregnancy, for the high degree of nonspecific binding of 125I-labelled OTA including labour, did cervical OTR mRNA expression approach binding within the placentomes in our study could not be the very high concentrations found at oestrus. determined. It is possible that another receptor subtype or an We were unable to detect mRNA encoding oxytocin oxytocin-binding protein is present. Alternatively, the iodine peptide in the ovine uterus at any stage of pregnancy using itself may become associated with binding proteins involved in in situ hybridization. The low concentrations measured by iodine transfer across the placenta (London et al, 1964; Boyd radioimmunoassay of tissue extracts are therefore likely to be et al, 1981). attributable to oxytocin present in the plasma. These results do Localization of mRNA encoding the oxytocin receptor in not support the existence of a paracrine system within the the endometrium was in both the luminal epithelium and ovine uterus, as has been demonstrated in humans and rats caruncular stroma. The luminal epithelium is the cell type (Lefebvre et al, 1992, 1993; Chibbar et al, 1993). responsible for PGF2a synthesis during luteolysis in nonpreg¬ nant ewes (Wathes and Lamming, 1995). Injection of a large The authors thank E. L. Sheldrick for assistance with the oxytocin dose (10 iu) of oxytocin at different stages throughout preg¬ receptor binding assay; R. Young, G. E. Rice and W. McClaren for nancy produces a small increase in peripheral PGFM concen¬ help with collection of parturient tissue; M. Manning for provision of trations in early pregnancy, but the size of the response the OTA; and D. Gunn for photography. increases significantly from mid-gestation onwards (Meier et al, 1995). This is related partly to an increase in oxytocin receptor concentrations at this time. The capacity of the uterus to References synthesize PGF2a, prostaglandin synthase (PGHS) activity and the tissue content is also enhanced as advances PGF2(1 gestation Ayad VJ, McGoff SA and Wathes DC (1990) Oxytocin receptors in the oviduct et et (Rice al, 1990; Boshier al, 1991; Evans et al, 1982), during the oestrous cycle of the ewe Journal of Endocrinology 124 353—359 possibly owing to an increase in the availability of free Ayad VJ, Matthews EL, Wathes DC, Parkinson TJ and Wild ML (1991) Auto¬ arachidonic acid (Meier et al, 1994). The increase in the radiographical localization of oxytocin receptors in the endometrium during the oestrous of the ewe Journal 130 199—206 oxytocin concentration in cycle of Endocrinology receptor intercotyledonary Boshier DP, Jacobs RA, Han VKM, Smith W, Riley SC and Challis JRG (1991) endometrium in both and sheep (this study) cows (Fuchs et al, Immunohistochemistry localization of prostaglandin H synthase in the sheep 1992a) is therefore likely to contribute to the stimulation of placenta from early pregnancy to term Biology of Reproduction 45 322-327 PGF2u release observed at term. The role of the oxytocin Boyd RDH, Channing JF, Stacey TE and Ward RHT (1981) Iodide transfer across the near term receptors in the caruncular stroma remains to be sheep placenta Journal of Physiology 312 23—24P capsule Broad KD, Kendrick KM, Sirinathsinghji DJS and Keverne EB (1993) Changes in as there was no increase labour. determined, during oxytocin immunoreactivity and mRNA expression in the sheep brain during Oxytocin receptors were also present in the myometrium pregnancy, parturition and lactation and in response to oestrogen and during pregnancy. Measurements of uterine EMG activity progesterone Journal of Neuroendocrinology 5 435—444 Challis Louis Robinson and Thorburn GD and revealed three phases of activity: an initial quiescent period JRG, TM, JS (1975) Progesterone oestradiol in pituitary, brain and uterine tissues of the Journal of from the start of the luteal until about 40, a more sheep phase day Endocrinology 69 451-452 active period of uterine activity at about 50 days of gestation Chibbar R, Miller FD and Mitchell BF (1993) Synthesis of oxytocin in amnion, and a third phase from week 10 of gestation until term, chorion and decidua may influence the timing of human parturition Journal of characterized by a lower and steady frequency of uterine Clinical Investigation 91 185-192. activity (Garcia-Villar et al, 1984). 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