Reprod. Fertil. Dev., 1992, 4, 193-203
Effect of Oxytocin on the Pattern of Electromyographic Activity in the Oviduct and Uterus of the Ewe around Oestrus
C. L. ~ i l b e r t ~ 'P. , J. crippsB and D. C . wathesAC Department of Anatomy, University of Bristol School of Medical Sciences, Bristol BS8 ITD, UK. Department of Veterinary Medicine, University of Bristol, Langford House, Langford, Bristol BS18 7DU, UK. Present address: AFRC Institute of Animal Physiology and Genetics Research, Babraham Hall, Babraham, Cambridge CB2 4AT, UK.
A
Abstract This study tested the hypothesis that the administration of oxytocin in doses equivalent to endogenous concentrations at and around oestrus could affect uterine and oviducal muscular activity at the time of gamete transport. Four ewes were fitted with recording electrodes in the left ampulla, ampullaryisthmic junction, utero-tuba1 junction and uterine horn. After surgical recovery, recordings from conscious free-standing animals were made for up to 10 h per day from Day -3 to Day + 3 relative to oestrus in each ewe. Daily blood samples were taken for progesterone radioimmunoassay, and a vasectomized ram used to assess oestrus. A range of physiological doses of oxytocin (10-100 mu), or control saline injections were given intravenously. Electromyographic (EMG) activity was measured before and after injections. Spontaneous activity throughout the reproductive tract was low on Day - 3 but increased to peak at oestrus (P< 0.05), when the pattern of activity consisted of short (2-10 s) co-ordinated high amplitude bursts (2-5 min-I). After oestrus, as overall activity declined, longer episodes of activity appeared but these occurred with a much slower frequency (1-4 h-I). Responsiveness to oxytocin was greatest on the day of oestrus at all electrode sites. Elevated responsiveness (relative to Day - 3, the late luteal phase) was seen from Day - 1 to Day + 2 at the ampullary-isthmic junction and uterus, but on the day of oestrus only at the ampulla and utero-tuba1 junction. Duration of increased EMG activity after oxytocin injection ranged from 5 to 20 min. These results show for the first time that the uterine and oviducal musculatures of the ewe in vivo reached a peak in sensitivity to physiological concentrations of oxytocin at oestrus. When combined with information on oxytocin receptor populations and endogenous circulating concentrations, this suggests that endogenous oxytocin could influence oviduct and myometrial activity at this time. Extra keywords: gamete, myometrium, myosalpinx.
Introduction Oxytocin receptors have been described in the reproductive tracts of oestrogen-treated rats (Soloff 1975) and rabbits (Maggi et al. 1988), and in cyclic sheep (Roberts et al. 1976; Sheldrick and Flint 1985; Ayad and Wathes 1989; Ayad et al. 1990), cattle (Fuchs et al. 1990) and women (Fuchs et al. 1985). In the ewe, studies have shown that concentrations of oxytocin receptors increased from low mid-luteal concentrations to reach a peak at oestrus followed by a decline to previous values in both the uterine and oviduct musculature (Sheldrick and Flint 1985; Ayad et al. 1990). However, a physiological role for these myometrial receptors at oestrus has not yet been determined.
C. L. Gilbert et al.
Spontaneous patterns of electromyographic (EMG) activity during the cycle have been described previously in the oviduct and uterus of the ewe and were found t o be of maximum intensity at oestrus (Ruckebusch and Bueno 1976; Garcia-Villar et al. 1985; Toutain et al. 1985). In addition, some studies on the effect of exogenous oxytocin on oviduct and uterine motility have been performed. Isthmic and uterine smooth muscle from ewes in oestrus was stimulated by oxytocin in vitro (Edqvist et al. 1975), and Noonan et al. (1978) showed that the oviduct was responsive to oxytocin in vivo, although high doses were used and the stage of the oestrous cycle unspecified. Ruckebusch and Pichot (1975) stated that spontaneous EMG output at the isthmus could be mimicked by oxytocin injection, with a larger dose required at oestrus than on Day 2. Stimulatory effects of oxytocin on the non-pregnant reproductive tract of other species have also been reported (pig, Zerobin and Sporri 1972; cow, Ruckebusch and Bayard 1975; man, Rorie and Newton 1965, Guiloff et al. 1974). However, a comprehensive study of the effects of physiological doses of oxytocin on the activity of different regions of the reproductive tract on specified days of the cycle throughout the oestrous period has not been published hitherto. Gilbert et al. (1991) have shown that spontaneous short-duration pulses of oxytocin can be measured in the plasma of ewes at oestrus. The present experiment was designed to investigate the electrical responsiveness of musculature from various regions of the reproductive tract in vivo to doses of oxytocin equivalent to endogenous concentrations during and around oestrus.
Materials and Methods Surgical Preparation Four ewes were anaesthetized using a mixture of halothane and oxygen and a laparotomy was performed through a ventral midline incision extending caudally between the mammary glands. The reproductive tract was exteriorized. Pairs of individually insulated braided steel wire extracellular electrodes (Cooner Wire Co., Chatsworth, CA, USA; Code AS633, overall diameter 0.33 mm, core resistance 60.3 ohms m-') were inserted at four sites on the left side of the tract in the ampulla, ampullary-isthmic junction, utero-tuba1 junction and uterine horn (Fig. 1).
2
Fig. 1. Drawing of the ventral surface of the uterus of a sheep to illustrate the placement of electrodes (n = 4 ewes); 1, ampulla; 2, ampullary-isthmic junction; 3, utero-tuba1 junction; 4, uterine horn. The electrodes used were pairs of insulated braided steel wires with insulation stripped over 1 mm, stitched and tied into the smooth muscle wall of the organ 1-2 mm apart. An additional earth wire was stitched into the body wall. Recordings were made daily for up to 8 h over 7 days around oestrus in each ewe.
Oxytocin and Oviduct EMG Activity a t Oestrus in the Ewe
Prior to insertion, each electrode had been prepared as follows. A 1-2-mm length of insulation was removed about 5 cm from the end of a 60-cm length of wire by means of a hot scalpel blade. A loop of silk suture material (4 '0' size) was tied firmly around the wire just proximal to this and the suture ends cut off short. All the wires for each electrode point were then passed through a 40-cm length of silastic tubing and both ends sealed with epoxy resin, leaving 15 cm of free wire protruding from the end destined to be implanted intra-abdominally. The whole assembly was then gas-sterilized. During surgery, secure placement of each wire was achieved by stitching it into the smooth muscle wall at the appropriate site using a round-bodied needle. The wire was drawn through until stopped by the loop of silk, leaving the bared area of wire buried in the muscle layers. Each wire was tied off and the free end cut short and insulated with quick-setting epoxy resin. Pairs of electrodes were positioned 1-2 mm apart. An additional electrode was stitched into the body wall to act as an earth. All electrodes, protected by the silastic tubing, were passed subcutaneously to a small incision on the left flank where they were exteriorized and fitted into a multiwire miniature plug ('D'-connector; RS Components, Corby, Northants, UK) which could be stored in a cloth pouch attached to the flank. 0.3 mg of buprenorphine (Temgesic, Reckitt and Colman, Hull, UK) was injected intramuscularly as a routine post-operative analgesic and animals left for 14 days before experimentation. This study was performed under the control and within the requirements of a licence issued by the UK Government's Animals' (Scientific Procedures) Inspectorate. Experimental Procedure EMG recordings were made in each ewe from 3 days before an anticipated oestrus until 3 days afterwards. The animals were tested twice a day for behavioural oestrus using a vasectomized ram and, to confirm the stage of the oestrous cycle, daily blood samples were taken for progesterone radioimmunoassay. The animals were connected to a Grass polygraph by means of a shielded multicore cable suspended from the ceiling; this allowed recordings t o be made from animals free from human interference. During recording the ewes stood in a rubber-wheeled crate (with which they had previously been familiarized) to improve electrical insulation. Water and hay were made available throughout. 50-pv calibration marks were made on each channel before and after recording. Chart speed was set to 15 mm min-'. On each day a 30-min recording period for measurement of spontaneous EMG activity was followed by injections of oxytocin (Syntocinon; Sandoz Pharmaceuticals, Frimley, Surrey, UK) given intravenously into the jugular vein of the ewe at intervals of not less than 30 min. Doses used were 5, 10, 25, 50 and 100 mU of oxytocin given in 1 mL of 0.9% NaCl and injected in random order. Control injections of vehicle alone were also given. Doses of 50 mU on the day of oestrus and 100 m u within one day of oestrus were not used, as they tended to produce intense and prolonged effects. Interpretation of EMG Output and Statistical Analysis To quantify the electrical output, traces were divided into 5-min sections and events greater than a 50-pv threshold (equivalent to background readings) were counted. This was done from 20 min before each injection (as a measure of spontaneous activity) to 20 min afterwards (as a measure of the effect of injection on subsequent activity), giving a total of eight values for each injection. For MINITAB and GENSTAT analyses (see below), all data were transformed to loglo(x+ 1), where x was the number of electrical events in each 5-min period. A comparison of the spontaneous EMG activity at each electrode placement on different days of the cycle was analysed using the pre-injection data in a balanced 2-way ANOVA (with 'ewes' and 'days' as factors) on the computer programme MINITAB. An overall analysis which included all the experimental data on the effect of oxytocin on EMG activity was performed on GENSTAT using multiple regression techniques, because the absence of higher dose injections near oestrus did not allow a balanced design for ANOVA. The influence of each of five explanatory variables on post-injection EMG activity was assessed. These were: (I) ewe; (2) mean pre-injection EMG activity; (3) dose of oxytocin; (4) time after injection (up to 20 min); (5) day relative to oestrus on which the injections were given. The influence of each of these variables was measured by adding them into the experimental model in the above order and looking for changes in the quality of fit of the data. This was assessed by comparing the reduction in residual mean square that each individual variable achieved with the final residual mean square, which included the contributions of all five variables.
C. L. Gilbert et al.
A further analysis which tested only for the effects of saline or 25 m u oxytocin on EMG activity was performed using Student's t-test. Progesterone Radioimmunoassay Plasma samples (0.25 mL) were extracted and assayed as described previously (Wathes et al. 1986). The minimum detectable dose was 0.1 ng rnLv1, and inter- and intra-assay coefficients of variation were 12.4 and 10.4% respectively.
Results All animals were undergoing oestrous cycles of normal length (judged by testing twice a day with a ram) prior to experimentation. At oestrus during the experimental cycle, the nadir in plasma progesterone profiles taken from individual animals coincided in each case with behavioural oestrus. The spontaneous electrical activity is described, as this was the baseline against which the effects of oxytocin were assessed. The mean spontaneous activity of all areas of the reproductive tract was maximal at behavioural oestrus (Figs 2 and 3). Results of regression
1.5
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Time relative to oestrus (days)
Time relative to oestrus (days)
Fig. 2. Summary of the spontaneous electromyograph (EMG) activity in the oviduct and uterus of four ewes through an oestrous period. Recordings were made from 3 days before to 3 days after behavioural oestrus from pairs of electrodes placed in four sites (as shown in Fig. 1) in the left-hand side of the tract. EMG results were quantified by counting all electrical events above a 5 0 - p threshold that occurred within a 5-min period. These periods were selected in groups of four (i.e. 20 min total) throughout any one day at random times. At least 24 five-min time bins were analysed each day. Statistical analysis using a blocked 2-way ANOVA was performed on each data set, after transformation of values to loglo(x+ 1), which is used as the scale for the ordinate axis. l.s.d., the least difference between any two mean values for statistical significance ( P < 0.05). a v. b indicates significant differences in spontaneous electrical activity between the day of oestrus (a) and other days (b). Significant differences do occur between other days but are not shown. Fig. 3. Representative traces of EMG output (raw unintegrated data) from a ewe (a) 2 days before behavioural oestrus and (b) on the day of oestrus; 1, ampulla; 2, ampullary-isthmic junction; 3, utero-tuba1 junction; 4, uterine horn. (a) Levels of activity are low. Output from the ampulla and ampullary-isthmic junction is closely related, although this coupling is not absolute as some spikes occur in one trace only. The utero-tuba1 junction displays regular low amplitude bursting. An injection of 50 m u of oxytocin i.v, failed to alter the activity in the oviduct, whereas a small increase in activity
Oxytocin and Oviduct EMG Activity at Oestrus in the Ewe
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50mU Oxytocin i.v.
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can be observed in the uterine horn in this instance. (b) Spontaneous activity in all channels at oestrus is greater than on Day - 2. Regular, high amplitude, co-ordinated bursts of activity are seen at all sites, superimposed upon which are episodes of more continuous low-level activity in the oviduct, lasting 2-5 min and occurring with a frequency of 1-4 h-'. (This pattern became more dominant in ensuing days as general levels of activity fell.) An injection of saline i.v. was unable to alter the output appreciably whereas 10 m u oxytocin i.v. [compared with 50 m u used in (a)] clearly enhanced the output in all channels.
C . L. Gilbert et al.
analysis suggested that at the utero-tuba1 junction and ampulla, spontaneous activity on the day of oestrus was significantly greater than on any other day, whereas at the ampullaryisthmic junction and uterine horn, significant elevations in mean activity over levels seen in the late luteal phase were maintained until at least 2 days after oestrus. Patterns of EMG activity consisted of low and generally discontinuous activity prior to oestrus increasing to regular high-amplitude co-ordinated bursts in all channels on the day of oestrus itself (Fig. 3). This latter output was characterized by sudden switching between high-intensity activity and quiescence, with a frequency of 2-3 bursts min-'. In addition to and superimposed upon these bursts, from oestrus onwards, the electrodes recorded episodes of continuous but lower amplitude activity lasting 2-5 min. The initiation and ending of these events was more gradual than the higher amplitude activity. They occurred simultaneously at all oviducal electrodes (but with reduced intensity at the utero-tuba1 junction), and were seen irregularly on the day of oestrus, increasing in frequency to 3-4 episodes h-' by Day 2. The high-amplitude, high-frequency bursts disappeared on Days 1-2, and overall activity declined steadily to pre-oestrous levels by Day 3. Examples of the effect of oxytocin on the EMG trace are shown in Fig. 3 and histograms that illustrate the effects of injecting saline vehicle or 25 m u oxytocin at the different electrode sites are shown in Fig. 4. This dose caused significant ( P < 0.05) elevations in EMG activity at all four electrode sites on the day of oestrus only; these were not seen in response to saline. The effect of injections at all doses and on all days on the activity of the tract was analysed by multiple regression techniques. This analysis was able to compare in a single model the influences of dose of oxytocin, time relative to injection and day of the cycle at each separate electrode site. This revealed differences between sites in the duration of stimulation, in the number of days around oestrus for which the sensitivity to injections was increased and in the dose-dependency of the effect. At all four sites oxytocin treatment caused a significant increase in EMG activity. In the ampulla and uterus there was a significant relationship between dose of oxytocin (10-100 m u ) and the post-injection EMG response ( P < 0.01). At the utero-tuba1 junction this relationship just failed to achieve significance (P < 0.1). However, at the ampullaryisthmic junction, dose of oxytocin and size of the post-injection response were not significantly related. The duration of response also varied between sites. At the ampulla and ampullary-isthmic junction EMG activity in response to oxytocin was significantly increased ( P < 0.01) above pre-treatment levels between 0 and 10 min after injection, whereas at both the utero-tuba1 junction and the uterus the response was more prolonged. At the utero-tuba1 junction activity had returned near to pre-treatment values within 20 min, whereas at the uterus there was no significant decrease in the stimulated activity throughout the 20-min period following oxytocin injection. At the ampulla and utero-tuba1 junction the sensitivity of the region to oxytocin injection was increased relative to that of the late luteal phase (Day - 3) for only one day, the day of oestrus itself ( P < 0.01; Table 1). However at the ampullary-isthmic junction and uterus the sensitivity relative to Day - 3 was increased for four days, throughout the period from Day -1 to Day +2.
Discussion These results demonstrate that oxytocin injected at physiological doses was able to increase EMG output from the oviduct and uterus of the ewe during and around oestrus. The response to oxytocin peaked on the day of oestrus itself, when concentrations of oxytocin receptors are maximal in both the myometrium (Sheldrick and Flint 1985; Ayad and Wathes 1989) and myosalpinx (Ayad et al. 1990). Recently Ayad et al. (1991) have shown, using autoradiography, that these receptors are found in both the endometrium and myometrium of the uterus but in the muscle wall only of the oviduct.
Oxytocin and Oviduct EMG Activity a t Oestrus in the Ewe
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Fig. 4. Responses of the reproductive tract at the four recording points used (Fig. 1) over 7 days to injections of (a) saline vehicle or (b)25 m u of oxytocin (n = 4 ewes); 1, ampulla; 2, ampullary-isthmic junction; 3, utero-tubal junction; 4, uterus. Results from Days 2 and 3 before, and Days 2 and 3 after oestrus were similar and have been combined. Solid bars represent pre-injection (spontaneous) EMG activity, meaned for the four 5-min periods immediately preceding injection (n > 16 for each bar). Open bars represent post-injection responses during the times indicated by the key (n = 4-6 for each bar). 1=time of injection. Error bars are s.e.m. 0.0, P < 0.001; 0 0 , P < 0.01; 0, P < 0.05, significantly different from pre-injection values (Student's t-test). See also Table 1. Table 1. Analysis of electrical responses in the reproductive tract of the ewe following systemic administration of oxytocin Results are based upon numbers of electrical events > 50 pv counted in four consecutive 5-min periods following oxytocin injection. Each electrode placement is considered separately. EMG activity in response to oxytocin on different days was assessed by a multiple regression model after effects of ewe, pre-injection means, oxytocin dose and time after injection had already been taken into account. Values on each day were compared to those on Day - 3 (late luteal phase) and significance values demonstrate an increased (or decreased when labelled 'less') sensitivity of the tract on the days indicated, relative to Day - 3 . *PO.05) Day relative to oestrus -2 -1 0 1 2 3
Ampulla
Ampullary-isthmic junction
Utero-tuba1 junction
Uterus
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C . L. Gilbert et al.
There are many qualitative descriptions of changing patterns of EMG activity during the oestrous cycle in the literature and these are remarkably consistent both between workers, and with the spontaneous EMG activity measured in the present experiments. However, this previous work has not systematically investigated the effects of oxytocin at physiological concentrations on the electrical activity of the reproductive tract in normally cyclic ewes. Plasma oxytocin concentrations at oestrus are generally low (Sheldrick and Flint 1981; Webb et al. 1981; Schams et al. 1982) and ovarian oxytocin content is minimal at this time (Wathes et al. 1986). However, Gilbert et al. (1991) have characterized release patterns for endogenous oxytocin at oestrus in more detail and demonstrated the existence of highamplitude (up to 31 pg mL-'), short-duration (1-4 min) pulses at this time; this finding is consistent with the results of McNeilly and Ducker (1972) and Mitchell et a[. (1982). These endogenous pulses (Gilbert et al. 1991) were of similar size and duration to pulses produced in anoestrous ewes when oxytocin was injected in the same concentrations as the present experiments (intravenous injections of 25 and 50 m u produced peak concentrations of 2.1 + 0.33 and 6.4 1 pg mL- (mean + s.e.) respectively 1-2 min later; V. J. Ayad and D. C. Wathes, unpublished observations). It may therefore be hypothesized that endogenous oxytocin pulses occurring in cyclic ewes near oestrus could affect uterine and oviduct EMG output in a similar way to the injections described here, and it is likely that changes in the excitability of reproductive tract muscle would be reflected in altered sperm and egg transport, which take place at this time. The lack of correlation between the endogenous oxytocin profile and EMG activity reported previously by Garcia-Villar et al. (1983) may be attributed to two factors. Firstly, the sampling frequency used would have been insufficient to detect reliably pulses of less than 5 min duration, and secondly, we show here that a single brief pulse of injected oxytocin may increase the EMG activity of the uterus (after a lag of about 1 min) for over 20 min. It is difficult to compare the overall intensity of response between different pairs of electrodes because local impedance and amplifier performance are important determinants of signal strength. However temporal variations in EMG activity at any one site reflect changes in muscle excitability and tentative predictions may be made as to the events being recorded. Phasic activity is likely to represent co-ordinated movements such as waves of peristalsis whereas more continuous activity may be an indicator of generally raised muscle tone or even spasm. Experiments employing simultaneous EMG and intraluminal pressure recordings (Harding et al. 1982) or strain-gauges placed between electrodes (Garcia-Villar et al. 1982) support these views. Nevertheless the relative contributions to any one part of the signal from longitudinal and circular muscle components remains unknown. An examination of data from the different electrode placements suggests that oxytocin did not exert a uniform effect on different regions of the reproductive tract. At the ampulla and uterine electrodes the dose of oxytocin was significantly related to the post-injection elevation in EMG output. However, the ampullary-isthmic junction and utero-tuba1 junctions showed no significant relationship between size of dose and intensity of response. In the utero-tuba1 junction this result was only just non-significant (P < 0.01), but in the case of the ampullary-isthmic junction the effect of dose did not approach significance although a stimulatory effect of oxytocin was clearly present (Fig. 4). The isthmic region is surrounded by a dense adrenergic nerve net (Brundin 1965) which may become excited as part of a sympathetic response to the presence of the experimenter, resulting in heightened muscle activity after any injection irrespective of dose. In support of this, a small (non-significant) but consistent elevation in activity above pre-injection values following administration of saline is seen on Day 0 at the ampullary-isthmic junction (Fig. 4). The duration of effect after injection was more transient in both the ampulla and ampullary-isthmic junction than in other areas. This may reflect lower overall receptor concentrations at oestrus (Ayad et al. 1990) than in the uterus (Ayad and Wathes 1989) or a different post-receptor response in this area.
*
'
Oxytocin and Oviduct EMG Activity at Oestrus in the Ewe
The variability in the number of days of elevated sensitivity to oxytocin is perhaps the most interesting aspect of these results. This effect is not clearly seen in Fig. 4 as the 25 m u dose shown there in general produces a significant response on only the day of oestrus. However, Table 1 reveals that when all doses are considered, the ampullary-isthmic junction and uterus have elevated sensitivities over 4 days around oestrus including 2 days after oestrus, whereas the ampulla and utero-tuba1 junction are more sensitive on the day of oestrus only. The timing of elevated responses to oxytocin at the ampulla electrode coincides with increased fimbrial tone to assist the envelopment of pre-ovulatory antral follicles. After ovulation, oviducal fluid concentrations of oxytocin are likely to be raised because of the contribution from follicular fluid and granulosa and cumulus oophorus cells shed with ova into the oviduct (Wathes et al. 1986). Transport of egg(s) to the ampullary-isthmic junction is rapid in most species examined and may be measured in minutes (Harper 1961; Hunter 1974; Blandau and Verdugo 1976) and it is here that fertilization occurs. Elevated responsiveness to oxytocin was maintained at the ampullary-isthmic junction until 2-3 days after oestrus, which is the time at which embryos are permitted to pass through to the uterus (Winterberger-Torres 1961; Holst 1974). By contrast the utero-tuba1 area displayed heightened sensitivity to oxytocin only on the day of oestrus, when, it has been suggested, this area and the distal isthmus act as a 'sperm reservoir' before allowing sperm to progress to fertilize the newly released egg on the day following oestrus (Hunter and Nichol 1983; Hunter 1988). It is possible that oxytocin contributes to elevated muscle tone in these areas at these times. The stimulatory effect of oxytocin was superimposed upon a background of spontaneous EMG activity which also peaked at oestrus. This result is in agreement with previous reports in all parts of the reproductive tract of the ewe (cervix, Garcia-Villar et al. 1982; uterus, Van der Weyden 1983; uterus and oviduct, Ruckebusch and Bueno 1976; oviduct, Ruckebusch and Pichot 1975) and cow (Ruckebusch and Bayard 1975). High concentrations of circulating oestrogen are known to result in elevated EMG activity in both ovariectomized (Porter and Lye 1983) and anoestrous ewes (Ayad et al. 1989), and oestrogens increased gap junction numbers between muscle cells in rats (Garfield et al. 1980) and actomyosin content of myometrial tissues in rabbits (Michael and Schofield 1969). It is likely that a proportion of the high spontaneous EMG activity seen at oestrus is due to circulating oestrogens. In summary, this study has shown that oxytocin at physiological concentrations is capable of influencing uterine and oviduct EMG activity. In addition, the sensitivity of the reproductive tract parallels previous reports of the receptor numbers present at these times. This suggests that oxytocin may have an important role to play in gamete transport, but as the relationship between EMG output and the movement of tube contents was not assessed, this conclusion remains speculative. The results of Wathes et al. (1989) showed that ewes immunized against oxytocin had a highly significant reduction in pregnancy rate 20 days after mating when compared with controls, and one explanation for this reduced fertility could be an influence of oxytocin on tuba1 transport. Acknowledgments
We thank Mrs S. Fennel1 and Mr P. Rees for expert technical assistance, Mr R. Francis and his staff for care of the animals, and the Wellcome Trust and the AFRC for financial support. References Ayad, V. J., and Wathes, D. C. (1989). Characterization of endometrial and myometrial oxytocin receptors in the non-pregnant ewe. J. Endocrinol. 123, 11-18. Ayad, V. J., McGoff, S. A., and Wathes, D. C. (1990). Oxytocin receptors in the oviduct during the oestrus cycle of the ewe. J. Endocrinol. 124, 353-9.
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Ayad, V. J., Guldenaar, S. E. F., and Wathes, D. C. (1991). Characterisation and localisation of oxytocin receptors in the uterus and oviduct of the non-pregnant ewe using an iodinated receptor antagonist. J. Endocrinol. 128, 187-95. Blandau, R. J., and Verdugo, P . (1976). An overview of gamete transport-comparative effects. In 'Symposium on Ovum Transport and Fertility Regulation'. (Eds M. J. K. Harper and C. J . Pauerstein.) pp. 138-46. (Scriptor: Copenhagen.) Brundin, J . (1965). Distribution and function of adrenergic nerves in the rabbit fallopian tube. Acta Physiol. Scand. Suppl. 259 66, 1-57. Edqvist, S., Einarsson, S., Gustafsson, B., Linde, C., and Lindell, J.-0. (1975). The in vitro and in vivo effects of prostaglandins E, and Fz and of oxytocin on the tubular genital tract of ewes. Znt. J. Fertil. 20, 234-8. Fuchs, A. R., Fuchs, F., and Soloff, M. S. (1985). Oxytocin receptors in nonpregnant human uterus. J. Clin. Endocrinol. Metab. 60, 37-41. Fuchs, A. R., Behrens, O., Helmer, H., Liu, C.-H., Barros, C. M., and Fields, M. J. (1990). Oxytocin and vasopressin receptors in bovine endometrium and myometrium during the estrous cycle and early pregnancy. Endocrinology 127, 629-36. Garcia-Villar, R., Toutain, P. L., More, J., and Ruckebusch, Y. (1982). Spontaneous motility of the cervix in cyclic and ovariectomized ewes and changes induced by exogenous hormones. J. Reprod. Fertil. 66, 317-26. Garcia-Villar, R., Toutain, P . L., Schams, D., and Ruckebusch, Y. (1983). Are regular activity episodes of the genital tract controlled by pulsatile releases of oxytocin? Biol. Reprod. 29, 1183-8. Garcia-Villar, R., Schams, D., Alvinerie, M., Laurentie, M. P., and Toutain, P. L. (1985). Activity of the genital tract and plasma levels of oxytocin and cortisol at the time of mating in the ewe. J. Endocrinol. 105, 323-9. Garfield, R. E., Kannan, M. S., and Daniel, E. E. (1980). Gap junction formation in myometrium; control by estrogens, progesterone and prostaglandins. Am. J. Physiol. 238, C81-C89. Gilbert, C. L., Jenkins, K., and Wathes, D. C. (1991). Pulsatile release of oxytocin into the circulation of the ewe at oestrus, mating and in the early luteal phase. J. Reprod. Fertil. 91, 337-46. Guiloff, E., Ibarra-Polo, A. A., and Gomez-Rogers, C. (1974). Effect of oxytocin on the contractility of the human oviduct in vivo. Fertil. Steril. 25, 946-53. Harding, R., Poore, E. R., Bailey, A., Thorburn, G. D., Jansen, C. A. M., and Nathanielsz, P. W. (1982). Electromyographic activity of the nonpregnant and pregnant sheep uterus. Am. J. Obstet. Gynecol. 142, 448-57. Harper, M. J. K. (1961). The mechanisms involved in the movement of newly ovulated eggs through the ampulla of the rabbit fallopian tube. J. Reprod. Fertil. 2, 522-4. Holst, P . J. (1974). The time of entry of ova into the uterus of the ewe. J. Reprod. Fertil. 36, 427-8. Hunter, R. H. F. (1974). Chronological and cytological details of fertilization and early embryonic development in the domestic pig, Sus scrofa. Anat. Rec. 178, 169-86. Hunter, R. H. F. (1988). The Fallopian tubes: their role in fertility and infertility. (Springer-Verlag: Berlin, Heidelberg and New York.) Hunter, R. H. F., and Nichol, R. (1983). Transport of spermatozoa in the sheep oviduct; preovulatory sequestering of cells in the caudal isthmus. J. Exp. Zool. 228, 121-8. Maggi, M., Genazzini, A. D., Giannini, S., Torrisi, C., Baldi, E., Di Tomasso, M., Munson, P. J., Rodbard, D., and Serio, M. (1988). Vasopressin and oxytocin receptors in vagina myometrium and oviduct of rabbits. Endocrinology 122, 2970-80. McNeilly, A. S., and Ducker, H. A. (1972). Blood levels of oxytocin in the female goat during coitus. J. Endocrinol. 54, 399-406. Michael, C. A., and Schofield, B. M. (1969). The influence of the ovarian hormones on the actomyosin content and the development of tension in uterine muscle. J. Endocrinol. 44, 501-11. Mitchell, M. D., Kraemer, D. L., Brennecke, S. P., and Webb, R. (1982). Pulsatile release of oxytocin during the oestrous cycle, pregnancy and parturition in sheep. Biol. Reprod. 27, 1169-73. Noonan, J. J., Adair, R. L., Halbert, S. A., Ringo, J. A., and Reeves, J. J. (1978). Quantitative assessment of oxytocin-stimulated oviduct contractions of the ewe by optoelectronic measurements. J. Anim. Sci. 47, 914-18. Porter, D. G., and Lye, S. J. (1983). Partial reversal of the myometrial progesterone 'block' in the non-pregnant ewe in vivo by oestradiol 17P. J. Reprod. Fertil. 67, 227-34.
Oxytocin and Oviduct EMG Activity at Oestrus in the Ewe
Roberts, J. S., McCracken, J. A., Gavagan, J. E., and Soloff, M. S. (1976). Oxytocin-stimulated release of prostaglandin F2 from ovine endometrium in vitro: correlation with oestrous cycle and oxytocin-receptor binding. Endocrinology 99, 1107-14. Rorie, D. K., and Newton, M. (1965). Response of isolated human fallopian tube to oxytocin: a preliminary report. Fertil. Steril. 16, 27-32. Ruckebusch, Y., and Bayard, F. (1975). Motility of the oviduct and uterus of the cow during the oestrous cycle. J. Reprod. Fertil. 43, 23-32. Ruckebusch, Y., and Pichot, D. (1975). Effects of adrenergic drugs on sheep oviduct motility. Eur. J. Pharmacol. 33, 193-6. Ruckebusch, Y., and Bueno, L. (1976). An electromyographic study of uterotubal activity in the ewe. J. Reprod. Fertil. 47, 221-7. Schams, D., Lahlou-Kassi, A., and Glatzel, P . (1982). Oxytocin concentrations in peripheral blood during the oestrous cycle and after ovariectomy in two breeds of sheep with low and high fecundity. J. Endocrinol. 92, 9- 13. Sheldrick, E. L., and Flint, A. P. F. (1981). Circulating concentrations of oxytocin during the oestrous cycle and early pregnancy in sheep. Prostaglandins 22, 631-6. Sheldrick, E. L., and Flint, A. P. F. (1985). Endocrine control of uterine oxytocin receptors in the ewe. J. Endocrinol. 106, 249-58. Soloff, M. S. (1975). Oxytocin receptors in rat oviduct. Biochem. Biophys. Res. Commun. 65, 205-12. Toutain, P . L., Marnet, P . G., Laurentie, M. P., Garcia-Villar, R., and Ruckebush, Y. (1985). Direction of uterine contractions during oestrus in ewes: a re-evaluation. Am. J. Physiol. 249, R410-R416. Van der Weyden, G. C. (1983). Myometrial activity in the non-pregnant texel ewe. Ph.D. Thesis, University of Utrecht, Netherlands. Wathes, D. C., Guldenaar, S. E. F., Swann, R. W., Webb, R., Porter, D. G., and Pickering, B. T. (1986). A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe. J. Reprod. Fertil. 78, 167-83. Wathes, D. C., Ayad, V. J., McGoff, S. A,, and Morgan, K. L. (1989). Effect of active immunization against oxytocin on gonadotrophin secretion and the establishment of pregnancy in the ewe. J. Reprod. Fertil. 86 653-64. Webb, R., Mitchell, M. D., Falconer, J., and Robinson, J. S. (1981). Temporal relationships between peripheral concentrations of oxytocin, progesterone and 13,14-dihydro-15-keto prostaglandin F during the oestrus cycle and early pregnancy in the ewe. Prostaglandins 22, 443-54. Winterberger-Torres, S. (1961). Mouvements des trompes et progression des oeufs chez la brebis. Ann. Biol. Anim. Biochim. Biophys. 1, 121-33. Zerobin, K., and Sporri, H. (1972). Motility of the bovine and porcine uterus and fallopian tube. Adv. Vet. Sci. Comp. Med. 16, 303-54.
Manuscript received 2 April 1991; revised 11 September 1991; accepted 10 January 1992
Effect of Oxytocin on the Pattern of Electromyographic Activity in the Oviduct and Uterus of the Ewe around Oestrus
C. L. ~ i l b e r t ~ 'P. , J. crippsB and D. C . wathesAC Department of Anatomy, University of Bristol School of Medical Sciences, Bristol BS8 ITD, UK. Department of Veterinary Medicine, University of Bristol, Langford House, Langford, Bristol BS18 7DU, UK. Present address: AFRC Institute of Animal Physiology and Genetics Research, Babraham Hall, Babraham, Cambridge CB2 4AT, UK.
A
Abstract This study tested the hypothesis that the administration of oxytocin in doses equivalent to endogenous concentrations at and around oestrus could affect uterine and oviducal muscular activity at the time of gamete transport. Four ewes were fitted with recording electrodes in the left ampulla, ampullaryisthmic junction, utero-tuba1 junction and uterine horn. After surgical recovery, recordings from conscious free-standing animals were made for up to 10 h per day from Day -3 to Day + 3 relative to oestrus in each ewe. Daily blood samples were taken for progesterone radioimmunoassay, and a vasectomized ram used to assess oestrus. A range of physiological doses of oxytocin (10-100 mu), or control saline injections were given intravenously. Electromyographic (EMG) activity was measured before and after injections. Spontaneous activity throughout the reproductive tract was low on Day - 3 but increased to peak at oestrus (P< 0.05), when the pattern of activity consisted of short (2-10 s) co-ordinated high amplitude bursts (2-5 min-I). After oestrus, as overall activity declined, longer episodes of activity appeared but these occurred with a much slower frequency (1-4 h-I). Responsiveness to oxytocin was greatest on the day of oestrus at all electrode sites. Elevated responsiveness (relative to Day - 3, the late luteal phase) was seen from Day - 1 to Day + 2 at the ampullary-isthmic junction and uterus, but on the day of oestrus only at the ampulla and utero-tuba1 junction. Duration of increased EMG activity after oxytocin injection ranged from 5 to 20 min. These results show for the first time that the uterine and oviducal musculatures of the ewe in vivo reached a peak in sensitivity to physiological concentrations of oxytocin at oestrus. When combined with information on oxytocin receptor populations and endogenous circulating concentrations, this suggests that endogenous oxytocin could influence oviduct and myometrial activity at this time. Extra keywords: gamete, myometrium, myosalpinx.
Introduction Oxytocin receptors have been described in the reproductive tracts of oestrogen-treated rats (Soloff 1975) and rabbits (Maggi et al. 1988), and in cyclic sheep (Roberts et al. 1976; Sheldrick and Flint 1985; Ayad and Wathes 1989; Ayad et al. 1990), cattle (Fuchs et al. 1990) and women (Fuchs et al. 1985). In the ewe, studies have shown that concentrations of oxytocin receptors increased from low mid-luteal concentrations to reach a peak at oestrus followed by a decline to previous values in both the uterine and oviduct musculature (Sheldrick and Flint 1985; Ayad et al. 1990). However, a physiological role for these myometrial receptors at oestrus has not yet been determined.
C. L. Gilbert et al.
Spontaneous patterns of electromyographic (EMG) activity during the cycle have been described previously in the oviduct and uterus of the ewe and were found t o be of maximum intensity at oestrus (Ruckebusch and Bueno 1976; Garcia-Villar et al. 1985; Toutain et al. 1985). In addition, some studies on the effect of exogenous oxytocin on oviduct and uterine motility have been performed. Isthmic and uterine smooth muscle from ewes in oestrus was stimulated by oxytocin in vitro (Edqvist et al. 1975), and Noonan et al. (1978) showed that the oviduct was responsive to oxytocin in vivo, although high doses were used and the stage of the oestrous cycle unspecified. Ruckebusch and Pichot (1975) stated that spontaneous EMG output at the isthmus could be mimicked by oxytocin injection, with a larger dose required at oestrus than on Day 2. Stimulatory effects of oxytocin on the non-pregnant reproductive tract of other species have also been reported (pig, Zerobin and Sporri 1972; cow, Ruckebusch and Bayard 1975; man, Rorie and Newton 1965, Guiloff et al. 1974). However, a comprehensive study of the effects of physiological doses of oxytocin on the activity of different regions of the reproductive tract on specified days of the cycle throughout the oestrous period has not been published hitherto. Gilbert et al. (1991) have shown that spontaneous short-duration pulses of oxytocin can be measured in the plasma of ewes at oestrus. The present experiment was designed to investigate the electrical responsiveness of musculature from various regions of the reproductive tract in vivo to doses of oxytocin equivalent to endogenous concentrations during and around oestrus.
Materials and Methods Surgical Preparation Four ewes were anaesthetized using a mixture of halothane and oxygen and a laparotomy was performed through a ventral midline incision extending caudally between the mammary glands. The reproductive tract was exteriorized. Pairs of individually insulated braided steel wire extracellular electrodes (Cooner Wire Co., Chatsworth, CA, USA; Code AS633, overall diameter 0.33 mm, core resistance 60.3 ohms m-') were inserted at four sites on the left side of the tract in the ampulla, ampullary-isthmic junction, utero-tuba1 junction and uterine horn (Fig. 1).
2
Fig. 1. Drawing of the ventral surface of the uterus of a sheep to illustrate the placement of electrodes (n = 4 ewes); 1, ampulla; 2, ampullary-isthmic junction; 3, utero-tuba1 junction; 4, uterine horn. The electrodes used were pairs of insulated braided steel wires with insulation stripped over 1 mm, stitched and tied into the smooth muscle wall of the organ 1-2 mm apart. An additional earth wire was stitched into the body wall. Recordings were made daily for up to 8 h over 7 days around oestrus in each ewe.
Oxytocin and Oviduct EMG Activity a t Oestrus in the Ewe
Prior to insertion, each electrode had been prepared as follows. A 1-2-mm length of insulation was removed about 5 cm from the end of a 60-cm length of wire by means of a hot scalpel blade. A loop of silk suture material (4 '0' size) was tied firmly around the wire just proximal to this and the suture ends cut off short. All the wires for each electrode point were then passed through a 40-cm length of silastic tubing and both ends sealed with epoxy resin, leaving 15 cm of free wire protruding from the end destined to be implanted intra-abdominally. The whole assembly was then gas-sterilized. During surgery, secure placement of each wire was achieved by stitching it into the smooth muscle wall at the appropriate site using a round-bodied needle. The wire was drawn through until stopped by the loop of silk, leaving the bared area of wire buried in the muscle layers. Each wire was tied off and the free end cut short and insulated with quick-setting epoxy resin. Pairs of electrodes were positioned 1-2 mm apart. An additional electrode was stitched into the body wall to act as an earth. All electrodes, protected by the silastic tubing, were passed subcutaneously to a small incision on the left flank where they were exteriorized and fitted into a multiwire miniature plug ('D'-connector; RS Components, Corby, Northants, UK) which could be stored in a cloth pouch attached to the flank. 0.3 mg of buprenorphine (Temgesic, Reckitt and Colman, Hull, UK) was injected intramuscularly as a routine post-operative analgesic and animals left for 14 days before experimentation. This study was performed under the control and within the requirements of a licence issued by the UK Government's Animals' (Scientific Procedures) Inspectorate. Experimental Procedure EMG recordings were made in each ewe from 3 days before an anticipated oestrus until 3 days afterwards. The animals were tested twice a day for behavioural oestrus using a vasectomized ram and, to confirm the stage of the oestrous cycle, daily blood samples were taken for progesterone radioimmunoassay. The animals were connected to a Grass polygraph by means of a shielded multicore cable suspended from the ceiling; this allowed recordings t o be made from animals free from human interference. During recording the ewes stood in a rubber-wheeled crate (with which they had previously been familiarized) to improve electrical insulation. Water and hay were made available throughout. 50-pv calibration marks were made on each channel before and after recording. Chart speed was set to 15 mm min-'. On each day a 30-min recording period for measurement of spontaneous EMG activity was followed by injections of oxytocin (Syntocinon; Sandoz Pharmaceuticals, Frimley, Surrey, UK) given intravenously into the jugular vein of the ewe at intervals of not less than 30 min. Doses used were 5, 10, 25, 50 and 100 mU of oxytocin given in 1 mL of 0.9% NaCl and injected in random order. Control injections of vehicle alone were also given. Doses of 50 mU on the day of oestrus and 100 m u within one day of oestrus were not used, as they tended to produce intense and prolonged effects. Interpretation of EMG Output and Statistical Analysis To quantify the electrical output, traces were divided into 5-min sections and events greater than a 50-pv threshold (equivalent to background readings) were counted. This was done from 20 min before each injection (as a measure of spontaneous activity) to 20 min afterwards (as a measure of the effect of injection on subsequent activity), giving a total of eight values for each injection. For MINITAB and GENSTAT analyses (see below), all data were transformed to loglo(x+ 1), where x was the number of electrical events in each 5-min period. A comparison of the spontaneous EMG activity at each electrode placement on different days of the cycle was analysed using the pre-injection data in a balanced 2-way ANOVA (with 'ewes' and 'days' as factors) on the computer programme MINITAB. An overall analysis which included all the experimental data on the effect of oxytocin on EMG activity was performed on GENSTAT using multiple regression techniques, because the absence of higher dose injections near oestrus did not allow a balanced design for ANOVA. The influence of each of five explanatory variables on post-injection EMG activity was assessed. These were: (I) ewe; (2) mean pre-injection EMG activity; (3) dose of oxytocin; (4) time after injection (up to 20 min); (5) day relative to oestrus on which the injections were given. The influence of each of these variables was measured by adding them into the experimental model in the above order and looking for changes in the quality of fit of the data. This was assessed by comparing the reduction in residual mean square that each individual variable achieved with the final residual mean square, which included the contributions of all five variables.
C. L. Gilbert et al.
A further analysis which tested only for the effects of saline or 25 m u oxytocin on EMG activity was performed using Student's t-test. Progesterone Radioimmunoassay Plasma samples (0.25 mL) were extracted and assayed as described previously (Wathes et al. 1986). The minimum detectable dose was 0.1 ng rnLv1, and inter- and intra-assay coefficients of variation were 12.4 and 10.4% respectively.
Results All animals were undergoing oestrous cycles of normal length (judged by testing twice a day with a ram) prior to experimentation. At oestrus during the experimental cycle, the nadir in plasma progesterone profiles taken from individual animals coincided in each case with behavioural oestrus. The spontaneous electrical activity is described, as this was the baseline against which the effects of oxytocin were assessed. The mean spontaneous activity of all areas of the reproductive tract was maximal at behavioural oestrus (Figs 2 and 3). Results of regression
1.5
b
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5
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Time relative to oestrus (days)
Time relative to oestrus (days)
Fig. 2. Summary of the spontaneous electromyograph (EMG) activity in the oviduct and uterus of four ewes through an oestrous period. Recordings were made from 3 days before to 3 days after behavioural oestrus from pairs of electrodes placed in four sites (as shown in Fig. 1) in the left-hand side of the tract. EMG results were quantified by counting all electrical events above a 5 0 - p threshold that occurred within a 5-min period. These periods were selected in groups of four (i.e. 20 min total) throughout any one day at random times. At least 24 five-min time bins were analysed each day. Statistical analysis using a blocked 2-way ANOVA was performed on each data set, after transformation of values to loglo(x+ 1), which is used as the scale for the ordinate axis. l.s.d., the least difference between any two mean values for statistical significance ( P < 0.05). a v. b indicates significant differences in spontaneous electrical activity between the day of oestrus (a) and other days (b). Significant differences do occur between other days but are not shown. Fig. 3. Representative traces of EMG output (raw unintegrated data) from a ewe (a) 2 days before behavioural oestrus and (b) on the day of oestrus; 1, ampulla; 2, ampullary-isthmic junction; 3, utero-tuba1 junction; 4, uterine horn. (a) Levels of activity are low. Output from the ampulla and ampullary-isthmic junction is closely related, although this coupling is not absolute as some spikes occur in one trace only. The utero-tuba1 junction displays regular low amplitude bursting. An injection of 50 m u of oxytocin i.v, failed to alter the activity in the oviduct, whereas a small increase in activity
Oxytocin and Oviduct EMG Activity at Oestrus in the Ewe
I
50mU Oxytocin i.v.
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can be observed in the uterine horn in this instance. (b) Spontaneous activity in all channels at oestrus is greater than on Day - 2. Regular, high amplitude, co-ordinated bursts of activity are seen at all sites, superimposed upon which are episodes of more continuous low-level activity in the oviduct, lasting 2-5 min and occurring with a frequency of 1-4 h-'. (This pattern became more dominant in ensuing days as general levels of activity fell.) An injection of saline i.v. was unable to alter the output appreciably whereas 10 m u oxytocin i.v. [compared with 50 m u used in (a)] clearly enhanced the output in all channels.
C . L. Gilbert et al.
analysis suggested that at the utero-tuba1 junction and ampulla, spontaneous activity on the day of oestrus was significantly greater than on any other day, whereas at the ampullaryisthmic junction and uterine horn, significant elevations in mean activity over levels seen in the late luteal phase were maintained until at least 2 days after oestrus. Patterns of EMG activity consisted of low and generally discontinuous activity prior to oestrus increasing to regular high-amplitude co-ordinated bursts in all channels on the day of oestrus itself (Fig. 3). This latter output was characterized by sudden switching between high-intensity activity and quiescence, with a frequency of 2-3 bursts min-'. In addition to and superimposed upon these bursts, from oestrus onwards, the electrodes recorded episodes of continuous but lower amplitude activity lasting 2-5 min. The initiation and ending of these events was more gradual than the higher amplitude activity. They occurred simultaneously at all oviducal electrodes (but with reduced intensity at the utero-tuba1 junction), and were seen irregularly on the day of oestrus, increasing in frequency to 3-4 episodes h-' by Day 2. The high-amplitude, high-frequency bursts disappeared on Days 1-2, and overall activity declined steadily to pre-oestrous levels by Day 3. Examples of the effect of oxytocin on the EMG trace are shown in Fig. 3 and histograms that illustrate the effects of injecting saline vehicle or 25 m u oxytocin at the different electrode sites are shown in Fig. 4. This dose caused significant ( P < 0.05) elevations in EMG activity at all four electrode sites on the day of oestrus only; these were not seen in response to saline. The effect of injections at all doses and on all days on the activity of the tract was analysed by multiple regression techniques. This analysis was able to compare in a single model the influences of dose of oxytocin, time relative to injection and day of the cycle at each separate electrode site. This revealed differences between sites in the duration of stimulation, in the number of days around oestrus for which the sensitivity to injections was increased and in the dose-dependency of the effect. At all four sites oxytocin treatment caused a significant increase in EMG activity. In the ampulla and uterus there was a significant relationship between dose of oxytocin (10-100 m u ) and the post-injection EMG response ( P < 0.01). At the utero-tuba1 junction this relationship just failed to achieve significance (P < 0.1). However, at the ampullaryisthmic junction, dose of oxytocin and size of the post-injection response were not significantly related. The duration of response also varied between sites. At the ampulla and ampullary-isthmic junction EMG activity in response to oxytocin was significantly increased ( P < 0.01) above pre-treatment levels between 0 and 10 min after injection, whereas at both the utero-tuba1 junction and the uterus the response was more prolonged. At the utero-tuba1 junction activity had returned near to pre-treatment values within 20 min, whereas at the uterus there was no significant decrease in the stimulated activity throughout the 20-min period following oxytocin injection. At the ampulla and utero-tuba1 junction the sensitivity of the region to oxytocin injection was increased relative to that of the late luteal phase (Day - 3) for only one day, the day of oestrus itself ( P < 0.01; Table 1). However at the ampullary-isthmic junction and uterus the sensitivity relative to Day - 3 was increased for four days, throughout the period from Day -1 to Day +2.
Discussion These results demonstrate that oxytocin injected at physiological doses was able to increase EMG output from the oviduct and uterus of the ewe during and around oestrus. The response to oxytocin peaked on the day of oestrus itself, when concentrations of oxytocin receptors are maximal in both the myometrium (Sheldrick and Flint 1985; Ayad and Wathes 1989) and myosalpinx (Ayad et al. 1990). Recently Ayad et al. (1991) have shown, using autoradiography, that these receptors are found in both the endometrium and myometrium of the uterus but in the muscle wall only of the oviduct.
Oxytocin and Oviduct EMG Activity a t Oestrus in the Ewe
-2/3
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Fig. 4. Responses of the reproductive tract at the four recording points used (Fig. 1) over 7 days to injections of (a) saline vehicle or (b)25 m u of oxytocin (n = 4 ewes); 1, ampulla; 2, ampullary-isthmic junction; 3, utero-tubal junction; 4, uterus. Results from Days 2 and 3 before, and Days 2 and 3 after oestrus were similar and have been combined. Solid bars represent pre-injection (spontaneous) EMG activity, meaned for the four 5-min periods immediately preceding injection (n > 16 for each bar). Open bars represent post-injection responses during the times indicated by the key (n = 4-6 for each bar). 1=time of injection. Error bars are s.e.m. 0.0, P < 0.001; 0 0 , P < 0.01; 0, P < 0.05, significantly different from pre-injection values (Student's t-test). See also Table 1. Table 1. Analysis of electrical responses in the reproductive tract of the ewe following systemic administration of oxytocin Results are based upon numbers of electrical events > 50 pv counted in four consecutive 5-min periods following oxytocin injection. Each electrode placement is considered separately. EMG activity in response to oxytocin on different days was assessed by a multiple regression model after effects of ewe, pre-injection means, oxytocin dose and time after injection had already been taken into account. Values on each day were compared to those on Day - 3 (late luteal phase) and significance values demonstrate an increased (or decreased when labelled 'less') sensitivity of the tract on the days indicated, relative to Day - 3 . *PO.05) Day relative to oestrus -2 -1 0 1 2 3
Ampulla
Ampullary-isthmic junction
Utero-tuba1 junction
Uterus
ns. n.s.
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n.s.
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*
* * n.s.
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*
*
*
*
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C . L. Gilbert et al.
There are many qualitative descriptions of changing patterns of EMG activity during the oestrous cycle in the literature and these are remarkably consistent both between workers, and with the spontaneous EMG activity measured in the present experiments. However, this previous work has not systematically investigated the effects of oxytocin at physiological concentrations on the electrical activity of the reproductive tract in normally cyclic ewes. Plasma oxytocin concentrations at oestrus are generally low (Sheldrick and Flint 1981; Webb et al. 1981; Schams et al. 1982) and ovarian oxytocin content is minimal at this time (Wathes et al. 1986). However, Gilbert et al. (1991) have characterized release patterns for endogenous oxytocin at oestrus in more detail and demonstrated the existence of highamplitude (up to 31 pg mL-'), short-duration (1-4 min) pulses at this time; this finding is consistent with the results of McNeilly and Ducker (1972) and Mitchell et a[. (1982). These endogenous pulses (Gilbert et al. 1991) were of similar size and duration to pulses produced in anoestrous ewes when oxytocin was injected in the same concentrations as the present experiments (intravenous injections of 25 and 50 m u produced peak concentrations of 2.1 + 0.33 and 6.4 1 pg mL- (mean + s.e.) respectively 1-2 min later; V. J. Ayad and D. C. Wathes, unpublished observations). It may therefore be hypothesized that endogenous oxytocin pulses occurring in cyclic ewes near oestrus could affect uterine and oviduct EMG output in a similar way to the injections described here, and it is likely that changes in the excitability of reproductive tract muscle would be reflected in altered sperm and egg transport, which take place at this time. The lack of correlation between the endogenous oxytocin profile and EMG activity reported previously by Garcia-Villar et al. (1983) may be attributed to two factors. Firstly, the sampling frequency used would have been insufficient to detect reliably pulses of less than 5 min duration, and secondly, we show here that a single brief pulse of injected oxytocin may increase the EMG activity of the uterus (after a lag of about 1 min) for over 20 min. It is difficult to compare the overall intensity of response between different pairs of electrodes because local impedance and amplifier performance are important determinants of signal strength. However temporal variations in EMG activity at any one site reflect changes in muscle excitability and tentative predictions may be made as to the events being recorded. Phasic activity is likely to represent co-ordinated movements such as waves of peristalsis whereas more continuous activity may be an indicator of generally raised muscle tone or even spasm. Experiments employing simultaneous EMG and intraluminal pressure recordings (Harding et al. 1982) or strain-gauges placed between electrodes (Garcia-Villar et al. 1982) support these views. Nevertheless the relative contributions to any one part of the signal from longitudinal and circular muscle components remains unknown. An examination of data from the different electrode placements suggests that oxytocin did not exert a uniform effect on different regions of the reproductive tract. At the ampulla and uterine electrodes the dose of oxytocin was significantly related to the post-injection elevation in EMG output. However, the ampullary-isthmic junction and utero-tuba1 junctions showed no significant relationship between size of dose and intensity of response. In the utero-tuba1 junction this result was only just non-significant (P < 0.01), but in the case of the ampullary-isthmic junction the effect of dose did not approach significance although a stimulatory effect of oxytocin was clearly present (Fig. 4). The isthmic region is surrounded by a dense adrenergic nerve net (Brundin 1965) which may become excited as part of a sympathetic response to the presence of the experimenter, resulting in heightened muscle activity after any injection irrespective of dose. In support of this, a small (non-significant) but consistent elevation in activity above pre-injection values following administration of saline is seen on Day 0 at the ampullary-isthmic junction (Fig. 4). The duration of effect after injection was more transient in both the ampulla and ampullary-isthmic junction than in other areas. This may reflect lower overall receptor concentrations at oestrus (Ayad et al. 1990) than in the uterus (Ayad and Wathes 1989) or a different post-receptor response in this area.
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Oxytocin and Oviduct EMG Activity at Oestrus in the Ewe
The variability in the number of days of elevated sensitivity to oxytocin is perhaps the most interesting aspect of these results. This effect is not clearly seen in Fig. 4 as the 25 m u dose shown there in general produces a significant response on only the day of oestrus. However, Table 1 reveals that when all doses are considered, the ampullary-isthmic junction and uterus have elevated sensitivities over 4 days around oestrus including 2 days after oestrus, whereas the ampulla and utero-tuba1 junction are more sensitive on the day of oestrus only. The timing of elevated responses to oxytocin at the ampulla electrode coincides with increased fimbrial tone to assist the envelopment of pre-ovulatory antral follicles. After ovulation, oviducal fluid concentrations of oxytocin are likely to be raised because of the contribution from follicular fluid and granulosa and cumulus oophorus cells shed with ova into the oviduct (Wathes et al. 1986). Transport of egg(s) to the ampullary-isthmic junction is rapid in most species examined and may be measured in minutes (Harper 1961; Hunter 1974; Blandau and Verdugo 1976) and it is here that fertilization occurs. Elevated responsiveness to oxytocin was maintained at the ampullary-isthmic junction until 2-3 days after oestrus, which is the time at which embryos are permitted to pass through to the uterus (Winterberger-Torres 1961; Holst 1974). By contrast the utero-tuba1 area displayed heightened sensitivity to oxytocin only on the day of oestrus, when, it has been suggested, this area and the distal isthmus act as a 'sperm reservoir' before allowing sperm to progress to fertilize the newly released egg on the day following oestrus (Hunter and Nichol 1983; Hunter 1988). It is possible that oxytocin contributes to elevated muscle tone in these areas at these times. The stimulatory effect of oxytocin was superimposed upon a background of spontaneous EMG activity which also peaked at oestrus. This result is in agreement with previous reports in all parts of the reproductive tract of the ewe (cervix, Garcia-Villar et al. 1982; uterus, Van der Weyden 1983; uterus and oviduct, Ruckebusch and Bueno 1976; oviduct, Ruckebusch and Pichot 1975) and cow (Ruckebusch and Bayard 1975). High concentrations of circulating oestrogen are known to result in elevated EMG activity in both ovariectomized (Porter and Lye 1983) and anoestrous ewes (Ayad et al. 1989), and oestrogens increased gap junction numbers between muscle cells in rats (Garfield et al. 1980) and actomyosin content of myometrial tissues in rabbits (Michael and Schofield 1969). It is likely that a proportion of the high spontaneous EMG activity seen at oestrus is due to circulating oestrogens. In summary, this study has shown that oxytocin at physiological concentrations is capable of influencing uterine and oviduct EMG activity. In addition, the sensitivity of the reproductive tract parallels previous reports of the receptor numbers present at these times. This suggests that oxytocin may have an important role to play in gamete transport, but as the relationship between EMG output and the movement of tube contents was not assessed, this conclusion remains speculative. The results of Wathes et al. (1989) showed that ewes immunized against oxytocin had a highly significant reduction in pregnancy rate 20 days after mating when compared with controls, and one explanation for this reduced fertility could be an influence of oxytocin on tuba1 transport. Acknowledgments
We thank Mrs S. Fennel1 and Mr P. Rees for expert technical assistance, Mr R. Francis and his staff for care of the animals, and the Wellcome Trust and the AFRC for financial support. References Ayad, V. J., and Wathes, D. C. (1989). Characterization of endometrial and myometrial oxytocin receptors in the non-pregnant ewe. J. Endocrinol. 123, 11-18. Ayad, V. J., McGoff, S. A., and Wathes, D. C. (1990). Oxytocin receptors in the oviduct during the oestrus cycle of the ewe. J. Endocrinol. 124, 353-9.
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Ayad, V. J., Guldenaar, S. E. F., and Wathes, D. C. (1991). Characterisation and localisation of oxytocin receptors in the uterus and oviduct of the non-pregnant ewe using an iodinated receptor antagonist. J. Endocrinol. 128, 187-95. Blandau, R. J., and Verdugo, P . (1976). An overview of gamete transport-comparative effects. In 'Symposium on Ovum Transport and Fertility Regulation'. (Eds M. J. K. Harper and C. J . Pauerstein.) pp. 138-46. (Scriptor: Copenhagen.) Brundin, J . (1965). Distribution and function of adrenergic nerves in the rabbit fallopian tube. Acta Physiol. Scand. Suppl. 259 66, 1-57. Edqvist, S., Einarsson, S., Gustafsson, B., Linde, C., and Lindell, J.-0. (1975). The in vitro and in vivo effects of prostaglandins E, and Fz and of oxytocin on the tubular genital tract of ewes. Znt. J. Fertil. 20, 234-8. Fuchs, A. R., Fuchs, F., and Soloff, M. S. (1985). Oxytocin receptors in nonpregnant human uterus. J. Clin. Endocrinol. Metab. 60, 37-41. Fuchs, A. R., Behrens, O., Helmer, H., Liu, C.-H., Barros, C. M., and Fields, M. J. (1990). Oxytocin and vasopressin receptors in bovine endometrium and myometrium during the estrous cycle and early pregnancy. Endocrinology 127, 629-36. Garcia-Villar, R., Toutain, P. L., More, J., and Ruckebusch, Y. (1982). Spontaneous motility of the cervix in cyclic and ovariectomized ewes and changes induced by exogenous hormones. J. Reprod. Fertil. 66, 317-26. Garcia-Villar, R., Toutain, P . L., Schams, D., and Ruckebusch, Y. (1983). Are regular activity episodes of the genital tract controlled by pulsatile releases of oxytocin? Biol. Reprod. 29, 1183-8. Garcia-Villar, R., Schams, D., Alvinerie, M., Laurentie, M. P., and Toutain, P. L. (1985). Activity of the genital tract and plasma levels of oxytocin and cortisol at the time of mating in the ewe. J. Endocrinol. 105, 323-9. Garfield, R. E., Kannan, M. S., and Daniel, E. E. (1980). Gap junction formation in myometrium; control by estrogens, progesterone and prostaglandins. Am. J. Physiol. 238, C81-C89. Gilbert, C. L., Jenkins, K., and Wathes, D. C. (1991). Pulsatile release of oxytocin into the circulation of the ewe at oestrus, mating and in the early luteal phase. J. Reprod. Fertil. 91, 337-46. Guiloff, E., Ibarra-Polo, A. A., and Gomez-Rogers, C. (1974). Effect of oxytocin on the contractility of the human oviduct in vivo. Fertil. Steril. 25, 946-53. Harding, R., Poore, E. R., Bailey, A., Thorburn, G. D., Jansen, C. A. M., and Nathanielsz, P. W. (1982). Electromyographic activity of the nonpregnant and pregnant sheep uterus. Am. J. Obstet. Gynecol. 142, 448-57. Harper, M. J. K. (1961). The mechanisms involved in the movement of newly ovulated eggs through the ampulla of the rabbit fallopian tube. J. Reprod. Fertil. 2, 522-4. Holst, P . J. (1974). The time of entry of ova into the uterus of the ewe. J. Reprod. Fertil. 36, 427-8. Hunter, R. H. F. (1974). Chronological and cytological details of fertilization and early embryonic development in the domestic pig, Sus scrofa. Anat. Rec. 178, 169-86. Hunter, R. H. F. (1988). The Fallopian tubes: their role in fertility and infertility. (Springer-Verlag: Berlin, Heidelberg and New York.) Hunter, R. H. F., and Nichol, R. (1983). Transport of spermatozoa in the sheep oviduct; preovulatory sequestering of cells in the caudal isthmus. J. Exp. Zool. 228, 121-8. Maggi, M., Genazzini, A. D., Giannini, S., Torrisi, C., Baldi, E., Di Tomasso, M., Munson, P. J., Rodbard, D., and Serio, M. (1988). Vasopressin and oxytocin receptors in vagina myometrium and oviduct of rabbits. Endocrinology 122, 2970-80. McNeilly, A. S., and Ducker, H. A. (1972). Blood levels of oxytocin in the female goat during coitus. J. Endocrinol. 54, 399-406. Michael, C. A., and Schofield, B. M. (1969). The influence of the ovarian hormones on the actomyosin content and the development of tension in uterine muscle. J. Endocrinol. 44, 501-11. Mitchell, M. D., Kraemer, D. L., Brennecke, S. P., and Webb, R. (1982). Pulsatile release of oxytocin during the oestrous cycle, pregnancy and parturition in sheep. Biol. Reprod. 27, 1169-73. Noonan, J. J., Adair, R. L., Halbert, S. A., Ringo, J. A., and Reeves, J. J. (1978). Quantitative assessment of oxytocin-stimulated oviduct contractions of the ewe by optoelectronic measurements. J. Anim. Sci. 47, 914-18. Porter, D. G., and Lye, S. J. (1983). Partial reversal of the myometrial progesterone 'block' in the non-pregnant ewe in vivo by oestradiol 17P. J. Reprod. Fertil. 67, 227-34.
Oxytocin and Oviduct EMG Activity at Oestrus in the Ewe
Roberts, J. S., McCracken, J. A., Gavagan, J. E., and Soloff, M. S. (1976). Oxytocin-stimulated release of prostaglandin F2 from ovine endometrium in vitro: correlation with oestrous cycle and oxytocin-receptor binding. Endocrinology 99, 1107-14. Rorie, D. K., and Newton, M. (1965). Response of isolated human fallopian tube to oxytocin: a preliminary report. Fertil. Steril. 16, 27-32. Ruckebusch, Y., and Bayard, F. (1975). Motility of the oviduct and uterus of the cow during the oestrous cycle. J. Reprod. Fertil. 43, 23-32. Ruckebusch, Y., and Pichot, D. (1975). Effects of adrenergic drugs on sheep oviduct motility. Eur. J. Pharmacol. 33, 193-6. Ruckebusch, Y., and Bueno, L. (1976). An electromyographic study of uterotubal activity in the ewe. J. Reprod. Fertil. 47, 221-7. Schams, D., Lahlou-Kassi, A., and Glatzel, P . (1982). Oxytocin concentrations in peripheral blood during the oestrous cycle and after ovariectomy in two breeds of sheep with low and high fecundity. J. Endocrinol. 92, 9- 13. Sheldrick, E. L., and Flint, A. P. F. (1981). Circulating concentrations of oxytocin during the oestrous cycle and early pregnancy in sheep. Prostaglandins 22, 631-6. Sheldrick, E. L., and Flint, A. P. F. (1985). Endocrine control of uterine oxytocin receptors in the ewe. J. Endocrinol. 106, 249-58. Soloff, M. S. (1975). Oxytocin receptors in rat oviduct. Biochem. Biophys. Res. Commun. 65, 205-12. Toutain, P . L., Marnet, P . G., Laurentie, M. P., Garcia-Villar, R., and Ruckebush, Y. (1985). Direction of uterine contractions during oestrus in ewes: a re-evaluation. Am. J. Physiol. 249, R410-R416. Van der Weyden, G. C. (1983). Myometrial activity in the non-pregnant texel ewe. Ph.D. Thesis, University of Utrecht, Netherlands. Wathes, D. C., Guldenaar, S. E. F., Swann, R. W., Webb, R., Porter, D. G., and Pickering, B. T. (1986). A combined radioimmunoassay and immunocytochemical study of ovarian oxytocin production during the periovulatory period in the ewe. J. Reprod. Fertil. 78, 167-83. Wathes, D. C., Ayad, V. J., McGoff, S. A,, and Morgan, K. L. (1989). Effect of active immunization against oxytocin on gonadotrophin secretion and the establishment of pregnancy in the ewe. J. Reprod. Fertil. 86 653-64. Webb, R., Mitchell, M. D., Falconer, J., and Robinson, J. S. (1981). Temporal relationships between peripheral concentrations of oxytocin, progesterone and 13,14-dihydro-15-keto prostaglandin F during the oestrus cycle and early pregnancy in the ewe. Prostaglandins 22, 443-54. Winterberger-Torres, S. (1961). Mouvements des trompes et progression des oeufs chez la brebis. Ann. Biol. Anim. Biochim. Biophys. 1, 121-33. Zerobin, K., and Sporri, H. (1972). Motility of the bovine and porcine uterus and fallopian tube. Adv. Vet. Sci. Comp. Med. 16, 303-54.
Manuscript received 2 April 1991; revised 11 September 1991; accepted 10 January 1992
