Role of progesterone and oestradiol in the regulation of uterine oxytocin receptors in ewes J.

Zhang,

P. G. Weston and J. E. Hixon

Department of Veterinary Biosciences, University of Illinois College of Veterinary Medicine, 2001 S. Lincoln, Urbana, IL 61801, USA

Summary. The interaction between oestrogen and progesterone in the regulation of the uterine oxytocin receptor in sheep was evaluated by measuring the binding of oxytocin to membrane preparations of caruncular and intercaruncular endometrium and myometrium. Ovariectomized ewes were assigned in groups of five to each cell of a 4 \m=x\ 2 factorial design. The four treatments were (a) vehicle (maize oil) for 12 days, (b) progesterone (10 mg day\m=-\1)for 9 days, (c) progesterone for 9 days followed by maize oil until day 12 and (d) progesterone for 12 days. The two oestradiol treatments consisted of the administration of implants in the presence or absence of oestradiol. The ewes were killed on day 10 (group b) or day 13 (groups a, c and d) for collection

of uterine tissues. The response of the caruncular and intercaruncular endometrium to the treatments was similar. In the absence of oestradiol, treatment with progesterone continuously for either 9 or 12 days reduced the concentration of the oxytocin receptor in comparison with both the control and the progesterone withdrawal group (in which values were similar). The presence of oestradiol reduced the receptor concentrations in control and both 9- and 12-day continuous progesterone treatment groups, but enhanced the concentration in the progesterone withdrawal group. The myometrial oxytocin receptors responded in a similar way to those in the endometrium to progesterone treatment alone, but the addition of oestradiol produced no further effect. In conclusion, progesterone and oestradiol caused downregulation of the endometrial oxytocin receptor. On the other hand, progesterone withdrawal, similar to that which occurs during luteolysis, increased receptor density in the presence of oestradiol. Progesterone may influence the response of the myometrium to oxytocin by causing a reduction in receptor density.

Keywords: endometrium; myometrium; oestradiol; oxytocin receptor; progesterone; sheep

Introduction

Prostaglandin F2a (PGF2(I) is considered

to

be the uterine

luteolytic factor in ewes (McCracken et

ai, 1972). The pulsatile pattern in the secretion of PGF2u, which begins on day 12 or 13 of the cycle (Thorburn et ai, 1973; Flint & Sheldrick, 1983; Hooper et ai, 1986), appears to be an important component of its luteolytic activity (Schramm et al., 1983). Because the secretion of PGF2a is an established response of the uterine endometrium to oxytocin (Sharma & Fitzpatrick, 1974; Mitchell et al., 1975; Roberts et al., 1976), interest has focused on the role of oxytocin and its uterine receptor in regulating the pulsatile secretion of PGF2(I (Flint et al., 1990). In ruminants endometrial concentrations of the oxytocin receptor are sensitive to the action of steroids, the density of receptors generally being lowest during the luteal phase of the cycle Department of Veterinary Medicine, Northwestern Agricultural University, Yangling, Shaanxi, People's Republic of China. tReprint requests. *Present address;

highest at oestrus (Roberts et ai, 1976; Sheldrick & Flint, 1985; Fuchs et ai, 1990). This pattern would suggest that progesterone may suppress and oestrogen may induce the receptor. This suggestion is supported by the findings that oestrogen treatment allows the expression of oxytocininduced PGF,a secretion in anoestrous or ovariectomized ewes (Sharma & Fitzpatrick, 1974; and

McCracken et ai, 1981) and that the administration of a luteolytic dose of oestradiol benzoate results in an increased density of the uterine oxytocin receptor (Hixon & Flint, 1987). The role of progesterone may be more complex. The initial effect of this steroid in ovariectomized ewes is to suppress the effects of oestrogen on oxytocin-induced secretion of PGF2a, but after 10 days progesterone enhances the effects of oestrogen (McCracken et al., 1981). Homanics & Silvia (1988) could not demonstrate effects of progesterone on oxytocin-induced secretion of PGF2a, as measured by concentrations of its metabolite, 13,14-dihydro-15-ketoprostaglandin F,u (PGFM) after 10 days, but they did observe PGF2cj secretion in response to oxytocin after 15 days of progesterone treatment, and this response was enhanced by oestradiol. These observations suggest short-term suppressive effects of progesterone on oxytocin-induced secretion of PGF2a which disappear with longer periods of treatment and an interaction between progesterone and oestradiol to increase the responsiveness of the uterus to oxytocin. The finding that endometrial concen¬ trations of the oxytocin receptor were higher after 12 days than after 5 days of progesterone treatment is consistent with the notion that uterine responsiveness to oxytocin may vary with the duration of progesterone treatment. However, because oestradiol changes the pattern of oxytocininduced secretion of PGF2a without affecting receptor density (Vallet et al., 1990), the interaction between progesterone and oestradiol to enhance PGF2u secretion may extend beyond changes in oxytocin receptor concentrations. This study evaluated the interaction between progesterone and oestradiol in the regulation of the uterine oxytocin receptor by quantifying the specific binding of oxytocin to membrane preparations of caruncular and intercaruncular endometrium and myometrium. Receptor concen¬ trations were measured in ovariectomized ewes on the 10th day of treatment with progesterone or oestrogen or both, as this appeared to be the minimum time required to observe the priming effect of progesterone. Receptor concentrations were also measured on the 13th day of steroid treatment because this is about the time of progesterone dominance during the luteal phase of the cycle. The study also evaluated the effects of progesterone withdrawal, similar to that observed at the end of the luteal phase of the cycle, on the density of oxytocin receptors.

Materials and Methods Animals and treatments

Forty ewes of mixed breeds were housed in pens and fed hay and water ad libitum. The ewes were ovariectomized early during the anoestrous season and at least 6 weeks before the start of treatments. Five ewes were randomly assigned to each of eight treatment groups in which four treatment protocols were evaluated in the presence and absence of oestradiol. The treatments were (a) vehicle (maize oil) for 12 days; (b) progesterone for 9 days; (c) progesterone for 9 days followed by progesterone withdrawal until day 12; and (d) progesterone for 12 days. An i.m. injection of progesterone (10 mg; Sigma, St Louis, MO, USA) or 2 ml of its vehicle (maize oil) was administered twice a day at 07:00 h and 19:00 h beginning on day 1 of the experimental period to produce circulating concentrations of progesterone similar to those observed during the midluteal phase of the cycle (Homanics & Silvia, 1988). On the morning of day I, implants filled with oestradiol (Sigma, St Louis, MO, USA) or empty implants (placebos) were placed s.c. in the axillary region. The implants (0-33 cm i.d. 0-46 cm o.d. 3-5 cm long, 3 cm packed with steroid; Karsch et al., 1973, 1980) were made with Medical Grade Silastic Tubing (Storz, St Louis, MO, USA). Before insertion, implants were equilibrated overnight in 50 ml of a medium containing 1% bovine serum albumin in phosphate-buffered saline with constant agitation. Ewes were killed with a lethal intravenous injection of T-6I (Hoechst Roussel, Somerville, NJ. USA) on the 13th day after initiation of the treatments, except for ewes that were assigned to group b, which were killed on the 10th day. The uteri were then removed. mm i.d. At least 24 h before the first blood sample was taken, a Silastic catheter ( 2-2 mm o.d.) was inserted into an external jugular vein under anaesthesia induced with Biotal (thiamylal sodium, BioCeutic Division, Boehringer Ingelheim, St Joseph, MO, USA). The free end of the catheter was passed under the skin to a canvas pouch tied to the wool of the animal in the dorsum of the shoulder region. The catheter was filled with saline containing heparin

(200iuml '; Grade II, sodium salt, Sigma, St Louis, MO, USA). Blood samples (10 ml) were collected by syringe once daily at 19:00h, except the last sample which was taken just before the ewes were killed in the morning of day 10 or day 13. The blood samples were immediately centrifuged for 10 min at 1000 g and 4 C and the plasma was removed and stored frozen ( 20"C) for analysis of progesterone and oestradiol. Two additional ovariectomized ewes, maintained under identical conditions, were treated with an intravaginal sponge containing medroxyprogesterone acetate (60 mg; Veramix sheep sponge, Upjohn, Tuco Products Company, Orangeville, Ontario, Canada). Before removing the sponges on day 11, 100 µg of oestradiol (100 µ of a 3-67 mmol 1 ' solution in maize oil) was administered subcutaneously. A second injection of oestradiol was given on day 12 and the animals were killed on day 13 for collection of uterine tissues. —

Radioimmunoassays Progesterone. Progesterone was assayed in 0-2 ml of plasma extracted with petroleum ether as described by Weston & Hixon ( 1980). The assay sensitivity was 0-12 ng ml ' (calculated from 2 sd below the zero mass added point). Intra- and interassay coefficients of variation were 9-3 and 17-5%, respectively. ~

Oestradiol. The antiserum (482-8A) was generously provided by O. D. Sherwood. The preparation of the antiand its specificity have been described by Downing & Sherwood (1985). Oestradiol was extracted twice by shaking 2-4 ml plasma for 30 min the first time and for 15 min the second time with 8 ml and 5 ml benzene, respectively (nanograde, Mallinckrodt, St Louis, MO, USA). The extracts were combined and dried under air at 45°C. The residue was washed twice with 0-5 ml benzene:methanol (90:10, v/v). The combined washings were transferred onto a column which was made with a 5¿-inch Pasteur capillary pipette containing 0-5 g swelled Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA); the columns had been washed with at least 20 ml (v/v) of benzene:methanol. Oestradiol was eluted by the addition of 5 ml benzene:methanol (v/v). The first 2 ml were discarded, and the last 4 ml of solvent passing through the column were collected. Mean ( + SEM) recovery of [3H]oestradiol after chromatography was 86-0 ± 11%. The eluate was dried under air, redissolved in 600 µ of 001 mol phosphate-buffered saline 1 ' (PBS, pH 7-4) containing thimerosal (01 g 1 ') and 01% gelatin (J. T. Baker, Phillipsburg, NJ, USA), and incubated over¬ night at 4C. The steroid solution (250 µ ) was transferred into duplicate test tubes containing 50 µ of antiserum (1:80 000 dilution in PBS). After incubation for 1 h at 4°C, 50 µ of PBS containing 18 fmol [2,4,6,7,16,17-3H]oestradiol (150 Ci mmol '; Amersham, Arlington Heights, IL, USA) was added, and the assay tubes were incubated for another 2-5 h at 4ÜC. The [3H]oestradiol was subjected to chromatography as previously outlined before it was used in the assay. The range of the standard curve was from 0-3 to 200 pg of oestradiol (Sigma, St Louis, MO, USA). Free oestradiol was separated from bound by the addition of 250 µ of a stock solution containing 0-5% charcoal (Norit A, Fisher Scientific, Fair Lawn, NJ, USA) and 005% Dextran T-70 (Pharmacia, Piscataway, NJ, USA) in PBS. After a 15 min incubation at 4°C, the tubes were centrifuged for 30 min at 1500#. The supernatant was decanted into vials, and 4-5 ml of a scintillation cocktail containing 70% toluene (Fisher Scientific, Fair Lawn, NJ, USA), 30% Triton X-100 (Mallinckrodt, St Louis, MO, USA), 0-4% 2,5-diphenyloxazole (Research Products International, Mt Prospect, IL, USA) and 0-01% 2,2'(l,4-phenylene)bis(4-methyl-5-phenyloxazole) (J. T. Baker, Phillipsburg, NJ, USA) was added. The amount of [3H]oestradiol was quantified in a Packard TriCarb Liquid Spectrometer. The sensitivity of the assay was 0-3 pg ml1. The estimate of oestradiol concentrations from samples of pooled plasma from ovariectomized ewes that were included in each assay was 1 16 ± 0-10 pg ml '. The addition of 5, 10 and 25 pg of unlabelled oestradiol resulted in the recovery of 4-4 + 0-4, 9-1 + IT and 21-0 + l-4pg, respectively. Intra- and interassay coefficients of variation were 161% and 18-0%, respectively. serum

Determination of uterine Uteri

were

collected and

oxytocin receptor concentrations placed

on

ice

immediately after

the animals

were

killed. About 4 g each of caruncular

endometrium, intercaruncular endometrium and myometrium were collected. Membrane fractions were prepared by ' homogenization in 25 mmol Tris-HCl 1 ', pH 7-6, containing 0-25 mol sucrose 1 and 1 mmol EDTA f, followed by differential centrifugation (Sheldrick & Flint. 1985). Membrane preparations were stored at 70"C until assay. membrane Oxytocin receptor concentrations were determined as described by Sheldrick & Flint (1985). Briefly, ' —

fractions representing 25 50 µg of protein were incubated with 5 nmol [3,5-3H-Tyr]oxytoxin 1 (specific activity 37-9 Ci mmol1; NEN Research Products, Boston, MA, USA) for 60 min in the presence of 1 mmol MnCl2 1 '. Nonspecific binding was assessed in the presence of 1 µ unlabelled oxytocin 1 ' (Peninsula Laboratories, Belmont, CA, USA). Free [3H]oxytocin was separated from bound by filtration through Millipore filters (Durapore, Type GVWP; Bedford, MA, USA) under vacuum. Receptor concentrations were determined at one saturating ligand concentration only. No information was therefore obtained on receptor affinity.

Assay validation. Membrane fractions were prepared from pooled samples of caruncular and intercaruncular endometrium and myometrium obtained from the uteri of two ewes treated with Veramix sponges and oestradiol. Membrane suspensions (containing 50 µg protein) were assayed at 4, 25 and 37°C for 5, 10, 30. 60 and 90 min. Specific binding was highest at 25°C and equilibrium was obtained by 60 min (Fig.' 1) which were the conditions selected for the assay. Oxytocin binding increased with [3H]oxytocin up to 50 nmol 1 (Fig. 1). Scatchard plots (Scatchard, 1949)

8000

3000

4000 O 2000

0-0

50

2-5

Oxytocin

concentration (nmol I

0-06

004

002

20

Bound

40

60

oxytocin (pmol

I

80

1)

Fig. 1. Effects on specific (total minus nonspecific) binding of [3H]oxytocin in ewes of (a) time and temperature (O, 4°C; ·, 25°C; , 37°C), (b) ligand concentration and (c) concentration of the receptor fraction protein. Binding data plotted by the method of Scatchard (1949) are illustrated in (d). of binding parameters were linear (r 0-98). The apparent dissociation constant (Ká) of 1-35 nmol 1 ' and binding capacity of 302-8 fmol mg~ ' protein are comparable to the values reported by others for these characteristics of the uterine oxytocin receptor (Sheldrick & Flint, 1985; Ayad & Wathes, 1989). Specific binding of [3H]oxytocin to uterine membranes increased linearly (r 0-99) as the amount of membrane protein increased from 0 to 200 µg (Fig. 1). Incubations of fixed amounts of pooled membrane protein (50 µg) and [3H]oxytocin (5 nmol 1 ') in the presence of 0 to 1 µ unlabelled oxytocin 1 resulted in the displacement of labelled oxytocin from the membrane preparations. Maximum displacement was observed with 01, 0-5 and 1 µ unlabelled oxytocin I"1 (data not shown). It was concluded that amounts of oxytocin in excess of 01 µ 1_1 were sufficient to displace all the bound [3H]oxytocin from the oxytocin receptors, and 1 µ 1~ ' of unlabelled oxytocin was chosen to estimate the nonspecific binding in this study. The tissue specificity of oxytocin binding was evaluated by the preparation of membrane fractions from the oviduct, caruncular and intercaruncular endometrium, myometrium, vagina, intestine, muscle and liver from three ewes that had been treated with progesterone (10 mg, twice a day) for 10 days. Receptor concentrations (fmol per mg protein) for oviduct, caruncular and intercaruncular endometrium and myometrium were 42-9 ± 70, 621 + 10-4, 46-4 ±8-0 and 11-7 + 30, respectively. Specific binding of oxytocin was not observed in the other tissues. These data are consistent with the identification of single, high-affinity oxytocin receptors in the uterus (Sheldrick & Flint, 1985; Ayad & Wathes, 1989) and the oviduct (Ayad el al., 1990) of the ewe. =

=

'

Statistical

analysis

Statistical

procedures with a statistical software package distinguish group differences. Plasma hormone concentrations were analysed as a two-way factorial design with repeated measures over time according to a analyses

were

carried out

using general

(SAS Institute Inc., 1982). Single degree of freedom

linear model

contrasts were used to

model that included the effects due to progesterone and oestradiol treatments, the interaction between oestradiol and progesterone, the residual variance due to subjects, time, the two-way interactions between progesterone or oestradiol treatment and time and the three-way interaction between progesterone, oestradiol and time. The estimates of error reported for circulating concentrations of progesterone and oestradiol (Figs 2 and 3) are the square root of the value obtained by dividing the mean square error by the number of observations in the mean (Steel & Torrie, 1980). Data for oxytocin receptor concentrations were analysed as a two-way factorial design according to a model that included effects due to progesterone and oestradiol treatments and the interaction between progesterone and oestradiol.

O—CK

-o—o

4

5

6

7

8

9

10

11

12

13

Day of treatment concentrations of oestradiol in ewes treated with oestradiol (o) or placebo (·) 2. Plasma Fig. implants, averaged over four progesterone treatments, in blood samples collected on each day of the experimental period. The sem was 1-2 pg ml"1.

30

(b)

2-5

20 o—o^_ /

15 1-0-

E

0-5 '

00

„„.^e-e-^î-e^S^clrSx^,

Fig. 3. Plasma concentrations of progesterone in ewes from blood samples collected on each day of the experimental period in groups treated with (a) maize oil for 12 days, (b) progesterone ( 10 mg day ' ) for 9 days, (c) progesterone for 9 days followed by progesterone withdrawal until day 12, (d) progesterone for 12 days, and oestradiol (o) or placebo (·) implants. The sem was "

0-2ngmr'.

Results

Systemic concentrations of oestradiol and progesterone Oestradiol. Concentrations of oestradiol among the four progesterone treatment protocols were similar (P > 0-40) and were combined for presentation (Fig. 2). Mean concentrations of oestradiol were higher (P < 0 01) in the groups that received the oestradiol-containing implant than in

groups that received the placebo. There was a sharp increase in oestradiol concentrations after administration of the implant, after which concentrations decreased to values that ranged between 4 and 8 pg ml"'. Oestradiol concentrations over the period of the experiment remained low ( 0-9).


0-9) in the myometrium (Fig. 5). When compared to the vehicle-treated groups, the effect of continuous progesterone treatment for 9 or 12 days was to reduce the density of myometrial oxytocin receptors (P < 001). These groups also had lower oxytocin receptor concentrations than the groups where progesterone was withdrawn after day 9 (P < 001) Withdrawal of progesterone had no effect on myometrial concentrations of the oxytocin receptor when compared with the vehicle-treated groups.

Discussion The circulating concentrations of progesterone and oestradiol that we observed were similar to those reported for the luteal phase of the oestrous cycle (Hansel et al., 1973; Karsch et al., 1980) and to those reported by Homanics & Silvia (1988) in their study of oxytocin-induced secretion in ovariectomized ewes. Withdrawal of progesterone resulted in declining progesterone concen¬ trations and increased oxytocin receptor concentrations in both endometrial tissues, and the greatest increase in receptor density occurred in the presence of oestradiol. A similar relationship between progesterone withdrawal and oestradiol on the density of oxytocin receptors in ovari¬ ectomized ewes was observed after stopping a 5 day infusion of progesterone (Leavitt et al., 1985). In this study, declining concentrations of progesterone were associated with increased nuclear oestrogen binding. Because a positive correlation was observed between nuclear oestrogen binding and oxytocin receptor concentrations, the increase in oxytocin receptor density may have been an oestradiol-mediated event (Leavitt et al., 1985). However, our finding that oestradiol reduced receptor concentrations in the groups treated continuously with progesterone or with maize oil would suggest a more complex role for oestradiol. In fact, our data suggest that oestradiol and

progesterone may act together to downregulate the receptor, which may explain why low concen¬ trations of the receptor are found during most of the luteal phase of the oestrous cycle in sheep and

cattle (Roberts et al., 1976; Sheldrick & Flint, 1985; Fuchs et ai, 1990). Oestradiol may be expected to increase receptor density because oestradiol increases oxytocin-induced secretion of PGF,U in anoestrous (Sharma & Fitzpatrick, 1974) and ovariectomized ewes (McCracken et ai, 1981). However, these effects of oestradiol may not be entirely mediated by changes in receptor density since Vallet et al., (1990) found that the administration of oestradiol changed the pattern of PGFM secretion associated with the administration of oxytocin without an increase in receptor density. Nevertheless, the fact that effects of oestradiol on receptor concentrations varied from downregulation (except after progesterone withdrawal, as observed in the current study) to no effect (Vallet et ai, 1990) to upregulation (Hixon & Flint, 1987) makes it difficult to define clearly the effects of oestradiol in the regulation of the oxytocin receptor. Administration of progesterone for 9 and 12 days reduced oxytocin receptor concentrations in caruncular and intercaruncular endometrium, which would suggest that the effect of progesterone is to suppress synthesis of the oxytocin receptor. When ovariectomized sheep were pretreated with a progestagen for 10 days and oestradiol for 2 days, progesterone administration for 12 days was associated with high concentrations of the oxytocin receptor, and the concentration after 12 days was greater than that after 5 days of treatment (Vallet et ai, 1990). The differences between our study and that of Vallet et al. (1990) in the effects of progesterone administration may be due to differences in the treatment protocols used. The finding that concentrations of the receptor were greater after 12 days than after 5 days of treatment (Vallet et ai, 1990) is interesting because it suggests that the initial effect of progesterone was downregulation of the receptor and that the uterus then became refractory to the suppressive effects of progesterone. The finding that oxytocininduced secretion of PGFM was observed after 15 days, but not after 10 days, of progesterone treatment (Homanics & Silvia, 1988) is consistent with this idea. In the current study, the most dramatic increase in receptor concentrations was observed after progesterone withdrawal in the presence of oestradiol. It is therefore possible that the high concentrations of the oxytocin receptor in the endometrium that have been reported from about the time of luteolysis through oestrus in ruminants (Roberts et al., 1976; Sheldrick & Flint, 1985; Fuchs et ai, 1990) may be due to a combination of factors that include refractoriness to the suppressive effects of progesterone, falling concentrations of progesterone due to luteolysis and oestradiol secretion by preovulatory follicles. Concentrations of endometrial oxytocin receptors were greater in maize oil-treated ewes than in ewes treated continuously with oestradiol and progesterone either alone or in combination. High concentrations of the oxytocin receptor in ovariectomized ewes that had not received any steroid treatments were also observed by Vallet et al. (1990). These findings are of interest because they suggest that the genetic mechanisms responsible for synthesis of the receptor are expressed in the absence of ovarian steroids. However, oxytocin failed to elicit the secretion of PGF2u under these conditions (Vallet et ai, 1990), which indicates that the receptors were not linked to the secretion of

PGF2a.

Concentrations of the oxytocin receptor in the myometrium were lower in the groups treated continuously with progesterone than in the vehicle-treated groups or following withdrawal of progesterone. This finding is consistent with the observations that the density of the receptor decreased during the luteal phase of the ovine oestrous cycle (Sheldrick & Flint, 1985). Oestradiol was without effect which would suggest that the increase in receptor density that occurs during oestrus (Sheldrick & Flint, 1985) may be due, in part, to falling concentrations of progesterone resulting from luteolysis. The failure of oestradiol to influence myometrial concentrations of the receptor is in contrast to the increase in receptor concentrations that were observed after the administration of oestradiol during the midluteal phase of the cycle (Hixon & Flint, 1987). These contrasting observations, together with data suggesting a negative correlation between circulating concentrations of oestradiol and concentrations of the receptor in bovine myometrium (Fuchs et ai, 1990), suggest a complex role for oestradiol in the regulation of the receptor. The failure

of oestradiol to influence concentrations of the oxytocin receptor in the myometrium, when contrasted to its effects in the endometrium, indicates that, under the conditions of this study, differences exist between the two uterine compartments in the regulation of the oxytocin receptor. Differences between the endometrium and myometrium in the regulation of the oxytocin receptor have also been suggested for the bovine uterus (Fuchs et al., 1990). In conclusion, the response of caruncular and intercaruncular endometrium to ovarian steroids was similar. Oestradiol treatment increased the concentration of the oxytocin receptor after progesterone withdrawal, but otherwise, the effect of oestradiol was to reduce the density of the receptor. Progesterone also reduced receptor concentrations when compared with vehicle-treated animals, and the lowest receptor concentrations were observed in the groups treated with both ovarian steroids. These findings suggest that progesterone and oestradiol serve to downregulate the receptor during the luteal phase of the cycle and that progesterone withdrawal with luteolysis contributes to an increase in receptor density during oestrus. Progesterone reduced the density of the receptor in the myometrium while oestradiol was without effect. Failure of oestradiol to influence myometrial concentrations of the receptor indicates that differences between the myometrium and endometrium may exist in the regulation of the density of the oxytocin receptor. We are indebted to A. P. F. Flint for his help in setting up the oxytocin receptor assay and for his many useful suggestions. We thank J. Bergren, D. Greathouse, J. Smith and D. Walker for their assistance. This work was supported by USDA Grant 87-CRCR-1-2537. References

Ayad, V.J., McGoff,

S.A. & Wathes, D.C. (1990) Oxy¬ tocin receptors in the oviduct during the oestrous cycle of the ewe. J. Endocr. 124, 353-359. Ayad, V.J. & Wathes, D.C. (1989) Characterization of endometrial and myometrial oxytocin receptors in the non-pregnant ewe. J. Endocr. 123, 11-18. Downing, S.J. & Sherwood, O.D. ( 1985) The physiological role of relaxin in the pregnant rat. III. The influence of relaxin on cervical extensibility. Endocrinology 116, 1215-1220. Flint, A.P.F. & Sheldrick, E.L. (1983) Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep. J. Reprod. Feri. 67, 215-225. Flint, A.P.F., Sheldrick, EX., McCann, T.J. & Jones, D.S.C. (1990) Luteal oxytocin: characteristics and control of synchronous episodes of oxytocin and PGF-2a secretion at luteolysis in ruminants. Dom. Anim. Endocrinol. 7, 111-124.

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dose of oestradiol benzoate on uterine oxytocin receptor concentrations, phosphoinositide turnover and prostaglandin F-2a secretion in sheep. J. Reprod. Fert. 79, 457-467. Homanics, G.E. & Silvia, W.J. (1988) Effects of pro¬ gesterone and estradiol-17ß on uterine secretion of prostaglandin F-2u in response to oxytocin in ovariectomized ewes. Biol. Reprod. 38, 804-811.

Hooper, S.B., Watkins, W.B. & Thorburn, G.D. (1986) Oxytocin, oxytocin-associated neurophysin, and pros¬ taglandin F-2a concentrations in the utero-ovarian vein of pregnant and nonpregnant sheep. Endocrin¬ ology 119,2590 2597. Karsch, F.J., Dierschke, D.J., Weick, R.F., Yamaji, T., Hotchkiss, J. & Knobil, E. (1973) Positive and nega¬ tive feedback control by estrogen of luteinizing hor¬ mone secretion in the rhesus monkey. Endocrinology 92, 799-804. Karsch, F.J., Legan, S.J., Ryan, K.D. & Foster, D.L. (1980) Importance of estradiol and progesterone in regulating LH secretion and estrous behavior during the sheep estrous cycle. Biol. Reprod. 23,404-413. Leavitt, W.W., Okulicz, W.C., McCracken, J.A., Schramm, W. & Robidoux, W.F., Jr (1985) Rapid recovery of nuclear estrogen receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal. J. Steroid Biochem. 22, 687-691. McCracken, J.A., Carlson, J.C.. Glew, M.E., Goding, J.R., Baird, D.T., Green, K. & Samuelsson, B. (1972) Prostaglandin F-2a identified as a luteolytic hormone in sheep. Nature 238, 129-134. McCracken, J.A., Schramm, W., Barcikowski, B. & Wilson, L., Jr (1981) The identification of prosta¬ glandin F-2a as a uterine luteolytic hormone and the

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Received 21 December 1990