Oxytocin binding in the uterus of the cycling mare

Refereed

OXYTOCIN BINDING IN THE UTERUS OF THE CYCLING MARE C.L. Stull 1, Phi) and J.W. Evans 2, PhD

SUMMARY

INTRODUCTION

Excised uteri from 22 reproductively sound cycling mares were divided into 4 groups according to the stage of the estrous cycle of each mare and analyzed for changes in the number of oxytocin binding sites and their affinity. The number of binding sites in the myometrial layer of the uterus was greater (P<.01) than in the endometrial layer, with an approximate 3-fold difference. The number of oxytocin binding sites was greater (P<.05) during Days 1417, post-ovulation, in both the endometrial and myometrial uterine tissue samples than during Days 2-5, Days 6-13 or Days 18-1. The affinity constants were greater (P<.01) in the endometrium than myometrium, but no difference (P>.05) was found in the four groups throughout the estrous cycle for endometrium or myometrium. In view of the known hormonal events occurring in the estrous cycle of the mare, along with in vitro data demonstrated with other species and the mare, the increase in oxytocin binding sites during Days 14-17 may enhance the production of the luteolytic hormone, prostaglandin (PFG ac~). This may be due to the increasing concentration of estradiol, which has a stimulatory effect on estrogen and oxytocin binding sites and thus promotes increased PGFzc~ synthesis. Authors' address: 1Department of Animal Science, University of California, Room218, ttart ttall, Davis, California95616. 2Animal Science Department, Texas A&M University, College Station, Texas77843 The authors gratefully acknowledge the technical advice of Dr. Melvyn Soloff along with the laboratory assistance of Shawn Robillard and Sam Balwin. The oxytocin was a gift from Sandoz Pharmaceuticals, E. Hanover,New Jersey.

Oxytocin is synthesized in the hypothalamus and stored with its specific neurophysin in the posterior pituitary. Recently, studies have shown that the gonads of some mammals, especially cows and ewes, may contain large amounts of oxytocin.l°,3s-When released into peripheral circulation, oxytocin causes contraction of the smooth muscle in the oviduct and uterus, and myoepithelial cells of the mammary gland. Oxytocin binding sites have been identified in the uterus. The location of the binding sitcs within uterine tissues may differ with species. In the rat, 35 and SOW, 36 binding sites were found only in the myometrium, while in the ewe, 23 and human, ~2 sites were found in both the endometrial and myometrial fractions. Using autoradiographic techniques, oxytocin binding sites were localized in smooth muscle cells of rat oviduct, while binding sites were not found in the luminal epithelium. 34 More specifically oxytocin binding sites appear to be located on the plasma membrane of the smooth muscle cells. 4 Concentration of uterine oxytocin binding sites change throughout the estrous cycle, gestation and parturition. In women, the number of uterine oxytocin binding sites increased throughout pregnancy and reached a maximum at parturition. 11 In rats, uterine oxytocin binding sites show a large surge in concentration at term and during labor, and then abruptly decline after birth.32 In ewes, the number of oxytocin binding sites rose to a peak at estrus in both the endometrium and myometrium.23 Oxytocin may interact with many other hormones during

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EQUINE VETERINARY SCIENCE

the estrous cycle.23,25,28,3°,33 Endometrial tissue from ewes stimulated with oxytocin, significantly increased its rate of PGF2ct production. 23 King and Evans (1984) investigated the influence of oxytocin on endometrial PFG2a production in vitro during the estrous cycle of the mare. 16 PGF2~t production level was maximal in tissue obtained on Day 14, around the time of luteolysis in the mare. When oxytocin stimulated PGF2ct production, a small increase above basal production occurred on Days 5 and 16 whereas a noticeable increase occurred on Day 20. No further investigations into the role of oxytocin receptors in the uterus of the mare have been made. This investigation was undertaken to examine the concentration and affinity of uterine oxytocin binding sites during the estrous cycle of the mare, and to determine its possible significance in accordance with other hormonal and physiological changes. The normal sequence of interactions between circulating hormones and their receptors must be understood before pathological changes, which may result in reduced fertility, can be evaluated and effectively treated.

follows from the characterization of the oxytocin binding sites in the rat oviduct.31 Known amounts of tissue diluted in 200 lal of assay buffer were incubated in the presence of varying amounts (1,550 to 87,000 dpm) of tritiated oxytocina (34.4 Ci/mmol) in 10 lal of assay buffer. Nonspecific binding was determined by the addition of 10 lal of excess non-radioactive oxytocin (250 rig) to the incubation solution. The total bound and non-specific bound tubes were done in triplicate at 9 different concentrations of tritiated oxytocin. Incubation was terminated by the addition of 10 ml of ice-cold assay buffer without gelatin. The suspended tissue was then collected on glass microfiber filter paper (Whatman GF/F) by a rapid filtration process. The filters were dried and placed in 5 ml of scintillation cocktailb and then counted by scintillation spectrometry. Specific binding was determined by subtracting counts for non-specific binding (tubes with excess oxytocin) from those for total binding. The data were analyzed by Scatchard plots z8 to determine the affinity constant (Ka) and binding site number concentration (Figure 1). Protein concentrations were performed using the BioRad Assayc.

MATERIALS AND METHODS

Tissue Collection and Preparation The uteri from 22 reproductively sound cycling mares were excised and frozen on dry ice at the time of slaughter and transported to the laboratory for processing and analysis. Daily teasing and rectal palpations were performed previous to slaughter to determine regularity of the cycle, ovulation and specific stage of the estrous cycle. Also ovaries were carefully examined for follicular and luteal developments after slaughter. Excised uteri were divided into four groups according to the determined stage of the estrous cycle as Days 18-1, Days 2-5, Days 6-13 and Days 14-17 post-ovulation, with 5, 4, 6 and 7 uteri per group, respectively. Each uterus was thawed in a water bath at 22°C. The endometrium was carefully dissected from the myometrium. Subsequently, the myometrium was scraped with a scapel to remove any remaining endometrium which was discarded. The separated tissues were then homogenized in 5 volumes of ice-cold buffer containing 10mM Tris HC l, 1mM EDTA and .5mM dithiothreitol (pH 7.4) in a Waring laboratory blender. The homogenate was strained through one layer of cheesecloth and centrifuged at 1000 x g for 10 mins at 4°C. The resulting pellet was discarded and the supematant centrifuged at 100,000 x g for 60 rnins at 4°(2 to obtain an isolated membrane fraction. The resulting pellet was weighed, resuspended in assay buffer (50 mM Tris-maleate, pH 7.6, 10mM MnC12 and .1% gelatin from swine skin), aliquoted, and stored frozen at -70°C. Binding Assay Procedure Both the endometrial and myometrial tissue samples of each mare were individually subjected to an oxytocin binding site assay. The binding site assay was modified as Volume 6, Number 3

Characterization of Assay The previously described assay was characterized for equine uterine tissue by assessing optimal incubation, and hormonal and binding site specificity using methods previously described.8 Incubation time and temperature were varied to determine optimal incubation conditions. Tissue concentration was varied from 2.5 mg/ml to 10 mg/ml. An aliquot of 200 gl of suspended tissue was used per assay tube. In the assessment of non-specific binding, amounts of non-radioactive oxytocin were varied to determine maximum inhibition. Specificity of binding tritiated oxytocin to plasma membrane binding sites in the endometrium and/or myometrium was evaluated by incubating membrane fractions (35 lag of protein) of nontarget tissues including heart, spleen, skeletal muscle, duodenum, lung, testes, liver, pancreas, ovary and kidney. Non-specific binding was evaluated by adding excess non-radioactive oxytocin to the incubation solution. Hormone specificity was assessed by the ability of nonradioactive hormones to inhibit the binding of vitiated oxytocin to binding sites. Tritiated oxytocin was held constant while increasing the amounts (1 ng to 10,000 rig) of rat TSH (NIH I-3), equine LH (Papkoff E98A), PMSG (Sigma Chemical Co.), LRF (U.S. Biochemical Co), ADH (Dr. L. Keil, NASA, Ames Research Center), Vasotocin (Dr. L. Keil, NASA, Ames Research center), and Angiotensin I and II (Sigma Chemical Co.).

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3H-OXYTOCIN BOUND (xlO -11 Mlpg protein) Figure 1. Scatchard plot to quantify the number (Rt) and affinity (Ka) of oxytocin binding sites in the endometrium (o) and myometrium (.) of the mare. The values of Ka, Rt and r, respectively, are 6.45 x 1011 x M -1, 0.32 x 1011M, and 0.94 for endometrium and 3.09 x 1021 x M -1, 0.67 x 10-11M and 0.98 for myometrium.

Statistical Analysis

Statistical analysis was performed using a one-way analysis of variance and Duncan's Multiple Range test37 comparing the concentration of oxytocin binding sites in the endometrium and myometrium of the uterus for all four cycle groups. A paired t-test was performed on affinity and binding site concentration data to compare endometrial tissue with myometrial samples. Correlation coefficients for Scatchard plots were determined by regression analysis. Data are expressed as mean + SEM. RESULTS

Incubation of equine uterine tissue in a water bath at 22°C was determined to provide optimal conditions for maximum oxytocin binding without proteolytic degradation (Table 1). Tissue concentration could be varied from 2.5 mg/ml to 10 rag/m1 while maintaining linearity of response, thus 5 mg/ml was utilized in the assays. While varying the amounts o f non-radioactive oxytocin for the assessment for non-specific binding, maximum inhibition was determined to occur at the concentrations in excess of 125 ng per tube. Therefore, to ensure an excess of nonradioactive oxytocin, 250 ng were added to each non-specific binding tube. Tissue specificity was determined by binding tritiated oxytocin to the target tissues, endometrium and myometrium, and also non-target tissues (Figure 2). Binding was observed in the tissues from the endometrium, myometrium and duodenum, but the remaining tissues showed no response. The assessment of hormone specificity 116

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by increasing the amounts of various non-radioactive hormones is represented in Figure 3. When compared to unlabelled oxytocin, at least a 100 fold greater concentration of VAS, LH and greater than 1,000 for the other unlabelled hormones was required to displace 50% of the binding of 3H-labelled oxytocin. Oxytocin binding sites were found in both the endometrium and myometrium layers of the mare's uterus. A single population of binding sites was found in each uterine layer indicated by a straight line on the individual Scatchard plots (Figure 1). Using a paired t-test, the concentration of myometrial oxytocin binding sites was greater (P<.01) than in the endometrium, with a mean difference of .397 x 10-HM/~tg protein + .053. There were approximately three times the concentration of binding sites in myometrium (.575 x 10-11M/lag protein + .062) than in TABLE 1 Specific Counts (dpm) Bound with Increasing Incubation Length at 22°C when 39,763 dpm Were Added Per Tube T i m e (Minutes)

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endometdum (.179 x 10-UM/~tg protein +.031) at all stages of the estrous cycle (Figure 4). The concentration of oxytocin binding sites in the endometrium or myometrium fluctuated throughout the estrous cycle (Figure 4). Oxytocin binding site concentration in the endometrium or myometrium was greater (P<.05) for the Days 14-17 group than in any of the other three groups (Days 18-1, Days 2-5, Days 6-13). No significant difference was found (P>.05) between the remaining three groups in either the endometrium or myometrium. The affinity (Ka) of the oxytocin binding sites was greater (P<.01) in the endometrium than the myometrium. The mean difference was 3.15 x 1011M-1 +.66. The Ka in the endometrium (6.04 x 10UM -z +.60) was about twice the Ka in the myometrium (2.89 x 10UM -1 +.24). Comparison of the 4 groups during the estrous cycle shows no difference (P>.05) in the Ka's for either the endometrium or myometrium (Figure 5). Therefore, the binding sites in the endometrium have a higher affinity for the oxytocin molecule than in the myometrium, but there was no fluctuation in affinity throughout the cycle in either of the tissue layers within the uterus.

DISCUSSION Oxytocin binding sites were found in equine tissues from the uterus and duodenum. The response of the duodenum was unexpected and may be partially attributed to its smooth muscle composition. The mare is similar to the ewe23 with respect to localization of oxytocin binding sites in both uterine layers. In contrast to the ewe, a greater number (P<.01) of oxytocin binding sites in the mare were located in the myometrium than the endometrium. Although, myometrial oxytocin binding sites are essential V o l u m e 6, N u m b e r 3

for contraction during parturition their role, if any, in the modulation of the mare's estrous cycle is unknown. The response of ovarian steroid hormones on uterine oxytocin binding sites has been examined in a number of species, but not in the mare. The stimulatory effect of estrogen and the inhibitory effect of progesterone on uterine oxytocin binding sites has been demonstrated in rats aa,33and rabbits.20,3o Estrogen also induces a rise in its own receptors..2,26 In the mare, an increase (F<.01) in oxytocin binding was observed during Days 14-17, post-ovulation. During this same phase of the estrous cycle, plasma estrogen concentrations were found to rise on Days 14-15 and reach a peak on Day 18, post-ovulation.iS,aa,22 Prostaglandin Faa production in the endometrium which is associated with luteolysis, peaks between Days 14-17 in the mare.SJ 9 Progesterone levels were found to decrease rapidly with the first minor elevation in a prostaglandin metabolite39 Thus, the endocrinology of the mare between Days 14-17 would support the observed increase in oxytocin binding. The physiological significance of the observed increase in oxytocin binding may parallel the role of oxytocin binding sites in the uterus of the ewe. Exogenous oxytocin had the ability to induce the secretion of PFG2a throughout the entire estrous cycle of the ewe, but a pronounced increase in PFG2a secretion occurred late in the luteal stage around Day 14.24 In a later study, the number of uterine oxytocin binding sites was found to increase late in the cycle of the ewe. 13 The interaction of oxytocin with its binding sites in the endometrium may evoke PFG2~ secretion,a3 The control of the oxytocin receptors in the ewe appears to be regulated by estrogen and progesterone concentrations. Experiments utilizing an autotransplanted uterus in ovariectomized ewes concluded that while progesterone inhibits the estrogen117

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Figure 4 Concentration of oxytocin binding sites in the endometrium (slashed bars) and myometrium (open bars) of the mare's uterus (mean + SEM). Day 0 is designated as the day of ovulation. Numbers within bars are number of mares per group. (*P<0.05 between myometrium groups; ** P<0.05 between endometrium groups).

induced oxytocin binding sites, it loses its inhibitory ability after about 10 days at which time the estrogen stimulatory effects on oxytocin binding sites was evidenced by increased PGF2c~ secretions.24 Similar events may also occur in the mare. Luteolysis failed to occur in response to exogenous oxytocin when given early in the mares' luteal phase and estrogen also failed to exhibit oxytocin induced luteolysis,z9 Failure of luteolysis to occur during the early luteal phase was probably due to the inhibitory effects of progesterone on oxytocin binding sites, and therefore, the lack of stimulation on PFG2a secretion and luteolysis. The influence of oxytocin on PFG2o~ production by the mare was studied by incubating endometrial tissue biopsied during various stages of the estrous cycle in the presence and absence of oxytocin.I6 Peak basal PFG2a production occurred on Day 14, around the time of luteolysis. While oxytocin induced PFG2a production started to increase on Day 16, maximal stimulation occurred on Day 20, declined by Day 5 and was minimal during Days 10-14. The maximal stimulation occurring on Day 20 may not be as physiologically significant, as the initial increase observed on Day 16. In the cattle and sheep there is evidence for luteal secretion of oxytocin,27,~0 but no investigations have been made in the mare. Flint and Sheldrick (1983) proposed a positive feedback loop between ovarian oxytocin and uterine PFG2a which would result in a rapid and complete luteal regression and termination of each digression of secretion.lO If this positive feedback loop occurs in the mare, it may provide an explanation for the ability of oxytocin to continue to induce PFG20~secretion although luteolysis has occurred36 118

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Fig. 5. Affinity of oxytocin binding sites in the endometrium (slashed bars) and myometrium (open bars) of the mare's uterus (mean + SEM). Day 0 is designated as the day of ovulation.

The affinity of oxytocin sites in the endometrium was greater than the myometrial binding sites in the mare. This may denote that the oxytocin molecule may preferentially bind to the endometrium binding sites compared with myometrium binding sites, but the difference may be of marginal physiological significance. The affinity does not change in either the endometrium or myometrium throughout the estrous cycle of the mare. Similar findings have been reported regarding uterine binding site affinity throughout the estrous cycle in the ewe23 and myometrial affinity during gestation in the rat. 1 REFERENCES 1. Alexandrova M, Soloff MS: Oxytocin receptors and partuftion. Ill. Increases in estrogen receptor and oxytocin receptor concentrations in tile rat myometrium during prostaglandin F2uinduced abortion. Endocrinology 106(3):739-743, 1980. 2. Clark JII, ltsueh AW, Peck .El: Regulation of estrogen receptor replenishment by progesterone. Ann NY Acad Sci 286:161-179, 1977. 3. Crankshaw DJ, Branda LA, Matlib MA, Daniel EE: Localization of the oxytocin receptor in the plasma membrane of rat myometrium. Eur J Biochem 86:481-86, 1978. 4. Crankshaw DJ, Romaniuk E, Bnmda LA: Identification and characterization of receptors for oxytocin in myometrium of the pregnant ewe. Gynecol Obstet Invest 14:202-213, 1982. 5. Douglas RIt, Ginther OJ: Effect ofprostaglandin F2u on length of diestrus in mares. Prostaglandins 2:265-268, 1972. 6. Douglas RH, Ginther OJ: Concentration of prostaglandin F2ct in uterine venous plasma of anesthetized mares during the estrous cycle and early pregnancy. Prostaglandins 11:251-260,1982. 7. Evans MJ, Irvine CIIG: The serum concentrations of FSIt, LH and progesterone during the oestrous cycle and early pregnancy in the mare. J Reprod Fertil (Suppl) 23:193-200, 1975. 8. Evans JW, Roser JF, Mikuckis GM: Comparison of the interaction of equine LtI and human chorionic gonadotropin to equine EQUINE V E T E R I N A R Y SCIENCE

teatieular receptor. JReprodFert Suppl 32:113-121, 1982. 9. Fields, PA, Eldridge RK, Fuchs AR, Roberts RF, Fields MJ: Human placental and bovine corpora luteal oxytocin. Endocrinology 112:1544-1546, 1983. 10. Flint APF, Sheldrick EL: Evidence for a systemic role for ovarian oxytocin in luteal regression in sheep. J Reprod Fert 67:215225, 1983. 11. Fuchs AR, Fuchs F, Husslein P, Soloff MS, Femstrom MJ: Oxytocin receptors and human parturition: A dual role for oxytocin in the initiation of labor. Science 21:1396-1398, 1982. 12. Fuchs AR, Fuchs F, Soloff MS: Oxytocin receptors in nonpregnant human uterus. J Clin Endo Met 60(1):37-41, 1985. 13. Fuchs AR, Periyasamy S, Alexandmva M, Soloff MS: Correlation between oxyocin receptor concentration and responsiveness to oxytoein in pregnant rat myometrium: Effects of ovarian steroids. Endo 113(3):742-749, 1983. 14. Hughes JP, Stabenfeldt GH, Evans JW: Clinical and endocrine aspects of the estrous cycle of the mare. Proc 18th Am Assoc Equine Pract 119-148, 1972. 15. King SS: The effect of steroids on endometrial prostaglandin F2~x in production in the equine. PhD. thesis, University of California, Davis, Calif. 1983. 16. King SS, Evans JW: Equine endometrial PFG2ct production in response to oxytocin and arachidonic acid during the normal estrous cycle and the spontaneously prolonged corpus luteum syndrome. 10th Intl Congr on Anita Reprod and A1. Vol 1:483, 1984. 17. McCraken JA, Schramm W, Bareikowski B, Wilson L: The identification of PGF2~x as a uterine luteolytic hormone in the sheep and the endocrine control of its synthesis. Acta Vet Scand Suppl 77:71-88, 1981. 18. Neely DP: Studies on the luteal function and prostaglandin release in the mare. PhD. thesis, University of California, Davis, Calif, 1979. 19. Neely DP, Kindahl H, Stabenfeldt GH, Edqvist LE, llughes JP: Prostaglandin release patterns in the mare: Physiological, pathophysiological, and therapeutic responses. J Reprod Fert (Suppl) 27:181189, 1979. 20. Nissenson R, Fouret G, llecter O: Opposing effects of estradiol and progesterone on oxytocin receptors in rabbit uterus. Proc Natl Acad Sci 74(4):2044-2048, 1978. 21. Noden PA, Oxender WD, Hafs lID: The cycle of oestrus, ovulation and plasma levels of hormones in the mare. J Reprod Fertil (Suppl) 23:189-192, 1975. 22. Pattison ML, Chen CL, King SL: Determinations of LIt and estradiol-1713 surge with reference to the time of ovulation in mares. Biol Reprod 7:136, 1972.

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23. Roberts JS, McCracken JA, Gavagan .rE, Soloff MS: Oxytocinstimulated release of prostaglandin F2.ct from ovine endometrium in vitro: Correlation with estrous cycle and oxytocin-receptor binding. Endocrinology 99(4):1107-1114, 1976. 24. Roberts JS, Barcikowskil B, Wilson L, Skames RC, McCraken JA: ttormonal and related factors affecting the release of prostaglandin F2a from the uterus. J. SteroM Biochem. 6:1091-1097, 1975. 25. Roberts JS, Share L: Inhibition by progesterone of oxytocin secretion during vaginal stimulation. Endocrinology 87:812-815, 1970. 26. Saraff M, Gorski J: Control of estrogen binding protein concentration under basal conditions and after estrogen administration. Biochem. 10(13):2557-2563, 1971. 27. Schams D, Schallenberger E, Legros JJ: Evidence for secretion of immunoreactive neurophysin I i n addition to oxytocin from the ovary in cattle. J Reprod Fert 73:165-171, 1985. 28. Scatchard G: The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660, 1949. 29. Sharma SC, Fitzpatrick RJ: Effect of oestradiol-1713 and oxytocin treatment of prostaglandin F alpha release in the anoestrous ewe. Prostagladins 6(2):97-105, 1974. 30. Small GP, Gavagan JE, Roberts JS: Oxytocin-stimulated production of prostaglandin FT.a by isolated endometrium of rabbit: . . . . . Modulation o V anan steroids. Prostaglandtns 15:103-112, 1978. 31. Soloff MS: Oxytocin receptors in rat oviduct. Biochem Biophys Res Comm 66:671-677, 1975. 32. Soloff MS, Alexandrova M, Femstrom M/: Oxytocin receptors: Triggers for parturition and lactation. Science 204:13131315, 1979. 33. Soloff MS, Femstrom MA, Periyasamy S, Soloff S, Baldwin S, Weider M: Regulations of oxytocin receptor concentration in rat uterine explants by estrogen and progesterone. Can J Biochem Cell Biol 61:625-630, 1983. 34. Soloff MS, Rees IID, Sar M, Stumpf WE: Autoradiographic localization of radioactivity from 3H oxytocin in the rat mammary gland and oviduct. Endocrinology 96:1477, 1975. 35. Soloff MS, Schroeder BT, Chakraborty J, Pcarlmutter AF: Characterization of oxytocin receptors in the uterus and mammary gland. Fed Proc 36:481-486, 1977. 36. Soloff MS, Swartz TL: Characterization of a proposed oxytocin receptor in the uterus of the rat and sow. J Biol Chem 249(5):13761381, 1974. 37. Steel RG, Torrie JIl: Principles and Procedures of Statistics. McGraw-Hill Book Co, New York, 1960. 38. Wathes DC, Swann R: Is oxytocin an ovarian hormone? Nature (London) 297:225-227, 1982

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