Prolactin binding sites on human chorion-decidua tissue

Prolactin binding sites on human chorion-decidua tissue LINDA

A.

CHARLES ALAN

McWEY, A.

D.

Charlottesville,

M.S.

SINGHAS,

ROGOL,

PH.D.

M.D.,

PH.D.

Virginia

An effective procedure has been developed and utilized to demonstrate the presence of prolactin receptors on the plasma membranes of human chorion-decidua cells. Particulate fractions from human chorion-decidua sedimenting between 1,500 and 45,000 x g display optimal binding of 1251-labeled ovine prolactin when incubated at a membrane protein concentration of 200 @ per assay tube for 2 hours at 22” C. Specific binding was increased by pretreatment of the membrane particles with 5M magnesium chloride to remove endogenous prolactin. These receptors show binding parameters (affinity, 0.92 x lo* L/mole; capacity, approximately 80 fmoleslmg) similar to those of lactogenic receptors in the rabbit mammary gland and, the rabbit and rat liver. The presence of prolactin receptors in human chorion-decidua suggests they may play a role in mediating local action(s) of prolactin such as involvement in the decidualization reaction or in maintaining fetal osmoregulation. (AM. J. OBSTET. GYNECOL. 144:283, 1982.)

ENDOMETRIAL STROMAL TISSUE transformed into decidua has been demonstrated to synthesize prolactin during normal gestation, 2, ii during the late luteal phase of the normal menstrual cycle, and during abnormal pregnancies involving ectopic implantation sites.gz io Human decidua also has been shown to synthesize and release significant quantities of prolactin in vitro.2* l3 Bromocriptine does not influence prolactin secretion from decidua although inhibition of protein synthesis by cycloheximide or puromycin strongly reduces both the release and the decidual tissue content of prolactin.2 Neither amnion nor chorion nor placental tissue, all of fetal origin, appear to synthesize prolactin in vitro.” However, prolactin was localized in the cytoplasm of

From the Department of Obstetrics and Gynecology, the Department of Pediatrics, and the Department of Pharmacology, The University of Virginia School of Medicine. ThzF work was supported by United Service, National Institute of Child Development Grant HD 13893 (to National Institute of Health Career K-04AMOOI53 (to A. D. R.). Received for publication Revised April

27, 1982.

Accepted May

6, 1982.

February

States Public Health Health and Human C. A. S.) and by Developnent Award 23, 1982.

Material and methods

Reprint requests: Dr. Charles A. Singhas, Department Obstetrics and Gynecology, Box 387, University of Virginia Hospital, Charlottesville, Virginia 22908. 000%9378/82/190283+06$00.60/O

cells within the amnion by immunofluorescence.” Later, with the same technique, Frame and co-workers’ were able to localize prolactin in the cytoplasm of cells within the chorion, with the most intense fluorescence in cells of the decidua. These findings suggest that the maternal decidua cell may be the site of prolactin localization and/or synthesis. Since binding to receptors is the first step in the action of protein hormones, demonstration of specific prolactin receptors would be an indication of possible biologic action. Substantial levels of specific binding of ovine prolactin to the deciduoma-bearing rat uterus have been previously reported, and prolactin also has been shown to be involved with decidua formation in the pseudopregnant and the pregnant rat uterus.i6 Binding of human prolactin has been described in plasma membrane fractions of placenta in the fetal rhesus monkey.‘j This finding is relevant to a study of prolactin receptors in chorion-decidua since the chorion laeve and the chorion frondosum, which make up much of the term placenta, are of identical origin. The aim of this study was to examine fractions of membrane from the human chorion-decidua for the presence of specific prolactin receptors.

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Co

Preparation of tissue and protein Fetal membranes were obtained within completion of normal term deliveries

determination. 30 minutes after (38 to 42 weeks’ 283

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Table I. Total binding of ‘2SI-labeled ovine prolactin to particulate fractions of chorion-decidua as a function of force required for sedimentation after 18 to 24 hours of incubation (n = 5)

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I

2-

Membrane

fraction

BIF With magnesium chloride

x 100 Withaut map&m

chloride

I-

O-

5ooo

15ooo CENTRIFUGAL

45000 FORCE

loopoo

(xQ)

Fig. 1. Specific binding of 1*51-labeled ovine prolactin to chorion-decidua fractions as a function of force required for sedimentation after 2 hours of incubation (n = 3). gestation). Membranes were trimmed from the placental disk, rinsed in normal saline, and maintained frozen at -20” C until used. For each experiment, frozen tissue was thawed in a 4” C water bath, and the superficial layer of chorion with adherent decidua was scraped into 0.3M sucrose buffer containing 10,000 Kallikrein inactivator units (KIU) of Trasylol, 0.25 mM phenylmethyl sulfonyl fluoride (PMSF), and 1.0 mM ethyleneglycoltetraacetic acid (EGTA) (Trasylol, PMSF, and EGTA were present in all buffers used). The tissue was homogenized in a Polytron PI-10 (Brinkman) at setting No. 7 for IO seconds with sample tubes surrounded by ice. In initial experiments, sucrose homogenates were centrifuged for 20 minutes at 500 x g; the pellet was saved, and the supernatant was recentrifuged at 1,500 X g for 20 minutes. This procedure was continued for successive centrifugations of 5,000 x g (30 minutes), 15,000 X g (30 minutes), 45,000 X g (45 minutes), and 100,000 x g (60 minutes) at 4“ C in order to determine optimal conditions for preparation of membrane particles for further studies. PeIlets were resuspended in 1 ml of 25 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl) buffer, pH 7.5, containing 10 mM magnesium chloride and reground in a Teflon-glass homogenizer. The membrane preparation was diluted to give a final concentration of 200 to 300 @g of protein per 0.1 ml as determined by the Bradford protein assay with bovine serum albumin used as standard. Aliquots of 1 ml were stored in 12 by 75 mm glass tubes at -70” C. In a similar manner, amniotic membrane pellets sedimenting between 1,500 and 45,000 X g were prepared from pooled placentas and stored. Removal of endogenous prolactin. In preliminary experiments, some of the resuspended pellets were treated with 2 ml of 5M magnesium chloride-O.IM Tris HCl, pH 7.5, for 15 minutes at 4” C, at which time

500 x 1,500 5,000 15,000 100,000

g x g x g x g x g

11.4 13.8 10.7 12.8 10.0

a.03 9.41 10.4 10.9 9.50

the suspension was diluted to 10 ml with 25 mM Tris HCl, pH 7.5, to facilitate subsequent sedimentation of the membranes. To remove excess magnesium chloride, this suspension was again centrifuged at 45,000 x g for 45 minutes. This procedure is designed to remove endogenously bound prolactin from its receptors.R Radioiodination of ovine prolactin. Ovine prolactin (oPRL-14, National Institute of Arthritis, Metabolism, and Digestive Disease) was radioiodinated by a stoichiometric method (modified from the chloramine-T procedure) and repurified by gel filtration on Sephadex G-100 to a specific activity of approximately 60 &iI~g.i2 Prolactin binding assay. Incubations were performed in triplicate at room temperature (22” C) in a Dubnoff metabolic shaking incubator with detachable plastic conical centrifuge tubes (6 ml). The reaction mixture consisted of 100 ~1 of membrane preparation, 100 ~1 of 1251-labeled ovine prolactin (50,000 to 100,000 cpm), 100 ~1 of unlabeled ovine prolactin (standard) when required, and 200 to 300 ~1 of 25 mM Tris HCl, pH 7.5, containing 10 mM magnesium chloride and 0.1% bovine serum albumin to give a final volume of 500 ~1 for all tubes. The mixture was allowed to preincubate 1 hour before the 1251-labeled ovine prolactin was added. The binding reaction was terminated by dilution with 2 to 3 ml of ice-cold buffer. Separation of bound from free hormone was achieved by centrifugation at 1,500 X g for 30 minutes at 4” C. The tubes were inverted, and the detachable conical bottoms containing the membrane pellets were dried and counted for radioactivity in a Beckman Gamma 4000 counter at approximately 70% counting efficiency. Total binding was measured in the absence of unlabeled hormone (B,), and nonspecific binding was determined by the amount of lz51 remaining bound in the presence of 10 pg of unlabeled hormone (NS). The percentage of specific binding at any concentration of unlabeled hormone was assessed

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by subtracting the nonspecific from the total binding (B - NS). As a control, parallel incubations of membranes purified from the livers of pregnant rats, supplied by Dr. H. G. Friesen (University of Manitoba), were performed for each of the binding assays. The binding parameters for this tissue have been previously reported. I5 Total and nonspecific binding was also examined in pellets from amniotic membrane. Binding of 1251-labeledovine prolactin versus membrane concentration. The effects of membrane protein concentrations of 25, 30, 100, 200, and 400 pg per assay tube on binding of “9-labeled ovine prolactin were studied to determine a reasonable protein concentration for later binding experiments. Time course of binding. In preliminary experiments, the chorion-decidua binding assay was terminated after 1, 2, 4, 8, 16, and 24 hours to establish optimal incubation time for further studies. Displacement of ‘251-labeled ovine prolactin. A standard displacement curve was performed by incubating tissue with unlabeled hormone at concentrations of 1, 3, 10, 30, 100, 300, and 1,000 rig/ml. Data obtained from the displacement curve were subjected to Scatchard analysis.

Prolaotin

binding

sites on chorion-daoidua

100 200 AMOUNT OF MEMBRANE

tissue

285

300 400 PROTEIN PER TUBE (pg)

Fig. 2. Effect of concentration of membrane protein on binding of ‘251-labeled ovine prolactin to chorion-decidua (one experiment, performed in triplicate) (I = SEM).

TOTAL

2

Results Dissociation of endogenous prolactin with magnesium chloride. In initial experiments, total binding of chorion-decidua membrane fractions treated with 5M magnesium chloride was compared with that of untreated membranes after 18 to 24 hours of incubation. A higher degree of total binding was observed in all of the magnesium-treated pellets (Table I). Following the demonstration that prolactin binding sites were present in magnesium chloride-treated decidua-chorion preparations, this technique became the standard procedure for all of the following membrane preparations. Optimization of membrane fractions for binding. To establish a technique for the preparation of a membrane fraction that yielded the greatest amount of lz51labeled ovine prolactin bound to chorion-decidua (expressed as a ratio of bound hormone : free hormone, or B/F), fractions sedimenting at 5,000, 15,000, 45,000, and 100,000 x g were compared for their ability to bind lz51-labeled ovine prolactin during a 2-hour incubation period. As shown in Fig. 1, the binding specificity of the 5,000, 15,000, and 45,000 X g pellets was not significantly different, although decreased binding was observed in the high-speed 100,000 X g fraction. For further experiments with chorion-decidua, the membrane particles sedimenting between 1,500 and 45,000 x g were used. Effect of membrane concentration on biding. Fig.

4

,

q-c,,

i OI 2

+-l--/w+ 8 ;ME (HOURS)

16

24

3. Time course of binding of l*%labeled ovine prolactin to chorion-decidua (45,000 x g fraction.) (n = 4).

Fig.

2 shows the effect of increasing amounts of choriondecidua membrane protein in specific binding (B, - NS) of ‘251-labeled ovine prolactin. The nonspecific binding averaged 84.7% of the total counts bound in the B, tubes. Binding was approximately linear over the range of 25 to 200 pg of protein; therefore, a membrane protein concentration averaging 200 pg/O.l ml was chosen for convenience for binding studies. Time course of binding. Specific binding was maximal at 1 to 4 hours, after which it decreased significantly

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100 -I z z 1 ,+j’ \ 5; z &! a-

80604020-

0.1

I 0.3

Fig. 4. Displacement of ‘Y-labeled (0) (means of six experiments)

, I 1.0 3.0 UNLABELED

ovine

prolactin

1 I 1 loo IO 30 oPRL ADDED (rig/ml) (LOG SCALE) by unlabeled

ovine

prolactin

1 300

1 ICOO

for chorion-decidua

and pregnant rat liver (o) (n = 9).

and remained low (Fig. 3). Nonspecific binding increased throughout the time study with lowest values at 1 to 2 hours of incubation. The parameter for selection of an optimal incubation period was the specific binding, which is the difference between total and nonspecific binding at each time interval. On the basis of these results, incubation at 22” C for 2 hours was adopted as the standard procedure. Displacement of 1251-labeled ovine prolactin by unlabeled ovine prolactin. With the use of the conditions determined above, a standard displacement curve by unlabeled ovine prolactin for the binding of lz51-labeled ovine prolactin to chorion-decidua was constructed (Fig. 4). Addition of increasing amounts of unlabeled ovine prolactin resulted in a substantial decrease in binding of labeled tracer with a decrease of more than 80% produced by 1 bg of unlabeled hormone. The affinity constant (K,) determined at 50% displacement of ‘251-labeled ovine prolactin by unlabeled hormone was 0.78 x log L/mole. The mean for nonspecific binding averaged 68.4% of the total counts bound in the B, tubes. Scatchard analysis revealed a binding capacity of approximately 80 fmoles/mg of protein for chorion decidua. Comparison to a known target organ. In parallel incubations performed on membranes prepared from the livers of pregnant rats, the displacement curve (K, = 0.85 x lo9 L/mole) (Fig. 4) was very similar to that determined for chorion-decidua and compared favorably with previous studies.4 The nonspecific binding averaged 27.2% of B,. Pooled data revealed an approximate binding capacity of 600 fmoles/mg of protein for rat liver.

to amnion. Binding of ‘251-labeled ovine prolactin In a series of four experiments performed with magnesium-treated membrane fractions incubated 2 hours at 22” C with ‘Y-labeled ovine prolactin, the total binding (B,) and the nonspecific binding (NS) were approximately equal. Therefore, all binding was considered nonspecific. Comment The present study strongly suggests the presence of prolactin receptors on the plasma membranes of human chorion-decidua cells. Prior to the initiation of this investigation, the existence of prolactin receptors had not been reported in this tissue. Consequently, it was considered important to establish conditions of preparation and assay that would allow for maximal binding of ovine prolactin to human chorion-decidua. The binding characteristics of prolactin to receptors on the chorion-decidua are very similar to those of other recognized prolactin target tissues. The binding affinity of 0.78 x lo9 L/mole compares favorably with 2.9 x lo9 L/mole for the rabbit mammary gland and 0.3 to 2.0 x lo9 L/mole for rat and rabbit liver receptors. The approximate binding capacity of 80 fmoles/ mg of protein is also similar to that reported for the rabbit mammary gland (58 fmoles/mg) but is slightly lower than the values reported for receptors in livers of rabbits and rats (250 to 315 fmoles/mg).‘4 From these data, the prolactin binding site in chorion-decidua fulfills the criteria for a high-affinity, low-capacity, specific polypeptide receptor. During the course of this study, Herington and coworkers5 briefly reported the presence of lactogen receptors on human chorion laeve. With ‘2”I-laheled

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human growth hormone used as the labeled ligand, they determined a high-affinity, low-capacity prolactin binding site having an affinity constant of 0.47 X 10’ L/mole and a capacity of 175 fmoles/mg. These data compare favorably with the values found in the current study. One of the difficulties in this investigation was the very high percentage of nonspecific binding (68% to 85%) of i251-labeled ovine prolactin to human chorion decidua in comparison to 27% nonspecific binding obtained for the rat liver. The possible reasons for the resulting low. percentage of specific binding are many. Placental membranes could vary among individuals both in their ability to bind prolactin and in their stability upon freezing. This may be due to differences in the labor history of the patients, such as duration, ease of delivery, or, most important, perinatal treatment with drugs. Degradation of receptors may have occurred when the membranes were frozen in saline before processing or when the fractions were frozen in buffer after processing. However, even when membranes were processed immediately after delivery and assayed for ligand binding, thereby avoiding storage at 4” C or freezing, difficulties were encountered in obtaining consistent binding data. It was initially determined that enzyme inhibitors in the buffer systems were required to detect binding in the chorion-decidua particles; no binding was observed in assays performed in the absence of inhibitors. Since Herington and associate? did not include nonspecific binding data for their system, it is difficult to compare fully all parameters involved in these two studies. Since high levels of prolactin are found in amniotic fluid, a portion of the prolactin receptors in target cells of the chorion-decidua may be occupied with endogenous lactogenic hormone. Although homogenization and plasma membrane fractionation by centrifugation provide an opportunity for dissociation of prolactin from its binding site, a large percentage of this hormone remains bound to its receptor because of the slow rate at which prolactin dissociates.7 With the technique reported by Kelly and co-workers,s in vitro desaturation of lactogenic hormone from its receptor by 5M magnesium chloride was used to estimate total prolactin receptor concentrations. An increase in the total to magnesium binding of ‘251-labeled ovine prolactin chloride-treated fractions was observed when compared to binding in untreated membrane fractions (Table I). These data indicate that very large concentrations of magnesium chloride uncovered additional prolactin binding sites presumably as a result of the release of endogenous prolactin from its receptors on the chorion decidua. As others have described for interactions of a variety

Prolactin

binding

sites

on chorion-decidua

tissue

287

of protein and polypeptide hormones with their target tissue binding sites, binding of ovine prolactin to human chorion-decidua depends on several factors, including membrane protein concentration, sedimentation force required to prepare membrane fractions, and duration of incubation. The use of the 1,500 to 45,000 X g pooled membrane fractions to study binding of 1251-labeled ovine prolactin was based on data indicating that 5,000, 15,000, and 45,000 x g fractions exhibited similar binding of ovine prolactin (Fig. 1). Specific binding of labeled hormone was maximal (1.53% to 1.62% of total 1251-labeled ovine prolactin added) after 1 to 2 hours of incubation. After this time the increase in nonspecific binding produced an overall decrease in specific binding (Fig. 3). Since binding of lactogenic hormones to membrane fractions from the livers of pregnant rats has previously been demonstrated, this tissue was selected as a control to determine appropriate assay conditions4 The binding affinity of 0.85 X log L/mole and the approximate binding capacity of 600 fmoleslmg determined for the rat liver in parallel incubations with chorion-decidua compare favorably with an affinity constant of 0.30 x lo9 L/mole and a binding site capacity of 314 fmoles/mg determined in a previous study by Herington and co-workers.’ Only a small amount of protein can be obtained from the amniotic epithelium of one placenta; therefore, it was necessary to pool several specimens to obtain sufficient membrane protein to study “j1-labeled ovine prolactin binding. The absence of binding of labeled hormone to amniotic membrane fractions is in agreement with the study by Herington and associates,” where binding of 1Z51-labeled human growth hormone to human amnion particles was negligible. Similarly, Frame and co-workers’ were unable to localize prolactin by immunofluorescence in the amniotic epithelium from human placentas, although localization of prolactin to the cytoplasm of cells within the chorion and decidua tissue was observed. The results of the current study suggest the presence of specific receptors for prolactin on human choriondecidua. During the course of investigation, this observation was confimed by Herington and co-worker9 and similar binding parameters were noted. Although the function(s) of these receptors is unknown, it may be speculated that their existence provides a mechanism for local action(s) of prolactin such as involvement in the decidualization reaction or maintaining fetal homeostasis. We acknowledge Dr. Henry Friesen for his donation of tissue used for control experiments. We also thank Ms. Patti Bunts, Ms. Lis Taylor, and Ms. Becky Weaver for their technical assistance.

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REFERENCES

1. Frame, L. T., Wiley, L., and Rogol, A. D.: Indirect imnunofluorescent localization of prolactin to the cytoplasm of decidua and trophoblast cells in human placental membranes at term, J, Clin. Endocrinol. Metab. 49:435, 1979. 2. Golander, A., Hurley, T., Barrett, J., Hizi, A., and Handwerger, S.: Prolactin synthesis by human chorion-decidual tissue: a possible source of prolactin in the amniotic fluid, Science 202:311, 1978. 3. Healy, D. L., Muller, H. K., and Burger, H. G.: Immunolluorescence shows localization of prolactin to human amnion, Nature. 265:642, 1977. 4. Herington, A. C., Phillips, L. S., and Daughaday, W. H.: Pituitary regulation of human growth hormone binding sites in rat liver membranes, Metab. Clin. Exp. 25:341, 1976. 5. Herington, A. C., Graham, I., and Healy, D. L.: The presence of lactogen receptors in human chorion laeve, J. Clin. Endocrinol. Metab. 51:1466. 1980. 6. Josimovich, J. B., Merisko, K., Boccella, L., and Tobon, H.: Binding of prolactin by fetal rhesus cell membrane fractions, Endocrinology. 100~556, 1977. 7. Kelly, P. A., Tsushima, T., Shiu, R. P. C., and Friesen, H. G.: Lactogenic and growth hormone-like activities in pregnancy determined by radioreceptor assay, Endocrinology. 99:765, 1976. 8. Kelly, P. A., Leblanc, G., and Djiane, J.: Estimation of

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total prolactin-binding sites after in vitro desaturation, Endocrinology. 104:1631, 1979. Maslar, I. A., and Riddick, D. H.: Prolactin production by human endometrium during the normal menstrual cycle, AM. J. OBSTET. GYNECOL. 135:751, 1979. Ma&r, I. A., Kaplan, B. M., Luciano, A. A., and Riddick, D. H.: Prolactin production by the endometrium of early human pregnancy, J. Clin. Endocrinol. Metab. 51:78, 1980. Riddick, D. H., Luciano, A. A., Kusmik, W. F., and Maslar, I. A.: De novo synthesis of prolactin by human decidua, Life Sci. 23:1913, 1978. Rogol, A. D., and Chrambach, A.: Radioiodinated human pituitary and amniotic fluid prolactins with preserved molecular integrity, Endocrinology 97:407, 1975. Rosenberg, S. M., Maslar, I. A., and Riddick, D. H.: Decidual production of prolactin in late gestation: further evidence for a decidual source of amniotic fluid prolactin, AM. J. OBSTET. GYNECOL. 138:681, 1980. Shiu, R. P. C., and Friesen, H. G.: Properties of a prolactin receptor from the rabbit mammary gland, Biochem. J. 140:301, 1974. Tsushima, T., and Friesen, H. G.: Radioreceptor assay for growth hormone,J. Clin. Endocrinol. Metab. 37:334, 1973. Williams, G. H., Hammond, J. M., Weisz, J., and Mortel, R.: Binding sites for lactogenic hormone in the rat uterus, Biol. Reprod l&69?, 1978.