0021 -972X/90/7003-0983$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society

Vol. 70, No. 3 Printed in U.S.A.

Progesterone and Human Chorionic Gonadotropin Do not Stimulate Placental Proteins 12 and 14 or Prolactin Production by Human Decidual Tissue in Vitro SONG GUANG REN AND GLENN D. BRAUNSTEIN Department of Medicine, Cedars-Sinai Medical Center, University of California School of Medicine, Los Angeles, California 90048

ABSTRACT. To investigate the regulation of the synthesis and secretion of placental proteins-12 (PP12) and -14 (PP14) and PRL, explants and enriched preparations of stromal cells and gland cells obtained from 10 human early pregnancy decidua were preincubated in medium for 24 h (baseline), followed by incubation in medium with or without progesterone (0.02-32 /imol/L), hCG (10 and 100 ng/ml), or cAMP (0.25-1 mmol/L) in an atmosphere of 5% CO2-95% air at 37 C for another 96-120 h. Media were changed each 24 h, and PP12, PP14, and PRL levels were determined by RIA. Decidual explants, as well as their isolated cells produced detectable levels of PP12, PP14, and PRL in vitro. The gland cells synthesized and secreted about 30 times more PP14 than did stromal cells. After 96-120 h of incubation, the production

of each protein by control cultures was increased 81-167% compared to the baseline (not significant). The secretion of these proteins in medium supplemented with progesterone or hCG was not significantly different from that in the control groups. 8-Bromo-cAMP significantly increased the secretion of PRL and PP12, but not PP14, by stromal cells compared to control values. We conclude that 1) PP14 is mainly produced by decidual gland cells; 2) progesterone at the concentrations used in our study does not stimulate production of PP12, PP14, and PRL in decidualized endometrium in vitro; 3) hCG does not stimulate the production of PP12 and PP14 in decidualized endometrium; and 4) 8-bromo-cAMP stimulates decidual stromal cell secretion of PRL and PP12. (J Clin Endocrinol Metab 70: 983-989,1990)

P

LACENTAL proteins-12 and -14 (PP12 and PP14), which have also been termed pregnancy-associated endometrial cti- and a2-globulins, as well as endometrial PRL are primarily synthesized and secreted by decidualized endometrium (1-3). These proteins may play an important role in implantation and the maintenance of normal pregnancy (4-7). Little is known about the factors that regulate the synthesis and secretion of decidual proteins (7). It has been suggested that these proteins are progesterone dependent, since the endometrial concentrations of PP12 and serum concentrations of PP14 rise during the luteal phase of the normal menstrual cycle (8-10), and the maternal serum levels of PP12 and PRL parallel the serum concentrations of progesterone during normal pregnancy (1, 7, 11). Some studies have suggested that the biosynthesis and secretion of PP12 and PRL by nonpregnant endometrium are regulated by progesterone (12-14). However, there have been conflicting data concerning the role of progesterone in the regulation of the production of these proteins in vivo and in vitro (15-19). Received April 25,1989. Address all correspondence and requests for reprints to: Dr. Glenn D. Braunstein, Department of Medicine, B118, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048.

The present study was undertaken to evaluate the effect of progesterone on the in vitro production of these proteins by decidualized endometrium obtained from early pregnancy. In addition, the pattern of secretion of PP14, as reflected by maternal serum concentrations throughout pregnancy, closely resembles that of hCG (7, 11). The effect of hCG on the production of PP14 by decidual tissue has not been reported previously. Therefore, the effect of hCG on decidual protein production was also evaluated. Materials and Methods Tissue preparations Specimens of decidua, obtained from 10 women undergoing therapeutic abortion between 6 and 12 weeks gestation, were transported to the laboratory within a few minutes after surgery. After removal of blood clots, the decidual tissue was rinsed thoroughly in Hank's Balanced Salt Solution (HBSS; Gibco, Grand Island, NY) containing 25 mM HEPES (Sigma, St. Louis, MO) and cut into 2-mm3 explants. Approximately 50mg wet weight portions of the explants were placed in 16-mm diameter polystyrene multiwell tissue culture plates (Costar, Cambridge, MA) for tissue culture. The remaining tissue was treated enzymatically in order to purify gland and stromal cells by modifications of the methods

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reported by Satyaswaroop et al. (20) and Braverman et al. (21). The tissue was treated with 0.25% collagenase-A (Boehringer, Indianapolis, IN) in HBSS with stirring at 37 C for 2 h. At the end of this period, the suspension of cells was filtered through a 30-^m pore size sieve (Spectrum, Los Angeles, CA). The gland cells remaining on the sieve were washed three times with HBSS and plated in the 16-mm polystyrene multiwell culture plates. The cells filtered through the sieve were collected by centrifugation, washed, and resuspended in 20% isotonic Percoll (Sigma). The stromal cells were separated from the suspension by gradient density centrifugation with 20-60% and 4055% Percoll gradients and collected from the fraction with a density of about 1.048 g/mL. The stromal cells were rinsed three times with HBSS and then placed in plastic dishes for 60 min to remove any fast adsorbing fibroblast-like cells. The remaining cell suspension was transferred to the culture plates. Incubation conditions Decidual explants, stromal cells, or gland cells were incubated in 1066 medium (Gibco, catalog no. 320-1535A) supplemented with 10% fetal bovine serum (Gibco), 25 mM HEPES, 1 U/mL insulin (Nordisk, Gentofte, Denmark), 10 mM glutamine (Irving, Santa Ana, CA), 100 U/mL penicillin, and 100 /ig/mL streptomycin (Gibco) in an atmosphere of 5% CO2-95% air at 37 C. In each experiment the decidual explants or the enriched preparations of stromal or gland cells were divided into control and treatment groups, with four wells used for each group. After the first 24 h of incubation, which also served as a baseline, progesterone (Sigma) dissolved in 0.06% ethanol, hCG (NIDDK, Bethesda, MD) diluted in medium, or 8-bromocAMP (Sigma) diluted in medium was added to the culture. The final concentrations of progesterone were 0.02, 0.1, 1, 2, 3.2, or 32 /zmol/L. These doses were chosen to cover the entire physiological luteal phase and pregnancy serum concentrations. Concentrations of hCG were 10 and 100 ng/mL, representing mean hCG levels in maternal serum on days 21-24 and 33-35 after the last menstrual period in the conception cycle (22). 8Bromo-cAMP concentrations were 0.25, 0.5, and 1 mmol/L. After addition of the treatment medium, the incubations were continued for another 96-120 h. Media were changed every 24 h. After centrifugation, the supernatant was stored at -20 C until measurement for PP12, PP14, and PRL. At the end of the incubation period, the wet weight of the explants and cell number for the gland and stromal cells in each well were determined. Viability was assessed in selected cultures at various intervals by examining the incorporation of [35S]methionine (ICN, Cleveland, OH) into trichloroacetic acid-percipitable protein after 2-h incubation with [35S]methionine. Cells killed by freezing and thawing were used as controls. RIAs PP12, PP14, and PRL were measured by modifications of the methods of Rutanen et al. (24), Julkunen et al. (25), and Sinha et al. (26), respectively. All samples from each individual experiment were assayed in a single assay. Purified PP12 (lot

JCE & M • 1990 Vol 70 • No 3

307/323) and PP14 (lot 136/154), rabbit anti-PP12 antiserum (lot 247A), and anti-PPl4 antiserum (lot 199ZA) were obtained from Dr. Hans Bohn, Behringwerk AG (Marburg, West Germany). The human PRL (hPRL) and rabbit anti-hPRL antiserum were provided by the National Hormone and Pituitary Program (NIDDK, Bethesda, MD). PP12, PP14, and hPRL were iodinated using the Iodogen method (27) (Pierce, Rockford, IL). Final concentrations of antibodies were 1:40,000 for PP12 and PP14 and 1:400,000 for hPRL. The antibody-bound tracers were precipitated using goat antirabbit 7-globulin antiserum (Biotek, Shawnee, Mission, KS) and 6% polyethylene glycol (mol wt, 8000; Sigma) for PP12 and hPRL, and 3% normal rabbit serum (Biotek) and goat antirabbit 7-globulin antiserum for PP14. The sensitivities of the assays were 7.8 ng/mL for PP12 and PP14, and 1.7 ng/mL for hPRL. The intraassay variations were 6% at 20 ng/mL for PP12, 8.8% at 120 ng/mL for PP14, and 6.3% at 19 ng/mL for PRL. None of the three proteins cross-reacted in the RIAs for the other two proteins. Degradation of progesterone and hCG added to the culture medium was assessed in several experiments through direct measurement of hCG and progesterone concentrations in the medium before and after 24 h of incubation using the PANTEX progesterone kit (Santa Monica, CA) according to manufacture's instructions and our previously described hCG RIA (22). Statistical analysis The results were expressed as nanograms per 100 mg wet wt of explants or 1 X 106 stromal or glandular cells during the period of incubation or as the percent increase over baseline in order to combine data from different experiments. Student's t test corrected for multiple observations was used for statistical analyses (23).

Results The baseline values for PP12, PP14, and PRL concentrations in the medium after 24 h of incubation for each experiment are given in Table 1, in which each value represents the mean level of all wells before the addition of progesterone or hCG. As shown in Table 1, the explants as well as the enriched cultures of stromal and gland cells produced detectable PP12, PP14, and PRL. For each decidual sample, the gland cells produced an average 30 times greater quantity of PP14 than stromal cells, suggesting that PP14 was produced mainly by the gland cells during pregnancy. There was substantial variation in baseline endometrial protein production, reflecting both biological variability and differences in gestational duration before therapeutic abortion. As shown in Figs. 1-4, there was a tendency for each of the proteins to progressively increase over time in culture. However, this increase was not statistically significant for any of the proteins or in the different culture systems. At the end of 96-120 h of incubation, the increase in the proteins in the control groups was 110%

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SYNTHESIS AND SECRETION OF PP12, PP14, AND PRL

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TABLE 1. Baseline concentrations (mean ± SEM) of PP12, PP14, and PRL in cultures of all experiments Cultures treated with hCG

Cultures treated with P Explants or cells (U) Explants (/ig/100 mg)

Stromal cell (ng/106 cells)

Gland cell (ng/106 cells)

Sample no. 4 5 6 7 10 11 4 6 7 8 10 21 22 3 6 7 9 10

PP12 7.8 ± 1.3 -

PP14 58.6 ± 10.4 -

45.2 ± 10.6 ± 3.3 ± 128.4 ±

4.2 1.3 0.3 1.4

67.7 ± 748.4 ± 472.5 ± 148.5 ±

7.8 82.9 75.3 17.5

17 ± 2 24 ± 4 289 ± 62

80 ±9

122 ± 55 15.7 ± 2.9 13.6 ± 1.9

12 57 96 21 77

±2 ±3 ±5 ±8 ±4

-

37,067 ± 2,385 ± 4,821 ± 327 ± 649 ±

32.2 ± 9.2 -

4 ±0.3 424 ± 90 58 ± 6 158 ± 17 12.7 ± 2.1 189 ± 26

PRL

9,736 162 682 38 42

0.6 ± 0.0 297.5 ± 53.1 51.8 ± 7.8 177.1 ± 54 0.6 ± 4.7 ± 52.3 ± 6.6 ± 0.6 ± 6.2 ± 4.3 ±

0.1 1.0 4.8 1.0 0.1 0.2 0.4

2.9 ± 0.4 -

47.5 ± 12 2.4 ± 0.2 1.1 ± 0.1

Sample no. 4 5 6 7 10 11

PP12 _

6.5 ± 0.6 -

-

469.1 ± 1 564.2 ± 66.9 155.4 ± 19.7

2.0 ± 0.3

7

66 ± 7 682 ± 105 117 ± 8

3 6 7 9 10

_

48.0 ± 6.7

9.6 ± 0.9 3.8 ± 0.3 158.4 ± 24.1

5 6 11 10 21 22

PP14

6 ±0.6

-

-

221 ± 43 84 ± 11 136 ± 8

-

-

11 ± 1

66,958 ± 14,691

-

-

106 ± 5

6,891 ± 1,034

-

-

101 ± 15

197 ± 24

-, Not determined.

in the explant culture system, 167% in the stromal cell culture, and 81% in the gland cell cultures. That the tissues examined remained viable was demonstrated by specific incorporation of labeled [35S]methionine into protein by the cultures. The incorporation ratios of labeled [35S]methionine were 13.4 ± 2%, 9.6 ± 1.7%, and 8.6 ± 1.7% for 24, 72, and 120 h of culture. Progesterone in concentrations from 0.02-32 /umol/L did not increase the production of PP12, PP14, or PRL compared to the control groups. As shown in Fig. 1, which illustrates the results with progesterone in concentrations of 3.2 and 32 /umol/L, there was no statistically significant difference in the release of PP12 or PP14 from individual cultures of decidual explants or the stromal and gland cell cultures. Two of 5 explant cultures demonstrated significant PRL increases in cultures treated with progesterone, but in both instances the lower dose of progesterone was associated with a greater increase in PRL than was the higher dose (Fig. 2). When the data from all experiments were expressed as the percent increase over baseline, no significant differences between control or progesterone-treated cultures were found. The combined data for the explant cultures, representing the mean of 20 wells from 5 experiments, are shown in Fig. 3. Similar results were found for the combined data for the gland and stromal cell cultures. During 24 h of incubation, 30-50% of the added progesterone was degraded, as assessed by RIA; therefore, medium was changed daily. hCG in concentrations of 10 or 100 ng/mL did not

have an effect on decidual explant tissue or cell culture production of PP12 or PP14, as shown in Fig. 4. No degradation of hCG was found during 24 h of incubation with the explants or cells. cAMP significantly increased the secretion of PRL and PP12 in stromal cells compared to controls (Table 2), again attesting to the viability of the cells. Discussion Our results demonstrate that both stromal and gland cells isolated from early pregnancy decidua produce PP12 and PP14 in vitro, and that PP14 is mainly produced by the gland cells. Furthermore, neither progesterone nor hCG stimulates the production of PP12 or PP14 in decidualized pregnancy endometrium in vitro, and progesterone has no effect on PRL production by this tissue. In contradistinction, cAMP stimulates the production of PP12 and PRL, but not PP14. The human endometrium contains progesterone receptors, and progesterone is required for endometrial differentiation to decidua. Our results showed that decidualized endometrial tissue produced PP12, PP14, and PRL in medium that did not contain progesterone and failed to be stimulated by the addition of progesterone. Serum levels of progesterone during the late secretory phase are about 40 nmol/L (10) and approximately 600 nmol/L in term pregnancy (11). The doses of progesterone used in our studies covered the full physiological luteal and pregnancy hormone concentrations, even in the face of 30-

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REN AND BRAUNSTEIN P P 1 2

DECIDUAL TISSUE EXPLANTS

JCE & M • 1990 Vol70«No3

DECIDUAL EXPLANTS

P P 1 4

200- #11

200 -

100-

300- # 6 200Hi

UJ (A < UJ OC

o z

96

120

24

96

120

100 -

• CONTROL • P (3.2pM) A P (32pM)

500-

200100-

INCUBATION HOURS

FIG. 1. The effect of progesterone (P) on the production of PP12 and PP14 in representative examples of explant, stromal cell, and gland cell cultures. The number in each panel refers to the culture number in Table 1.

50% degradation of progesterone in the culture system, as measured by RIA. The doses of progesterone chosen for our study are similar to those used by others (10). Identical results were found when medroxyprogesterone acetate in doses of 0.02-2 /imol/L were used in place of progesterone (our unpublished observation). These data support the hypothesis proposed by Bell (7) that the effect of progesterone may only be facilitative, maintaining decidual differentiation, and that production of these proteins is related to the progesterone-dependent differentiation of the endometrium and not directly to progesterone. The factors responsible for regulation of the synthesis and secretion of these proteins by the decidualized endometrium are presently unknown. PP14 has been localized by immunocytochemical techniques to the secretory glandular epithelium of the first trimester endometrium (7, 28). Our studies, which quantitate the production of PP14 by both enriched preparations of stromal and gland cells in culture, are consistent with the immunocytochemical data. Since the decidual glandular structures involute after the first trimester (29), the decrease in PP14 levels during the second and

200-

* 10

100—I—

24

—i—

48

—i—

72

96

120

INCUBATION HOURS

FIG. 2. The effect of progesterone (P) on production of PRL in separate decidual explant cultures. The number of the culture refers to the numbers in Table 1. *, P < 0.05.

third trimesters may reflect the regression of the glands as pregnancy advances. Immunohistological studies have localized PP12 to both the secretory glandular epithelial cells and decidualized stromal cells of the endometrium (30), findings that are concordant with our results which demonstrated approximately equal quantities of PP12 released into the medium from each of these cell types. PRL has been localized to the decidua by means of immunohistochemical techniques (21, 30, 31). Since the concentrations of PRL present in the medium from cultures of both stromal and gland cells in our study were approximately equal, it is likely that both cell types are

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SYNTHESIS AND SECRETION OF PP12, PP14, AND PRL PP12

200

P P 1 2

987 P P 1 4

DECIDUAL TISSUE EXPLANTS #5

200 100 100 200 • • •

CONTROL P (3.2pM) P (32uM)

100

Progesterone and human chorionic gonadotropin do not stimulate placental proteins 12 and 14 or prolactin production by human decidual tissue in vitro.

To investigate the regulation of the synthesis and secretion of placental proteins-12 (PP12) and -14 (PP14) and PRL, explants and enriched preparation...
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