0013.7227/92/1305-2897$03.00/O Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 5 Printed in U.S.A.
Society
M. YAMAGUCHI, Department
H. ENDO,
of Biology, University
G. THORDARSON, of California,
Lactogen-I and Mouse
L. OGREN,
Santa Cruz, California
ABSTRACT.
The primary objective of this study was to develop a cell culture system for assessing effects of putative secretagogues on mouse PL-I (mPL-I) secretion. Trophoblast from days 7 to 11 of pregnancy was dispersed in collagenase, and the cells were fractionated on a Percoll gradient and plated on collagen gels in serum-free medium. Cells from days 7-9 of pregnancy yielded five bands on Percoll gradients and those from days 10 and 11 yielded six. mPL-I was present in four of the bands of cells from each day of pregnancy. Cells from day 7 of pregnancy that banded at a density of 1.044 g/ml secreted the largest amount of mPL-I during 5 days of culture. The mPL-I concentration of the medium of these cells increased for the first 3 or 4 days of culture and then declined on the fifth day. mPLII could not be detected in the medium until the third or fourth day of culture, and its concentration increased thereafter. Cell viability was about 90% at the time of plating, remained at about 80% between days 1 and 4, and then declined on day 5. The cell type that produced mPL-I was identitied with the reverse hemolytic plaque assay and by staining with anti-mPL-I anti-
T
WO PLs are produced by the mouse placenta: mouse PL (mPL)-I (1) and mPL-II (2). The mPLs are structurally related to one another (3) and are both produced by trophoblast giant cells (4). The hormones share at least some PRL-like biological and receptorbinding activities (5-7), but they differ in their gestational profiles in maternal serum. mPL-I appears in the maternal blood on day 6 of pregnancy. Its concentration increases to high values on days 9 and 10 and then declines to very low values for the remainder of pregnancy (8). mPL-II first appears in the maternal circulation on day 9 of pregnancy (9), and its concentration increases until about day 14 and then levels off in some strains of mice or continues to increase for the remainder of pregnancy in others (9, 10). Several factors that regulate the concentration of Received November 21, 1991. Address all correspondence and requests for reprints to: Dr. Frank Talamantes, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064. *This work was supported by NIH Grants GM-08132 and HD14966.
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serum. Both methods indicated that mPL-I was produced by giant cells. The ability of the cells to respond to putative secretagogues was examined using mPL-II and progesterone. mPL-II, at concentrations ranging between 10 rig/ml and 10 rg/ml, had no effect on the mPL-I concentration of the medium when it was present for up to 3 days of culture, which suggests that mPL-II does not inhibit mPL-I secretion in uitro. Incubation of the cells in the presence of 100-1000 rig/ml progesterone caused a dose- and time-dependent reduction in the mPL-I concentration of the medium and a decrease in the number of cells that stained with anti-mPL-I antiserum. The effect of progesterone on both endpoints was not apparent until the second day of treatment. These data suggest that progesterone inhibits mPL-I secretion at least in part by inhibiting the differentiation of mPL-I-producing giant cells. The fact that the mPL-I-producing cells responded to progesterone indicates that this culture system will be useful in assessing effects of putative secretagogues on mPL-I secretion. (Endocrinology 130: 28972905,1992)
mPL-II in maternal serum have been identified. These include litter size (lo), feto-placental genotype (lo), maternal nutritional status (ll), progesterone (12), and GH (13). In contrast, little is known about the regulation of mPL-I secretion, Recent studies have shown that the maternal serum mPL-I concentration is proportional to litter size (8) and that the pituitary gland suppressesthe mPL-I concentration of the maternal serum (14). The present study was undertaken to develop a cell culture system that can be used for examining the regulation of mPL-I secretion.
Materials
and Methods
Animals
Pregnant SwissWebster mice were purchasedfrom Simonsen Laboratories (Gilroy, CA). The presenceof a vaginal plug was used as an indicator of day 0 of pregnancy. The animals were maintained on a 14-h light, 10-h dark lighting cycle (lights on at 0600 h). Food and water were available ad libitum. The use of animals for this study was approved by the University Animal Care Committee.
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Modulation of Mouse Placental Vitro: Effects of Progesterone Lactogen-II*
MOUSE Hormones
PLACENTAL
and reagents
Cell dissociation
and culture
Conceptuseswere collected on days 7-11 of pregnancy. The fetus and decidua basaliswere removed from each conceptus. The remaining tissue was minced finely and dispersedwith collagenaseusing a modification of a method previously describedby this laboratory (15). The tissue was incubated in 30 ml dissociation medium (medium 199, 20 mM HEPES, 10 mM NaHC03, 50 pg gentamicin sulfate/ml, pH 7.4) containing 0.1% collagenaseand 0.002% DNAse at 37 C for I-2 h. The incubation wascarried out in a water bath-shaker (Gyrorotory Water Bath Shaker, New Brunswick Scientific, Edison, NJ) at shaker setting 5. The tissuewas centrifuged at 800 x g for 5 min, and fresh dissociation medium without enzymes was added. The tissue was dispersedby repeated pipetting and then filtered through 150 pm Nitex. The cell suspensionwas then centrifuged at 800 x g for 5 min, resuspendedin a small volume of dissociation medium containing two or three drops of 0.04% DNAse, and centrifuged through a 40% Percoll density gradient for 15 min at 800 x g. The density of the gradient at various positionswasdeterminedwith density marker beads.The bands of cells were collected from the gradient with a Pasteur pipet and washedin 4-5 volumes of dissociation medium to remove the Percoll. The cells were resuspendedin NCTC-135 containing 20 mM HEPES, 25 mM NaHCO:l, 1.65 mM cysteine, and 50 rg gentamicin sulfate/ml (culture medium) at a concentration of 0.5 x lo6 cells/ml, as estimated with a hemocytometer. The cellswere plated at a density of 2.0 to 3.0 x 10” cells/cm’ onto collagengelsin plastic multiwell plates (Corning GlassWorks, Corning, NY). The cells were incubated at 37 C under an atmosphereof 95% sir/5% CO, for up to 5 days. The day the cells were plated was considered day 0. The medium was changeddaily and stored at -20 C until assayed.The cells were harvested for DNA assayby dissolving the collagen gelsin 4.3 N acetic acid at 37 C for 30 min and then collecting the cells by centrifugation at 800 X g for 5 min. When effects of secretagogueson mPL-I secretion were assessed, the cells were plated and allowed to attach for 2 h. The medium was then replaced with medium containing a
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putative secretagogueor with control medium. Progesterone was dissolved in absolute ethanol and then diluted in culture medium so that the final ethanol concentration was lessthan 0.1%. Ethanol was added to control medium at the sameconcentration. Reverse hemolytic
plaque assay
The reverse hemolytic plaque assay was performed as describedpreviously with slight modifications (15). The cellswere removedfrom the collagengelsby incubation in culture medium containing 0.1% collagenase,0.1% trypsin, and 0.002%DNAse at 37 C for 30 min, followed by centrifugation at 800 x g for 5 min. Equal volumes of protein A-coated ovine erythrocytes (18% suspension)and placental cells in NCTC-135 containing 0.1% BSA (1 X 10fi cells/ml) were mixed, infused into an incubation chamber consisting of poly-L-lysine-coated glass microscopeslides, and incubated for 50 min at 37 C in 95% sir/5% CO,. The chamber was washed with NCTC-135 containing 0.1% BSA. Rabbit anti-mPL-I antiserum, diluted 1:lOO (vol/vol) in NCTC-135 containing 0.1% BSA, wasthen infused. After a 4-h incubation at 37 C, guinea pig serum, diluted I:30 to 1:50 (vol/vol) in NCTC-135 containing 0.1% BSA, was added. The slideswere incubated for 20 min under the same conditions, and the reaction was stopped by infusing 1.8% glutaraldehyde in isotonic saline. The cells were stained with 1% toluidine blue in isotonic saline and examined microscopically for the presenceof plaques.No plaque formation occurred when anti-mPL-I antiserum was replaced with normal rabbit serum or when guinea pig serum, the source of complement, was omitted. Immunocytochemistry
The cells were removed from the collagen gels as described for the reverse hemolytic plaque assay. They were plated in culture medium containing 10% fetal calf serum in 24- or 96well plastic plates without collagen at a density of 2.0-3.0 x lo” cells/cm’ and incubated in 95% sir/5% CO, at 37 C for 2 h to allow attachment. The cellswerewashedwith 10 mM sodium phosphate, 150 mM NaCl, pH 7.4, and then fixed in Bouin’s solution. The cells were stained for mPL-I using an avidinbiotin immunoperoxidasekit as previously described(16), except that 10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.5% Tween 20 (vol/vol) was used as the buffer. Anti-mPL-I antiserum wasusedat a dilution of l:lOOO-1:1500. The cells were not counterstained. They were stored in 70% ethanol. The specificity of the methodwasassessed by replacing the anti-mPL-I antiserum with normal rabbit serum and by saturating the anti-mPL-I antiserum with recombinant mPLI (20 pg/ml, overnight incubation at 4 C). Incubating the antimPL-I antiserum with mPL-II or mGH before use did not affect staining. Placental
extraction
The tissue was homogenizedin five volumes (wt/vol) of 100 NH,HCO:,, 10 mM EDTA, 10 mM EGTA, pH 9.3, and centrifuged for 5 min at maximum speedin a Beckman (Palo Alto, CA) Microfuge 12. The supernatant was stored at -20 C until assayed. mM
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mPL-II and recombinant mPL-I were purified as previously described(2, 5). Rabbit antiserum to recombinant mPL-I has been described (5). Guinea pig serum was purchased from GIBCO (Grand Island, NY). Collagenase(Clostridium histolyticum, type 1, CLS) was from Worthington Biochemical Co. (Malvern, PA). Percoll and density marker beadswere obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Bouin’s solution, bovine pancreatic deoxyribonuclease(DNAse) type 1 (EC 3.1.21.1),calf thymus DNA, BSA, gentamicin sulfate, polyL-lysine, progesterone, staphylococcal protein A, fetal calf serum, Medium 199 (Hank’s salts), and NCTC-135 were from SigmaChemical Co. (St. Louis, MO). Ovine erythrocytes were purchasedfrom the Colorado Serum Co. (Denver, CO). Avidinbiotin immunoperoxidase kits were purchased from Vector Laboratories (Burlingame, CA). Nitex was purchased from Tetko Inc. (Elmsford, NY). NCS wasobtained from Amersham Corp. (Arlington Heights, IL). L-[3,4,5-“H(N)]-leucine (156 Ci/ mmol) was from New England Nuclear (Boston, MA).
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RIAs and DNA assay
Protein
synthesis
The effect of progesteroneon protein synthesis was determined by incubating cells plated in 24-well plates in the presenceof 25 &i [3H] -1eucinefor 24 h. The cellular proteins were solubilized, and they and the proteins in the medium were precipitated as describedby Thrailkill et al. (18). The precipitates were collected by centrifugation at 10,000X g for 15 min, solubilized in NCS, and the radioactivity was determined by liquid scintillation counting. Statistical
analysis
The data were analyzed for homogeneity of variance with Bartlett’s test. When significant heterogeneity of variance was present, the data were log transformed before additional analysiswas carried out. The nontransformed data are shown in the figures.The data on effects of mPL-II on mPL-I secretionwere analyzed by analysis of variance for repeatedmeasuresdesign. All of the other data were analyzed by analysis of variance for completely randomizeddesign.Subsequentanalysiswascarried out with Dunnett’s test or Duncan’s multiple range test as appropriate. A P value of lessthan 0.05 wasconsideredsignificant. Results Tissue content of mPL-I and mPL-II The concentration of mPL-I in the placenta was much higher on days 9 and 10 of pregnancy than on days 7,8, or 11 [mean f SE placental mPL-I COntent (rig/placenta): day 7, 14.4 f 5.7; day 8, 34.6 f 9.7; day 9, 279.2 f 25.7; day 10, 250.1 f 13.5; day 11, 88.2 f 11.1 (n = six pools of placentas, where each pool comprises all of the placentas from 1 dam); P < 0.05, days 9,10 > day 11 > days 7, 81. The placental mPL-II concentration increased steadily after day 8 [mean + SE placental mPL-II content (ng/ placenta): day 7, 6.0 + 0.2; day 8, 7.8 k 2.3; day 9, 19.3 + 5.2; day 10, 428.1 f 61.7; day 11, 837.6 f 61.0 (n = six pools of placentas, where each pool comprises all of the placentas from 1 dam); P < 0.05, day 11 > day 10 > day 9 > days 7, 81. Banding of mPL-I-containing
cells in Percoll gradients
Placental cells from days 10 and 11 of pregnancy formed six bands when fractionated on 40% Percoll gradients [density (g/ml): band 1, 1.028; band 2, 1.032; band 3,1.044; band 4,1.052; band 5,1.072; band 6, 1.1061.
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Cells from days 7,8, and 9 formed five bands having the same densities as bands 1,2,3,4, and 6 above. The mPLI concentration of the cells in each of the bands on days 7-11 of pregnancy is shown in Fig. 1. Although the mPLI concentration of the cells in bands 1 and 2 was high, especially on days 9 and 10 of pregnancy, these bands contained a large number of dead cells and cellular debris, and cell viability was only about 50% as assessed by trypan blue exclusion. The cells in these bands were not characterized further. Bands 5 and 6 contained primarily red blood cells and nuclei, and these bands were also not characterized further. Cell viability in bands 3 and 4 was about 90 and 95%, respectively, and subsequent analysis was carried out on cells from these bands. Time-course of mPL-I and mPL-II secretion
The time-course of mPL-I and mPL-II secretion was examined in cells from days 7, 8, and 9 of pregnancy. Tissue from days 10 and 11 of pregnancy was excluded because preliminary data indicated that mPL-I could not be detected in the medium after several days of culture. The time-course of mPL-I secretion by cells in band 3 from days 7-9 of pregnancy is shown in Fig. 2. The mPLI concentration of the medium of cells obtained from day 7 of pregnancy increased until the third day and then fell by the fifth day of culture. The behavior of cells from day 8 of pregnancy was similar to that of cells from day 7, but it was somewhat variable between cultures and the increase in mPL-I concentration early in the culture period was not always present. The mPL-I concentration of the medium of cells obtained from day 9 of pregnancy decreased during the entire culture period. The total amount of mPL-I released during 5 days of culture was greater for cells from day 7 of pregnancy than for cells
1.026
1.032
1.044
1.052
1.072
1.106
Density (g/ml) FIG. 1. Concentration of mPL-I in bands of placental cells from days 7-11 of pregnancy obtained after enzymatic dispersion and separation on a 40% Percoll gradient. The data are from one cell preparation that was fractionated on one Percoll gradient. Similar results were obtained with three other cell preparations.
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mPL-I and mPL-II concentrations were measured with highly specific RIAs as previously describedby this laboratory (8, 9). When the mPL-I or mPL-II concentration of a sample fell below the minimum concentration that could be detected in the assay,it was assignedthe value of the minimum detectable dosefor statistical evaluation. The DNA content of the cellswas determined by the method of Hinegardner (17) using calf thymus DNA asthe standard.
LACTOGEN-I
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2900 50
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,
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Time (days) 2. The time-course of mPL-I secretion by cells in band 3 of the Percoll gradient from days 7-9 of pregnancy. The cells were plated in 96-well plates at a density of 0.6 x lo5 cells per well. Each point represents the mean -t SE of 6 wells. The mPL-I concentration of the medium was less than 3.25 ng/rg DNA for cells from day 9 of pregnancy on days 3-5 of culture. The data shown are from one experiment. Similar results were obtained in two other experiments performed on two different cell preparations.
FIG.
from days 8 or 9 [mean + SE total mPL-I in the medium (ng/pg DNA): day 7, 143.90 k 13.41; day 8, 71.17 f 4.66; day 9, 25.07 f 2.32 (n = 5-6); P < 0.05, day 7 > day 8 > day 91. The time-course of mPL-I secretion by cells in band 4 was very similar to that of cells in band 3 except that the mPL-I concentration of the medium was lower (data not shown), and cells in band 3 were consequently selected for further analysis. mPL-II could not be detected in the medium of cells in band 3 from day 7 of pregnancy until the third or fourth day of culture (Fig. 3). mPL-II appeared in the medium of cells from day 8 of pregnancy on the third day of culture, and it was present on the first day of culture in the medium of cells from day 9 of pregnancy (data not shown). The mPL-II concentration of the medium increased during the culture period. Since cells from day 7 of pregnancy secreted the largest amount of mPL-I, and the concentration of mPL-I in the medium increased or remained constant for 3-4 days of culture, they were selected for use in screening putative secretagogues of mPL-I. The DNA content of the cells in band 3 from day 7 of pregnancy remained relatively constant for the first 4 days of culture and then decreased significantly on the fifth day [mean + SE DNA content (micrograms/well): day 1, 0.947 + 0.062; day 2, 0.878 f 0.057; day 3, 0.884 f 0.043; day 4, 0.882 f 0.024; day 5, 0.734 f 0.016 (n = 46); P < 0.05, days l-4 > day 51. The viability of the cells, as assessedby trypan exclusion, was about 80% for the first 4 days and then decreased on day 5 to about 60%. Most of the dead cells were large and binucleate.
I
Time
I
I
I
3
4
5
lo
6
(days)
3. The time-course of mPL-I and mPL-II secretion by cells in band 3 of the Percoll gradient from day 7 of pregnancy. The cells were plated in 96-well plates at a density of 0.6 x lo” cells per well. Each point represents the mean + SE of 6 wells. The mPL-II concentration of the medium was less than 0.78 ng/rg DNA on days 1-3 of culture. The data shown are from one experiment. Similar results were obtained in two other experiments performed on two different cell preparations. FIG.
Identification
of mPL-I-secreting cells
mPL-I-secreting cells in the culture were identified by immunostaining (Fig. 4) and the reverse hemolytic plaque assay (Fig. 5). Two types of mPL-I cells were detected by both methods: small, round mononucleate cells having a diameter of about 25-50 microns and large, flat, polygonal mono- or binucleate cells. The size of the latter cells increased from about 60 microns on the first day of culture to about loo-140 microns on the third. The number of cells that immunostained with antiserum to mPL-I increased between days 1 and 2 (Table 1). Most of the cells that immunostained with anti-mPL-I antiserum on day 1 of culture were small round cells, but by day 3, most of the immunostaining cells were of the larger type. Effect of progesterone on mPL-I secretion Incubation of the cells in the presence of 1 pg/ml progesterone resulted in a highly significant reduction in the mPL-I concentration of the medium; in most, but not all cultures, incubation with 100 rig/ml of progesterone also reduced the mPL-I concentration of the medium (Fig. 6). Significant inhibition of mPL-I secretion occurred by 48 h of treatment when cells were incubated with 1 pg/ml of progesterone (Table 1). Incubation of cells in the presence of 1 pg/ml progesterone resulted in a significant decrease in the number of mPL-I-plaqueforming cells in the reverse hemolytic plaque assay [mean f SE number of plaques at 72 h (plaques per slide): control, 6.83 f 0.95; progesterone, 0.83 f 0.31 (n = 6); P < O.OOl] and a reduction in the number of cells that immunostained with anti-mPL-I antiserum (Table 1).
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The cells that stained with anti-mPL-I antiserum tended to be smaller in progesterone-treated cultures than in control cultures (Fig. 7). The effect of progesterone on mPL-I synthesis was examined using 72 h of treatment with 300 rig/ml of progesterone. The concentration of mPL-I in the medium plus cellular homogenate after the final 24 h of treatment was significantly lower in the progesterone-treated wells than in the control wells [mean & SE total mPL-I in the medium plus cellular homogenate (rig/ml): control, 23.06 + 1.18; progesterone, 16.13 f 0.60 (n = 6); P < 0.051. Seventy-two hours of treatment with 300 rig/ml of progesterone had no effect on the concentration of newly synthesized trichloroacetic acid-precipitable proteins measured during the final 24 h of treatment [mean f SE newly synthesized trichloroacetic acid-precipitable protein (cpm x 10-5/well): control, 20.71 +: 1.43; progesterone, 19.88 +: 1.24 (n = 6); P > 0.051, which suggests that progesterone did not cause a general inhibition of protein synthesis. Progesterone treatment did not affect cell viability or the DNA content of the cells [mean f SE DNA content at 72 h (micrograms/well): control, 0.317 +: 0.028; 10 ng progesterone/ ml, 0.353 f 0.021; 100 ng progesterone/ml, 0.329 f 0.021; 1000 ng progesterone/ml, 0.353 f: 0.026 (n = 6); P > 0.051. Effect of mPL-II
on mPL-I
secretion
Incubation of cells in the presence of mPL-II, in concentrations ranging from 10 rig/ml to 10 pg/ml, for up to 3 days had no effect on the mPL-I concentration of the medium (Table 2). Discussion Placenta from day 7 of pregnancy yielded a heterogeneous population of cells that secreted mPL-I and mPL-
II. The mPL-I-secreting cells were identified by immunostaining and reverse hemolytic plaque assay. Both methods indicated the presence of two types of mPL-I cells: large mono- and binucleate cells having a morphology similar to that reported for trophoblast giant cells in culture (15, 19) and smaller round mononucleate cells. Since mPL-I (4, 20) and rat PL-I (21) have been localized in the conceptus exclusively to trophoblast giant cells, it is likely that the smaller round cells are giant cells that have not attained a mature morphology. The observation that the number of small round cells that immunostained for mPL-I declined between days 1 and 3 of culture, while the number of mPL-I-positive large cells increased, is consistent with this hypothesis. The cells that produced mPL-II were not identified but they are presumed to be giant cells. mPL-II has been localized exclusively to this cell type in the mouse conceptus at midpregnancy (4, 16, 22), and mPL-II was secreted in vitro by giant cells isolated from ll-dayspregnant mice (15). Recent data from our laboratory indicate that mPL-I and mPL-II can be secreted by the same cell (Yamaguchi M. and F. Talamantes, in preparation). The increase in mPL-I secretion by cells from day 7 of pregnancy for the first 3 or 4 days of culture appears to result from the differentiation of mPL-I-producing cells as well as an increase in mPL-I secretion by individual cells, since the number of cells that immunostained for mPL-I increased between the first and second days of culture, while the mPL-I concentration of the medium increased daily through days 3 or 4. The increase in mPL-II secretion over time in culture probably also resulted at least partly from giant cell differentiation. We have previously shown that in a culture of mouse
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FIG. 4. Immunocytochemistry of cells from band 3 of the Percoll gradient from day I of pregnancy on the third day of culture. The cells were stained with antimPL-I antiserum as described in the text. No staining was present when the anti-mPL-I antiserum was replaced with normal rabbit serum. Note that both small and large cells stained for mPL-I. Magnification, X260.
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TABLE 1. Effect of progesterone on the mPL-I concentration medium and cell differentiation mPL-I concentration of the medium (rig/ml)
Day
Control
1992 No 5
of the
Number of mPL-I-staining cells (cells/well) Control
Progesterone
46.0 + 4.7" 80.8 + 4.7b 78.8 + 6.3b
43.6 + 5.0 52.2 rt 5.7* 45.5 + 3.4*
1
17.98 zk 2.61”
18.87 + 3.03”
2 3 4 5
57.61 + 7.34b 93.43 + 8.58' 55.08 rt 3.52b 32.65 + 1.97d
23.74 15.01 5.68 3.25
f 2.81"* AZ 1.70a* k l.48b* k 0.30b*
ND ND ND ND 1 x lo5 cells were plated in 96-well plates and incubated in control medium or medium containing 1 pg/ml progesterone. The mPL-I concentration of the medium was determined for 6 different wells on days l-5 of culture. Cells from 5 of the wells on days l-3 of culture were stained with anti-mPL-I antiserum and counted. Each value represents the mean -C SE. Differences between days of culture for each treatment are indicated by letters, where means with different letters differ significantly, P < 0.05. ND, not done. * Indicates a significant difference between control and progesterone-treated cells, P < 0.01.
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0 0
10
100
1000
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FIG. 5. Micrograph of mPL-I-releasing cells in the reverse hemolytic plaque assay. Details of the method are in the text. Note the size difference between the plaque-forming cells in the panels. Magnification, x260.
placental cells from day 11 of pregnancy, the number of mPL-II-producing cells increased significantly during the first 72 h after plating (15). The signal or signals that stimulate the differentiation of mPL-I- and mPLII-producing giant cells are not known. Although significant differentiation of these cells occurred in vitro in the apparent absence of in uiuo signals, it is possible that these cells were already committed to this pathway in uiuo. The cause of the decline in mPL-I secretion after the fourth day of culture is not clear. A decline in cell viability occurred at this time, which may have been a contributing factor. It is also possible that once giant cells have started to produce mPL-I, they are “programmed” to synthesize the hormone for only some finite
FIG. 6. Effect of progesterone concentration on the mPL-I concentration of the medium; 0.6 x lo5 cells per well were plated in 96-well plates. Progesterone was added 2 h after plating. The medium was changed daily, and the mPL-I concentration of the medium was determined after 72 h of progesterone treatment. Each value represents the mean + SE of 6 wells. (*), Significantly different from control, P < 0.05. The data shown are from a representative experiment. The experiment was replicated more than three times. Significant inhibition of mPL-I secretion occurred with 100 ng progesterone/ml in about 75% of the trials. The lowest concentration of progesterone that consistently inhibited mPL-I secretion was 300 rig/ml (data not shown).
time period. The presence or absence of unknown factors in the medium may also have played a role in reducing mPL-I production. Since an increase in mPL-II secretion roughly coincided with the decline in mPL-I secretion on each of the days of pregnancy examined, we assessed the effect of mPL-II on mPL-I secretion using cells from day 7 of pregnancy. Incubation of the cells in the presence of a wide range of mPL-II concentrations for up to 72 h had no effect on the mPL-I concentration of the medium. These data suggest that the decline in mPL-I secretion
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Progesterone
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TABLE 2. Effect of mPL-II on the mPL-I concentration of the medium (w/ml) mPL-II, rig/ml
Day 1 5.15 4.82 3.92 3.80 5.22
+ 0.89 + 0.85 rfr0.71 f 0.77 zk 0.64
Day 2 15.37 18.38 15.30 14.59 18.74
zk 1.49 + 2.92 + 2.26 + 1.64 z!z2.12
Day 3 33.27 43.41 38.33 38.16 41.33
+ f + + f
3.62 6.61 3.63 4.43 3.38
Cells (0.6 x 105) were plated in 96-well plates and incubated in the presence or absence of mPL-II. Each value represents the mean -C SE of 6 wells. The values for days 1,2, and 3 of culture are from the same wells for each treatment.
that occurred in vitro was not caused by mPL-II. In-uiuo, the mPL-I concentration of the maternal serum also declines as that of mPL-II increases (8, 9). Although mPL-II did not affect the secretion of mPL-I in uitro, the possibility remains that mPL-II could affect mPL-I production by an indirect mechanism in vivo. The ability of the cells to produce mPL-I in uitro did not parallel the amount of mPL-I in the tissue prior to cell dispersion. The mPL-I concentration of the placenta was greatest on days 9 and 10 of pregnancy, but mPL-I production by these cells declined from the beginning of the culture period. In contrast, mPL-I secretion was sustained at the highest levels for the longest time in cells from day 7 of pregnancy, when placental mPL-I concentration is very low. These data suggest that once giant cells have differentiated in uiuo to produce large amounts of mPL-I, its synthesis continues for only a relatively short time in vitro, at least under these culture conditions. The signals that reduce mPL-I gene expression in cells from days 9 and 10 of pregnancy are probably carried over from in uiuo since the maternal serum mPLI concentration (8), placental mPL-I concentration (this study), and steady state levels of mPL-I mRNA (3, 14) all decline rapidly after day 10 of pregnancy. A similar situation may exist for the rat placenta. The production of PL-I by rat trophoblast giant cells declined significantly after 1 day of culture when cells were obtained during the peak in maternal serum PL-I concentration (23, 24). Treatment of cells from day 7 of pregnancy with progesterone resulted in a time- and dose-dependent inhibition of mPL-I secretion. The lowest concentration of progesterone that consistently inhibited mPL-I secretion was 300 rig/ml, which is 4-5 times higher than the maximum maternal serum progesterone concentration that is attained in late pregnancy in this strain of mice FIG. 7. Immunocytochemistry of cells incubated in the absence (top) or presence of 1 pg/ml progesterone for 72 h (middle). The cells were stained with anti-mPL-I antiserum (top and middle) or normal rabbit serum (bottom) as described in the text. Note the size difference between the immunostained cells in the top and middle panels. Magnification, x260.
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Acknowledgments We thank Drs. Robert Soares for their comments
Bigsby, Ronald and advice.
Kensinger,
and
Michael
References 1. Colosi P, Ogren L, Thordarson G, Talamantes F 1987 Purification and partial characterization of two prolactin-like glycoprotein hormone complexes from the midpregnant mouse conceptus. Endocrinology 120:2500-2511 2. Colosi P, Marr G, Lopez J, Haro L, Ogren L, Talamantes F 1982 Isolation, purification, and charactertization of mouse placental lactogen. Proc Nat1 Acad Sci USA 79:771-775 3. Colosi P, Talamantes F, Linzer DIH 1987 Molecular cloning and expression of mouse placental lactogen I complementary deoxyribonucleic acid. Mol Endocrinol 1:767-776 4. Faria TN, Ogren L, Talamantes F, Linzer DIH, Soares MJ 1991 Localization of placental lactogen-I in trophoblast giant cells of the mouse placenta. Biol Reprod 44:327-331 5. Colosi P, Ogren L, Southard JN, Thordarson G, Linzer DIH, Talamantes F 1988 Biological, immunological, and binding properties of recombinant mouse placental lactogen-I. Endocrinology -. 123:2662-2667 6. Thordarson G. Villalobos R. Colosi P. Southard J. Oaren L. Talamantes F 1986 The lactogenic response of cultured mouse mammary epithelial cells to mouse placental lactogen. J Endocrinol 109:263-274 7. MacLeod KR, Smith WC, Ogren L, Talamantes F 1989 Recombinant mouse placental lactogen-I binds to lactogen receptors in mouse liver and overy: partial characterization of the ovarian receptor. Endocrinology 125:2258-2266 8. Ogren L, Southard JN, Colosi P, Linzer DIH, Talamantes F 1989 Mouse placental lactogen-I: RIA and gestational profile in maternal serum. Endocrinoloav 125:2253-2257 9. Soares MJ, Colosi P, Talamantes F 1982 The development and characterization of a homolgous radioimmunoassay for mouse placental lactoaen. Endocrinolotrv 110:668-670 10. Soares MJ,-Talamantes F 1983 Genetic and litter size effects on serum placental lactogen in the mouse. Biol Reprod 29:165-171 11. Fielder PJ, Ogren L, Edwards D, Talamantes F 1987 Effects of fasting on serum lactogenic hormone concentrations during midand late pregnancy in mice. Am J Physiol 253 (Endocrinol Metab 16):E40-E44 12. Soares MJ, Talamantes F 1985 Placental lactogen secretion in the mouse: in vitro responses and ovarian and hormonal influences. J Exp Zoo1 234:97-104 13. Kishi K, Ogren L, Southard J, Talamantes F 1988 Pituitary factors regulating mouse placental lactogen-II secretion during the last half of pregnancy in mice. Endocrinology 122:2309-2317 14. Lopez MF, Carrion FA, Talamantes F 1991 Pituitary-placental interaction: hypophysectomy modulates the secretion of mouse placental lactogen-I. Endocrinology 129:2325-2328 15. Thordarson G, Folger P, Talamantes F 1987 Development of a placental cell culture system for studying the control of mouse placental lactogen secretion. Placenta 8573-585 16. Hall J, Talamantes F 1984 Immunocytochemical localization of mouse placental lactogen in the mouse placenta. J Histochem Cytochem 32:379-382 17. Hinegardner RT 1971 An improved fluorometric assay for DNA. Anal Biochem 30:197-201 18. Thrailkill KM, Golander A, Underwood LE, Richards RG, Handwerger S 1989 Insulin stimulates the synthesis and release of pro&tin from human decidual cells. Endocrinology 124:3010-3014 19. Zuckermann FA. Head JR 1986 Isolation and characterization of trophoblast from murine placenta. Placenta 7:349-364 20. Nieder GL, Jennes L 1990 Production of mouse placental lactogenI by trophoblast giant cells in utero and in uitro. Endocrinology 1262809-2814 21. Faria TN, Deb S, Kwok SCM, Talamantes F, Soares MJ 1990 Ontogeny of placental lactogen-I and placental lactogen-II expres-
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(13). However, since progesterone is produced by giant cells of the mouse placenta (25), it is likely that its concentration within the placenta is higher than that in the maternal serum, and the concentrations used in this study may not have been overwhelmingly unphysiological. Moreover, since the cells were exposed to progesterone prior to tissue dispersion (25), their sensitivity to progesterone in vitro may have been lessened relative to that of cells in ho. The effect of progesterone appeared to be due both to inhibition of the differentiation of mPL-I-producing cells and to inhibition of mPL-I secretion by individual cells. The number of cells that immunostained with anti-mPLI antiserum remained about the same for 3 days in cultures treated with progesterone, whereas it almost doubled in control cultures, which suggests that progesterone prevented the appearance of new mPL-I-producing cells. By the third day of culture, the progesteronetreated mPL-I-producing cells were also frequently smaller than the control cells. Since control giant cells increased in size between the first and third days of culture, progesterone may have also arrested or slowed the growth of cells that had already differentiated to produce mPL-I. An effect of progesterone on the secretion of mPL-I by individual cells is suggested by the observation that the percent reduction in the mPL-I concentration of the medium of progesterone-treated cells was greater than the percent reduction in the number of mPL-I-producing cells treated with progesterone. Similarly, progesterone had a greater inhibitory effect on the number of plaque-forming cells in the reverse hemolytic plaque assay than it did on the number of cells that immunostained for mPL-I. Although the reverse hemolytic plaque assay provides an assessment of hormone release by individual cells, it is not known whether progesterone specifically inhibited mPL-I release or whether the inhibition of mPL-I release was secondary to a decrease in hormone synthesis, which also occurred. Since the effect of progesterone on both mPL-I cell differentiation and the mPL-I concentration of the medium was not apparent until the second day of culture, the inhibition of mPL-I synthesis and/or release may have been related to the inhibition of cell differentiation. Progesterone also inhibits the secretion of mPL-II (12) and rat PL-II (26). Its mechanism of action has been examined in the rat (26). In these studies, PL-II secretion was inhibited by progesterone only in placental labyrinth cell cultures, which were undergoing giant cell differentiation, and not in cultures of already differentiated giant cells. Thus, the mechanism of action of progesterone on the secretion of both PLs of rodents appears similar. The importance of progesterone relative to other factors in regulating the differentiation of PL-producing giant cells remains to be determined.
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the two forms of rat placental lactogen (rPL): rPL-I and rPL-II. Endocrinology 111:1862-1866 25. Sherman MI 1983 Endocrinology of rodent trophoblast cells. In: Loke YW, Whyte A (eds) Biology of Trophoblast. Elsevier Science Publishers BV, Amsterdam, pp 401-467 26. Soares MJ, Glasser SR 1987 Placental lactogen production and functional differentiation of rat trophoblast cells in vitro. J Reprod Fertil79:335-341
Pituitary
Congress
The Third International Pituitary Congress devoted to basic and clinical updates will be held at the Ritz Carlton Hotel, Marina Del Ray, California, June 13-15, 1993. The Congress will immediately follow The Endocrine Society meeting in Las Vegas. Major topics to be presented include: Molecular Pathogenesis of Pituitary Tumors, Growth Factors in the Pituitary, Clinical Use of Growth Hormone in Adults, Hypothalamic Peptides, Novel Pituitary Diagnostic and Therapeutic Techniques, Pituitary Receptors, and Medical Management of Adenomas. For further information, please contact: Carolyn Lavitt, Coordinator, Continuing Medical Education Office, Room 2727, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 900481869; Tel. (310) 855-5547, Fax. (310) 857-1778.
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sion in the devleoping rat placenta. Dev Biol 141:279-291 22. Lee SJ, Talamantes F, Wilder E, Linzer DIH, Nathans D 1986 Trophoblastic giant cells of the mouse placenta as the site of proliferin synthesis. Endocrinology 122:1761-1768 23. Soares MJ, Julian JA, Glasser SR 1985 Trophoblast giant cell release of placental lactogens: temporal a.ld regional characteristics. Dev Biol 107:520-526 24. Robertson MC, Gillespie B, Friesen HG 1982 Characterization of
LACTOGEN-I
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Society
M. YAMAGUCHI, Department
H. ENDO,
of Biology, University
G. THORDARSON, of California,
Lactogen-I and Mouse
L. OGREN,
Santa Cruz, California
ABSTRACT.
The primary objective of this study was to develop a cell culture system for assessing effects of putative secretagogues on mouse PL-I (mPL-I) secretion. Trophoblast from days 7 to 11 of pregnancy was dispersed in collagenase, and the cells were fractionated on a Percoll gradient and plated on collagen gels in serum-free medium. Cells from days 7-9 of pregnancy yielded five bands on Percoll gradients and those from days 10 and 11 yielded six. mPL-I was present in four of the bands of cells from each day of pregnancy. Cells from day 7 of pregnancy that banded at a density of 1.044 g/ml secreted the largest amount of mPL-I during 5 days of culture. The mPL-I concentration of the medium of these cells increased for the first 3 or 4 days of culture and then declined on the fifth day. mPLII could not be detected in the medium until the third or fourth day of culture, and its concentration increased thereafter. Cell viability was about 90% at the time of plating, remained at about 80% between days 1 and 4, and then declined on day 5. The cell type that produced mPL-I was identitied with the reverse hemolytic plaque assay and by staining with anti-mPL-I anti-
T
WO PLs are produced by the mouse placenta: mouse PL (mPL)-I (1) and mPL-II (2). The mPLs are structurally related to one another (3) and are both produced by trophoblast giant cells (4). The hormones share at least some PRL-like biological and receptorbinding activities (5-7), but they differ in their gestational profiles in maternal serum. mPL-I appears in the maternal blood on day 6 of pregnancy. Its concentration increases to high values on days 9 and 10 and then declines to very low values for the remainder of pregnancy (8). mPL-II first appears in the maternal circulation on day 9 of pregnancy (9), and its concentration increases until about day 14 and then levels off in some strains of mice or continues to increase for the remainder of pregnancy in others (9, 10). Several factors that regulate the concentration of Received November 21, 1991. Address all correspondence and requests for reprints to: Dr. Frank Talamantes, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064. *This work was supported by NIH Grants GM-08132 and HD14966.
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serum. Both methods indicated that mPL-I was produced by giant cells. The ability of the cells to respond to putative secretagogues was examined using mPL-II and progesterone. mPL-II, at concentrations ranging between 10 rig/ml and 10 rg/ml, had no effect on the mPL-I concentration of the medium when it was present for up to 3 days of culture, which suggests that mPL-II does not inhibit mPL-I secretion in uitro. Incubation of the cells in the presence of 100-1000 rig/ml progesterone caused a dose- and time-dependent reduction in the mPL-I concentration of the medium and a decrease in the number of cells that stained with anti-mPL-I antiserum. The effect of progesterone on both endpoints was not apparent until the second day of treatment. These data suggest that progesterone inhibits mPL-I secretion at least in part by inhibiting the differentiation of mPL-I-producing giant cells. The fact that the mPL-I-producing cells responded to progesterone indicates that this culture system will be useful in assessing effects of putative secretagogues on mPL-I secretion. (Endocrinology 130: 28972905,1992)
mPL-II in maternal serum have been identified. These include litter size (lo), feto-placental genotype (lo), maternal nutritional status (ll), progesterone (12), and GH (13). In contrast, little is known about the regulation of mPL-I secretion, Recent studies have shown that the maternal serum mPL-I concentration is proportional to litter size (8) and that the pituitary gland suppressesthe mPL-I concentration of the maternal serum (14). The present study was undertaken to develop a cell culture system that can be used for examining the regulation of mPL-I secretion.
Materials
and Methods
Animals
Pregnant SwissWebster mice were purchasedfrom Simonsen Laboratories (Gilroy, CA). The presenceof a vaginal plug was used as an indicator of day 0 of pregnancy. The animals were maintained on a 14-h light, 10-h dark lighting cycle (lights on at 0600 h). Food and water were available ad libitum. The use of animals for this study was approved by the University Animal Care Committee.
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Modulation of Mouse Placental Vitro: Effects of Progesterone Lactogen-II*
MOUSE Hormones
PLACENTAL
and reagents
Cell dissociation
and culture
Conceptuseswere collected on days 7-11 of pregnancy. The fetus and decidua basaliswere removed from each conceptus. The remaining tissue was minced finely and dispersedwith collagenaseusing a modification of a method previously describedby this laboratory (15). The tissue was incubated in 30 ml dissociation medium (medium 199, 20 mM HEPES, 10 mM NaHC03, 50 pg gentamicin sulfate/ml, pH 7.4) containing 0.1% collagenaseand 0.002% DNAse at 37 C for I-2 h. The incubation wascarried out in a water bath-shaker (Gyrorotory Water Bath Shaker, New Brunswick Scientific, Edison, NJ) at shaker setting 5. The tissuewas centrifuged at 800 x g for 5 min, and fresh dissociation medium without enzymes was added. The tissue was dispersedby repeated pipetting and then filtered through 150 pm Nitex. The cell suspensionwas then centrifuged at 800 x g for 5 min, resuspendedin a small volume of dissociation medium containing two or three drops of 0.04% DNAse, and centrifuged through a 40% Percoll density gradient for 15 min at 800 x g. The density of the gradient at various positionswasdeterminedwith density marker beads.The bands of cells were collected from the gradient with a Pasteur pipet and washedin 4-5 volumes of dissociation medium to remove the Percoll. The cells were resuspendedin NCTC-135 containing 20 mM HEPES, 25 mM NaHCO:l, 1.65 mM cysteine, and 50 rg gentamicin sulfate/ml (culture medium) at a concentration of 0.5 x lo6 cells/ml, as estimated with a hemocytometer. The cellswere plated at a density of 2.0 to 3.0 x 10” cells/cm’ onto collagengelsin plastic multiwell plates (Corning GlassWorks, Corning, NY). The cells were incubated at 37 C under an atmosphereof 95% sir/5% CO, for up to 5 days. The day the cells were plated was considered day 0. The medium was changeddaily and stored at -20 C until assayed.The cells were harvested for DNA assayby dissolving the collagen gelsin 4.3 N acetic acid at 37 C for 30 min and then collecting the cells by centrifugation at 800 X g for 5 min. When effects of secretagogueson mPL-I secretion were assessed, the cells were plated and allowed to attach for 2 h. The medium was then replaced with medium containing a
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putative secretagogueor with control medium. Progesterone was dissolved in absolute ethanol and then diluted in culture medium so that the final ethanol concentration was lessthan 0.1%. Ethanol was added to control medium at the sameconcentration. Reverse hemolytic
plaque assay
The reverse hemolytic plaque assay was performed as describedpreviously with slight modifications (15). The cellswere removedfrom the collagengelsby incubation in culture medium containing 0.1% collagenase,0.1% trypsin, and 0.002%DNAse at 37 C for 30 min, followed by centrifugation at 800 x g for 5 min. Equal volumes of protein A-coated ovine erythrocytes (18% suspension)and placental cells in NCTC-135 containing 0.1% BSA (1 X 10fi cells/ml) were mixed, infused into an incubation chamber consisting of poly-L-lysine-coated glass microscopeslides, and incubated for 50 min at 37 C in 95% sir/5% CO,. The chamber was washed with NCTC-135 containing 0.1% BSA. Rabbit anti-mPL-I antiserum, diluted 1:lOO (vol/vol) in NCTC-135 containing 0.1% BSA, wasthen infused. After a 4-h incubation at 37 C, guinea pig serum, diluted I:30 to 1:50 (vol/vol) in NCTC-135 containing 0.1% BSA, was added. The slideswere incubated for 20 min under the same conditions, and the reaction was stopped by infusing 1.8% glutaraldehyde in isotonic saline. The cells were stained with 1% toluidine blue in isotonic saline and examined microscopically for the presenceof plaques.No plaque formation occurred when anti-mPL-I antiserum was replaced with normal rabbit serum or when guinea pig serum, the source of complement, was omitted. Immunocytochemistry
The cells were removed from the collagen gels as described for the reverse hemolytic plaque assay. They were plated in culture medium containing 10% fetal calf serum in 24- or 96well plastic plates without collagen at a density of 2.0-3.0 x lo” cells/cm’ and incubated in 95% sir/5% CO, at 37 C for 2 h to allow attachment. The cellswerewashedwith 10 mM sodium phosphate, 150 mM NaCl, pH 7.4, and then fixed in Bouin’s solution. The cells were stained for mPL-I using an avidinbiotin immunoperoxidasekit as previously described(16), except that 10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.5% Tween 20 (vol/vol) was used as the buffer. Anti-mPL-I antiserum wasusedat a dilution of l:lOOO-1:1500. The cells were not counterstained. They were stored in 70% ethanol. The specificity of the methodwasassessed by replacing the anti-mPL-I antiserum with normal rabbit serum and by saturating the anti-mPL-I antiserum with recombinant mPLI (20 pg/ml, overnight incubation at 4 C). Incubating the antimPL-I antiserum with mPL-II or mGH before use did not affect staining. Placental
extraction
The tissue was homogenizedin five volumes (wt/vol) of 100 NH,HCO:,, 10 mM EDTA, 10 mM EGTA, pH 9.3, and centrifuged for 5 min at maximum speedin a Beckman (Palo Alto, CA) Microfuge 12. The supernatant was stored at -20 C until assayed. mM
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mPL-II and recombinant mPL-I were purified as previously described(2, 5). Rabbit antiserum to recombinant mPL-I has been described (5). Guinea pig serum was purchased from GIBCO (Grand Island, NY). Collagenase(Clostridium histolyticum, type 1, CLS) was from Worthington Biochemical Co. (Malvern, PA). Percoll and density marker beadswere obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Bouin’s solution, bovine pancreatic deoxyribonuclease(DNAse) type 1 (EC 3.1.21.1),calf thymus DNA, BSA, gentamicin sulfate, polyL-lysine, progesterone, staphylococcal protein A, fetal calf serum, Medium 199 (Hank’s salts), and NCTC-135 were from SigmaChemical Co. (St. Louis, MO). Ovine erythrocytes were purchasedfrom the Colorado Serum Co. (Denver, CO). Avidinbiotin immunoperoxidase kits were purchased from Vector Laboratories (Burlingame, CA). Nitex was purchased from Tetko Inc. (Elmsford, NY). NCS wasobtained from Amersham Corp. (Arlington Heights, IL). L-[3,4,5-“H(N)]-leucine (156 Ci/ mmol) was from New England Nuclear (Boston, MA).
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RIAs and DNA assay
Protein
synthesis
The effect of progesteroneon protein synthesis was determined by incubating cells plated in 24-well plates in the presenceof 25 &i [3H] -1eucinefor 24 h. The cellular proteins were solubilized, and they and the proteins in the medium were precipitated as describedby Thrailkill et al. (18). The precipitates were collected by centrifugation at 10,000X g for 15 min, solubilized in NCS, and the radioactivity was determined by liquid scintillation counting. Statistical
analysis
The data were analyzed for homogeneity of variance with Bartlett’s test. When significant heterogeneity of variance was present, the data were log transformed before additional analysiswas carried out. The nontransformed data are shown in the figures.The data on effects of mPL-II on mPL-I secretionwere analyzed by analysis of variance for repeatedmeasuresdesign. All of the other data were analyzed by analysis of variance for completely randomizeddesign.Subsequentanalysiswascarried out with Dunnett’s test or Duncan’s multiple range test as appropriate. A P value of lessthan 0.05 wasconsideredsignificant. Results Tissue content of mPL-I and mPL-II The concentration of mPL-I in the placenta was much higher on days 9 and 10 of pregnancy than on days 7,8, or 11 [mean f SE placental mPL-I COntent (rig/placenta): day 7, 14.4 f 5.7; day 8, 34.6 f 9.7; day 9, 279.2 f 25.7; day 10, 250.1 f 13.5; day 11, 88.2 f 11.1 (n = six pools of placentas, where each pool comprises all of the placentas from 1 dam); P < 0.05, days 9,10 > day 11 > days 7, 81. The placental mPL-II concentration increased steadily after day 8 [mean + SE placental mPL-II content (ng/ placenta): day 7, 6.0 + 0.2; day 8, 7.8 k 2.3; day 9, 19.3 + 5.2; day 10, 428.1 f 61.7; day 11, 837.6 f 61.0 (n = six pools of placentas, where each pool comprises all of the placentas from 1 dam); P < 0.05, day 11 > day 10 > day 9 > days 7, 81. Banding of mPL-I-containing
cells in Percoll gradients
Placental cells from days 10 and 11 of pregnancy formed six bands when fractionated on 40% Percoll gradients [density (g/ml): band 1, 1.028; band 2, 1.032; band 3,1.044; band 4,1.052; band 5,1.072; band 6, 1.1061.
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Cells from days 7,8, and 9 formed five bands having the same densities as bands 1,2,3,4, and 6 above. The mPLI concentration of the cells in each of the bands on days 7-11 of pregnancy is shown in Fig. 1. Although the mPLI concentration of the cells in bands 1 and 2 was high, especially on days 9 and 10 of pregnancy, these bands contained a large number of dead cells and cellular debris, and cell viability was only about 50% as assessed by trypan blue exclusion. The cells in these bands were not characterized further. Bands 5 and 6 contained primarily red blood cells and nuclei, and these bands were also not characterized further. Cell viability in bands 3 and 4 was about 90 and 95%, respectively, and subsequent analysis was carried out on cells from these bands. Time-course of mPL-I and mPL-II secretion
The time-course of mPL-I and mPL-II secretion was examined in cells from days 7, 8, and 9 of pregnancy. Tissue from days 10 and 11 of pregnancy was excluded because preliminary data indicated that mPL-I could not be detected in the medium after several days of culture. The time-course of mPL-I secretion by cells in band 3 from days 7-9 of pregnancy is shown in Fig. 2. The mPLI concentration of the medium of cells obtained from day 7 of pregnancy increased until the third day and then fell by the fifth day of culture. The behavior of cells from day 8 of pregnancy was similar to that of cells from day 7, but it was somewhat variable between cultures and the increase in mPL-I concentration early in the culture period was not always present. The mPL-I concentration of the medium of cells obtained from day 9 of pregnancy decreased during the entire culture period. The total amount of mPL-I released during 5 days of culture was greater for cells from day 7 of pregnancy than for cells
1.026
1.032
1.044
1.052
1.072
1.106
Density (g/ml) FIG. 1. Concentration of mPL-I in bands of placental cells from days 7-11 of pregnancy obtained after enzymatic dispersion and separation on a 40% Percoll gradient. The data are from one cell preparation that was fractionated on one Percoll gradient. Similar results were obtained with three other cell preparations.
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mPL-I and mPL-II concentrations were measured with highly specific RIAs as previously describedby this laboratory (8, 9). When the mPL-I or mPL-II concentration of a sample fell below the minimum concentration that could be detected in the assay,it was assignedthe value of the minimum detectable dosefor statistical evaluation. The DNA content of the cellswas determined by the method of Hinegardner (17) using calf thymus DNA asthe standard.
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,
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lo-
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Time (days) 2. The time-course of mPL-I secretion by cells in band 3 of the Percoll gradient from days 7-9 of pregnancy. The cells were plated in 96-well plates at a density of 0.6 x lo5 cells per well. Each point represents the mean -t SE of 6 wells. The mPL-I concentration of the medium was less than 3.25 ng/rg DNA for cells from day 9 of pregnancy on days 3-5 of culture. The data shown are from one experiment. Similar results were obtained in two other experiments performed on two different cell preparations.
FIG.
from days 8 or 9 [mean + SE total mPL-I in the medium (ng/pg DNA): day 7, 143.90 k 13.41; day 8, 71.17 f 4.66; day 9, 25.07 f 2.32 (n = 5-6); P < 0.05, day 7 > day 8 > day 91. The time-course of mPL-I secretion by cells in band 4 was very similar to that of cells in band 3 except that the mPL-I concentration of the medium was lower (data not shown), and cells in band 3 were consequently selected for further analysis. mPL-II could not be detected in the medium of cells in band 3 from day 7 of pregnancy until the third or fourth day of culture (Fig. 3). mPL-II appeared in the medium of cells from day 8 of pregnancy on the third day of culture, and it was present on the first day of culture in the medium of cells from day 9 of pregnancy (data not shown). The mPL-II concentration of the medium increased during the culture period. Since cells from day 7 of pregnancy secreted the largest amount of mPL-I, and the concentration of mPL-I in the medium increased or remained constant for 3-4 days of culture, they were selected for use in screening putative secretagogues of mPL-I. The DNA content of the cells in band 3 from day 7 of pregnancy remained relatively constant for the first 4 days of culture and then decreased significantly on the fifth day [mean + SE DNA content (micrograms/well): day 1, 0.947 + 0.062; day 2, 0.878 f 0.057; day 3, 0.884 f 0.043; day 4, 0.882 f 0.024; day 5, 0.734 f 0.016 (n = 46); P < 0.05, days l-4 > day 51. The viability of the cells, as assessedby trypan exclusion, was about 80% for the first 4 days and then decreased on day 5 to about 60%. Most of the dead cells were large and binucleate.
I
Time
I
I
I
3
4
5
lo
6
(days)
3. The time-course of mPL-I and mPL-II secretion by cells in band 3 of the Percoll gradient from day 7 of pregnancy. The cells were plated in 96-well plates at a density of 0.6 x lo” cells per well. Each point represents the mean + SE of 6 wells. The mPL-II concentration of the medium was less than 0.78 ng/rg DNA on days 1-3 of culture. The data shown are from one experiment. Similar results were obtained in two other experiments performed on two different cell preparations. FIG.
Identification
of mPL-I-secreting cells
mPL-I-secreting cells in the culture were identified by immunostaining (Fig. 4) and the reverse hemolytic plaque assay (Fig. 5). Two types of mPL-I cells were detected by both methods: small, round mononucleate cells having a diameter of about 25-50 microns and large, flat, polygonal mono- or binucleate cells. The size of the latter cells increased from about 60 microns on the first day of culture to about loo-140 microns on the third. The number of cells that immunostained with antiserum to mPL-I increased between days 1 and 2 (Table 1). Most of the cells that immunostained with anti-mPL-I antiserum on day 1 of culture were small round cells, but by day 3, most of the immunostaining cells were of the larger type. Effect of progesterone on mPL-I secretion Incubation of the cells in the presence of 1 pg/ml progesterone resulted in a highly significant reduction in the mPL-I concentration of the medium; in most, but not all cultures, incubation with 100 rig/ml of progesterone also reduced the mPL-I concentration of the medium (Fig. 6). Significant inhibition of mPL-I secretion occurred by 48 h of treatment when cells were incubated with 1 pg/ml of progesterone (Table 1). Incubation of cells in the presence of 1 pg/ml progesterone resulted in a significant decrease in the number of mPL-I-plaqueforming cells in the reverse hemolytic plaque assay [mean f SE number of plaques at 72 h (plaques per slide): control, 6.83 f 0.95; progesterone, 0.83 f 0.31 (n = 6); P < O.OOl] and a reduction in the number of cells that immunostained with anti-mPL-I antiserum (Table 1).
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The cells that stained with anti-mPL-I antiserum tended to be smaller in progesterone-treated cultures than in control cultures (Fig. 7). The effect of progesterone on mPL-I synthesis was examined using 72 h of treatment with 300 rig/ml of progesterone. The concentration of mPL-I in the medium plus cellular homogenate after the final 24 h of treatment was significantly lower in the progesterone-treated wells than in the control wells [mean & SE total mPL-I in the medium plus cellular homogenate (rig/ml): control, 23.06 + 1.18; progesterone, 16.13 f 0.60 (n = 6); P < 0.051. Seventy-two hours of treatment with 300 rig/ml of progesterone had no effect on the concentration of newly synthesized trichloroacetic acid-precipitable proteins measured during the final 24 h of treatment [mean f SE newly synthesized trichloroacetic acid-precipitable protein (cpm x 10-5/well): control, 20.71 +: 1.43; progesterone, 19.88 +: 1.24 (n = 6); P > 0.051, which suggests that progesterone did not cause a general inhibition of protein synthesis. Progesterone treatment did not affect cell viability or the DNA content of the cells [mean f SE DNA content at 72 h (micrograms/well): control, 0.317 +: 0.028; 10 ng progesterone/ ml, 0.353 f 0.021; 100 ng progesterone/ml, 0.329 f 0.021; 1000 ng progesterone/ml, 0.353 f: 0.026 (n = 6); P > 0.051. Effect of mPL-II
on mPL-I
secretion
Incubation of cells in the presence of mPL-II, in concentrations ranging from 10 rig/ml to 10 pg/ml, for up to 3 days had no effect on the mPL-I concentration of the medium (Table 2). Discussion Placenta from day 7 of pregnancy yielded a heterogeneous population of cells that secreted mPL-I and mPL-
II. The mPL-I-secreting cells were identified by immunostaining and reverse hemolytic plaque assay. Both methods indicated the presence of two types of mPL-I cells: large mono- and binucleate cells having a morphology similar to that reported for trophoblast giant cells in culture (15, 19) and smaller round mononucleate cells. Since mPL-I (4, 20) and rat PL-I (21) have been localized in the conceptus exclusively to trophoblast giant cells, it is likely that the smaller round cells are giant cells that have not attained a mature morphology. The observation that the number of small round cells that immunostained for mPL-I declined between days 1 and 3 of culture, while the number of mPL-I-positive large cells increased, is consistent with this hypothesis. The cells that produced mPL-II were not identified but they are presumed to be giant cells. mPL-II has been localized exclusively to this cell type in the mouse conceptus at midpregnancy (4, 16, 22), and mPL-II was secreted in vitro by giant cells isolated from ll-dayspregnant mice (15). Recent data from our laboratory indicate that mPL-I and mPL-II can be secreted by the same cell (Yamaguchi M. and F. Talamantes, in preparation). The increase in mPL-I secretion by cells from day 7 of pregnancy for the first 3 or 4 days of culture appears to result from the differentiation of mPL-I-producing cells as well as an increase in mPL-I secretion by individual cells, since the number of cells that immunostained for mPL-I increased between the first and second days of culture, while the mPL-I concentration of the medium increased daily through days 3 or 4. The increase in mPL-II secretion over time in culture probably also resulted at least partly from giant cell differentiation. We have previously shown that in a culture of mouse
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FIG. 4. Immunocytochemistry of cells from band 3 of the Percoll gradient from day I of pregnancy on the third day of culture. The cells were stained with antimPL-I antiserum as described in the text. No staining was present when the anti-mPL-I antiserum was replaced with normal rabbit serum. Note that both small and large cells stained for mPL-I. Magnification, X260.
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TABLE 1. Effect of progesterone on the mPL-I concentration medium and cell differentiation mPL-I concentration of the medium (rig/ml)
Day
Control
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Number of mPL-I-staining cells (cells/well) Control
Progesterone
46.0 + 4.7" 80.8 + 4.7b 78.8 + 6.3b
43.6 + 5.0 52.2 rt 5.7* 45.5 + 3.4*
1
17.98 zk 2.61”
18.87 + 3.03”
2 3 4 5
57.61 + 7.34b 93.43 + 8.58' 55.08 rt 3.52b 32.65 + 1.97d
23.74 15.01 5.68 3.25
f 2.81"* AZ 1.70a* k l.48b* k 0.30b*
ND ND ND ND 1 x lo5 cells were plated in 96-well plates and incubated in control medium or medium containing 1 pg/ml progesterone. The mPL-I concentration of the medium was determined for 6 different wells on days l-5 of culture. Cells from 5 of the wells on days l-3 of culture were stained with anti-mPL-I antiserum and counted. Each value represents the mean -C SE. Differences between days of culture for each treatment are indicated by letters, where means with different letters differ significantly, P < 0.05. ND, not done. * Indicates a significant difference between control and progesterone-treated cells, P < 0.01.
30
z
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75 5 5 n E
10
0 0
10
100
1000
Progesterone (rig/ml)
FIG. 5. Micrograph of mPL-I-releasing cells in the reverse hemolytic plaque assay. Details of the method are in the text. Note the size difference between the plaque-forming cells in the panels. Magnification, x260.
placental cells from day 11 of pregnancy, the number of mPL-II-producing cells increased significantly during the first 72 h after plating (15). The signal or signals that stimulate the differentiation of mPL-I- and mPLII-producing giant cells are not known. Although significant differentiation of these cells occurred in vitro in the apparent absence of in uiuo signals, it is possible that these cells were already committed to this pathway in uiuo. The cause of the decline in mPL-I secretion after the fourth day of culture is not clear. A decline in cell viability occurred at this time, which may have been a contributing factor. It is also possible that once giant cells have started to produce mPL-I, they are “programmed” to synthesize the hormone for only some finite
FIG. 6. Effect of progesterone concentration on the mPL-I concentration of the medium; 0.6 x lo5 cells per well were plated in 96-well plates. Progesterone was added 2 h after plating. The medium was changed daily, and the mPL-I concentration of the medium was determined after 72 h of progesterone treatment. Each value represents the mean + SE of 6 wells. (*), Significantly different from control, P < 0.05. The data shown are from a representative experiment. The experiment was replicated more than three times. Significant inhibition of mPL-I secretion occurred with 100 ng progesterone/ml in about 75% of the trials. The lowest concentration of progesterone that consistently inhibited mPL-I secretion was 300 rig/ml (data not shown).
time period. The presence or absence of unknown factors in the medium may also have played a role in reducing mPL-I production. Since an increase in mPL-II secretion roughly coincided with the decline in mPL-I secretion on each of the days of pregnancy examined, we assessed the effect of mPL-II on mPL-I secretion using cells from day 7 of pregnancy. Incubation of the cells in the presence of a wide range of mPL-II concentrations for up to 72 h had no effect on the mPL-I concentration of the medium. These data suggest that the decline in mPL-I secretion
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TABLE 2. Effect of mPL-II on the mPL-I concentration of the medium (w/ml) mPL-II, rig/ml
Day 1 5.15 4.82 3.92 3.80 5.22
+ 0.89 + 0.85 rfr0.71 f 0.77 zk 0.64
Day 2 15.37 18.38 15.30 14.59 18.74
zk 1.49 + 2.92 + 2.26 + 1.64 z!z2.12
Day 3 33.27 43.41 38.33 38.16 41.33
+ f + + f
3.62 6.61 3.63 4.43 3.38
Cells (0.6 x 105) were plated in 96-well plates and incubated in the presence or absence of mPL-II. Each value represents the mean -C SE of 6 wells. The values for days 1,2, and 3 of culture are from the same wells for each treatment.
that occurred in vitro was not caused by mPL-II. In-uiuo, the mPL-I concentration of the maternal serum also declines as that of mPL-II increases (8, 9). Although mPL-II did not affect the secretion of mPL-I in uitro, the possibility remains that mPL-II could affect mPL-I production by an indirect mechanism in vivo. The ability of the cells to produce mPL-I in uitro did not parallel the amount of mPL-I in the tissue prior to cell dispersion. The mPL-I concentration of the placenta was greatest on days 9 and 10 of pregnancy, but mPL-I production by these cells declined from the beginning of the culture period. In contrast, mPL-I secretion was sustained at the highest levels for the longest time in cells from day 7 of pregnancy, when placental mPL-I concentration is very low. These data suggest that once giant cells have differentiated in uiuo to produce large amounts of mPL-I, its synthesis continues for only a relatively short time in vitro, at least under these culture conditions. The signals that reduce mPL-I gene expression in cells from days 9 and 10 of pregnancy are probably carried over from in uiuo since the maternal serum mPLI concentration (8), placental mPL-I concentration (this study), and steady state levels of mPL-I mRNA (3, 14) all decline rapidly after day 10 of pregnancy. A similar situation may exist for the rat placenta. The production of PL-I by rat trophoblast giant cells declined significantly after 1 day of culture when cells were obtained during the peak in maternal serum PL-I concentration (23, 24). Treatment of cells from day 7 of pregnancy with progesterone resulted in a time- and dose-dependent inhibition of mPL-I secretion. The lowest concentration of progesterone that consistently inhibited mPL-I secretion was 300 rig/ml, which is 4-5 times higher than the maximum maternal serum progesterone concentration that is attained in late pregnancy in this strain of mice FIG. 7. Immunocytochemistry of cells incubated in the absence (top) or presence of 1 pg/ml progesterone for 72 h (middle). The cells were stained with anti-mPL-I antiserum (top and middle) or normal rabbit serum (bottom) as described in the text. Note the size difference between the immunostained cells in the top and middle panels. Magnification, x260.
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Acknowledgments We thank Drs. Robert Soares for their comments
Bigsby, Ronald and advice.
Kensinger,
and
Michael
References 1. Colosi P, Ogren L, Thordarson G, Talamantes F 1987 Purification and partial characterization of two prolactin-like glycoprotein hormone complexes from the midpregnant mouse conceptus. Endocrinology 120:2500-2511 2. Colosi P, Marr G, Lopez J, Haro L, Ogren L, Talamantes F 1982 Isolation, purification, and charactertization of mouse placental lactogen. Proc Nat1 Acad Sci USA 79:771-775 3. Colosi P, Talamantes F, Linzer DIH 1987 Molecular cloning and expression of mouse placental lactogen I complementary deoxyribonucleic acid. Mol Endocrinol 1:767-776 4. Faria TN, Ogren L, Talamantes F, Linzer DIH, Soares MJ 1991 Localization of placental lactogen-I in trophoblast giant cells of the mouse placenta. Biol Reprod 44:327-331 5. Colosi P, Ogren L, Southard JN, Thordarson G, Linzer DIH, Talamantes F 1988 Biological, immunological, and binding properties of recombinant mouse placental lactogen-I. Endocrinology -. 123:2662-2667 6. Thordarson G. Villalobos R. Colosi P. Southard J. Oaren L. Talamantes F 1986 The lactogenic response of cultured mouse mammary epithelial cells to mouse placental lactogen. J Endocrinol 109:263-274 7. MacLeod KR, Smith WC, Ogren L, Talamantes F 1989 Recombinant mouse placental lactogen-I binds to lactogen receptors in mouse liver and overy: partial characterization of the ovarian receptor. Endocrinology 125:2258-2266 8. Ogren L, Southard JN, Colosi P, Linzer DIH, Talamantes F 1989 Mouse placental lactogen-I: RIA and gestational profile in maternal serum. Endocrinoloav 125:2253-2257 9. Soares MJ, Colosi P, Talamantes F 1982 The development and characterization of a homolgous radioimmunoassay for mouse placental lactoaen. Endocrinolotrv 110:668-670 10. Soares MJ,-Talamantes F 1983 Genetic and litter size effects on serum placental lactogen in the mouse. Biol Reprod 29:165-171 11. Fielder PJ, Ogren L, Edwards D, Talamantes F 1987 Effects of fasting on serum lactogenic hormone concentrations during midand late pregnancy in mice. Am J Physiol 253 (Endocrinol Metab 16):E40-E44 12. Soares MJ, Talamantes F 1985 Placental lactogen secretion in the mouse: in vitro responses and ovarian and hormonal influences. J Exp Zoo1 234:97-104 13. Kishi K, Ogren L, Southard J, Talamantes F 1988 Pituitary factors regulating mouse placental lactogen-II secretion during the last half of pregnancy in mice. Endocrinology 122:2309-2317 14. Lopez MF, Carrion FA, Talamantes F 1991 Pituitary-placental interaction: hypophysectomy modulates the secretion of mouse placental lactogen-I. Endocrinology 129:2325-2328 15. Thordarson G, Folger P, Talamantes F 1987 Development of a placental cell culture system for studying the control of mouse placental lactogen secretion. Placenta 8573-585 16. Hall J, Talamantes F 1984 Immunocytochemical localization of mouse placental lactogen in the mouse placenta. J Histochem Cytochem 32:379-382 17. Hinegardner RT 1971 An improved fluorometric assay for DNA. Anal Biochem 30:197-201 18. Thrailkill KM, Golander A, Underwood LE, Richards RG, Handwerger S 1989 Insulin stimulates the synthesis and release of pro&tin from human decidual cells. Endocrinology 124:3010-3014 19. Zuckermann FA. Head JR 1986 Isolation and characterization of trophoblast from murine placenta. Placenta 7:349-364 20. Nieder GL, Jennes L 1990 Production of mouse placental lactogenI by trophoblast giant cells in utero and in uitro. Endocrinology 1262809-2814 21. Faria TN, Deb S, Kwok SCM, Talamantes F, Soares MJ 1990 Ontogeny of placental lactogen-I and placental lactogen-II expres-
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(13). However, since progesterone is produced by giant cells of the mouse placenta (25), it is likely that its concentration within the placenta is higher than that in the maternal serum, and the concentrations used in this study may not have been overwhelmingly unphysiological. Moreover, since the cells were exposed to progesterone prior to tissue dispersion (25), their sensitivity to progesterone in vitro may have been lessened relative to that of cells in ho. The effect of progesterone appeared to be due both to inhibition of the differentiation of mPL-I-producing cells and to inhibition of mPL-I secretion by individual cells. The number of cells that immunostained with anti-mPLI antiserum remained about the same for 3 days in cultures treated with progesterone, whereas it almost doubled in control cultures, which suggests that progesterone prevented the appearance of new mPL-I-producing cells. By the third day of culture, the progesteronetreated mPL-I-producing cells were also frequently smaller than the control cells. Since control giant cells increased in size between the first and third days of culture, progesterone may have also arrested or slowed the growth of cells that had already differentiated to produce mPL-I. An effect of progesterone on the secretion of mPL-I by individual cells is suggested by the observation that the percent reduction in the mPL-I concentration of the medium of progesterone-treated cells was greater than the percent reduction in the number of mPL-I-producing cells treated with progesterone. Similarly, progesterone had a greater inhibitory effect on the number of plaque-forming cells in the reverse hemolytic plaque assay than it did on the number of cells that immunostained for mPL-I. Although the reverse hemolytic plaque assay provides an assessment of hormone release by individual cells, it is not known whether progesterone specifically inhibited mPL-I release or whether the inhibition of mPL-I release was secondary to a decrease in hormone synthesis, which also occurred. Since the effect of progesterone on both mPL-I cell differentiation and the mPL-I concentration of the medium was not apparent until the second day of culture, the inhibition of mPL-I synthesis and/or release may have been related to the inhibition of cell differentiation. Progesterone also inhibits the secretion of mPL-II (12) and rat PL-II (26). Its mechanism of action has been examined in the rat (26). In these studies, PL-II secretion was inhibited by progesterone only in placental labyrinth cell cultures, which were undergoing giant cell differentiation, and not in cultures of already differentiated giant cells. Thus, the mechanism of action of progesterone on the secretion of both PLs of rodents appears similar. The importance of progesterone relative to other factors in regulating the differentiation of PL-producing giant cells remains to be determined.
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the two forms of rat placental lactogen (rPL): rPL-I and rPL-II. Endocrinology 111:1862-1866 25. Sherman MI 1983 Endocrinology of rodent trophoblast cells. In: Loke YW, Whyte A (eds) Biology of Trophoblast. Elsevier Science Publishers BV, Amsterdam, pp 401-467 26. Soares MJ, Glasser SR 1987 Placental lactogen production and functional differentiation of rat trophoblast cells in vitro. J Reprod Fertil79:335-341
Pituitary
Congress
The Third International Pituitary Congress devoted to basic and clinical updates will be held at the Ritz Carlton Hotel, Marina Del Ray, California, June 13-15, 1993. The Congress will immediately follow The Endocrine Society meeting in Las Vegas. Major topics to be presented include: Molecular Pathogenesis of Pituitary Tumors, Growth Factors in the Pituitary, Clinical Use of Growth Hormone in Adults, Hypothalamic Peptides, Novel Pituitary Diagnostic and Therapeutic Techniques, Pituitary Receptors, and Medical Management of Adenomas. For further information, please contact: Carolyn Lavitt, Coordinator, Continuing Medical Education Office, Room 2727, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 900481869; Tel. (310) 855-5547, Fax. (310) 857-1778.
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sion in the devleoping rat placenta. Dev Biol 141:279-291 22. Lee SJ, Talamantes F, Wilder E, Linzer DIH, Nathans D 1986 Trophoblastic giant cells of the mouse placenta as the site of proliferin synthesis. Endocrinology 122:1761-1768 23. Soares MJ, Julian JA, Glasser SR 1985 Trophoblast giant cell release of placental lactogens: temporal a.ld regional characteristics. Dev Biol 107:520-526 24. Robertson MC, Gillespie B, Friesen HG 1982 Characterization of
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