0013.7227/92/1314-1595$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Production Lactogen-II M. YAMAGUCHI, F. TALAMANTES
Vol. 131, No. 4 Printed in U.S.A.
Society
of Mouse Placental by the Same Giant L.
OGREN,
H.
ENDO,
G.
Lactogen-I Cell*
THORDARSON,
R. M.
BIGSBY,
and Placental
AND
Department of Biology (M. Y., L.O., H.E., G. T., F. T.), U nzversity . of California, Santa Cruz, California and the Department of Obstetrics and Gynecology (R. M. B.). Indiana University School of Medicine, Indianapolis, Indiana 46202-5196
95064;
ABSTRACT In previous studies, mouse placental lactogen I (mPL-I) and mPLII were localized to trophoblast giant cells in the placenta at midpregnancy. The present study was undertaken to determine whether mPLI and mPL-II are produced by two distinct populations of giant cells or by the same cells. A heterogeneous population of cells that included trophoblast giant cells was obtained by enzymatic dispersion and Percoll gradient centrifugation of placentas from days 7 and 9 of pregnancy. Cells from day 7 of pregnancy were cultured in serum-free medium for 5 days, and cells that contained mPL-I, mPL-II, or both mPL-I and mPL-II were identified by double-staining immunocytochemistry. The percentage of PL cells that contained both mPL-I and mPL-II increased from about 30% on the first day of culture to about 90% on the third, and then declined to zero by day 5. Between 50% and 60% of the PL cells contained only mPL-I on the first 2 days of culture, and then the percentage of PL cells containing only mPL-I declined. The percentage of cells that contained only mPL-II was low
for 3 days (~10%) and then increased to about 80% of the PLcontaining cells by day 5. Cells from day 9 of pregnancy were analyzed for the release of mPL-I and/or mPL-II by sequential reverse hemolytic plaque assay. Cells that released only one of the PLs, as well as those that released both PLs, were identified. A shift was present in the type of PL released by the cells when they were followed for two consecutive days of culture. On day 1, most of the plaque-forming cells released only mPL-I, but by day 2, the fraction of plaque-forming cells that released only mPL-I declined whereas the fraction that released only mPL-II increased. Cells that released only mPL-I on the first day of culture and both mPL-I and mPL-II or only mPL-II on the second dav of culture were observed. These data suggest that under these culture conditions, PL cells follow a pathway in which they initiallv nroduce only mPL-I, then both mPL-i and GPL-II, and finally onl; APL-II. In uiuo, there is a shift at midpregnancy in the type of PL that is produced by the mouse placenta, and these data suggest that this shift results, at least partly, from a change in gene expression in one population of giant cells. (Endocrinology 131: 1595-1602, 1992)
T
increases steadily after its appearance at midpregnancy (3, 14). The gestational profiles of the two PLs are of interest becausethere is a clear switch at midpregnancy in the type of PL expressedby the placenta. Immunohistochemical and in situ hybridization analyses have localized both PL-I and PL-II to trophoblast giant cells in the mouse and rat (4, 5, 15-20), but it is not known whether a single giant cell produces both of the hormones. Theoretically, the shift at midpregnancy in the type of PL produced by the placenta could result from the disappearance of a population of cells that produces PL-I and the differentiation of a second population that produces PL-II, or it could result from a change in gene expression in one population of cells, or from both processes.In the present study we have used a system for primary culture of cells from the midpregnant mouse placenta (21, 22) to demonstrate that mPL-I and mPL-II are produced by the same giant cell, which suggeststhat the midpregnancy shift in PL production can be attributed, at least partly, to a change in gene expressionin one population of giant cells.
WO placental lactogens (PLs), designated PL-I and PLII, are produced by the mouse (1, 2) and rat placenta (3). The PL-Is are glycosylated single-chain polypeptides that exist as multiple molecular weight forms, with M, ranging between about 29 and 42 kilodaltons (kDa) (2, 4,5). The PL11sare nonglycosylated single-chain polypeptides having an M, of about 25 kDa; they share significant amino acid sequence homology with their respective PL-I (3, 5-8). The known functions of mouse (m) and rat (r) PL-I and PL-II are PRL-like (2, 3, 9, lo), but the gestational profiles of the hormones in maternal blood differ significantly. mPL-I appears in maternal serum on day 6 of pregnancy. Its concentration increasesto very high values on days 9 and 10, and then declines rapidly (11). mPL-II appears in the maternal circulation on day 9 (12). Its concentration increases until about day 14 and then remains relatively constant in some mouse strains or increases until the end of pregnancy in others (13). The gestational profiles of rPL-I and rPL-II in maternal blood are very similar to those of the respective mouse hormones. A large peak in maternal serum rPL-I is present at midpregnancy, and the concentration of rPL-II Received April 15, 1992. Address 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 HD-14966 and GM-08132 (to F. T.).
Materials Cell dissociation Conceptuses Webster mice
and Methods
and culture
were collected on days 7 and 9 of pregnancy (Simonsen Laboratories, Gilroy, CA; vaginal
1595
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from Swiss plug = day
RELEASE
1596
OF mPLs B Y THE SAME CELL
0 of pregnancy). The fetus and decidua basalis were discarded, and the remaining tissue was minced and incubated in dissociation medium [Medium 199 (Hank’s salts), 20 mM HEPES, 10 mM NaHC03, 50 rg streptomycin/ml, 50 U penicillin G/ml, pH 7.21 containing 0.1% (wt/ vol) collagenase (Closhidium histolyticum, type 1, CLS; Worthington Biochemical Co., Malvem, PA) and 0.002% (wt/vol) bovine pancreatic DNAse (type 1, EC3.1.21.1; Sigma, St. Louis, MO) at 37 C for 1 h. For a typical cell preparation, 30 ml medium were used for pooled tissue from 10 animals. After centrifugation at 600 X g for 5 min, the tissue was dispersed in calciumand magnesium-free Hanks solution containing 0.1% (wt/vol) BSA by repeated pipetting, and the cells were filtered through 150-r Nitex (Tetko, Inc., Elmsford, NY). The cell suspension was centrifuged as above, resuspended in 2 ml dissociation medium containing 0.015% DNAse, and then fractionated on a 40% Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient. Cells shown to banding at a density of 1.044 g/ m 1, which were previously contain mPL-I and mPL-II (22), were collected and washed in 5 vol dissociation medium. The cells were resuspended to a concentration of 0.5 x lo6 cells/ml in culture medium (NCTC-135 containing 20 mM HEPES, 25 mM NaHC03, 1.65 mM cysteine, 50 rg streptomycin/ml, 50 U penicillin G/ml, pH 7.2) that was supplemented with 10% (vol/vol) fetal calf serum. The cells were plated at a density of 2.0 to 3.0 X 10s cells/cm’ in multiwell plates or 6.0 x lo4 cells/cm’ in two-well tissue culture chamber slides. Before plating in the tissue culture chamber slides, the cells were incubated in dissociation medium containing 0.1% (wt/vol) trypsin, 0.01% (wt/vol) EDTA, 0.002% DNAse for 30 min at 37 C to obtain a preparation of single cells. After plating in either type of dish, the cells were allowed to attach, and the medium was removed and culture medium containing 0.1% BSA was added. The cells were incubated at 37 C under an atmosphere of 95% sir/5% CO, for up to 5 days. The day the cells were plated was considered day 0. The medium was changed daily and stored at -20 C.
Immunocytochemistry Cells were plated in multiwell plates, Before staining they were washed with 10 rnM sodium phosphate, 150 rnM NaCl, pH 7.4, and fixed in Bouin’s solution. The cells were stained for mPL-I or mPL-II using an avidin-biotin immunoperoxidase kit (Vector Laboratories, Burlingame, CA) as previously described (22). Antisera to mPL-II and recombinant mPL-I were generated in rabbits and have been described (10, 12). Double-staining immunocytochemistry was used to determine whether both mPL-I and mPL-II were present in a single cell using an approach described by Frawley et al. (23) for identifying hormonesecreting pituitary cells. When the cells were stained sequentially with both antisera, they were first stained with one of the antisera and then incubated with nonbiotinylated anti-rabbit immunoglobulin G (IgG) (1:25, vol/vol) in order to saturate any primary antibody that did not bind to biotinylated antirabbit IgG. The cells were then stained with the second antiserum, and the number of stained cells was counted. In control experiments, the dilution of nonbiotinylated antirabbit IgG that was used almost completely abolished subsequent staining by biotinylated antirabbit IgG. The effectiveness of the blocking procedure with antirabbit IgG was also demonstrated by the fact that when cells were stained with either anti-mPL-I or anti-mPL-II, blocked, and then stained with nonimmune rabbit serum, staining intensity did not increase significantly in the second round of staining. To rule out possible effects of sequence and repeated staining on the number of cells that stained, the antisera were applied in different order and also simultaneously. The cells were stored in 70% ethanol. Anti-mPL-I and anti-mPL-II antisera were used at dilutions of 1:1500 (vol/vol) and 1:500 (vol/vol), respectively. When the antisera were used individually in double-staining experiments, nonimmune rabbit serum was added to the antiserum solution so that the total concentration of rabbit serum (antiserum plus nonimmune serum) was the same for each antiserum solution. The specificity of the method was verified by replacing the primary antiserum with non-immune rabbit serum and by saturating the anti-mPL-I and anti-mPL-11 antisera with recombinant mPL-I (20 pg; Ref. 10) and mPLII (10 rg; Ref. l), respectively. Incubation of anti-mPL-I antiserum with mPL-II or mGH (10 pg; Ref. 24) and anti-mPL-II antiserum with mPLI or mGH before use did not affect staining.
Sequential
Endo. Voll31.
reverse hemolytic
1992 No 4
plaque assay
The sequential hemolytic plaque assay was carried out to determine whether one cell could release both mPL-I and mPL-II. It was performed as described by Frawley et al. (23) with some modifications. Cells were plated in two-well tissue culture chamber slides. The day after plating, the cells were washed three times with culture medium containing 0.1% BSA, and the cover of the tissue culture chamber slides was removed, and assay chambers were constructed with etched grid coverslips (Bellco Glass, Inc., Vineland, NJ). The cells were washed two more times as above, and a mixture of protein A-coated ovine red blood cells [I:10 (vol/vol); Colorado Serum Co., Denver, CO], rabbit serum (anti-mPL-I antiserum, anti-mPL-II antiserum, or nonimmune serum; 1:200), and guinea pig serum [1:50 (vol/vol); GIBCO, Grand Island, NY] in culture medium containing 0.1% BSA was added. After a 30-min or 2-h incubation in 95% sir/5% COz at 37 C, plaque formation was checked, and the slides were photographed to record the location of the plaqueforming cells. The blood cells were washed out with culture medium containing 0.1% BSA, and the assay was repeated several times using different antiserum. When cells were followed for 2 days, they were incubated overnight in culture medium containing 0.1% BSA. Plaque formation did not occur when guinea pig serum, the source of complement, was omitted or when anti-mPL-I antiserum or anti-mPL-II antiserum was replaced with nonimmune rabbit serum.
Immunocytochemistry
after the reverse hemolytic
plaque assay
The reverse hemolytic plaque assay was performed as above using anti-mPL-I antiserum at a dilution of 1:400. The lower concentration of primary antiserum was used to reduce background in the immunostaining. Blood cells were washed out of the chambers with culture medium containing 0.1% BSA and the cells were fixed in Bouin’s solution. The cells were incubated with antirabbit IgG (1:25) to saturate any remaining anti-mPL-I antibodies, and the cells were then immunostained for mPLII as described above.
RIAs mPL-I and mPL-II concentrations were determined with highly specific RIAs as previously described (11, 12). mPL-II did not cross-react in the mPL-I RIA, and mPL-I did not cross-react in the RIA for mPL-II.
Results Immunocytochemistry
Double-staining immunocytochemistry for mPL-I and mPL-II was carried out on cells from day 7 of pregnancy on each day of a 5-day culture. The cells that stained for mPLI and/or mPL-II were trophoblast giant cells (data not shown; 21, 22). The time-course of mPL-I and mPL-II secretion by these cells has been reported (22). Briefly, the mPL-I concentration of the medium increased until the third day of culture and then fell by the fifth day. mPL-II could not be detected in the medium until the third day and then its concentration increased steadily (data not shown). The cells were stained with anti-mPL-I or anti-mPL-II antiserum applied individually or with both antisera applied sequentially, and the number of cells that stained when both antisera were applied sequentially was compared with the sum of the numbers of cells that stained when the antisera were used individually. When the cells were stained sequentially with anti-mPL-I antiserum followed by anti-mPL-II antiserum, the number of stained cells was lower than the sum of the numbers of cells that stained when each antiserum was used alone on the first 4 days of culture (Table l), which suggeststhat some
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RELEASE OF mPLs BY THE SAME CELL TABLE
1. Number of cells that immunostained
for mPL-I and/or mPL-II
Antiserum
Day of culture
mPL-I”
1 2 3 4 5
22.0 42.0 41.5 11.5 2.8
+ + f rt rt
1.1 2.7 2.7 3.0 0.9
10.0 22.8 39.3 16.8 11.3
* + + + +
as a function of time in culture No. of cells
used individually
containing only mPLId
No. of cells containing only mPLII’
No. of cells containing both mPL-I and mPL-II’
32.0 64.8 80.8 28.3 14.1
14.0 21.5 4.2 3.5 3.5
2.0 2.3 2.0 8.8 12.0
8.0 20.5 37.3 8.0 0
Sum of antisera
mPL-IP
mPL-I
+ mPL-II’
24.0 44.3 43.5 20.3 14.8
2.0 1.8 2.5 2.2 2.3
f * f + +
1.8 2.9 3.2 2.6 2.5
1597
Cells (105) from day 7 of pregnancy were plated in 96-well plates and incubated for up to 5 days. The cells were immunostained for mPL-I, mPL-II, or both hormones each day. Each value represents the mean f SE of four wells. DNumber of cells that stained when anti-mPL-I antiserum was used alone. * Number of cells that stained when anti-mPL-II antiserum was used alone. ’ Number of cells that stained when both antisera were applied sequentially. ’ The mean number of cells that theoretically contained only mPL-I was calculated as: [(number stained with both antisera applied sequentially) - (number stained with anti-mPL-II antiserum used alone)]. ’ The mean number of cells that theoretically contained only mPL-II was calculated as: [(number stained with both antisera applied sequentially) - (number stained with anti-mPL-I antiserum used alone)]. ‘The mean number of cells that theoretically contained both hormones was calculated as: [(number stained with anti-mPL-I antiserum used alone
+ number
stained
with
anti-mPL-II
antiserum
used
alone)
- (number
of the immunostained cells contained both mPL-I and mPLII. The number of cells that stained with each antiserum treatment was used to calculate the theoretical number (Table 1) and percentage (Fig. 1) of cells that contained one or both PLs. The number and percentage of cells that theoretically
contained both of the PLs increased until day 3 and then declined to zero by day 5. The number and percentage of cells that theoretically contained only mPL-I was relatively high on the first 2 days of culture and then declined, whereas the number and percentage of cells that theoretically contained only mPL-II was low for 3 days and then increased. Similar results using other preparations of cells were obtained when sequential staining was carried out using the antimPL-I and anti-mPL-II antisera in reverse order (data not shown) and when the cells were stained simultaneously with both antisera on the third day of culture [mean + SE number of stained cells (cells per well): anti-mPL-I alone, 49.0 + 3.3;
l ao-
mPL-II
stained
with
both
antisera)].
anti-mPL-II alone, 50.0 + 3.1; anti-mPL-I and anti-mPL-II simultaneously, 52.0 + 3.2; theoretical percentage of cells that contained both hormones, 90.4%; theoretical percentage of cells that contained only mPL-I, 3.8%; theoretical percentage of cells tht contained only mPL-II, 5.8% (n = 6 wells)]. Reverse hemolytic
plaque assay
Cells from day 9 of pregnancy were used to determine whether individual cells released both mPL-I and mPL-II. Cells from day 9 were chosen for these experiments because the amounts of mPL-I and mPL-II released did not differ greatly and the release of both hormones could be detected easily. The time course of mPL-I and mPL-II secretion by these cells, as determined by RIA, is shown in Fig. 2. The results of a representative sequential plaque assay are shown in Fig. 3. The cells were first incubated with anti-mPL-I antiserum for 2 h, and a plaque-forming cell was identified (Fig. 3a). The anti-mPL-I antiserum was washed out and the cells were incubated with anti-mPL-II antiserum, and after a
O
25
m
25 *
$
60A
E 8 & P
E 3l 5 ? a. E
40 -
mPL-I mPL-II
:/!-I -20
-15 -10
=
E -5l 5 = d E
-5
0
1
2
3
4
5
6
Time (days) FIG. 1. Theoretical
percentage of cells from day 7 of pregnancy that
contained mPL-I, mPL-II, or both hormones as a function of time in culture. Values were calculated from the data shown in Table 1 by dividing the number of cells that contained each PL by the total number of PL-containing cells on the same day of culture.
0 1 0
1
2
3
4
5
10 6
Time (days) FIG. 2. Time course of mPL-I and mPL-II secretion by cells from day 9 of pregnancy. Cells were plated in two-well tissue culture chamber slides at a density of 6.0 X 10’ cells/cm2. The medium was collected daily and assayed for mPL-I and mPL-II by RIA. Each point represents the mean + SE mPL-I or mPL-II concentration of the medium (n =
wells).
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4
1598
RELEASE
OF mPLs
BY THE
SAME
CELL
Endo. Voll31
l
1992 No 4
FIG. 3. Micrograph of plaque-forming cells in the sequential reverse hemolytic plaque assay. Cells from day 9 of pregnancy were plated in two-well tissue culture chamber slides at a density of 6.0 X lo4 cells/cm*. Twenty-four hours after plating, the cells were subjected to seven sequential reverse hemolytic plaque assays. Anti-mPL-I antiserum was used in assays 1, 3, and 4 (a, c, d), anti-mPL-II antiserum was used in assays 2, 5, and 6 (b, e, f), and nonimmune rabbit serum was used in assay 7 (g). Assays 1,2,4,6, and 7 (a, b, d, f, g) were carried out for 2 h, and assays 3 and 5 (c, e) were carried out for 30 min. Note the presmce of a plaque-forming cell at the intersection of the grid borders in panels a, b, d, and f. This cell did not form a plaque in panels c, e, and g. Magnification, 260x.
a plaque formed around the same cell (Fig. 3b), indicating that it released mPL-II in addition to mPL-I. A third plaque assay was then carried out using anti-mPL-I antiserum. A plaque did not form after a 30-min incubation (Fig. 3c) but it was apparent after 2 h (Fig. 3d). The delay in the appearance of the plaque indicates that a significant amount of mPL-I was not retained around the cell after reagents from the first two assays were washed out. The assay was then repeated two more times, first using anti-
2-h incubation,
mPL-II
antiserum
and
finally
using
nonimmune
rabbit
serum. A plaque did not form after a 30-min incubation with anti-mPL-II antiserum (Fig. 3e), but one was present after 2 h (Fig. 3f). A plaque did not form after a 2-h incubation with nonimmune rabbit serum (Fig. 3g), indicating that earlier
plaque formation did not result from nonspecific effects of sequential application of the assay.In a separate experiment, cells that formed plaques after incubation with anti-mPL-I antiserum were immunostained for mPL-II. Fifty to 70% of
the plaque-forming cells were immunostained, which also suggeststhat formation of plaques in the presence of antimPL-II antiserum in the experiment above were not artifactual. In order to obtain information about the fraction of cells that releasedone or both of the hormones, sequential plaque assays were carried out on the first and second days of culture on two separatecultures. The number and percentage of plaque-forming cells that releasedmPL-I, mPL-II, or both hormones on each day in each culture is shown in Table 2. Although the percentage of plaque-forming cells that released each PL on each day varied between cultures, cells that released only mPL-I or mPL-II and cells that released both hormones were present in each culture on each day. On the first day of culture, the majority of plaque-forming cells in both cultures released only mPL-I. On the second day of culture, the percentage of plaque-forming cells that releasedonly mPL-I decreasedrelative to the percentage on
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RELEASE TABLE
2. Number
and percentage
of plaque-forming
OF mPLs BY THE SAME CELL cells that
released
mPL-I,
mPL-II,
or both
1599 hormones
in the reverse
hemolytic
plaque
assay Day
Hormone
mPL-I mPL-I mPL-II
released
+ mPL-II
Culture
1
1 Culture
Day 2
NO.
%
NO.
%
Mean %
40 6 8
74.1 11.1 14.8
42 10 29
51.9 12.3 35.8
63.0 11.7 25.3
Culture
2
Culture
1
2
No.
%
No.
%
Mean %
16 8 19
37.2 18.6 44.2
20 10 43
27.4 13.7 58.9
32.3 16.1 51.6
Cells (6.0 x lO’/cm’) from day 9 of pregnancy were plated in tissue culture chamber slides and incubated for 1 day. Sequential reverse hemolytic plaque assays for mPL-I and mPL-II were carried out, using a 2-h incubation for each assay. The number of plaque-forming cells was recorded, and the cells were incubated for an additional 24 h. A second series of sequential hemolytic plaque assays was performed, and the number of plaque-forming cells was recorded. The percentage of each type of plaque-forming cell was calculated by dividing the number of cells releasing that hormone by the total number of plaque-forming cells for each culture on each day of culture.
day 1, whereas the percentage of plaque-forming ceils that released only mPL-II increased. The percentage of plaqueforming cells that released both mPL-I and mPL-II did not change much between days 1 and 2. The data shown in Table 2 for day 2 of culture do not distinguish between cells that had also formed plaques on the first day of culture and those that formed plaques for the first time on day 2. In order to follow the fate of individual plaque-forming cells with time in culture, the location of each of the cells above that formed a plaque on the first day of culture was noted and those cells were examined for plaque formation on the second day (Table 3). About 28% of the plaque-forming cells from day 1 formed plaques on day 2; it is not known whether the remaining cells stopped releasing PLs or whether they died. Most of the plaqueforming cells that released only mPL-I on day 1 continued to release only mPL-I on day 2, but several others released both hormones or only mPL-II. One plaque-forming cell that released both hormones on day 1 continued to releaseboth hormones on day 2, while 2 others that released both hormones on the first day released only mPL-II on the second day. Plaque-forming cells that released only mPL-II on the first day continued to release only mPL-II on the second; in no casedid these cells begin to releasemPL-I on the second day. A representative portion of a slide showing plaqueTABLE
3. Fate
of individual
plaque-forming Culture
Day Hormone
mPL-I
mPL-II
40
+ mPL-II
6
8
These data demonstrate that primary cultures of mouse trophoblast cells from the first half of pregnancy contain giant cells that produce and releaseboth mPL-I and mPL-II, as well as giant cells that contain and releaseonly one of the PLs. The immunocytochemical analysis of cells from day 7 of pregnancy indicates that changes occurred over time in culture in the number and percentage of each type of PLcontaining cell in the population. On the first two days of culture, the majority of PL cells from day 7 of pregnancy contained only mPL-I. Their number and percentage declined markedly between days 2 and 3. This decline was accompanied by a large increase in the number and percentage of cells that contained both mPL-I and mPL-II, and by day 3, almost all of the PL cells contained both hormones. On the fourth day of culture the number and percentage of cellsthat contained both hormones declined, and by the fifth day, none of the cells contained both PLs. The fall in the percentage of PL cells that contained both hormones was accompanied by an increase in the number and percentage of cells that contained only PL-II. The temporal relationships
in culture Culture
Day
No.
time
Discussion
1
1
released
mPL-I
cells over
forming cells that releaseddifferent hormones on 2 days of culture is shown in Fig. 4.
Hormone mPL-I mPL-I mPL-II None mPL-I mPL-I mPL-II None mPL-I mPL-I mPL-II None
released
2
Day
No. 9
+ mPL-II
+ mPL-II
+ mPL-II
5 3 23 1 0 1 4 0 0 1 7
Hormone
released
mPL-I
mPL-I
mPL-II
On day 1 of culture, the location of each plaque-forming cell in Table 2 was noted. reverse hemolytic plaque assays were repeated. The slides were examined for plaque previous day, and the number of plaques for each hormone was recorded.
Day
No. 42
+ mPL-II
2
1
10
29
Hormone mPL-I mPL-I mPL-II None mPL-I mPL-I mPL-II None mPL-I mPL-I mPL-II None
2
released + mPL-II
+ mPL-II
+ mPL-II
No. 8 1 1 32 1 1 1
7 0 0 5 24
The cells were incubated for an additional 24 h and sequential formation in each location where a plaque had formed on the
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RELEASE
1600
OF mPLs
FIG. 4. Fate of individual mPL-I- and mPL-II-releasing cells on consecutive days of culture. Cells from day 9 of preg nancy were plated in two-well tissue culture chamber slides at a density of 6.0 X lo4 cells/cm’. Sequential reverse hemolytic plaque assays for mPL-I (a) and mPL-II (b) were carried out 24 h after plating, and cells that released one or both PLs were identified in grids 51 and 6. A second series of sequential reverse hemolytic plaque assays for mPL-I (c) and mPL-II (d) was carried out 48 h after plating, and grids 51 and 6 were examined for the presence of plaque-forming cells. The schematic (e) shows the location and fate of the plaque-forming cells in panels a-d. Note that each of the cells depicted underwent a change in the type of PL it released between days 1 and 2. Magnification, 130x.
BY
THE
&B
-/
/I /
SAME
CELL
mPL-I
0
mPL-II
0
mPL-I and mPL-II
0
neither
DAY
Endo * 1992 Voll31. No 4
1
DAY 2
e between the decline in the percentage of cells that contained only mPL-I and the increase in dual hormone producers and between the decline in dual hormone producers and the increasein the percentage of cells that contained only mPLII suggestthat under these culture conditions, PL cells may
undergo a cycle in which they first produce mPL-I, then both mPL-I and mPL-II, and finally only mPL-II. Whether all of the PL cells in the population followed this cycle could not be determined becauseit was not possible to follow the fate of individual cells immunohistochemically. The fact that the
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RELEASE
OF mPLs BY THE SAME CELL
number of cells that contained both hormones on the third day of culture was about 3 times greater than the number that contained only mPL-II on day 5 suggests that a significant fraction of the dual hormone producers ceased PL production after day 3. It is possible that some of the increase in the number of giant cells that contained only mPL-II toward the end of the culture period resulted from differentiation of a subpopulation that never produced mPL-I. Similarly, the small numbers of cells containing only mPL-I or mPL-II that were present at the end and beginning of the 5day culture, respectively, could be subpopulations that produce only one of the PLs. The data from the reverse hemolytic plaque assay suggest that a shift also occurred during time in culture in the type of PL that is releasedby giant cells. When the total number of plaque-forming cells that released each type of PL was examined, there was a decline between days 1 and 2 in the fraction of plaque-forming cells that released only mPL-I and an increase in the fraction that released only mPL-II. When the fate of individual plaque-forming cells was followed, it was clear that some cells that released only mPL-I on the first day of culture released both hormones or only mPL-II on the secondday. The latter observation is consistent with the hypothesis that at least some PL-producing cells undergo a shift in PL production from mPL-I to both hormones to mPL-II. Although the number of cells examined was relatively small, of the 37 plaque-forming cells that releasedonly mPL-II on the first day of culture, none released mPL-I on the second day, which suggests that once giant cells have differentiated to the extent that they release only mPL-II, they may not release mPL-I again, at least under these culture conditions. Unfortunately, we were unable to immunostain cells satisfactorily after performing sequential hemolytic plaque assays and could not determine whether cells that releasedonly mPL-II contained mPL-I. The fact that individual giant cells in this culture system undergo a shift in the type of PL they produce suggeststhat the pattern of PL expression that occurs in viva at midpregnancy can be attributed, at least partly, to a change in gene expression by individual giant cells. Whether all giant cells express both PLs in viva during midpregnancy remains to be determined. In situ hybridization and immunohistochemical analysis of conceptusesfrom midpregnant mice (19) and rats (4) have localized PL-I and PL-II to giant cells in two regions of the placenta, the chorioallantoic placenta (polar giant cells) and the choriovitelline placenta (mural giant cells). Studies in the rat have demonstrated that there are differences between polar and mural giant cells in both the day of pregnancy when PL expression begins and the amounts of PLs produced (4, 25). It is not known if regional differences also exist in the ability of individual giant cells to express both PLS. Although giant cells that stained for mPL-II were present as early as the first day of incubation in cultures from day 7 of pregnancy, mPL-II could not be detected in the culture medium until the third or fourth day (data not shown; 22). This was probably not due to the presence of too few mPL-
1601
II-producing cells since mPL-II could be detected easily in the medium on the fifth day of culture when the number of mPL-II-containing cells and their staining intensity (data not shown) was similar to those on day 1. These observations suggestthat at this stage of pregnancy mPL-II is stored for several days before it is releasedin significant amounts and that factors that signal mPL-II releasemay appear later than the stimulus for hormone synthesis. The apparent storage of mPL-II by giant cells at this stage of pregnancy differs from the situation later in pregnancy when most newly synthesized mPL-II is rapidly released(26). Little is known about the regulation of PL-I and PL-II gene expression.Preliminary data from our laboratory (Yamaguchi M., and F. Talamantes, in preparation) indicate that EGF stimulates mPL-I production but inhibits mPL-II production in the same culture system used here, which suggeststhat EGF may be involved in regulating the switch in gene expression from PL-I to PL-II. The fact that giant cells from day 7 of pregnancy undergo a shift in the type of PL they produce in vitro suggeststhat the culture system used in this study will be useful for examining the molecular events that underlie the shift from PL-I to PL-II production at midpregnancy. Acknowledgment We thank Dr. Stephen hemolytic plaque assay.
Frawley
for
his advice
about
the
reverse
References 1. Colosi P, Marr G, Lopez J, Haro L, Ogren L, Talamantes F 1982 Isolation, purification, and characterization of mouse placental lactogen. Proc Nat1 Acad Sci USA 79:771-775 2. Colosi P. Oeren 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 3. Robertson MC, Gillespie B, Friesen HG 1982 Characterization of the two forms of rat placental lactogen (rPL): rPL-I and rPL-II. Endocrinology 111:1862-1866 4. Faria TN, Deb S, Kwok SCM, Talamantes F, Soares MJ 1990 Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta. Dev Biol 141:279-291 5. Robertson MC, Croze F, Schroedter IC, Friesen HG 1990 Molecular cloning and expression of rat placental lactogen-I complementary deoxyribonucleic acid. Endocrinology 127:702-710 6. Colosi P, Talamantes F, Linzer DIH 1987 Molecular cloning and expression of mouse placental lactogen I complementary deoxynbonucleic acid. Mol Endocrinol 1:767-776 7. Duckworth ML, Kirk KL, Friesen HG 1986 Isolation and identification of a cDNA clone of rat placental lactogen II. J Biol Chem 261:10871-10878 8. Jackson LL, Colosi P, Talamantes F, Linzer DIH 1986 Molecular cloning of mouse placental lactogen cDNA. Proc Nat1 Acad Sci USA 83:8496-8500 9. Thordarson G, Villalobos R, Colosi P, Southard J, Ogren L, Talamantes F 1986 The lactogenic response of cultured mouse mammary epithelial cells to mouse placental lactogen. J Endocrinol 109:263-274 10. 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 11. Ogren L, Southard JN, Colosi P, Linzer DIH, Talamantes F 1989 Y
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RELEASE
1602
OF mPLs
Mouse serum.
placental lactogen-I: RIA and gestational profile in maternal Endocrinology 125:2253-2257 12. Soares MJ, Colosi P, Talamantes F 1982 The development and characterization of homologous radioimmunoassay for mouse placental lactogen. Endocrinology 110:668-670 13. Soares MJ, Talamantes F 1983 Genetic and litter size effects on serum placental lactogen in the mouse. Biol Reprod 29:165-171 14. Robertson MC, Friesen HG 1981 Two forms of rat placental lactogen revealed by radioimmunoassay. Endocrinology 108:2388-
2390 15. Hall J, Talamantes mouse tochem 16.
F 1984 Immunocytochemical
placental lactogen 32:379-382
Lee SJ, Talamantes
in the mouse
F, Wilder
placenta.
WJ, Deb S, Kwok
19. Faria TN, Ogren L, Talamantes
22. Yamanuchi
of Cy-
DIH, Nathans D 1988 as the site of proli-
24.
SCM, Joslin J, Soares MJ 1989
Differential expression of placental lactogen-II and prolactin-like protein-A in the rat chorioallantoic placenta. Endocrinology 125:1565-1574 18. Duckworth ML, Schroedter IC, Friesen HG 1990 Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization. Placenta 11:143-155
F, Linzer DIH, Soares MJ 1991
SAME
Endo. Voll31.
CELL
1992 No 4
Localization of placental lactogen-I in trophoblast giant cells of the mouse placenta: Biol Reprod 44:327-331 20. Nieder GL. Iennes L 1990 Production of mouse ulacental lactoeenI by trophbblast giant cells in utero and in v&o. Endocrinology 126:2809-2814 21. Thordarson G, Folger P, Talamantes F 1987 Development of a placental cell culture system for studying the control of mouse placental lactogen secretion. Placenta 8;575-585
23. E, Linzer
Trophoblastic giant cells of the mouse placenta ferin synthesis. Endocrinology 122:1761-1768
17. Campbell
localization J Histochem
BY THE
25. 26.
M. Endo H. Thordarson
G. Oeren L. Talamantes
F
1992 hodulation of mouse placental lactog&I secretion in vitro: effects of progesterone and mouse placental lactogen-II. Endocrinology 130:2897-2905 Frawley LS, Boockfor FR, Hoeffler JP 1985 Identification by plaque assays of a pituitary cell type that secretes both growth hormone and prolactin. Endocrinology 116:734-737 Colosi P, Talamantes F 1981 The amino acid composition of secreted mouse prolactin, growth hormone, and hamster prolactin: the presence of one tryptophan in mouse prolactin. Arch Biochem Biophys 212:759-761 Soares MJ, Julian JA, Glasser SR 1985 Trophoblast giant cell release of placental lactogens: temporal and regional characteristics. Dev Biol 107:520-526 Basch CV, Talamantes F 1986 In vitro kinetics of synthesis and release of mouse placental lactogen. Endocrinology 119:1939-1947
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Production Lactogen-II M. YAMAGUCHI, F. TALAMANTES
Vol. 131, No. 4 Printed in U.S.A.
Society
of Mouse Placental by the Same Giant L.
OGREN,
H.
ENDO,
G.
Lactogen-I Cell*
THORDARSON,
R. M.
BIGSBY,
and Placental
AND
Department of Biology (M. Y., L.O., H.E., G. T., F. T.), U nzversity . of California, Santa Cruz, California and the Department of Obstetrics and Gynecology (R. M. B.). Indiana University School of Medicine, Indianapolis, Indiana 46202-5196
95064;
ABSTRACT In previous studies, mouse placental lactogen I (mPL-I) and mPLII were localized to trophoblast giant cells in the placenta at midpregnancy. The present study was undertaken to determine whether mPLI and mPL-II are produced by two distinct populations of giant cells or by the same cells. A heterogeneous population of cells that included trophoblast giant cells was obtained by enzymatic dispersion and Percoll gradient centrifugation of placentas from days 7 and 9 of pregnancy. Cells from day 7 of pregnancy were cultured in serum-free medium for 5 days, and cells that contained mPL-I, mPL-II, or both mPL-I and mPL-II were identified by double-staining immunocytochemistry. The percentage of PL cells that contained both mPL-I and mPL-II increased from about 30% on the first day of culture to about 90% on the third, and then declined to zero by day 5. Between 50% and 60% of the PL cells contained only mPL-I on the first 2 days of culture, and then the percentage of PL cells containing only mPL-I declined. The percentage of cells that contained only mPL-II was low
for 3 days (~10%) and then increased to about 80% of the PLcontaining cells by day 5. Cells from day 9 of pregnancy were analyzed for the release of mPL-I and/or mPL-II by sequential reverse hemolytic plaque assay. Cells that released only one of the PLs, as well as those that released both PLs, were identified. A shift was present in the type of PL released by the cells when they were followed for two consecutive days of culture. On day 1, most of the plaque-forming cells released only mPL-I, but by day 2, the fraction of plaque-forming cells that released only mPL-I declined whereas the fraction that released only mPL-II increased. Cells that released only mPL-I on the first day of culture and both mPL-I and mPL-II or only mPL-II on the second dav of culture were observed. These data suggest that under these culture conditions, PL cells follow a pathway in which they initiallv nroduce only mPL-I, then both mPL-i and GPL-II, and finally onl; APL-II. In uiuo, there is a shift at midpregnancy in the type of PL that is produced by the mouse placenta, and these data suggest that this shift results, at least partly, from a change in gene expression in one population of giant cells. (Endocrinology 131: 1595-1602, 1992)
T
increases steadily after its appearance at midpregnancy (3, 14). The gestational profiles of the two PLs are of interest becausethere is a clear switch at midpregnancy in the type of PL expressedby the placenta. Immunohistochemical and in situ hybridization analyses have localized both PL-I and PL-II to trophoblast giant cells in the mouse and rat (4, 5, 15-20), but it is not known whether a single giant cell produces both of the hormones. Theoretically, the shift at midpregnancy in the type of PL produced by the placenta could result from the disappearance of a population of cells that produces PL-I and the differentiation of a second population that produces PL-II, or it could result from a change in gene expression in one population of cells, or from both processes.In the present study we have used a system for primary culture of cells from the midpregnant mouse placenta (21, 22) to demonstrate that mPL-I and mPL-II are produced by the same giant cell, which suggeststhat the midpregnancy shift in PL production can be attributed, at least partly, to a change in gene expressionin one population of giant cells.
WO placental lactogens (PLs), designated PL-I and PLII, are produced by the mouse (1, 2) and rat placenta (3). The PL-Is are glycosylated single-chain polypeptides that exist as multiple molecular weight forms, with M, ranging between about 29 and 42 kilodaltons (kDa) (2, 4,5). The PL11sare nonglycosylated single-chain polypeptides having an M, of about 25 kDa; they share significant amino acid sequence homology with their respective PL-I (3, 5-8). The known functions of mouse (m) and rat (r) PL-I and PL-II are PRL-like (2, 3, 9, lo), but the gestational profiles of the hormones in maternal blood differ significantly. mPL-I appears in maternal serum on day 6 of pregnancy. Its concentration increasesto very high values on days 9 and 10, and then declines rapidly (11). mPL-II appears in the maternal circulation on day 9 (12). Its concentration increases until about day 14 and then remains relatively constant in some mouse strains or increases until the end of pregnancy in others (13). The gestational profiles of rPL-I and rPL-II in maternal blood are very similar to those of the respective mouse hormones. A large peak in maternal serum rPL-I is present at midpregnancy, and the concentration of rPL-II Received April 15, 1992. Address 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 HD-14966 and GM-08132 (to F. T.).
Materials Cell dissociation Conceptuses Webster mice
and Methods
and culture
were collected on days 7 and 9 of pregnancy (Simonsen Laboratories, Gilroy, CA; vaginal
1595
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from Swiss plug = day
RELEASE
1596
OF mPLs B Y THE SAME CELL
0 of pregnancy). The fetus and decidua basalis were discarded, and the remaining tissue was minced and incubated in dissociation medium [Medium 199 (Hank’s salts), 20 mM HEPES, 10 mM NaHC03, 50 rg streptomycin/ml, 50 U penicillin G/ml, pH 7.21 containing 0.1% (wt/ vol) collagenase (Closhidium histolyticum, type 1, CLS; Worthington Biochemical Co., Malvem, PA) and 0.002% (wt/vol) bovine pancreatic DNAse (type 1, EC3.1.21.1; Sigma, St. Louis, MO) at 37 C for 1 h. For a typical cell preparation, 30 ml medium were used for pooled tissue from 10 animals. After centrifugation at 600 X g for 5 min, the tissue was dispersed in calciumand magnesium-free Hanks solution containing 0.1% (wt/vol) BSA by repeated pipetting, and the cells were filtered through 150-r Nitex (Tetko, Inc., Elmsford, NY). The cell suspension was centrifuged as above, resuspended in 2 ml dissociation medium containing 0.015% DNAse, and then fractionated on a 40% Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradient. Cells shown to banding at a density of 1.044 g/ m 1, which were previously contain mPL-I and mPL-II (22), were collected and washed in 5 vol dissociation medium. The cells were resuspended to a concentration of 0.5 x lo6 cells/ml in culture medium (NCTC-135 containing 20 mM HEPES, 25 mM NaHC03, 1.65 mM cysteine, 50 rg streptomycin/ml, 50 U penicillin G/ml, pH 7.2) that was supplemented with 10% (vol/vol) fetal calf serum. The cells were plated at a density of 2.0 to 3.0 X 10s cells/cm’ in multiwell plates or 6.0 x lo4 cells/cm’ in two-well tissue culture chamber slides. Before plating in the tissue culture chamber slides, the cells were incubated in dissociation medium containing 0.1% (wt/vol) trypsin, 0.01% (wt/vol) EDTA, 0.002% DNAse for 30 min at 37 C to obtain a preparation of single cells. After plating in either type of dish, the cells were allowed to attach, and the medium was removed and culture medium containing 0.1% BSA was added. The cells were incubated at 37 C under an atmosphere of 95% sir/5% CO, for up to 5 days. The day the cells were plated was considered day 0. The medium was changed daily and stored at -20 C.
Immunocytochemistry Cells were plated in multiwell plates, Before staining they were washed with 10 rnM sodium phosphate, 150 rnM NaCl, pH 7.4, and fixed in Bouin’s solution. The cells were stained for mPL-I or mPL-II using an avidin-biotin immunoperoxidase kit (Vector Laboratories, Burlingame, CA) as previously described (22). Antisera to mPL-II and recombinant mPL-I were generated in rabbits and have been described (10, 12). Double-staining immunocytochemistry was used to determine whether both mPL-I and mPL-II were present in a single cell using an approach described by Frawley et al. (23) for identifying hormonesecreting pituitary cells. When the cells were stained sequentially with both antisera, they were first stained with one of the antisera and then incubated with nonbiotinylated anti-rabbit immunoglobulin G (IgG) (1:25, vol/vol) in order to saturate any primary antibody that did not bind to biotinylated antirabbit IgG. The cells were then stained with the second antiserum, and the number of stained cells was counted. In control experiments, the dilution of nonbiotinylated antirabbit IgG that was used almost completely abolished subsequent staining by biotinylated antirabbit IgG. The effectiveness of the blocking procedure with antirabbit IgG was also demonstrated by the fact that when cells were stained with either anti-mPL-I or anti-mPL-II, blocked, and then stained with nonimmune rabbit serum, staining intensity did not increase significantly in the second round of staining. To rule out possible effects of sequence and repeated staining on the number of cells that stained, the antisera were applied in different order and also simultaneously. The cells were stored in 70% ethanol. Anti-mPL-I and anti-mPL-II antisera were used at dilutions of 1:1500 (vol/vol) and 1:500 (vol/vol), respectively. When the antisera were used individually in double-staining experiments, nonimmune rabbit serum was added to the antiserum solution so that the total concentration of rabbit serum (antiserum plus nonimmune serum) was the same for each antiserum solution. The specificity of the method was verified by replacing the primary antiserum with non-immune rabbit serum and by saturating the anti-mPL-I and anti-mPL-11 antisera with recombinant mPL-I (20 pg; Ref. 10) and mPLII (10 rg; Ref. l), respectively. Incubation of anti-mPL-I antiserum with mPL-II or mGH (10 pg; Ref. 24) and anti-mPL-II antiserum with mPLI or mGH before use did not affect staining.
Sequential
Endo. Voll31.
reverse hemolytic
1992 No 4
plaque assay
The sequential hemolytic plaque assay was carried out to determine whether one cell could release both mPL-I and mPL-II. It was performed as described by Frawley et al. (23) with some modifications. Cells were plated in two-well tissue culture chamber slides. The day after plating, the cells were washed three times with culture medium containing 0.1% BSA, and the cover of the tissue culture chamber slides was removed, and assay chambers were constructed with etched grid coverslips (Bellco Glass, Inc., Vineland, NJ). The cells were washed two more times as above, and a mixture of protein A-coated ovine red blood cells [I:10 (vol/vol); Colorado Serum Co., Denver, CO], rabbit serum (anti-mPL-I antiserum, anti-mPL-II antiserum, or nonimmune serum; 1:200), and guinea pig serum [1:50 (vol/vol); GIBCO, Grand Island, NY] in culture medium containing 0.1% BSA was added. After a 30-min or 2-h incubation in 95% sir/5% COz at 37 C, plaque formation was checked, and the slides were photographed to record the location of the plaqueforming cells. The blood cells were washed out with culture medium containing 0.1% BSA, and the assay was repeated several times using different antiserum. When cells were followed for 2 days, they were incubated overnight in culture medium containing 0.1% BSA. Plaque formation did not occur when guinea pig serum, the source of complement, was omitted or when anti-mPL-I antiserum or anti-mPL-II antiserum was replaced with nonimmune rabbit serum.
Immunocytochemistry
after the reverse hemolytic
plaque assay
The reverse hemolytic plaque assay was performed as above using anti-mPL-I antiserum at a dilution of 1:400. The lower concentration of primary antiserum was used to reduce background in the immunostaining. Blood cells were washed out of the chambers with culture medium containing 0.1% BSA and the cells were fixed in Bouin’s solution. The cells were incubated with antirabbit IgG (1:25) to saturate any remaining anti-mPL-I antibodies, and the cells were then immunostained for mPLII as described above.
RIAs mPL-I and mPL-II concentrations were determined with highly specific RIAs as previously described (11, 12). mPL-II did not cross-react in the mPL-I RIA, and mPL-I did not cross-react in the RIA for mPL-II.
Results Immunocytochemistry
Double-staining immunocytochemistry for mPL-I and mPL-II was carried out on cells from day 7 of pregnancy on each day of a 5-day culture. The cells that stained for mPLI and/or mPL-II were trophoblast giant cells (data not shown; 21, 22). The time-course of mPL-I and mPL-II secretion by these cells has been reported (22). Briefly, the mPL-I concentration of the medium increased until the third day of culture and then fell by the fifth day. mPL-II could not be detected in the medium until the third day and then its concentration increased steadily (data not shown). The cells were stained with anti-mPL-I or anti-mPL-II antiserum applied individually or with both antisera applied sequentially, and the number of cells that stained when both antisera were applied sequentially was compared with the sum of the numbers of cells that stained when the antisera were used individually. When the cells were stained sequentially with anti-mPL-I antiserum followed by anti-mPL-II antiserum, the number of stained cells was lower than the sum of the numbers of cells that stained when each antiserum was used alone on the first 4 days of culture (Table l), which suggeststhat some
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RELEASE OF mPLs BY THE SAME CELL TABLE
1. Number of cells that immunostained
for mPL-I and/or mPL-II
Antiserum
Day of culture
mPL-I”
1 2 3 4 5
22.0 42.0 41.5 11.5 2.8
+ + f rt rt
1.1 2.7 2.7 3.0 0.9
10.0 22.8 39.3 16.8 11.3
* + + + +
as a function of time in culture No. of cells
used individually
containing only mPLId
No. of cells containing only mPLII’
No. of cells containing both mPL-I and mPL-II’
32.0 64.8 80.8 28.3 14.1
14.0 21.5 4.2 3.5 3.5
2.0 2.3 2.0 8.8 12.0
8.0 20.5 37.3 8.0 0
Sum of antisera
mPL-IP
mPL-I
+ mPL-II’
24.0 44.3 43.5 20.3 14.8
2.0 1.8 2.5 2.2 2.3
f * f + +
1.8 2.9 3.2 2.6 2.5
1597
Cells (105) from day 7 of pregnancy were plated in 96-well plates and incubated for up to 5 days. The cells were immunostained for mPL-I, mPL-II, or both hormones each day. Each value represents the mean f SE of four wells. DNumber of cells that stained when anti-mPL-I antiserum was used alone. * Number of cells that stained when anti-mPL-II antiserum was used alone. ’ Number of cells that stained when both antisera were applied sequentially. ’ The mean number of cells that theoretically contained only mPL-I was calculated as: [(number stained with both antisera applied sequentially) - (number stained with anti-mPL-II antiserum used alone)]. ’ The mean number of cells that theoretically contained only mPL-II was calculated as: [(number stained with both antisera applied sequentially) - (number stained with anti-mPL-I antiserum used alone)]. ‘The mean number of cells that theoretically contained both hormones was calculated as: [(number stained with anti-mPL-I antiserum used alone
+ number
stained
with
anti-mPL-II
antiserum
used
alone)
- (number
of the immunostained cells contained both mPL-I and mPLII. The number of cells that stained with each antiserum treatment was used to calculate the theoretical number (Table 1) and percentage (Fig. 1) of cells that contained one or both PLs. The number and percentage of cells that theoretically
contained both of the PLs increased until day 3 and then declined to zero by day 5. The number and percentage of cells that theoretically contained only mPL-I was relatively high on the first 2 days of culture and then declined, whereas the number and percentage of cells that theoretically contained only mPL-II was low for 3 days and then increased. Similar results using other preparations of cells were obtained when sequential staining was carried out using the antimPL-I and anti-mPL-II antisera in reverse order (data not shown) and when the cells were stained simultaneously with both antisera on the third day of culture [mean + SE number of stained cells (cells per well): anti-mPL-I alone, 49.0 + 3.3;
l ao-
mPL-II
stained
with
both
antisera)].
anti-mPL-II alone, 50.0 + 3.1; anti-mPL-I and anti-mPL-II simultaneously, 52.0 + 3.2; theoretical percentage of cells that contained both hormones, 90.4%; theoretical percentage of cells that contained only mPL-I, 3.8%; theoretical percentage of cells tht contained only mPL-II, 5.8% (n = 6 wells)]. Reverse hemolytic
plaque assay
Cells from day 9 of pregnancy were used to determine whether individual cells released both mPL-I and mPL-II. Cells from day 9 were chosen for these experiments because the amounts of mPL-I and mPL-II released did not differ greatly and the release of both hormones could be detected easily. The time course of mPL-I and mPL-II secretion by these cells, as determined by RIA, is shown in Fig. 2. The results of a representative sequential plaque assay are shown in Fig. 3. The cells were first incubated with anti-mPL-I antiserum for 2 h, and a plaque-forming cell was identified (Fig. 3a). The anti-mPL-I antiserum was washed out and the cells were incubated with anti-mPL-II antiserum, and after a
O
25
m
25 *
$
60A
E 8 & P
E 3l 5 ? a. E
40 -
mPL-I mPL-II
:/!-I -20
-15 -10
=
E -5l 5 = d E
-5
0
1
2
3
4
5
6
Time (days) FIG. 1. Theoretical
percentage of cells from day 7 of pregnancy that
contained mPL-I, mPL-II, or both hormones as a function of time in culture. Values were calculated from the data shown in Table 1 by dividing the number of cells that contained each PL by the total number of PL-containing cells on the same day of culture.
0 1 0
1
2
3
4
5
10 6
Time (days) FIG. 2. Time course of mPL-I and mPL-II secretion by cells from day 9 of pregnancy. Cells were plated in two-well tissue culture chamber slides at a density of 6.0 X 10’ cells/cm2. The medium was collected daily and assayed for mPL-I and mPL-II by RIA. Each point represents the mean + SE mPL-I or mPL-II concentration of the medium (n =
wells).
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4
1598
RELEASE
OF mPLs
BY THE
SAME
CELL
Endo. Voll31
l
1992 No 4
FIG. 3. Micrograph of plaque-forming cells in the sequential reverse hemolytic plaque assay. Cells from day 9 of pregnancy were plated in two-well tissue culture chamber slides at a density of 6.0 X lo4 cells/cm*. Twenty-four hours after plating, the cells were subjected to seven sequential reverse hemolytic plaque assays. Anti-mPL-I antiserum was used in assays 1, 3, and 4 (a, c, d), anti-mPL-II antiserum was used in assays 2, 5, and 6 (b, e, f), and nonimmune rabbit serum was used in assay 7 (g). Assays 1,2,4,6, and 7 (a, b, d, f, g) were carried out for 2 h, and assays 3 and 5 (c, e) were carried out for 30 min. Note the presmce of a plaque-forming cell at the intersection of the grid borders in panels a, b, d, and f. This cell did not form a plaque in panels c, e, and g. Magnification, 260x.
a plaque formed around the same cell (Fig. 3b), indicating that it released mPL-II in addition to mPL-I. A third plaque assay was then carried out using anti-mPL-I antiserum. A plaque did not form after a 30-min incubation (Fig. 3c) but it was apparent after 2 h (Fig. 3d). The delay in the appearance of the plaque indicates that a significant amount of mPL-I was not retained around the cell after reagents from the first two assays were washed out. The assay was then repeated two more times, first using anti-
2-h incubation,
mPL-II
antiserum
and
finally
using
nonimmune
rabbit
serum. A plaque did not form after a 30-min incubation with anti-mPL-II antiserum (Fig. 3e), but one was present after 2 h (Fig. 3f). A plaque did not form after a 2-h incubation with nonimmune rabbit serum (Fig. 3g), indicating that earlier
plaque formation did not result from nonspecific effects of sequential application of the assay.In a separate experiment, cells that formed plaques after incubation with anti-mPL-I antiserum were immunostained for mPL-II. Fifty to 70% of
the plaque-forming cells were immunostained, which also suggeststhat formation of plaques in the presence of antimPL-II antiserum in the experiment above were not artifactual. In order to obtain information about the fraction of cells that releasedone or both of the hormones, sequential plaque assays were carried out on the first and second days of culture on two separatecultures. The number and percentage of plaque-forming cells that releasedmPL-I, mPL-II, or both hormones on each day in each culture is shown in Table 2. Although the percentage of plaque-forming cells that released each PL on each day varied between cultures, cells that released only mPL-I or mPL-II and cells that released both hormones were present in each culture on each day. On the first day of culture, the majority of plaque-forming cells in both cultures released only mPL-I. On the second day of culture, the percentage of plaque-forming cells that releasedonly mPL-I decreasedrelative to the percentage on
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RELEASE TABLE
2. Number
and percentage
of plaque-forming
OF mPLs BY THE SAME CELL cells that
released
mPL-I,
mPL-II,
or both
1599 hormones
in the reverse
hemolytic
plaque
assay Day
Hormone
mPL-I mPL-I mPL-II
released
+ mPL-II
Culture
1
1 Culture
Day 2
NO.
%
NO.
%
Mean %
40 6 8
74.1 11.1 14.8
42 10 29
51.9 12.3 35.8
63.0 11.7 25.3
Culture
2
Culture
1
2
No.
%
No.
%
Mean %
16 8 19
37.2 18.6 44.2
20 10 43
27.4 13.7 58.9
32.3 16.1 51.6
Cells (6.0 x lO’/cm’) from day 9 of pregnancy were plated in tissue culture chamber slides and incubated for 1 day. Sequential reverse hemolytic plaque assays for mPL-I and mPL-II were carried out, using a 2-h incubation for each assay. The number of plaque-forming cells was recorded, and the cells were incubated for an additional 24 h. A second series of sequential hemolytic plaque assays was performed, and the number of plaque-forming cells was recorded. The percentage of each type of plaque-forming cell was calculated by dividing the number of cells releasing that hormone by the total number of plaque-forming cells for each culture on each day of culture.
day 1, whereas the percentage of plaque-forming ceils that released only mPL-II increased. The percentage of plaqueforming cells that released both mPL-I and mPL-II did not change much between days 1 and 2. The data shown in Table 2 for day 2 of culture do not distinguish between cells that had also formed plaques on the first day of culture and those that formed plaques for the first time on day 2. In order to follow the fate of individual plaque-forming cells with time in culture, the location of each of the cells above that formed a plaque on the first day of culture was noted and those cells were examined for plaque formation on the second day (Table 3). About 28% of the plaque-forming cells from day 1 formed plaques on day 2; it is not known whether the remaining cells stopped releasing PLs or whether they died. Most of the plaqueforming cells that released only mPL-I on day 1 continued to release only mPL-I on day 2, but several others released both hormones or only mPL-II. One plaque-forming cell that released both hormones on day 1 continued to releaseboth hormones on day 2, while 2 others that released both hormones on the first day released only mPL-II on the second day. Plaque-forming cells that released only mPL-II on the first day continued to release only mPL-II on the second; in no casedid these cells begin to releasemPL-I on the second day. A representative portion of a slide showing plaqueTABLE
3. Fate
of individual
plaque-forming Culture
Day Hormone
mPL-I
mPL-II
40
+ mPL-II
6
8
These data demonstrate that primary cultures of mouse trophoblast cells from the first half of pregnancy contain giant cells that produce and releaseboth mPL-I and mPL-II, as well as giant cells that contain and releaseonly one of the PLs. The immunocytochemical analysis of cells from day 7 of pregnancy indicates that changes occurred over time in culture in the number and percentage of each type of PLcontaining cell in the population. On the first two days of culture, the majority of PL cells from day 7 of pregnancy contained only mPL-I. Their number and percentage declined markedly between days 2 and 3. This decline was accompanied by a large increase in the number and percentage of cells that contained both mPL-I and mPL-II, and by day 3, almost all of the PL cells contained both hormones. On the fourth day of culture the number and percentage of cellsthat contained both hormones declined, and by the fifth day, none of the cells contained both PLs. The fall in the percentage of PL cells that contained both hormones was accompanied by an increase in the number and percentage of cells that contained only PL-II. The temporal relationships
in culture Culture
Day
No.
time
Discussion
1
1
released
mPL-I
cells over
forming cells that releaseddifferent hormones on 2 days of culture is shown in Fig. 4.
Hormone mPL-I mPL-I mPL-II None mPL-I mPL-I mPL-II None mPL-I mPL-I mPL-II None
released
2
Day
No. 9
+ mPL-II
+ mPL-II
+ mPL-II
5 3 23 1 0 1 4 0 0 1 7
Hormone
released
mPL-I
mPL-I
mPL-II
On day 1 of culture, the location of each plaque-forming cell in Table 2 was noted. reverse hemolytic plaque assays were repeated. The slides were examined for plaque previous day, and the number of plaques for each hormone was recorded.
Day
No. 42
+ mPL-II
2
1
10
29
Hormone mPL-I mPL-I mPL-II None mPL-I mPL-I mPL-II None mPL-I mPL-I mPL-II None
2
released + mPL-II
+ mPL-II
+ mPL-II
No. 8 1 1 32 1 1 1
7 0 0 5 24
The cells were incubated for an additional 24 h and sequential formation in each location where a plaque had formed on the
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RELEASE
1600
OF mPLs
FIG. 4. Fate of individual mPL-I- and mPL-II-releasing cells on consecutive days of culture. Cells from day 9 of preg nancy were plated in two-well tissue culture chamber slides at a density of 6.0 X lo4 cells/cm’. Sequential reverse hemolytic plaque assays for mPL-I (a) and mPL-II (b) were carried out 24 h after plating, and cells that released one or both PLs were identified in grids 51 and 6. A second series of sequential reverse hemolytic plaque assays for mPL-I (c) and mPL-II (d) was carried out 48 h after plating, and grids 51 and 6 were examined for the presence of plaque-forming cells. The schematic (e) shows the location and fate of the plaque-forming cells in panels a-d. Note that each of the cells depicted underwent a change in the type of PL it released between days 1 and 2. Magnification, 130x.
BY
THE
&B
-/
/I /
SAME
CELL
mPL-I
0
mPL-II
0
mPL-I and mPL-II
0
neither
DAY
Endo * 1992 Voll31. No 4
1
DAY 2
e between the decline in the percentage of cells that contained only mPL-I and the increase in dual hormone producers and between the decline in dual hormone producers and the increasein the percentage of cells that contained only mPLII suggestthat under these culture conditions, PL cells may
undergo a cycle in which they first produce mPL-I, then both mPL-I and mPL-II, and finally only mPL-II. Whether all of the PL cells in the population followed this cycle could not be determined becauseit was not possible to follow the fate of individual cells immunohistochemically. The fact that the
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RELEASE
OF mPLs BY THE SAME CELL
number of cells that contained both hormones on the third day of culture was about 3 times greater than the number that contained only mPL-II on day 5 suggests that a significant fraction of the dual hormone producers ceased PL production after day 3. It is possible that some of the increase in the number of giant cells that contained only mPL-II toward the end of the culture period resulted from differentiation of a subpopulation that never produced mPL-I. Similarly, the small numbers of cells containing only mPL-I or mPL-II that were present at the end and beginning of the 5day culture, respectively, could be subpopulations that produce only one of the PLs. The data from the reverse hemolytic plaque assay suggest that a shift also occurred during time in culture in the type of PL that is releasedby giant cells. When the total number of plaque-forming cells that released each type of PL was examined, there was a decline between days 1 and 2 in the fraction of plaque-forming cells that released only mPL-I and an increase in the fraction that released only mPL-II. When the fate of individual plaque-forming cells was followed, it was clear that some cells that released only mPL-I on the first day of culture released both hormones or only mPL-II on the secondday. The latter observation is consistent with the hypothesis that at least some PL-producing cells undergo a shift in PL production from mPL-I to both hormones to mPL-II. Although the number of cells examined was relatively small, of the 37 plaque-forming cells that releasedonly mPL-II on the first day of culture, none released mPL-I on the second day, which suggests that once giant cells have differentiated to the extent that they release only mPL-II, they may not release mPL-I again, at least under these culture conditions. Unfortunately, we were unable to immunostain cells satisfactorily after performing sequential hemolytic plaque assays and could not determine whether cells that releasedonly mPL-II contained mPL-I. The fact that individual giant cells in this culture system undergo a shift in the type of PL they produce suggeststhat the pattern of PL expression that occurs in viva at midpregnancy can be attributed, at least partly, to a change in gene expression by individual giant cells. Whether all giant cells express both PLs in viva during midpregnancy remains to be determined. In situ hybridization and immunohistochemical analysis of conceptusesfrom midpregnant mice (19) and rats (4) have localized PL-I and PL-II to giant cells in two regions of the placenta, the chorioallantoic placenta (polar giant cells) and the choriovitelline placenta (mural giant cells). Studies in the rat have demonstrated that there are differences between polar and mural giant cells in both the day of pregnancy when PL expression begins and the amounts of PLs produced (4, 25). It is not known if regional differences also exist in the ability of individual giant cells to express both PLS. Although giant cells that stained for mPL-II were present as early as the first day of incubation in cultures from day 7 of pregnancy, mPL-II could not be detected in the culture medium until the third or fourth day (data not shown; 22). This was probably not due to the presence of too few mPL-
1601
II-producing cells since mPL-II could be detected easily in the medium on the fifth day of culture when the number of mPL-II-containing cells and their staining intensity (data not shown) was similar to those on day 1. These observations suggestthat at this stage of pregnancy mPL-II is stored for several days before it is releasedin significant amounts and that factors that signal mPL-II releasemay appear later than the stimulus for hormone synthesis. The apparent storage of mPL-II by giant cells at this stage of pregnancy differs from the situation later in pregnancy when most newly synthesized mPL-II is rapidly released(26). Little is known about the regulation of PL-I and PL-II gene expression.Preliminary data from our laboratory (Yamaguchi M., and F. Talamantes, in preparation) indicate that EGF stimulates mPL-I production but inhibits mPL-II production in the same culture system used here, which suggeststhat EGF may be involved in regulating the switch in gene expression from PL-I to PL-II. The fact that giant cells from day 7 of pregnancy undergo a shift in the type of PL they produce in vitro suggeststhat the culture system used in this study will be useful for examining the molecular events that underlie the shift from PL-I to PL-II production at midpregnancy. Acknowledgment We thank Dr. Stephen hemolytic plaque assay.
Frawley
for
his advice
about
the
reverse
References 1. Colosi P, Marr G, Lopez J, Haro L, Ogren L, Talamantes F 1982 Isolation, purification, and characterization of mouse placental lactogen. Proc Nat1 Acad Sci USA 79:771-775 2. Colosi P. Oeren 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 3. Robertson MC, Gillespie B, Friesen HG 1982 Characterization of the two forms of rat placental lactogen (rPL): rPL-I and rPL-II. Endocrinology 111:1862-1866 4. Faria TN, Deb S, Kwok SCM, Talamantes F, Soares MJ 1990 Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta. Dev Biol 141:279-291 5. Robertson MC, Croze F, Schroedter IC, Friesen HG 1990 Molecular cloning and expression of rat placental lactogen-I complementary deoxyribonucleic acid. Endocrinology 127:702-710 6. Colosi P, Talamantes F, Linzer DIH 1987 Molecular cloning and expression of mouse placental lactogen I complementary deoxynbonucleic acid. Mol Endocrinol 1:767-776 7. Duckworth ML, Kirk KL, Friesen HG 1986 Isolation and identification of a cDNA clone of rat placental lactogen II. J Biol Chem 261:10871-10878 8. Jackson LL, Colosi P, Talamantes F, Linzer DIH 1986 Molecular cloning of mouse placental lactogen cDNA. Proc Nat1 Acad Sci USA 83:8496-8500 9. Thordarson G, Villalobos R, Colosi P, Southard J, Ogren L, Talamantes F 1986 The lactogenic response of cultured mouse mammary epithelial cells to mouse placental lactogen. J Endocrinol 109:263-274 10. 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 11. Ogren L, Southard JN, Colosi P, Linzer DIH, Talamantes F 1989 Y
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RELEASE
1602
OF mPLs
Mouse serum.
placental lactogen-I: RIA and gestational profile in maternal Endocrinology 125:2253-2257 12. Soares MJ, Colosi P, Talamantes F 1982 The development and characterization of homologous radioimmunoassay for mouse placental lactogen. Endocrinology 110:668-670 13. Soares MJ, Talamantes F 1983 Genetic and litter size effects on serum placental lactogen in the mouse. Biol Reprod 29:165-171 14. Robertson MC, Friesen HG 1981 Two forms of rat placental lactogen revealed by radioimmunoassay. Endocrinology 108:2388-
2390 15. Hall J, Talamantes mouse tochem 16.
F 1984 Immunocytochemical
placental lactogen 32:379-382
Lee SJ, Talamantes
in the mouse
F, Wilder
placenta.
WJ, Deb S, Kwok
19. Faria TN, Ogren L, Talamantes
22. Yamanuchi
of Cy-
DIH, Nathans D 1988 as the site of proli-
24.
SCM, Joslin J, Soares MJ 1989
Differential expression of placental lactogen-II and prolactin-like protein-A in the rat chorioallantoic placenta. Endocrinology 125:1565-1574 18. Duckworth ML, Schroedter IC, Friesen HG 1990 Cellular localization of rat placental lactogen II and rat prolactin-like proteins A and B by in situ hybridization. Placenta 11:143-155
F, Linzer DIH, Soares MJ 1991
SAME
Endo. Voll31.
CELL
1992 No 4
Localization of placental lactogen-I in trophoblast giant cells of the mouse placenta: Biol Reprod 44:327-331 20. Nieder GL. Iennes L 1990 Production of mouse ulacental lactoeenI by trophbblast giant cells in utero and in v&o. Endocrinology 126:2809-2814 21. Thordarson G, Folger P, Talamantes F 1987 Development of a placental cell culture system for studying the control of mouse placental lactogen secretion. Placenta 8;575-585
23. E, Linzer
Trophoblastic giant cells of the mouse placenta ferin synthesis. Endocrinology 122:1761-1768
17. Campbell
localization J Histochem
BY THE
25. 26.
M. Endo H. Thordarson
G. Oeren L. Talamantes
F
1992 hodulation of mouse placental lactog&I secretion in vitro: effects of progesterone and mouse placental lactogen-II. Endocrinology 130:2897-2905 Frawley LS, Boockfor FR, Hoeffler JP 1985 Identification by plaque assays of a pituitary cell type that secretes both growth hormone and prolactin. Endocrinology 116:734-737 Colosi P, Talamantes F 1981 The amino acid composition of secreted mouse prolactin, growth hormone, and hamster prolactin: the presence of one tryptophan in mouse prolactin. Arch Biochem Biophys 212:759-761 Soares MJ, Julian JA, Glasser SR 1985 Trophoblast giant cell release of placental lactogens: temporal and regional characteristics. Dev Biol 107:520-526 Basch CV, Talamantes F 1986 In vitro kinetics of synthesis and release of mouse placental lactogen. Endocrinology 119:1939-1947
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