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THE JOURNAL OF B~L~CICAL CHEMISTRY
Vol. 251, No. 10, Issue of May 25. pp. 2915-2951. 1976
Printed in U.S.A.
Synthesis of Human Placental Lactogen and Human Chorionic
Gonadotropin by Polyribosomes and Messenger RNAs from
Early and Full Term Placentas*
(Received for publication, November 24, 1975)
MEERA CHATTEWEE,
B.
SUREN BALIGA, AND HAMISH
N.
MUNRO
From the Physiological Chemistry Laboratories, Department
of
Nutrition and Food Science,
Massachusetts Institute
of
Technology, Cambridge, Massachusetts 02139
Synthesis of human placental lactogen (hPL) and of human chorionic gonadotropin (hCG) by
membrane-bound and free polyribosomes from early and from full term human placentas was
investigated by in
vitro
release of the nascent hormone peptides, followed by immunoprecipitation and
electrophoresis in sodium dodecyl sulfate gels, and by specific binding of Y-labeled hPL antibody to
nascent peptide chains. In addition, messenger RNAs were extracted from total, free, and membrane-
bound placental polyribosomes and their capacities for hPL and hCG synthesis were measured in a
heterologous cell-free system prepared from wheat germ.
Membrane-bound polyribosomes from full term placentas were several times more active in the
synthesis of both peptide hormones than were free polyribosomes. By binding 12”I-labeled hPL antibody
to nascent chains on the polyribosomes, it was determined that hPL is made by clusters of seven to nine
ribosomes. About 8% of the nascent peptide chains released by incubation of polyribosomes from full
term placentas was accounted for by hPL, and 2% by hCG. In contrast, no chains of hPL were released by
polyribosomes from lo-week placentas, whereas 11% of the total released chains were accounted for by
hCG. When messenger RNAs prepared from the polyribosomes of 20. and 40.week placentas were used to
stimulate protein synthesis in a wheat germ system, hPL accounted for 0.4 and 2%, respectively, of total
protein synthesis, while hCG was 8 and 2%, respectively. This confirmed the relative proportions
observed for nascent chains on the polyribosomes of early and late placentas. Unexpectedly, translation
of mRNA from free polyribosomes yielded as much hPL and two-thirds as much hCG as did translation of
mRNA from bound polyribosomes. We conclude that the decreased blood levels of hCG and increased
blood leveis of hPL with advancing gestational age reflect the relative in.
vitro
rates of synthesis of these
hormones by placental polyribosomes, the abundance of which is determined by availability of their
respective messenger RNAs at different times in gestation.
During gestation, the human placenta synthesizes and
secretes two unique protein hormones, human chorionic gonad-
otropin and human placental lactogen. hCG’ can be detected
in maternal serum and urine as early as 7 days after fertiliza-
tion. It reaches peak levels during the first trimester of
pregnancy but continues to be secreted in lesser amounts by
the full term placenta (1). On the other hand, hPL becomes
detectable in plasma from about 6 weeks of gestation and rises
progressively with time to reach a plateau around 34 weeks (2,
3).
A few studies have been made of in
vitro
synthesis of these
hormones by preparations of human placenta. Friesen et al. (4,
5) found that hPL represented 5 to 10% of the proteins released
into the incubation medium by slices of full term placentas.
* This investigation was supported by Grant AM 15364-05 from the
United States Public Health Service.
‘The abbreviations used are: hCG, human chorionic gonadotropin;
hPL, human placental lactogen; SDS, sodium dodecyl sulfate.
Boime and Boguslawski (6) showed by gel separation that hPL
accounted for 10% of nascent peptide chains on the polyribo-
somes of term placentas and 5% on those of first trimester
placentas. Using a more discriminating radioimmunoassay
technique, they (7) later observed a 4-fold greater synthesis of
hPL by term than by first trimester placentas. Synthesis of
hCG by first trimester placental tissue slices incubated
in vitro
has been demonstrated by several investigators (S-10). Maruo
et al. (11, 12) estimated that after a 3-day incubation of
trophoblast slices almost 60% of the protein released into the
medium was hCG. No comparisons of
in vitro
hCG synthesis
between first trimester and term placentas appear to have been
made.
We have investigated the possibility that the synthesis of
these two protein hormones is regulated inversely during
placental development by variations in relative abundance of
mRNA for each hormone. Using early and full term placentas,
we have examined the distribution of nascent chains of hPL
2945
This is an Open Access article under the CC BY license.
2946 Synthesis
of
hPL and hCG by Placental Polyribosomes and mRNA
and hCG in the membrane-bound and free ribosome popula-
tions and have extracted the mRNAs of these polyribosomes
and translated them in a wheat germ system. In agreement
with evidence regarding the site of synthesis of other secreted
proteins, nascent chains for both placental peptide hormones
were much more abundant on membrane-bound than on free
ribosomes. The relative abundance of nascent chains and
mRNA for hCG was greater in early than in late placentas,
whereas this was reversed for hPL. We conclude that the
availability of mRNA at different stages of pregnancy deter-
mines the amounts of hPL and hCG formed.
EXPERIMENTAL PROCEDURES
Human placental lactogen (95% pure) was purchased from Nutri-
tional Biochemicals, human chorionic gonadotropin, lactoperoxidase,
and rabbit anti-bovine serum albumin from Calbiochem, and rabbit
anti-hCG serum from Miles Laboratories. L-[4,5-3H]Leucine, specific
activity 30 to 50 Ci/mmol, and carrier-free NaY were obtained from
New England Nuclear. Oligo(dT)-cellulose was purchased from Col-
laborative Research, Inc. All other chemicals used were of analytical
grade.
Human term placentas were obtained immediately after normal
deliveries and were immersed in ice-cold buffer, containing 50 mM
Tris-HCl, pH 7.6, 25 rn~ KCl, 106 mM NH&l, 10 mM MgCl, (Tris/K+/
NH,+/MgZ+ buffer), and 0.15 M NaCl, for transport to the laboratory.
They were trimmed of cord and membranes, cut into convenient pieces
which were washed in Tris/K+/NH,+/MgZ+ buffer, and finally homoge-
nized in a Waring Blendor in an equal volume (w/v) of Tris/K+/NH,+/
Mg2+ buffer containing 0.25 M sucrose. Early placentas were obtained
within 30 min from therapeutic abortions by saline (0.9% NaCl solution)
induction at 16 to 20 weeks or by suction curettage at around 10 weeks.
The trophoblast from the latter was separated out under a dissecting
microscope, several being pooled to provide an adequate sample.
Preparation of Total, Free, and Membrane-bound Placental
Polyribosomes-Total placental ribosomes and free and membrane-
bound polysomes were prepared as previously described by Laga et al.
(13) except for the following modifications. (a) The buffer used was
Tris/K+/NH,+/Mg2+. (b) The homogenization medium contained 15
mg/lOO ml of sodium heparin in addition to 0.5 mM EDTA to inhibit
ribonuclease. (c) A discontinuous gradient of 1.38 M, 2.0 M, and 2.3 M
sucrose was used in the preparation of free and membrane-bound
polysomes. (d) All polysome preparations were centrifuged through the
discontinuous gradient made up in buffer containing 0.5 M NH&l.
Analysis of polysome profiles was performed as previously described
(13).
Incubation of Polysomes with ‘251-labeled hPL Antibody-Antisera
against electrophoretically pure hPL were prepared in rabbits and the
y-globulin fraction was obtained by ammonium sulfate precipitation.
Removal of RNase by DEAE- and CM-cellulose chromatography and
then enzymatic iodination of the y-globulins were carried out as
described by Palacios et al. (14). The final concentration of y-globulin
was 5 mg/ml and the specific activity was 1.8 x lo6 cpm/mg.
Ten A,,, units of polysomes were incubated with ‘251-labeled hPL
antibody in buffer (50 mM Tris-HCl, pH 7.6/80 mM NH&l/5 rnM MgCl,
with 15 mg/lOO ml of heparin) in a final total volume of 0.5 ml. The
amount of antibody added varied from 10 to 250 pg of protein. Binding
of 100 kg of anti-hPL to nascent peptides on total term placental
polysomes was explored at 0” and 25” for 30, 60, 90, and 120 min. Since
binding did not increase substantially beyond 60 min of incubation at
0” and the polysomes tended to disaggregate within 30 min at 25’, the
procedure finally adopted was incubation at 0” for 60 min.
The binding of ‘2SI-antibody to nascent hPL chains was used for two
purposes. (a) To determine the size of polysome clusters synthesizing
hPL, 100 ~1 of the above reaction mixture was layered over a 10 to 50%
linear sucrose gradient and centrifuged in a Spinco L-2 ultracentrifuge,
SW 50.1 rotor, at 40,000 rpm (150,000 x g) for 80 min at 4’. While
recording the polysome profile (13), fractions of 140 ~1 were collected
and ‘Y was counted in a Packard 3002 gamma scintillation spectro-
photometer. (6) To determine whether hPL is made on free or
membrane-bound ribosomes, the incubation mixture of total, free, or
bound polysomes and ‘Y-anti-hPL was layered over a discontinuous
gradient of 0.5
M,
2.0 M, and 2.3 M sucrose and centrifuged in a Spinco
L-2 ultracentrifuge, Ti 50 rotor, 45,000 rpm (105,000 x g) for 4 hours at
4’. The pellet was recovered, counted, and then resuspended and its
polysome profile was recorded to obtain the amount of ribosomes
present. When Y-anti-hPL was spun through the same gradient
without prior reaction with ribosomes, no lzsI activity was pelleted.
Incubation of Placental Polysomes in Cell-free Amino Acid-incor-
porating System-Protein synthesis by placental polysomes was as-
sayed in reaction mixtures of the following final concentrations: 50 mM
Tris-HCl, pH 7.6, 80 mrvr NH&l, 5 rnM MgCl,, 2 rnM ATP, 0.5 mM
GTP, 2 rnM dithiothreitol, 50 PM each of 19 nonradioactive amino acids,
0.5 PCi of [3H]leucine, 100 pg of rat liver pH 5 enzyme protein, and 100
to 200 pg of placental ribosomes in a final volume of 0.1 ml. Reaction
mixtures were increased to 0.5 ml for isolation of specific products. A
rat liver pH 5 fraction was used because it was found to be more active
than placental pH 5 preparations (15).
After incubation at 37” for 60 min, the reaction mixtures were
chilled to 0” and centrifuged in a Spinco L-2 ultracentrifuge, Ti 50
rotor, at 45,000 rpm (105,000 x g) for 60 min to precipitate polysomes
with attached nascent chains, leaving only released chains in solution.
Incorporation of radioactivity into trichloroacetic acid-precipitable
protein was measured in a 5. or 10.~1 aliquot of the supernatant
fraction by the method of Mans and Novelli (16). Specific hormone
peptides were isolated from the remainder of the supernatant fraction
as described below.
Preparation and Translation of Placental Poly(A)-containing
RNA-Crude total, free, and bound placental RNA was prepared from
polysomes essentially by the procedure described by Aviv and Leder
(17) except that 1% SDS was added directly to the polysome
suspension buffer which also contained 15 mg/lOO ml of sodium
heparin to inhibit RNase activity. Oligo(dT)-cellulose chromatography
of the crude RNA preparations was performed as described by Aviv
and Leder (17) except that 20 rnM N-2.hydroxyethylpiperazine-N-2.
ethanesulfonic acid buffer, pH 7.4, was used instead of Tris-HCl. The
eluted poly(A)-containing RNA was frozen directly and stored at -60”.
Messenger RNA was translated in wheat germ S,, extract prepared
according to Roberts and Paterson (18). The reaction mixture (18)
included 4 PCi of [3H]leucine/50 ~1 (1 to 2
PM)
as the label. In addition,
the system was supplemented with 40
PM
spermine which increases the
efficiency of translation of mRNA (19). Incorporation of radioactivity
into protein was measured by the Mans and Novelli method (16).
Preparation of mRNA by Dissociation of Placental Ribosomes-The
procedure followed was essentially that of Ernst and Arnstein (20).
Washed placental ribosomes were made 33 rnM in EDTA and cen-
trifuged in a Spinco L-2 ultracentrifuge, Ti 50 rotor, at 45,000 rpm
(105,060 x g) for 2 hours. The supernatant, containing mRNP particles
dissociated from ribosome subunits, was made 1% in N-lauryl sarco-
sine and layered on top of 5 to 30% sucrose gradients (5.0 ml volume).
These were centrifuged at 45,000 rpm for 90 min in an SW 50.1 rotor,
and the absorption of the gradient at 260 nm was recorded by a Gilford
2000 recording spectrophotometer. Fractions of 0.5 ml were collected
and were later pooled into three batches, representing the upper (1.5
ml), middle (2.0 ml), and lower (1.5 ml) portions of the profile.
Absorbance of the pooled fractions at 260 and 280 nm was recorded.
RNA was precipitated by making the solutions 2% in potassium
acetate and adding 2 volumes of ethanol. Each section of the gradient
was tested separately in the wheat germ cell-free system for its ability
to stimulate incorporation of labeled leucine into protein.
Specific Product Analysis-hPL and hCG synthesized by placental
polysomes or messenger RNA in uitro were precipitated with a specific
antibody and subjected to SDS-polyacrylamide gel electrophoresis.
The technique used is similar to that described by Taylor and Schimke
(21) and Palmiter (22). Ten micrograms of carrier hPL or hCG and
sufficient excess of the specific antibody were added to aliquots of the
105,000 x g supernatant of the incubation mixtures in the presence of
1% 7-deoxycholic acid, 1% Triton X-100, 10 rnM nonradioactive leucine,
0.15
M
NaCl, and 10
mM
phosphate buffer, pH 7.5, a detergent solution
designed to prevent nonspecific binding of [3H]leucine to the antibody.
The mixture was incubated at 37” for 2 hours, and then at 4” overnight.
The following day, the immunoprecipitates were washed twice in the
above detergent solution (5x concentrated) and once by centrifuging
through a discontinuous gradient of 0.5 M and 1.2 M sucrose in
detergent to remove any nonspecifically bound [3H]leucine. The
resulting precipitates were dissolved in 25 ~1 each of a solution
containing 60 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol (v/v), 5%
p-mercaptoethanol, and 0.001% bromphenol blue and subjected to
electrophoresis for 4 hours at 175 volts in a linear 12 to 15% gradient
polyacrylamide gel slab prepared as described by Maize1 (23). The gel
was stained in 0.2% Coomassie blue and destained to disclose protein
bands. Radioactivity was measured in 3.3.mm slices by dissolving
Synthesis
of
hPL and hCG by Placental Polyribosomes and mRNA
them in 0.1 ml of H,O, (at 55” for several hours), taking up the aqueous
phase in 0.7 ml of Protosol (New England Nuclear) by incubation for 1
hour at 55”, and counting the mixture in 10 ml of toluene/2,5-
diphenyloxazole/l,4-bis [2-(5.phenyloxazolyl) ]-benzene scintillation
fluid in an Isocap model 300 (Nuclear Chicago) liquid scintillation
counter.
2947
were free polysomes, while mixed placental polysomes were
intermediate. The table shows excellent agreement in this
distribution at the two levels of antibody added.
Magnitude
of
in Vitro Synthesis
of
hPL by Placental
Polyribosomes-The
proportion of total protein synthesis rep-
resented by hPL was next examined, using [3H]leucine incor-
poration
in uitro
in the presence of rat liver pH 5 fraction to
label nascent chains on placental polysomes, followed by
specific immunoprecipitation. The efficiency of recovery of
labeled immunoprecipitate was measured in a separate experi-
ment by using hPL labeled with lZ51. The final recovery
averaged 80.5% and, more important, the standard deviation of
eight replicates was 14.6%. This implies considerable
reproducibility in spite of incomplete recovery.
RESULTS
Size and Class
of
Polyribosomes Synthesizing
hPL-The size
of the polyribosome aggregate making hPL was identified by
the binding of 12SI-labeled anti-hPL to mixed placental poly-
somes and their separation on sucrose gradients (Fig. 1). The
label bound to polysomes was principally associated with
intermediate-sized (seven to nine) polysomes. With increasing
amounts of labeled antibody, the radioactive peak increased in
height. However, resolution reached a limit at 200 /~g of
antibody when radioactivity associated with excess unbound
y-globulin trailed down from the top of the gradient. Control
polyribosomes prepared from rat liver showed no binding of
anti-hPL. As a further control procedure, placental polysomes
were preincubated with unlabeled anti-hPL in excess and then
mixed with labeled anti-hPL, when the radioactivity remained
entirely in the supernatant fraction. However, binding of
lZ51-anti-hPL was not prevented by preincubating the poly-
somes with unlabeled anti-bovine serum albumin.
The relative binding of ‘251-labeled antibody to membrane-
attached and free ribosomes was then examined. Polyribo-
somes of each type prepared from full term placentas were
incubated with either 100 or 200 pug of anti-hPL protein. They
were then pelleted through a discontinuous sucrose gradient
and the amount of lZ51 activity recovered was counted. About
50% of the ribosomes in the original incubation mixture were
reproducibly recovered in the pellet, regardless of class. The
lZ51 activity recovered was then divided by the amount of
ribosomes in the pellet. Table I shows that the labeled
antibody bound to the two classes of placental polysomes to
different extents. Membrane-attached polysomes were approx-
imately 4 times more active in binding the hPL antibody than
8
0
0 5 IO 15 20 25 30 35
FRACTION NUMBER
FIG. 1. Binding of ‘Y-anti-hPL to placental polysomes. After
incubation with different amounts of ‘%labeled anti-hPL, total term
placental polysomes were separated on 10 to 50% linear sucrose
gradients. The absorbance at 260 nm was recorded (. .) and IS51 was
counted in each fraction collected (-). The peaks at Fraction 25
represent counts recovered after addition of 50 pg (2), 100 pg (3), and
150 pg (4) of ‘ZSI-anti-hPL, respectively, to the incubation. Note that
sample (l), which shows no peak at Fraction 25, was obtained by
incubation of 50 pg of Y-anti-hPL with rat liver polysomes. The data
are representative of eight replications of the experiment.
The immunoprecipitates were then resolved on SDS gels in
which hPL and the two antibody subunits were identified by
protein staining. After membrane-attached, free, or total
piacental polyribosomes had been incubated with [3H]leucine,
the gels showed a large peak of radioactivity co-migrating with
standard hPL marker
(M,
= 21,600), membrane-bound ribo-
somes exhibiting the greatest incorporation and free the least
(Fig. 2). In addition, two smaller radioactive peaks were
associated with the light and heavy chains of the y-globulin
added at the end of the incubations. These represent nonspe-
cific radioactivity other than labeled hPL chains, since rat liver
polyribosomes incubated with [3H]leucine and subjected to
anti-hPL precipitation showed no activity in the hPL area, but
still exhibited these peaks.
Table II shows the percentage of [3H]leucine incorporated
into hPL relative to incorporation of radioactivity into total
protein under these conditions. hPL accounted for approxi-
mately 8% of incorporation into peptides released from total
term placental polysomes. Incorporation into hPL by bound
polysomes was 10% and by free polysomes was 3%. Polysomes
derived from lo-week placentas failed to incorporate [3H Jleu-
tine into hPL in detectable amounts.
In Vitro Synthesis
of
hPL by Placental Messenger
RNA-The capacity of free and membrane-bound ribosomes to
synthesize hPL was further examined by incubating the
poly(A)-containing RNAs extracted from each polyribosome
class of full term and early placentas with the wheat germ
TABLE I
Binding of ‘Z51-unti-hPL to different classes of placental polysomes
Ten A,,, units of polysomes were incubated with 100 or 200 pg of
“‘1-anti-hPL at 0” for 60 min. The polysomes were precipitated
through a discontinuous sucrose gradient. The absorption of the pellet
at 260 nm was determined and its ‘*? activity was counted and
converted to micrograms of antibody. In the last column, this amount
of bound antibody is expressed per A 260 unit of polysomes. The data are
representative of six replications of the experiment.
Equiv- Amount
m-anti- Class Ribosomes Y alent of
hPL of recovered bound amount anti-
added ribosomes in to of hod)
pellet pellet anti- bound/
body A260
fig A,,, cpm x 10 = PR MIA 280
100 Total 5.24 14.8 8.2 1.57
Free 5.15 5.1 2.8 0.55
Bound 5.09 21.9 12.2 2.39
200 Total 5.65 31.3 17.4 3.08
Free 5.91 10.9 6.1 1.02
Bound 4.89 40.5 22.5 4.61
2948 Synthesis
of
hPL and hCG by Placental Polyribosomes and mRNA
t,,'
Ol 5 IO 15 20 25 30 35 40 42
BOTTOM GEL SLICE NUMBER ,33rnrn) TOP
FIG. 2. Radioactivity profile on an SDS-polyacrylamide gradient
gel of immunoprecipitated Wlabeled hPL synthesized by in vitro
incubation of total, free, or membrane-bound full term placental
polysomes. Placental ribosomes were incubated in the presence of rat
liver pH 5 enzyme and [3H]leucine, and 3H-labeled hPL was precipi-
tated from released products as described under “Experimental
Procedures.” The immunoprecipitate was resolved on SDS gels and
radioactivity was measured in gel slices. The figure is representative of
six replications of the experiment.
TABLE II
In uitro synthesis ofhPL by placentalpolysomes
Placental polysomes were incubated in the presence of rat liver
pH 5 enzyme and [3H]leucine. Total protein counts released into the
supernatant were measured and ‘H-labeled hPL was precipitated and
isolated on SDS gels as described under “Experimental Procedures.”
The amount of radioactivity recovered in hPL is expressed as a per-
centage of total peptide incorporation. The mean * S.D. is based on
the number of experiments shown in parentheses for each class of
polysomes.
Total
protein
Incorporation into
hPL as cc total
peptide incorporation
Rat liver (total)
Full term placentas
Total
Free
Bound
Early placentas
Total
cpm
27,200 0.98 * 0.09 (4)
34,200 7.73 i 0.4 (6)
36,000 3.14 i 0.2 (6)
36,800 9.88 + 0.4 (6)
37,000 0 (3)
cell-free protein-synthesizing system. The optimum ion con-
centrations for both early and late placental mRNAs were
determined to be 2.5 mM Mg2+ and 70 mM K+. At these
concentrations, amino acid incorporation into protein was
stimulated approximately lo-fold by placental mRNA in this
system. The cell-free products synthesized in vitro were
analyzed on SDS gels. Uptake of radioactivity into hPL
stimulated by total term placental mRNA was about 2% of
that into total protein (Table III). In contrast to the free and
membrane-bound polyribosome data, no difference in this
percentage was found between messengers from free and bound
ribosomes.
In order to determine whether the placental mRNA isolated
by oligo(dT)-cellulose chromatography was representative of
all placental mRNAs, we compared this mRNA with mRNA
prepared by EDTA dissociation of total full term placental
ribosomes. The polysomes were treated sequentially with
EDTA and N-lauryl sarcosine, and the mRNA thus isolated
was separated on a 5 to 30% sucrose gradient. Upper, middle,
and lower portions of the spun gradient were tested separately
for their ability to stimulate incorporation into protein in the
TABLE III
Synthesis ofhPL by placental messenger RNAs in wheat germ cell-free
system
Placental messenger RNAs extracted from the different classes of
polyribosomes were incubated with wheat germ S,, extract and
[3H]leucine. Total protein counts were measured and “H-labeled hPL
synthesized was determined as described under “Experimental Proce-
dures.” The results are expressed as in Table II; the number of
replicates is shown in parentheses.
Source of
&WA
Total
protein
Incorporation into
hPL as Y total
peptide incorporation
None added
Full term placentas
Total poly(A)”
Total ribosomalb
Free
Bound
Early placentas
Total poly(A)
cpm
12,500 0.18 * 0.02 (4)
74,000 1.96 + 0.25 (8)
62,000 2.35 i 0.20 (3)
62,000 2.27 zt 0.35 (6)
76,000 2.18 zt 0.30 (6)
60,000 0.49 * 0.07 (3)
a mRNA prepared by oligo(dT)-cellulose chromatography.
’ mRNA prepared by EDTA dissociation of ribosomes.
wheat germ system. The results are shown in Table IV, along
with the 260/280 nm absorbance ratios of the three fractions.
The middle section of the gradient, which showed the max-
imum absorption at 260 nm, also demonstrated the highest
activity for in uitro incorporation, almost equal to that of
poly(A)-containing mRNA. When the products synthesized by
this mRNA were analyzed, it was found that [3H]leucine was
incorporated into hPL to the same extent as with poly(A)-con-
taining mRNA (Table III).
The presence of mRNA for hPL was also tested in early
placenta. Total mRNA, prepared with oligo(dT)-cellulose from
20.week placentas, stimulated uptake of radioactivity into hPL
equivalent to 0.4% of total peptide incorporation by the system
(Table III). This contrasts with the lack of incorporation into
hPL by lo-week placental polysomes described earlier (Table
II) but is in agreement with the increase in hPL secretion which
occurs between 10 and 20 weeks gestation.
Synthesis
of
hCG by Placental Polyribosomes and Messen-
ger RNAs-The synthesis of hCG by term and early placental
polyribosomes and messenger RNAs was also examined to
provide a comparison with hPL. The specific product was
isolated from incubation mixtures with anti-hCG, which ap-
pears to recognize both subunits of this protein. The protein
portion of the a subunit has an estimated molecular weight of
10,200 (24) and that of the p subunit, 16,000 (25), so that they
migrate close to each other on 12 to 15%> polyacrylamide gels.
This is evidenced by the two overlapping peaks of radioactivity
obtained on SDS gel electrophoresis of the immuno-
precipitated products (Fig. 3). As in the case of incubations for
hPL, radioactivity was also present at the positions marked by
the light and heavy chains of the antibody. In Table V, hCG
synthesis as a percentage of total protein synthesized by the
different classes of polyribosomes and of messenger RNAs is
shown. Percentage of total peptide uptake is given for the N
and p subunits separately and for the double peak of hCG.
Each subunit accounted for approximately 1% of total counts
incorporated into released peptides by total term placental
polysomes, so that total hCG accounted for 2%. Free polysomes
were about one-fifth as active as bound polysomes in taking up
Synthesis
of
hPL and hCG by Placental Polyribosomes and mRNA 2949
[3H]leucine into either the 01 or p subunits or total hCG. In
contrast, hCG represented about 11% of total peptide synthe-
sized by total early placental polysomes, each subunit account-
ing for some 4 to 5% of the radioactivity.
Placental messenger RNAs were then tested for their capac-
ity to synthesize hCG. Poly(A)-containing mRNA from total
term placental polyribosomes incorporated about 2% of total
protein radioactivity into hCG; mRNAs prepared from free
polysomes were about two-thirds as active as those from bound
polysomes. Early (20-week) placental mRNA stimulated hCG
synthesis 4-fold compared with mRNA from term placentas
(8% uersus 2% of total protein incorporation, respectively).
TABLE IV
Analysis of mRNA prepared by ribosome dissociation with EDTA
Total term placental polysomes were dissociated with EDTA. The
mRNP was deproteinized and the released mRNA was separated on a
linear 5 to 30% sucrose gradient and collected in three portions: upper
(1.5 ml), middle (2.0 ml), and lower (1.5 ml). The table shows the
260/280 nm absorbance ratios, the total A,,, absorbance for each
portion of the gradient, and the total incorporation into protein when
the RNAs from these fractions were used to stimulate the wheat germ
cell-free system. Values for mRNA prepared by oligo(dT)-cellulose
chromatography are shown for comparison. The experiment was
repeated three times.
mRSA
preparation
IL”:!,,
Total incor-
AdA,,, A ml porated
fractmn into
protein
None
Ribosome dissociation
and gradient separation
Upper portion
Middle portion
Lower portion
Oligo(dT)-cellulose
1.2
1.6
1.4
1.8
A uncts
0.19
0.65
0.27
cpmlp~ RNA
3,000
4,000
20,000
16,750
24,000
DISCUSSION
This study has been aimed at an examination of the
relationship between placental mRNA activity at different
stages of placental maturation and the amounts of peptide
hormones secreted by the placenta. Although early and late
placental ribosomes showed the same total in vitro incorpora-
tion of labeled amino acids in the presence of rat liver pH 5
fraction (Tables II and V), the proportions of the total counts
due to hCG and hPL are different at these two times. In the
case of polysomes from early placentas, 11% of the total
incorporation into released peptides was accounted for by hCG
but no activity could be detected for hPL. In contrast,
polyribosomes from full term placentas incorporated 2% of
total protein counts into hCG and 8% into hPL. In interpreting
such data, it should be noted that total polyribosomes tend to
contain a disproportionate number of free ribosomes because of
the loss of many membrane-bound
polysomes during initial
removal of nuclei and cell debris (26).
BOTTOM GEL SLiCE NUMBER ,33mml TOP
FIG. 3. Radioactivity profile on an SDS-polyacrylamide gradient
gel of immunoprecipitated Wlabeled hCG synthesized by in vitro
incubation of total, free, or membrane-bound full term placental
polysomes. Placental ribosomes were incubated in the presence of rat
liver pH 5 enzyme and [3H]leucine, and 3H-labeled hCG was precipi-
tated from released products as described under “Experimental
Procedures.” The immunoprecipitate was resolved on SDS gels and
radioactivity was measured in gel slices. The hCG a: and p subunits are
partly resolved. The figure is representative of four replications of the
experiment.
TABLE V
In vitro synthesis of hCG by polyribosomes and messenger RNAs from
full
term and early placentas
Polysomes and mRNAs were incubated as described under “Experi- samples at maximum activity for the hCG-cu band and the hCG-fi band
mental Procedures.” Total protein counts were measured and the are given. Total incorporation into hCG was calculated from counts
amount of 3H-labeled hCG was determined on SDS gels. The percent- contained in the entire peak (see Fig. 3). The number of experiments
age of total incorporated counts + S.D. represented by l-cm gel with each class of polysomes and mRNA is shown in parentheses.
Source of Total ‘& hCG-a of 7c hCG-8 of ‘8 total hCG of
polysomes or protein total peptide total peptide total peptide
mRNA incorporation incorporation incorporation
Polysomes
Rat liver
Full term placentas
Total
Free
Bound
Early placentas
Total
mRNA
None added
Full term placentas
Total
Free
Bound
Early placentas
Total
32,000 0.17 + 0.06 (4)
26,000 1.19
l
0.13 1.01 i 0.24 2.61 + 0.43 (4)
34,400 0.51 * 0.14 0.40 * 0.13 1.07 zt 0.25 (4)
24,400 1.99 * 0.21 1.74 i 0.25 4.46 i 0.76 (4)
28,600 4.62 i 0.27 4.16 i 0.22 10.58 i 0.60 (3)
14,000
64,000 0.81 A 0.19 0.74 * 0.22
75,200 0.73 * 0.22 0.63 + 0.23
66,500 1.09 + 0.16 1.04 * 0.17
60,000 3.47 * 0.18 3.08 * 0.06 7.66 i 0.28 (3)
0.11
l
0.02 (2)
2.00 zt 0.23 (4)
1.72 f 0.30 (4)
2.60 i 0.26 (4)
2950 Synthesis
of
hPL and hCG by Placental Polyribosomes and mRNA
The increase in the proportion of nascent hPL chains on
polyribosomes as the placenta matures is confirmed by other
evidence. Boime and Boguslawski (6) reported 5% of the total
protein incorporation into hPL by ribosomes from first trimes-
ter placentas and 10% with ribosomes from full term placentas.
Using a sensitive radioimmunoassay to measure the amount of
hPL released by placental ribosomes in vitro, Boime and
Boguslawski (7) later found 4 times more hPL on ribosomes
from term than from first trimester placentas. No values have
previously been reported for hCG synthesis by either early or
late placental ribosomes in uitro.
Our findings agree with the small amount of hPL detected in
serum at 10 weeks (less than 1 pg/ml), compared with serum
from women at full term (about 6 kg/ml) (27). In contrast,
serum hCG levels fall from about 50 i.u./ml at 10 weeks of
gestation to 10 to 15 i.u./ml at full term (1). Thus, the
proportion of nascent chains on the polyribosomes from early
and late placentas correlates with the levels of hPL and hCG
found in the blood and it can be concluded that the changing
blood levels of these hormones are regulated by their rates of
synthesis. Furthermore, the relative proportions of mRNA for
hPL (0.4% and 2%) and hCG (8% and 2%) found by us in early
and late placentas, respectively, also reflect the inverse serum
pattern for the two peptide hormones. We can accordingly
deduce that the relative rates of synthesis of hPL and hCG at
different stages in placental development are determined by
the amount of mRNA for each hormone.
Some aspects of the mechanism of protein secretion are
relevant to our findings. A number of secreted proteins are
known to be made in larger precursor form (28). In agreement
with this, Boime et al. (29) have reported the synthesis of a
product of 25,000 molecular weight when placental RNA was
translated by a wheat germ preparation, whereas hPL after
secretion has a molecular weight of 21,600. However, we find
that the specific immunoprecipitated hPL peptides synthe-
sized by placental messenger RNA in the wheat germ system
migrate on SDS gels along with authentic hPL (M, = 21,600).
As further evidence, the size of placental polysomes binding
‘2”I-labeled hPL antibody is consistent with mRNA of a size
coding for 190 amino acids, the number in the finalized hPL
molecule. No evidence of larger precursor peptides for the hCG
subunits translated by placental mRNA was obtained when
the products of translation were resolved on SDS gels.
In general, secreted proteins are made by membrane-bound
ribosomes in various types of cells, whereas free ribosomes
make proteins retained by the cell (30). Occasionally secreted
proteins are reported to be made to a significant extent on free
ribosomes, such as the synthesis of immunoglobulins by
antibody-forming cells (31-33). However, Cioli and Lennox
(34) showed that this could sometimes be accounted for by
incomplete removal of membrane-bound ribosomes during the
separation of these two classes. In our placental studies, we
used the separation procedure of Cioli and Lennox to obtain
more complete segregation of free and membrane-bound ribo-
somes and also employed two different methods to identify
peptide hormone synthesis on the polysomes, namely binding
of 1251-labeled specific antibody and incorporation of 3H-
labeled amino acid into peptides. Nevertheless, the free
ribosome population in mature placentas still showed about
one-fifth the capacity for hPL and hCG synthesis observed
with membrane-bound ribosomes. This is greater than the
proportion found by ver Eecke et a-l. (35), who observed only
one-twentieth the frequency of nascent hPL chains on free
ribosomes compared with bound ribosomes. Since we observed
the same relative distribution between free and membrane-
bound ribosomes prepared from early and late placentas, we
cannot conclude that the full term placenta shows the presence
of some nascent hPL chains among the free ribosome popula-
tion because of breakdown of tissue function related to
placental aging. Thus, we must conclude either that the best
current separation procedures found to be successful for other
tissues are inadequate for separating the two polysome classes
in placenta, or else that the activity in the free ribosome
population represents polysomes that have become detached
from membrane during preparation, or finally that some of the
message for hPL and hCG is indeed translated without
ribosome attachment to membrane.
The preferential synthesis of secreted proteins by mem-
brane-bound ribosomes makes it likely that the mRNAs for
these secreted proteins are mainly located in this polyribosome
population. However, Shafritz (36) has claimed that mRNA for
albumin is equally abundant among free and membrane-
bound polyribosomes from rat liver, a finding we are unable to
confirm (37). In term placenta, we find a somewhat higher
proportion of hCG messenger in the mRNA extracted from
bound ribosomes, but a similar concentration of hPL mRNA in
both bound and free ribosomes. The interpretation of these
results is uncertain. It will be noted that hPL comprises a
much smaller proportion of total protein synthesized by mRNA
than by polysomes (2% uersus 8%), whereas the corresponding
figures for hCG are both 2%. This indicates less efficiency of
hPL synthesis by mRNA. The two peptide chains of hCG are
appreciably smaller than that of hPL and the more complete
translation of hCG messenger than of the larger hPL messenger
is in agreement with the known tendency of the wheat germ
system to abort peptide chain synthesis of certain proteins (38,
39). It is also possible that the wheat germ system recognizes
less hPL than hCG mRNA. Whichever is the correct explana-
tion, the restricted translation of hPL mRNA makes it difficult
to compare the relative amounts of this mRNA in membrane-
bound and free ribosomes.
Note
Added
in Proof-Since this manuscript was submitted,
Hubert and Cedard (40) have reported that human placental
mRNA synthesizes hPL of IV, between 20,000 and 22,000 in a
rabbit reticulocyte cell-free system with 3 times the activity in
membrane-bound placental ribosomes, while Boime et al. (41)
confirm that there are greater levels of hPL mRNA in term
placenta than in first trimester placental tissue.
Acknowleclgments-We are grateful to Dr. Shirley Driscoll
and the nursing staff of the Boston Hospital for Women
(Lying-In Division) for arranging the collection of placentas, to
Ms. Rusty Murray and Prof. John Stanbury, Massachusetts
Institute of Technology, for help with the immunological and
iodination techniques, and to Dr. Josef Zahringer for assistance
with slab gel electrophoresis.
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