THE .JOURNAL OF BIOI.OGICAL CHEMISTRY Vol. 251, No. 3, Issue of Fehmry 10, pp. 820-825, 1976

Printed in U.S.A.

Synthesis of Human Placental Lactogen Messenger RNA as a Function of Gestation*

(Received for publication, June 9, 1975)

IRVING BOIME, DIANA MCWILLIAMS, ELZBIETA SZCZESNA, AND MARK CAMEL

From the Department of Obstetrics and Gynecology, Division of Oncology and the Department of

Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

It was shown previously that 4 to 5 times more human placental lactogen (hPL) was synthesized in cell-free extracts from term placentae than in comparable extracts prepared from first trimester tissue. In an attempt to define what accounts for this differential rate of synthesis RNA was prepared from first trimester and term placentae. Following purification through an oligo(dT)-cellulose column, these RNA preparations were tested for their ability to direct the synthesis of the hPL precursor in the wheat germ cell-free system. With similar amounts of first trimester and term mRNA, the overall efficiency as defined by the stimulation of total amino acid incorporation was comparable. However, there was approximately 4 times more hPL synthesized in the presence of term RNA. This was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic fingerprinting.

The peak of the hPL precursor messenger activity sedimented at 12 to 13 S on sucrose gradient.

Analysis of the RNA by formamide-polyacrylamide gel electrophoresis further supported this value. The data indicate that the increased synthesis of hPL at term reflects greater levels of hPL mRNA in

term tissue than in first trimester tissue. The data also show that the overall in uiuo levels of hPL can be correlated not only with the increase in placental syncytial mass during pregnancy but also in the greater proportion of hPL synthesized per g of tissue. The latter results from the continual differentiation of the placenta occurring throughout gestation.

One of the major peptide hormones released by the human placenta is human placental lactogen (hPL).’ Although its precise role during gestation has not been clearly defined, its synthesis reflects the normal function of the placenta.

Third trimester (term) placenta secretes greater amounts of hPL. into maternal serum than does first trimester placenta. Most investigators have agreed that the enhanced levels of the hormone at term reflect a proportional increase in the mass of placental tissue (l-4). Placental mass is approximately propor- tional to the levels of hPL in maternal sera and in one study it was shown that in tissue slices from first trimester and term placentae the fraction of hPL in newly synthesized proteins is

constant (5). One might therefore have expected that in cell-free extracts from the same amount of tissue the level of hPI. synthesized would be the same. Instead, from the same quantity of tissue, more hPI. was synthesized per g of tissue in term cell-free extracts than in comparable extracts derived from first trimester tissue (6-9). These results suggested that the synthesis of hPL is not only dependent on placental weight but other factors as well. In an effort to clarify this point the

amount of hPL mRNA in first trimester and term placenta was determined by quantitating the amount of hormone synthe-

* This work was aided in part by Grant M74.114 from the Population Council and Grant AM-16865 from the National Institutes of Health.

The abbreviations used are: hPL, human placental lactogen; Hepes, N-2-hydroxyethylpiperazine-W-Z-ethanesulfonic acid.

sized in a wheat germ cell-free system. The data indicate that purified term mRNA directs the synthesis of much more hPL than did equivalent quantities of purified first trimester mRNA.

EXPERIMENTAL PROCEDURE

Materials-[[3SS]Methionine (specific activity 300 Ci/mmol) was obtained from Amersham/Searle. Human placental lactogen (95’X pure) was purchased from Nutritional Biochemicals. Sucrose was purchased from Schwarz/Mann and wheat germ was purchased from the ADM Mill Co. (Shawnee Mission, Kansas).

Isolation of Cell-free Extracts-Ribosomes and cell sap were pre- pared from placenta and ascites tumor cells as described previously i&9,.

The 30,000 x g supernatant (S-30) derived from wheat germ was prepared according to Roberts and Patterson (lo), except that the preincubation step was omitted.

Preparation. of Placental RNA-Term placental tissue was washed extensively to remove as much blood as possible and then pressed through a 1.5mm grid (8). The tissue was then homogenized in 1.5 volumes of buffer containing 50 rn~ Tris-HCI (pH 7.7), 5 IIIM KCl, 5 rn~ M&l,, 7 rn~ P-mercaptoethanol, 880 rn~ sucrose, and 0.5 rn~ EDTA. Homogenization was carried out in the cold for approximately 3 min with a motor-driven stainless steel pestle and glass vessel. Following centrifugation at 8500 x g for 10 min the supernatant was diluted 2-fold with homogenizing buffer and brought to 0.1 M Tris-HCl (pH 8.5), 0.1 M NaCl, and 1% sodium dodecyl sulfate. Immediately after clarification an equal volume of a cold mixture of phenol/ chloroform/isoamyl alcohol (X)/50/0.5) saturated with 0.1 M Tris-HCl (pH 8.5) was added. Following extraction of the supernatant for 20 min

820

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Biosynthesis of Human Placental Lactogen mRNA 821

at room temperature, the phases were separated by centrifugmg at 8000 x g for 10 min. The aqueous phase was re-extracted twice more with an equal volume of the above phenol mixture. The resulting aqueous phase was made 2% in potassium acetate (pH 5.5), and the RNA precipitated overnight with 2 volumes of ethanol (prechilled to -20”). The RNA suspension was then centrifuged at 7000 x g for 10 min and the pellets were washed with a mixture of ethanol/O.2 M NaCl (2/l, v/v).

RNA from first trimester tissue was prepared in the same way except that the tissue was not put through the tissue press but homogemzed directly. The RNA was then dissolved in sterile H,O. The A&A,,, of this RNA was approximately 1.7, and the yield of RNA per g of term or first trimester placenta was 5 to 8 A,,, (0.25 to 0.40 mg).

We have recently found that more hPL mRNA is recovered when the placenta is obtained from cesarean section than from normal dellvery. Thus, all term placentae used in tins study were from cesarean section. Also the amount of mRNA recovered is much less when polysomes are extracted mstead of the total cytoplasmlc extract. All mRNA was extracted from cytoplasmic extracts for the experiments described. The crude placental RNA was purified further on an oligo(dT)-cel- lulose affmlty column as described previously (11). The yield of adsorbed RNA was 1 to 2% of the input total cellular RNA.

Assays for Protean Synthesis. Wheat Germ-Assays contained in a final volume of 50 ~1, approximately 10 ~1 of nonpreincubated S-30 (lo), 20 mM Hepes (pH 7.6), 2.5 rnM dithlothreitol, 1.5 mM ATP, 1.5 rnM phosphocreatine, 2 fig of creatine kmase, 0.3 mM GTP, 20 FM unlabeled ammo acids, 64 mM KCl, 4.0 mM magnesium acetate, and 0.5 PM [3sS]methionine. The reactions were then processed as previ- ously described (15).

Analysu of [35S]Methionine-labeled hPL-Sodium dodecyl sulfate- polyacrylamide gel electrophoretic and tryptic peptide analyses of hPL were performed essentially as previously reported (6). However, instead of a 7 to 28% gradient of acrylamide, 20% acrylamide gels were used.

Sucrose Gradzent Analysis of RNA-Total placental RNA was resolved on 5 to 20°C sucrose gradients containing 0.1 M Tns-HCl (pH 7.8)/0.1 rnM EDTA as described by Honjo et al. (12). The RNA was dissolved in sterile water, heated for 5 min at 65”, rapIdly chilled, and then the RNA was layered on the gradient. The gradients were centrifuged in a Beckman SW-41 rotor at 35,000 rpm for 18 hours. Fractions of 0.30 ml were collected and the absorbance at 260 nm was determined.

Formamide-Acrylamide Gel Electrophoresis of RNA-Placental RNA was further resolved by acrylamide gel electrophoresis in form- amide according to modifications by Orkm et al. (13) of the procedures of Pinder et al. (14). Formamide (100 ml) which was deionized with 5 g of Dowex RG-501-X8 mixed bed resin was made 20 rnM in phosphate with solid NaH,PO, and Na,HPO, and the pH brought to 6.5. Tins buffered formamlde was mixed with acrylamide-bisacrylamide polym- erizing solution and the pH of the resultmg mixture brought to 6.5. This solution was placed in Plexiglas tubes to polymerize, and 20 mM

phosphate-buffered formamide was layered over the acrylamide; then the top of the tube was covered with parafilm. The tubes could then be stored m the cold room for up to 3 weeks. Aliquots of RNA solutions which had been dialyzed to remove salt were lyophilized. The samples were dissolved in 10 to 15 rl of 5 rnM barbital-buffered formamide containing 20% sucrose and 0.05% bromphenol blue. The gels were placed in an electrophoresis apparatus containing 20 mM phosphate buffer in the anodal and cathodal chambers.

The gels were run initially at 50 volts for 0.5 hour and then at 100 volts for 5 to 10 hours. Gels were removed from the tubes and stained overnight m Stamsall (Eastman Catalog No. 2718) and destained in water.

RESULTS

Crude RNA isolated from term placenta has been shown to direct the synthesis of hPL in cell-free extracts. hPL itself is formed in extracts of ascites tumor cells, while a protein heavier than hPL but containing hPL sequences is formed in wheat germ cell-free extracts (15). The approximate size of the hPL mRNA was determined by resolving the total placental RNA on a 5 to 20% sucrose gradient (Fig. 1). Five fractions from regions of the gradient were tested for total protein

synthesis activity in wheat germ S-30 (closed circles), and each

* 3.0.

2.5

2.0.

2 1.5.

1.0’

0.5.

IO 15 20 25 30

I II IIIm‘sI

FIG. 1. Sucrose gradient centrifugation of total RNA (approxi- mately 25 mg) derived from term placenta. O-O, ultraviolet absorption; O-O, protein synthetic activity in the wheat germ cell-free system of ethanol-precipitated RNA from the indicated pooled fractions (I to V). Approximately 6 pg of RNA were added to 100 bl of reaction mixture containing [Ylmethionine, and identical aliquots (equivalent protein) were applied to a sodium dodecyl sulfate-polya- crylamide (20%) gel. The marker corresponding to 22,200 is purified hPL.

of these was run on an acrylamide gel to assess synthesis of the

precursor (arrow). The peak of the hPL precursor messenger activity sedimented at 12 to 13 S. When the RNA in this region was combined and centrifuged again in a 5 to 20% sucrose gradient, the hPL mRNA retained the same sedimentation rate.

Analysis of RNA by formamide-polyacrylamide gel electro- phoresis further supported the size of the RNA as 12 to 13 S (Fig. 2). The appearance of a band corresponding to this molecular weight was associated with peak Fraction III from the sucrose gradient shown in Fig. 1, whereas this band was absent in Fraction I which contained no detectable hPL mRNA

activity (Fig. 2B). Furthermore, when placental RNA was purified twice through an oligo(dT)-cellulose column, the major discrete band migrated at about 13 S (Fig. W). The gel data suggested that this RNA band had the mRNA activity. To confirm this, the (dT)-purified RNA was fractionated on a 5 to 20% sucrose gradient. Again, a major peak was observed

sedimenting at about 13 S. All hPL mRNA activity was associated with this fraction.

Assuming that the molecular weight of the mRNA can be

estimated by comparing sedimentation values and molecular weights for similarly analyzed RNAs, the size of hPL mRNA is approximately 300,000. This suggests that the information encoded in this mRNA is more than adequate to code for the hPL precursor (M, = 25,000).

For comparing the levels of hPL mRNA in first trimester and term placentae the poly(A)-containing RNA isolated from the tissue was purified on an oligo(dT)-cellulose column (11).

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822 Biosynthesis of Human Placental Lactogen mRNA

FIG. 2. Distribution of term placental RNA in formamide gels. A-l indicates the migration of23 S and 16 S ribosomal RNA, In lane A-2, 5 pg of RNA purified twice through an oligo(dT)-cellulose column were applied. Lanes B-l and B-2 received 16 ~(g of Fraction I and 7.5 pg of Fraction III, respectively, from the sucrose gradient displayed in Fig. 1.

Ordinarily, 25 mg of crude RNA were applied to the column, and the recovery of the adsorbed fraction (poly(A)RNA) represented 1 to 2% of the input RNA. Similar amounts of the adsorbed mRNA fractions from the two gestational periods stimulated total amino acid incorporation in the wheat germ S-30 to about the same extent (Fig. 3). Therefore, the efficiency of the two RNAs is comparable. In addition, the sucrose gradient profiles of the total cellular RNA derived from first trimester and term placentae were very similar.

The products synthesized in response to these adsorbed RNA fractions were examined by sodium dodecyl sulfate-polyacryl- amide gel electrophoresis (Fig. 4). Although the same amount of radioactivity from each preparation was applied to the gel, it is clear that much more of the hPL precursor is synthesized in response to term mRNA than to first trimester mRNA. The bands corresponding to the precursor were cut out of the gels and the radioactivity was determined. In the case of the term preparation the band contained approximately 20% of the total radioactivity applied to the gel; in the case of the first trimest,er preparation the figure was only about 5%.

The region of the gel containing the labeled hPL precursor was eluted from a preparative gel, mixed with purified unla- beled hPL, digested with trypsin, and the resulting peptides analyzed by two-dimensional chromatography and electropho- resis. The fingerprint was sprayed with ninhydrin in order to localize the peptides from the purified carrier and exposed to x-ray film (Fig. 5).

hPL contains six tryptic peptides each of which contains 1 methionine residue (16, 17). The product of the wheat germ system programmed with term RNA contained methionine- labeled tryptic peptides that were coincident with authentic hPL peptides (Panels A and B, Fig. 5). Panel B is the autoradiograph of Panel A. These labeled peptides were not present on maps containing carrier hPL and the proteins synthesized in the absence of mRNA (Fig. 5, Panel D) or in the presence of globin mRNA (data not shown).

Of the six methionine tryptic peptides, peptide 1 is closest to

I * I . 2 I 012345

ro RNA

FIG. 3. Protein synthesis in response to placental RNA fractions treated by oligo(dT)-cellulose chromatography. Fifty-microliter reac- tion mixtures were incubated for 60 min.

FIG. 4. Sodium dodecyl sulfate-polyacrylamide (20%) gel of prod- ucts synthesized in the presence of adsorbed RNA fractions isolated from term (2) and first trimester (3) placentae. Lane 1 indicates a reaction mixture incubated in the absence of mRNA. Approximately 4 fig of adsorbed RNA were added to 100 ~1 of reaction mixture and the following amounts of radioactivity were applied: (1) 20,000 cpm; (2) 206,000 cpm; and (3) 185,000 cpm.

the NH, terminus of hPL (6). The methionine in this pep- tide represents the 14th amino acid residue from the NH,- terminal end (16, 17). (It is not clear why there is an asym- metric distribution of radioactivity in the overlapping peptides. It appears that there is a progressive dilution of radioactivity from the amino to carboxy end of the protein, since from amino acid analyses, peptidas 2 and 3 are located near the carboxy end of hPL. This effect is probably related to the efficiency of the wheat germ system because in highly active extracts the asymmetry of the labeled peptides is reduced. Possibly some of the asymmetry can be attributed to the inclusion in the protein band of some incomplete prematurely released peptides from ribosomes in less efficient extracts (24).) Thus, the level of pep- tide 1 will probably best reflect the number of RNA molecules that have initiated and thus the proportion of hPL mRNA. Furthermore, since the RNA is translated in a heterologous cell-free system, any differences between first trimester and

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Biosynthesis of Human Placental Lactogen mRNA 823

FIG. 5. Two-dimensional tryptic fingerprint analyses of a mixture of carrier hPL and labeled protein synthesized in the presence (A and B) and absence (C and D) of term RNA. The reaction product was mixed with carrier. Panels B and D are the corresponding autoradio- graphs of Panels A and C which have been stained with ninhydrin. The

term mRNA activities would not likely be related to levels of the various cytoplasmic protein synthesis factors. Peptide 1 was cut out from two-dimensional fingerprints and counted. More of the labeled peptide was synthesized when the wheat germ S-30 was programmed with term mRNA than with first tri- mester RNA (Table I). The radioactivity seen in the absence of RNA was the result of nonspecific smearing of endogenous pep- tides. There was no discrete radioactive spot corresponding to peptide 1 observed in the absence of RNA (Fig. 5).

It was conceivable that the lower level of hPL mRNA in first trimester tissue may be related to a selective loss of hPL mRNA during isolation, To test this point equal quantities of first trimester and term tissue were mixed and the total RNA extracted from the resulting homogenate. The RNA was also extracted from the corresponding individual tissues. Equiva- lent subsaturating amounts of the unfractionated RNA were added to the wheat germ system (Fig. 6). The bands corre- sponding to the precursor were cut out of the gels and the radioactivity determined. In the term and first trimester

ninhydrin-positive peptide of hPL which shows the same mobility as the labeled peptide migrating near six in Panel D is not an overlapping peptide. Approximately 200,000 cpm were applied to Panel A and 60,000 cpm applied to Panel C. NIN refers to ninhydrin stained map.

preparations the band contained approximately 18% and 2%, respectively, of the total radioactivity applied to the gel. In the mixed preparation the figure was approximately 11%. There- fore, the presence of first trimester tissue did not appreciably alter the recovery of total hPL mRNA activity (taking into account dilution by first trimester RNA). Thus, the lower level of hPL mRNA recovered from first trimester tissue was not an artifact of preparation. Also this experiment shows that: (a) there were no specific translational inhibitors in the first trimester RNA sample which would lower the translation efficiency of hPL mRNA; and (b) the difference in hPL mRNA activity in unfractionated RNA from term and first trimester

tissue is comparable to that seen in oligo(dT)-purified mRNA. Thus, the results with the latter were not related to differences in recovery of poly(A)-containing RNA.

DISCUSSION

It had been shown earlier that 4 to 5 times more hPL was synthesized in cell-free extracts from term tissue than from the

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824 Biosynthesis of Human Placental Lactogen mRNA

TABLE I

Level of methionine tryptic peptide 1 synthesized in wheat germ S-30 in presence of oligo(dZ’j-purified placental RNA

The 0.25-ml reaction mixture containing wheat germ S-30 was incubated in the presence of indicated amounts of oligo(dT)-purified placental RNA and [%]methionine. Following a 90-min incubation, the in uitro-labeled protein was precipitated, mixed with 5 mg of purified hPL, denatured, and two-dimensional maps were prepared as described in the text. The peptide corresponding to spot 1 in Fig. 5 was cut out and counted.

Experiment RNA added

Counts per mm applied to map

counts per inin in peptide I

w

pglO.25 ml

I None 275,000 200 First trimester 505,000 3,270 0.65

(8.0) Term 900,000 27,100 3.0 (8.0)

II None 30,000 72 First trimester 260,000 1,770 0.68

(8.0) Term 330,000 7,642 2.3 (8.0)

FIG. 6. Sodium dodecyl sulfate-polyacrylamide gel of products syn- thesized in the presence of unfractionated RNAs derived from 10 g of first trimester (FT), and term tissues, and from a mixture of 10 g of first trimester + 10 g of term tissue (FT + term). Approximately 20 pg of each RNA preparation were added to 100 &reaction mixtures. The following amounts of radioactivity were applied; (-RNA) 6,500 cpm; (FT) 30,000 cpm; (term) 40,000 cpm; (FT + term) 40,000 cpm.

same amount of first trimester tissue; but it was not clear if this increase was the result of a translational control mecha- nism or an increase in specific hPL mRNA. The data presented here suggest that the enhanced synthesis of hPL in term cell-free systems is largely the result of an increased proportion of the corresponding mRNA. This is shown with mRNA purified by oligo(dT)-cellulose chromatography which pro- duced both an increased amount of labeled hPL and an increased level of the methionine tryptic peptide closest to the NH, terminus.

Both by sucrose gradient and formamide acrylamide gel

analyses, the size of the hPL mRNA from term tissue appears to be about 13 S. When RNA derived from first trimester tissue was resolved on a 5 to 20% sucrose gradient, the hPL activity also sedimented at about 13 S (data not shown). Thus, the apparent size of the mRNA remained unchanged between first trimester and term.

It appears that the differential serum level of hPL seen during gestation are largely the result of changes in synthesis rate of the hormone, although these data cannot exclude some additional regulatory component exerted on the secretion of hPL. The data presented are not compatible with earlier observations that the proportion of hPL synthesized in first trimester and term placental slices was similar (5). It is possible that the sensitivity and specificity of the radioim- munoassay used in the earlier study were insufficient to detect the difference shown here.

The enhanced synthesis of hPL at term is consistent with the hypothesis that the placenta is continually differentiating. The relatively immature placental cell, the cytotrophoblast, is the generative cell for the more differentiated syncytium (18-21), and the latter is apparently the region where hPL is synthe- sized (22). During gestation there is an increase in the proportion of syncytium per g of placental villi (23). Thus, a greater synthesis of the hormone per g of tissue at term probably reflects this increase. In other words, the overall in uiuo levels of hPL can be correlated not only with the increase in placental syncytial mass during pregnancy but also in a greater proportion of hPL synthesized per g of tissue.

Acknowledgments-The authors are grateful to Dr. David Swan for helping us with the formamide gels. We would also like to thank Miss Kathy Neely for her excellent typing assistance.

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Biosynthesis of Human Placental Lactogen mRNA 825

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I Boime, D McWilliams, E Szczesna and M CamelSynthesis of human placental lactogen messenger RNA as a function of gestation.

1976, 251:820-825.J. Biol. Chem. 

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