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

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

Isolation of Cell-free Extracts-Ribosomes and cell sap were prepared 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 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 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)-cellulose 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. The reactions were then processed as previously described (15).
Analysu of [35S]Methionine-labeled hPL-Sodium dodecyl sulfatepolyacrylamide 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 formamide 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 polymerizing 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.

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)  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-polyacrylamide 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 unlabeled hPL, digested with trypsin, and the resulting peptides analyzed by two-dimensional chromatography and electrophoresis. 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 methioninelabeled 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)  the NH, terminus of hPL (6). The methionine in this peptide represents the 14th amino acid residue from the NH,terminal end (16, 17). (It is not clear why there is an asymmetric 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 peptide 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 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 autoradiographs 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 trimester RNA (Table I). The radioactivity seen in the absence of RNA was the result of nonspecific smearing of endogenous peptides. 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. Equivalent subsaturating amounts of the unfractionated RNA were added to the wheat germ system (Fig. 6). The bands corresponding 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%. Therefore, 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 but it was not clear if this increase was the result of a translational control mechanism 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 produced 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 radioimmunoassay 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 synthesized (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.