- Email: [email protected]
Placental transfer of orotic acid, uridine, and UMP
lacer-d
er
nd 1. Comparison
T.
TERRY
DONG SAMANTHA Pittsburgh,
of acid-soluble
HAYASHI, I-1.
SHIN,
and
acid-insoluble
counts
M.D. PH.D.
WIAND,
B.A.
Pennsylvania
The transplacental passage and the incorpor-ation into RNA 01 %Z-labeled erotic acid, uridine, and 5’-UMP have been studied in pregnant rats from 14 through 19 days of gestation by analyzing acid-soluble and acid-insoluble material from the maternal liver, the placentas, and the fetuses. The rapid transfer of precursors is observed only ajter the seventeenth day of pregnancy. Further studies show the labeling of fetal, placental, and maternal liver RNA with t4C-erotic acid; however, with ‘JC-uridine as a precursor, labeling of only fetal and placental RNA is observed, with no significant labeling of the maternal liver RNA.
P R E v 1 o cT s works in our laboratory established that a variety of labeled precursors of RNA is transferred into the fetuses of the near-term pregnant rats1 The present investigation was carried out in an attempt to correlate the placental transfer of RNA precursors with the biosynthesis of RNA in fetuses, placentas, and the maternal liver. For this purpose a method was devised to distinguish the transferred precursors of RNA in the acid-so’luble form from those in the acid-insoluble form. The results indicate that the transplacental passage and the incorporation of labeled precursors of RNA into the acid-insoluble material, namely, RNA, in the maternal liver, the placenta, and the fetus are influenced by such factors as the type of RNA precursors, the incubation time, and the duration of From the Department of Obstetrics Gynecology, University of Pittsburgh School of Medicine, Magee-Womens
pregnancy. These results negative results previously investigato~rs.2~ 3 Methods
and
help explain the reported by other
materials
Y-erotic acid, lGuridine, and l*Curidine-5’-monaphosphate (UMP) used for the study were obtained from the Schwarz RioResearch, Inc. With each precursor, three sets of experiments were conducted on 14, 17, and 19 day pregnant rats of SpragueDawley strain; the dated pregnant rats were obtained commercialIy. The animals were injected intraperitoneally with 10 ,uc of a precursor and killed by cervical dislocation at 5, 30, and 60 minutes after the injectio’n. The maternal liver, the placentas, and the fetuses were immediately removed and placed separately in a 20 to 80 ml. ice-cold sodium acetate buffer, O.lM, pH 5.0. The tissues were homogenized for one minute at top speed in a Virtis Homogenizer. One-tenth volume of 50 per cent trichloroacetic acid was added to each homogenate. The homogenates were kept in ice for 10 minutes and centrifuged
and
HOSpitaE.
This project was supported by Grant HD No. 01890 from the National Institutes of Health, United States Public Health Service. 1144
Volume Number
102 8
Placental
transfer
of
erotic
acid,
8 9 0
3llSSll I
JO I
~i’3lllNlW
Eld I
SINnO I
I
I
uridine,
and
UMP
1145
46
Hayashi,
Shin,
and
Wiand
3fFSSll
jQ
61
3lflNllrU
t13d
SlNtl03
Volume Number
102 8
3tlSSll
3nssu
JO
JO
Placental
transfer
of erotic
I/
31llNIW
t13d
SlNnO3
61
31flNIW
ki3d
SINno
acid,
uridine,
and
UMP
1147
1148
Hayashi,
Shin,
and
Wiand
at 10,900 x g for 10 minutes.
The precipitates were washed twice with 10 ml. of 5 per cent trichloroacetic acid; the supernatants were combined to the original 10,000 x g supernatant. The radioactivity of this pooled supernatant represents the acid-soluble count and was determined with a Packard liquid scintillation counter by placing 0.5 ml. aliquot of the supernatant in a vial that contained 8.0 ml. of Bray’s solvent.* In order to measure the acid-insoluble count, the precipitate was suspended in 10 ml. of 10 per cent trichlo’roacetic acid and heated for 15 minutes in a boiling water bath to hydrotlyze the RNA. The preparation was centrifuged at 10,000 x g for 10 minutes, and the radioactivity of the supernatant containing the soluble hydrolysis product of RNA was determined as described above for the determination of the acid-soluble count. The supernatants representing either the acid-soluble preparation or the hydrolyzed
acid-insomluble material obtained from 19 day pregnant rats after 60 minutes of labeling were treated five times with equal volume of cold ether and Iyophilized. The Iyophilized preparatioas were dissolved in 1.0 to 2.0 ml. of water and aliquots were spoltted on a chro. matography paper of Whatman No. 1. n. Butanol-acetic acid solvent system” was used to separate erotic acid, UMP, uridine, and uracil. Apprompriate spots were cut out and placed in the bottom of the vials that contained 8.0 ml. of Bray’s solvent to determine radioactivity. Results
The resuits. of erotic acid experiments that are shown in Fig. 1 represent the studies with rats of 14, 17, and 19 days of pregnancy, respectively. The radioactivity is represented as counts per minute per gram of tissue rather than the total radioactivity because of the marked differences in placental and fetal
---
0
0
402
269
I--
0 276
0 609
0 3746
FETUS ACID Rf =.I0 Fig. from acid within area rexnt
-
LIVER PLACENTA ACID-SOLUBLE ------a SUPERNATANT
UMP
URIDINE
Rf =.03
Rf =.32
FETUS LIVER PLACENTA +----ACID-INSOLUBLE ---@ SUPERNATANT
URACIL Rf =.43
2. Radioscan of chromatogram of supernatants obtained from both the acid-soiuble and the hydrolyzed acid-insoluble material after intraperitoneal injection of 10 /KZ of 14C-orotic in a 19 day pregnant rat. Butanol-acetic acid solvent system was used. The numbers individual circles represent counts per 10 minutes as recorded by placing encircled in the bottom of a counting via1 with 8.0 mI. of Bray’s solution. The shaded areas repultraviolet absorbing spots of the standards employed.
Volume Number
102 8
Placental
3flSSll
JO
6/3UlNIW
transfer
Wd
of
SlNfl03
erotic
acid,
uridine,
and
UMP
1149
1150
Hayashi,
Shin, and Wand
weights depending orz the length of gestation. The actual total colunts per minute obtained from each tissue after 60 minutes of incubation are represented in the graphs. The experiments conducted on 14 day pregnant rats indicate that there is no significant transfer of labeled precursors of RNA into the placenta and the fetus; oln the contrary, the maternal Iiver shows extremely high radioactivity. The experiments conducted on 17 day pregnant ra,ts reveal a significant placental transfer of 14C-orotic acid with minimal incorporation into the acid-insoluble material in the fetus and the placenta. Here again the maternal liver shows the bulk of the radioactivity. The most striking results are observed with 19 day pregnant rats. There is unquestionably greater placental transfer, as seen in the to’tal counts per minute trans.. ferred per gram of tissue. Furthermore, the acid-insoluble count patterns indicate that there is a significant incorporation of the injected precurs#ors of RNA into the RNA of the placenta and the fetus with concomitant
December Am. J. Obst.
15, 1968 & Gym.
fall in the acid-insoluble count of the maternal liver. The pattern of the radioscan omfthe paper chromatogram on the supernatants from the 19 day pregnant rats is shown in Fig. 2. In all tissues the highest radioactivity is associated with the erotic acid and UMP spots, indicating an active conversion of the erotic acid to UMP, which is a direct intermediate precursor of RNA. The finding that the hydrolysis of the acid-insoluble precipitate yields labeled UMP is confirmatory evidence that the acid-insoluble material does represent an RNA-like material. Furthermore, results not presented here showed that this material is sensitive to treatment with RNase but not with DNase. The considerable amount of radioactivity associated with uracil and uridine spots after injection of 14Cerotic acid indicates a very high activity of nucleotidase a,nd nucleos’idase in the liver. The results of uridine experiments with 19 day pregnant rats are shown in Fig. 3. Here results with rats of earlier dated pregnancies are not included because of a min-
---
0 269
ORQTIC LIVER PLACENTA UMP FETUS ACID + ACID-SOLUBLE SUPERNATANT Rf =.03 Rfz.32 Rf =.I0 Fig. 4. Radioscan of chromatogram of supernatants after I9 day pregnant rat. See legend for Fig. 2 for details.
FETUS LIVER PLACENTA @--ACID-INSOLUBLE --+ SUPERNATANT
URAClL
injection
in a
of 10 pc of I%-uridine
Rf =.%9
Volume Number
102 8
Placental
transfer of erotic acid, uridine,
and UMP
(At
19 day
p!egnancy
e-o
FETUS
b-Q ^
PtACENTA MATERNAL
5
LIVER
60
30 MINUTES
I
I
19 day
,
pregnancy
30,000
0-o b-4 o---o
FETUS PLACENTA MATERNAL
LIVER
20,000
10,000
5,000 16,630 d 3,992 43g6,3lc
B 5 Fig. 5. A and B, Radioactivity day pregnant rats. Acid-soluble
A,
30 MINUTES after intraperitoneal injection of counts; acid-insoluble counts.
B,
60 X0 PC of
“C-UMP
in
19
115
1152
Hapshi,
Shin,
and
Wiand
imal incorporation of the precursor into RNA of tissues. In direct contrast to the results obtained with erotic acid experiments, uridine, after 60 minutes elf incubation, showed much greater incorporation into the fetal and the placental RNA than into the RNA of the maternal liver. The lo,w incorporation of the labeled uridine into the RNA of the materna.I liver suggests that the maternal liver does not have a significant activity of the enzyme that converts 14C-uridine to l&C-UMP, whereas a high level of this enzyme is operative in the fetus and the placenta. Consistent with this speculation is the finding, as shown in Fig. 4, that the acidsoluble supernatant fro’m the maternal liver showed no sigafter injection of 14C-uridine nificant radioactivity in UMP, while preparations from the fetus and the placenta had a very high UMP radioa.ctivity. The unusually high radioactivity of the uracil spot of the acid-solluble supernatant of the placenta indicates high nucleosidase activity in the placenta.G Studies on placental transfer of UMP with 19 day pregnant rats are shown in Fig. 5. The radioactivity represents entirely the acid-soluble counts, since the acid-insoluble radioactivity was not significant. There was gradual increase in placental and fetal soluble counts as the pregnancy approached term.
The three specific precursors were chosen on the basis of the results observed in our earlier studies in placental transfer.l Uridine and UMP series showed unusually high activity in the fetus, suggesting rapid transfer or incorporation; whereas, erotic acid displayed unusually high radioactivity in the maternal liver acco,mpanied by less radioactivity in the fetus. Our previous study was designed primarily to measure only placental transfer and not incorporation into the RNA. In the present work the use of sodium acetate buffer, pH 5.0, and low temperature was to inhibit ribonuclease activity; thus, our acid-insoluble counts will approximate
December Am. J. Obst.
15, 1968 & Gynec.
the labeling of RNA quantitatively, even though there is no additional information with regard to the type of RNA involved. The acid-insoluble material. is considered to be RNA because it yielded nucleotides on acid hydrolysis and it is sensitive to RNase but not to DNase. The erotic acid experiments clearly indicated the rapid increase in placental transfer from the fourteenth through nineteenth day of pregnancy. Obviously there is but minimal transfer at the fourteenth day and the most dramatic increase is observed near the nineteenth day of gestation. Thus, any effective in viva labeling of the fetal and the placental RNA must be performed near the nineteenth day of pregnancy in rats. This observation may explain the paucity of fetal labeling observed by Bresnick, Lanclos, and Gonzales” using 16 to 18 day pregnant rats. Likewise, the inability of Dancis and Balis3 to detect any labeling of fetal RNA with W-labeled a,denine must have been due to the adminis’tration of the labeled precursor in the first half of pregnancy. In a somewhat related work, Atlas, Bondi, and Cronkite’ injected tritiated thymidine into mice early in pregnancy but were not able to demonstrate any labeling of DNA in the embryos and observed labeling only after the eleventh day of pregnancy. Rather surprising was our finding that UMP is transferred moderately across the placenta from the seventeenth through the nineteenth day of pregnancy, even though it is a phosphorylated compound. Our radioscan of chromatogram did not indicate any hydrolysis or dephosphorylation. The intricate mechanism involved in the placental transfer of UMP may be different from that observed with other membranes. The Iack of any appreciable incorporation of UMP into maternal and fetd tissues, however, does reflect the difficulty encountered by a phosphorylated compound in crossing a cellular membrane. The most striking result in our uridine experiments was the enhanced RNA labeling in the fetal tissue followed by the placental
Volume Number
102 8
Placental
tissue. This aNbs,ervation is in direct contrast with erotic a.cid studies, in which all three tissues demonstrated significant incorporation of the precursor into RNA. Such results suggest that the RNA metabolism in
REFERENCES
1. Hayashi,
T. T., and Garvey, B.: AM. J. OBST. & GYNEC. In press. 2. Bresnick, E., Lanclos, K., and Gonzales, E.: Biochem. Biophys. Acta 108: 568, 1965. 3. Dancis, J., and Balis, M. E.: J. Biol. Chem. 207: 367, 1954. 4. Bray, G. A..: Anal. Biochem. 1: 279, 1960.
transfer
of
erotic
acid,
uridine,
and
UMP
1153
the fetus and the placenta may be different from that in the maternal liver in the capacity to utilize uridine as a specific precursor for RNA synthesis.
5.
Smith, I., editor: Chomatographic and Electrophoretic Techniques, New York, 1961, Interscience Publishers, Inc., vol. I, p. 233. 6. Hayashi, T. T., Baldridge, R. C.; Olmsted, P. S. and Kimmel, D. L.: AM. J. OBST. & GYNEC. 88: 470, 1964. 7. Atlas, M., Bondi, V. P., and Cronkite, E. P.: J, Histochem. Cytochem. 8: 171, 1960.
er
nd 1. Comparison
T.
TERRY
DONG SAMANTHA Pittsburgh,
of acid-soluble
HAYASHI, I-1.
SHIN,
and
acid-insoluble
counts
M.D. PH.D.
WIAND,
B.A.
Pennsylvania
The transplacental passage and the incorpor-ation into RNA 01 %Z-labeled erotic acid, uridine, and 5’-UMP have been studied in pregnant rats from 14 through 19 days of gestation by analyzing acid-soluble and acid-insoluble material from the maternal liver, the placentas, and the fetuses. The rapid transfer of precursors is observed only ajter the seventeenth day of pregnancy. Further studies show the labeling of fetal, placental, and maternal liver RNA with t4C-erotic acid; however, with ‘JC-uridine as a precursor, labeling of only fetal and placental RNA is observed, with no significant labeling of the maternal liver RNA.
P R E v 1 o cT s works in our laboratory established that a variety of labeled precursors of RNA is transferred into the fetuses of the near-term pregnant rats1 The present investigation was carried out in an attempt to correlate the placental transfer of RNA precursors with the biosynthesis of RNA in fetuses, placentas, and the maternal liver. For this purpose a method was devised to distinguish the transferred precursors of RNA in the acid-so’luble form from those in the acid-insoluble form. The results indicate that the transplacental passage and the incorporation of labeled precursors of RNA into the acid-insoluble material, namely, RNA, in the maternal liver, the placenta, and the fetus are influenced by such factors as the type of RNA precursors, the incubation time, and the duration of From the Department of Obstetrics Gynecology, University of Pittsburgh School of Medicine, Magee-Womens
pregnancy. These results negative results previously investigato~rs.2~ 3 Methods
and
help explain the reported by other
materials
Y-erotic acid, lGuridine, and l*Curidine-5’-monaphosphate (UMP) used for the study were obtained from the Schwarz RioResearch, Inc. With each precursor, three sets of experiments were conducted on 14, 17, and 19 day pregnant rats of SpragueDawley strain; the dated pregnant rats were obtained commercialIy. The animals were injected intraperitoneally with 10 ,uc of a precursor and killed by cervical dislocation at 5, 30, and 60 minutes after the injectio’n. The maternal liver, the placentas, and the fetuses were immediately removed and placed separately in a 20 to 80 ml. ice-cold sodium acetate buffer, O.lM, pH 5.0. The tissues were homogenized for one minute at top speed in a Virtis Homogenizer. One-tenth volume of 50 per cent trichloroacetic acid was added to each homogenate. The homogenates were kept in ice for 10 minutes and centrifuged
and
HOSpitaE.
This project was supported by Grant HD No. 01890 from the National Institutes of Health, United States Public Health Service. 1144
Volume Number
102 8
Placental
transfer
of
erotic
acid,
8 9 0
3llSSll I
JO I
~i’3lllNlW
Eld I
SINnO I
I
I
uridine,
and
UMP
1145
46
Hayashi,
Shin,
and
Wiand
3fFSSll
jQ
61
3lflNllrU
t13d
SlNtl03
Volume Number
102 8
3tlSSll
3nssu
JO
JO
Placental
transfer
of erotic
I/
31llNIW
t13d
SlNnO3
61
31flNIW
ki3d
SINno
acid,
uridine,
and
UMP
1147
1148
Hayashi,
Shin,
and
Wiand
at 10,900 x g for 10 minutes.
The precipitates were washed twice with 10 ml. of 5 per cent trichloroacetic acid; the supernatants were combined to the original 10,000 x g supernatant. The radioactivity of this pooled supernatant represents the acid-soluble count and was determined with a Packard liquid scintillation counter by placing 0.5 ml. aliquot of the supernatant in a vial that contained 8.0 ml. of Bray’s solvent.* In order to measure the acid-insoluble count, the precipitate was suspended in 10 ml. of 10 per cent trichlo’roacetic acid and heated for 15 minutes in a boiling water bath to hydrotlyze the RNA. The preparation was centrifuged at 10,000 x g for 10 minutes, and the radioactivity of the supernatant containing the soluble hydrolysis product of RNA was determined as described above for the determination of the acid-soluble count. The supernatants representing either the acid-soluble preparation or the hydrolyzed
acid-insomluble material obtained from 19 day pregnant rats after 60 minutes of labeling were treated five times with equal volume of cold ether and Iyophilized. The Iyophilized preparatioas were dissolved in 1.0 to 2.0 ml. of water and aliquots were spoltted on a chro. matography paper of Whatman No. 1. n. Butanol-acetic acid solvent system” was used to separate erotic acid, UMP, uridine, and uracil. Apprompriate spots were cut out and placed in the bottom of the vials that contained 8.0 ml. of Bray’s solvent to determine radioactivity. Results
The resuits. of erotic acid experiments that are shown in Fig. 1 represent the studies with rats of 14, 17, and 19 days of pregnancy, respectively. The radioactivity is represented as counts per minute per gram of tissue rather than the total radioactivity because of the marked differences in placental and fetal
---
0
0
402
269
I--
0 276
0 609
0 3746
FETUS ACID Rf =.I0 Fig. from acid within area rexnt
-
LIVER PLACENTA ACID-SOLUBLE ------a SUPERNATANT
UMP
URIDINE
Rf =.03
Rf =.32
FETUS LIVER PLACENTA +----ACID-INSOLUBLE ---@ SUPERNATANT
URACIL Rf =.43
2. Radioscan of chromatogram of supernatants obtained from both the acid-soiuble and the hydrolyzed acid-insoluble material after intraperitoneal injection of 10 /KZ of 14C-orotic in a 19 day pregnant rat. Butanol-acetic acid solvent system was used. The numbers individual circles represent counts per 10 minutes as recorded by placing encircled in the bottom of a counting via1 with 8.0 mI. of Bray’s solution. The shaded areas repultraviolet absorbing spots of the standards employed.
Volume Number
102 8
Placental
3flSSll
JO
6/3UlNIW
transfer
Wd
of
SlNfl03
erotic
acid,
uridine,
and
UMP
1149
1150
Hayashi,
Shin, and Wand
weights depending orz the length of gestation. The actual total colunts per minute obtained from each tissue after 60 minutes of incubation are represented in the graphs. The experiments conducted on 14 day pregnant rats indicate that there is no significant transfer of labeled precursors of RNA into the placenta and the fetus; oln the contrary, the maternal Iiver shows extremely high radioactivity. The experiments conducted on 17 day pregnant ra,ts reveal a significant placental transfer of 14C-orotic acid with minimal incorporation into the acid-insoluble material in the fetus and the placenta. Here again the maternal liver shows the bulk of the radioactivity. The most striking results are observed with 19 day pregnant rats. There is unquestionably greater placental transfer, as seen in the to’tal counts per minute trans.. ferred per gram of tissue. Furthermore, the acid-insoluble count patterns indicate that there is a significant incorporation of the injected precurs#ors of RNA into the RNA of the placenta and the fetus with concomitant
December Am. J. Obst.
15, 1968 & Gym.
fall in the acid-insoluble count of the maternal liver. The pattern of the radioscan omfthe paper chromatogram on the supernatants from the 19 day pregnant rats is shown in Fig. 2. In all tissues the highest radioactivity is associated with the erotic acid and UMP spots, indicating an active conversion of the erotic acid to UMP, which is a direct intermediate precursor of RNA. The finding that the hydrolysis of the acid-insoluble precipitate yields labeled UMP is confirmatory evidence that the acid-insoluble material does represent an RNA-like material. Furthermore, results not presented here showed that this material is sensitive to treatment with RNase but not with DNase. The considerable amount of radioactivity associated with uracil and uridine spots after injection of 14Cerotic acid indicates a very high activity of nucleotidase a,nd nucleos’idase in the liver. The results of uridine experiments with 19 day pregnant rats are shown in Fig. 3. Here results with rats of earlier dated pregnancies are not included because of a min-
---
0 269
ORQTIC LIVER PLACENTA UMP FETUS ACID + ACID-SOLUBLE SUPERNATANT Rf =.03 Rfz.32 Rf =.I0 Fig. 4. Radioscan of chromatogram of supernatants after I9 day pregnant rat. See legend for Fig. 2 for details.
FETUS LIVER PLACENTA @--ACID-INSOLUBLE --+ SUPERNATANT
URAClL
injection
in a
of 10 pc of I%-uridine
Rf =.%9
Volume Number
102 8
Placental
transfer of erotic acid, uridine,
and UMP
(At
19 day
p!egnancy
e-o
FETUS
b-Q ^
PtACENTA MATERNAL
5
LIVER
60
30 MINUTES
I
I
19 day
,
pregnancy
30,000
0-o b-4 o---o
FETUS PLACENTA MATERNAL
LIVER
20,000
10,000
5,000 16,630 d 3,992 43g6,3lc
B 5 Fig. 5. A and B, Radioactivity day pregnant rats. Acid-soluble
A,
30 MINUTES after intraperitoneal injection of counts; acid-insoluble counts.
B,
60 X0 PC of
“C-UMP
in
19
115
1152
Hapshi,
Shin,
and
Wiand
imal incorporation of the precursor into RNA of tissues. In direct contrast to the results obtained with erotic acid experiments, uridine, after 60 minutes elf incubation, showed much greater incorporation into the fetal and the placental RNA than into the RNA of the maternal liver. The lo,w incorporation of the labeled uridine into the RNA of the materna.I liver suggests that the maternal liver does not have a significant activity of the enzyme that converts 14C-uridine to l&C-UMP, whereas a high level of this enzyme is operative in the fetus and the placenta. Consistent with this speculation is the finding, as shown in Fig. 4, that the acidsoluble supernatant fro’m the maternal liver showed no sigafter injection of 14C-uridine nificant radioactivity in UMP, while preparations from the fetus and the placenta had a very high UMP radioa.ctivity. The unusually high radioactivity of the uracil spot of the acid-solluble supernatant of the placenta indicates high nucleosidase activity in the placenta.G Studies on placental transfer of UMP with 19 day pregnant rats are shown in Fig. 5. The radioactivity represents entirely the acid-soluble counts, since the acid-insoluble radioactivity was not significant. There was gradual increase in placental and fetal soluble counts as the pregnancy approached term.
The three specific precursors were chosen on the basis of the results observed in our earlier studies in placental transfer.l Uridine and UMP series showed unusually high activity in the fetus, suggesting rapid transfer or incorporation; whereas, erotic acid displayed unusually high radioactivity in the maternal liver acco,mpanied by less radioactivity in the fetus. Our previous study was designed primarily to measure only placental transfer and not incorporation into the RNA. In the present work the use of sodium acetate buffer, pH 5.0, and low temperature was to inhibit ribonuclease activity; thus, our acid-insoluble counts will approximate
December Am. J. Obst.
15, 1968 & Gynec.
the labeling of RNA quantitatively, even though there is no additional information with regard to the type of RNA involved. The acid-insoluble material. is considered to be RNA because it yielded nucleotides on acid hydrolysis and it is sensitive to RNase but not to DNase. The erotic acid experiments clearly indicated the rapid increase in placental transfer from the fourteenth through nineteenth day of pregnancy. Obviously there is but minimal transfer at the fourteenth day and the most dramatic increase is observed near the nineteenth day of gestation. Thus, any effective in viva labeling of the fetal and the placental RNA must be performed near the nineteenth day of pregnancy in rats. This observation may explain the paucity of fetal labeling observed by Bresnick, Lanclos, and Gonzales” using 16 to 18 day pregnant rats. Likewise, the inability of Dancis and Balis3 to detect any labeling of fetal RNA with W-labeled a,denine must have been due to the adminis’tration of the labeled precursor in the first half of pregnancy. In a somewhat related work, Atlas, Bondi, and Cronkite’ injected tritiated thymidine into mice early in pregnancy but were not able to demonstrate any labeling of DNA in the embryos and observed labeling only after the eleventh day of pregnancy. Rather surprising was our finding that UMP is transferred moderately across the placenta from the seventeenth through the nineteenth day of pregnancy, even though it is a phosphorylated compound. Our radioscan of chromatogram did not indicate any hydrolysis or dephosphorylation. The intricate mechanism involved in the placental transfer of UMP may be different from that observed with other membranes. The Iack of any appreciable incorporation of UMP into maternal and fetd tissues, however, does reflect the difficulty encountered by a phosphorylated compound in crossing a cellular membrane. The most striking result in our uridine experiments was the enhanced RNA labeling in the fetal tissue followed by the placental
Volume Number
102 8
Placental
tissue. This aNbs,ervation is in direct contrast with erotic a.cid studies, in which all three tissues demonstrated significant incorporation of the precursor into RNA. Such results suggest that the RNA metabolism in
REFERENCES
1. Hayashi,
T. T., and Garvey, B.: AM. J. OBST. & GYNEC. In press. 2. Bresnick, E., Lanclos, K., and Gonzales, E.: Biochem. Biophys. Acta 108: 568, 1965. 3. Dancis, J., and Balis, M. E.: J. Biol. Chem. 207: 367, 1954. 4. Bray, G. A..: Anal. Biochem. 1: 279, 1960.
transfer
of
erotic
acid,
uridine,
and
UMP
1153
the fetus and the placenta may be different from that in the maternal liver in the capacity to utilize uridine as a specific precursor for RNA synthesis.
5.
Smith, I., editor: Chomatographic and Electrophoretic Techniques, New York, 1961, Interscience Publishers, Inc., vol. I, p. 233. 6. Hayashi, T. T., Baldridge, R. C.; Olmsted, P. S. and Kimmel, D. L.: AM. J. OBST. & GYNEC. 88: 470, 1964. 7. Atlas, M., Bondi, V. P., and Cronkite, E. P.: J, Histochem. Cytochem. 8: 171, 1960.