[CANCER RESEARCH 32, 979-982,
May 1972)
Synthesis of a-Fetoprotein by Liver, Yolk Sac, and
Gastrointestinal Tract of the Human Conceptus1
David Gitlin, Anita Perricelli, and Géraldine
M. Gitlin
Department of Pediatrics, University of Pittsburgh School of Medicine and the Children's Hospital, Pittsburgh, Pennsylvania ¡5213
SUMMARY
The synthesis of serum a-fetoprotein was studied in 16
human embryos and fetuses between 4.2 and 18 weeks of
gestation by incubation of selected tissues in l4C-labeled
amino acids followed by immunoelectrophoresis of the culture
fluids and radioautography.
Relatively large amounts of
radioactive a-fetoprotein were found in each of the liver
cultures and in cultures of the developing yolk sac. Smaller
amounts of labeled a-fetoprotein were observed in almost all
of the gastrointestinal tract cultures. Labeled a-fetoprotein
was formed in the kidney cultures from 1 of 9 conceptuses
and in only 1 of 14 placentas cultured. None of the cultures of
lung, thymus, pancreas, skeletal muscle, amnion, chorion, or
blood produced detectable amounts of a-fetoprotein.
protein are also synthesized
organs.
MATERIALS
in other embryonic
and fetal
AND METHODS
Conceptus Studies. a-Fetoprotein synthesis was investigated
in cultures of tissues obtained from the 16 conceptuses listed
in Table 1. The pregnancy in each instance had been
interrupted
for entirely psychological reasons, and the
conceptuses
were grossly normal in appearance.
The
gestational age of each conceptus was determined from the
crown-rump length with the use of Streeter's data for embryos
measuring 30 mm or less (24) and Patton's data for
conceptuses of 35 mm or more (21). Those conceptuses of 8.5
weeks of gestation or less were delivered vaginally by forceps
extraction following cervical dilation, and all conceptuses
older than 8.5 weeks of gestation were delivered by
INTRODUCTION
hysterotokotomy.
Human serum a-fetoprotein
(7, 9, 10, 12, 18, 25) is
Tissues. In all but one instance, individual organs or tissues
normally a product only of the conceptus. The protein is were cultured separately. The youngest embryo, C8-68, was
known to be synthesized by the developing liver beginning at too small to permit adequate dissection for separate organs at
least as early as 6 weeks of gestation (10), but this synthesis the time, and therefore, the entire embryo was minced, mixing
ceases at or near birth, whether birth is premature or at term, the tissues. In 2 conceptuses, C5-68 and C9-68, only the yolk
and the a-fetoprotein present in the circulation of the neonate
sac was cultured. Lungs, liver, skeletal muscle, amnion,
then disappears with a half-life of approximately 3.5 days chorion, and placenta were obtained from each of the other
(10). The presence of a-fetoprotein in the serum of a patient
conceptuses. In most of the conceptuses, the stomach and the
older than 1 year of age has been considered to be diagnostic intestinal tract were minced and cultured together, but in 2
of either a primary hepatoma or an undifferentiated gonadal instances, H9-67 and H3-67, portions of both the stomach and
teratoma (1, 5, 17, 20, 26, 27). In children under 1 year of the small intestine were cultured separately. Kidneys from 9 of
age, however, the anomalous presence of serum a-fetoprotein
the 11 conceptuses of 7.5 weeks of gestation and older were
studied for a-fetoprotein synthesis (Table 1), as were the
may also be associated
with certain
nonneoplastic
hepatopathies as well (17).
pancreas, spleen, and thymus from all conceptuses of 9.5
Since a-fetoprotein synthesis in the fetal hepatocyte is weeks of gestation and older. In addition to the 2 yolk sacs
normally repressed at or near birth, later synthesis of the noted earlier, the yolk sac of 4 other conceptuses were also
protein by a hepatoma probably represents genie derepression
studied (Table 1). Aliquots of umbilical cord blood from 2
in anomalous cells arising from the hepatocyte. On the other fetuses, C7-68 and C1-68, were incubated with culture
hand, the association of a-fetoprotein with some primitive medium as with the other tissues.
gonadal tumors (17, 26) and with rare instances of
Tissue Culture. The medium used for tissue culture was
gastrointestinal carcinoma (4, 8, 16, 19) have been more Eagle's basal medium formulated with Hanks' solution (Grand
difficult to understand. In the study reported here, it was Island Biological Co., Grand Island, N. Y.) and containing 5%
found that although the liver is the principal source of horse serum. Each organ or tissue selected for culture was
a-fetoprotein in the human conceptus, lesser amounts of this minced with scissors and incubated in roller tubes at 37°with
2 ml of culture medium containing 2 to 5 piCi of amino acids
'Supported by Grants HD-00652 and HD-01031 from the National uniformly labeled with 14C. A mixture of l4C-labeled
L-amino acids, which were obtained from algal hydrolysates
Institute of Child Health and Human Development, USPHS.
Received December 27, 1971 ¡acceptedFebruary 11, 1972.
and which had a specific activity of 1 mCi/mg, was used for
MAY 1972
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1972 American Association for Cancer Research.
979
D. Gitlin, A. Perricelli, and G. M. Gitlin
Table l
The conceptuses studied and the presence of labeled a-fetoprotein in culture fluids after incubation of
tissues with ' *C-labeled amino acids
Yolk Gastrointestinal
age Kidney4.2
(wk)
Liver
sac
tract
ConceptusC8-68C2-68C4-68C5-68C9-68Hl-67C3-68Cl
length
(mm)6.41020202030353545547070111145155160Gestational
+++")4.5
(all organs mixed =
-H-++5.5
-H--H-
++++5.5
++-H-C
++++
CJ.J*7
Ç
J8.5
/ .
++++8.5
+++O
-M-++
1-68H6-67HlO-67H2-67C7-68H8-67H9-67Cl-68H3-67Crown-rump
7.J10.5Ç
-H-H-11.5
+++1
+14
1.5
++++
++17
+-M-H17.5
+-H-i
o
1otrtr_—0+trtr-++—tr—tr.__—±0-0-0tr0±±0Placenta00tr—-00000000000
0 0 to ++++, relative intensity of a-fetoprotein precipitation
detected; tr, faint but definite line observed; -, not done.
line on radioautography:
0, line not
those tissues with codes beginning with H,and L-leucine-14C,
which had a specific activity of 210 ¿iCi/mmole,was used with
those conceptuses with identification codes beginning with
"C." Incubation of the cultures began within 10 to 30 min
after delivery of the conceptus. After incubation for 2 to 4
days with constant rotation, the cultures were dialyzed against
water for 3 days, lyophilized, then redissolved in 0.1 ml of 0.1
M borate buffer (pH 8.6), and centrifuged. As controls, fetal
serum instead of tissue was incubated in culture medium with
and without radiolabeled amino acids, and the incubation
mixtures were handled in exactly the same manner as the
tissue cultures. Aliquots of fetal tissue incubated in culture
medium without the labeled amino acids served as additional
controls.
Radioimmunoelectrophoresis.
Aliquots of fetal serum were
added to the concentrated culture supernatants, the fetal
serum providing carrier a-fetoprotein
for any labeled
a-fetoprotein
present
in
the
culture
fluid.
Immunoelectrophoresis
(23) of each culture fluid was then
performed in duplicate or triplicate with a gel of 1.5 g of agar
in 100 ml of 0.1 M borate buffer at pH 8.6. The slides were
allowed to develop with antisera for 3 to 7 days in a humid
chamber, were then washed in 3 to 4 changes of 0.1 M NaCl
solution over a 3-day period, dried, and finally inverted on
sheets of RS Pan film (Eastman Kodak, Rochester, N. Y.) for
8 to 12 weeks for radioautography, with lead bricks on top of
the slides as weights.
Antisera. The preparation and characterization of the rabbit
antiserum against fetal serum proteins used in this study has
been described elsewhere (10). Aliquots of this antiserum were
rendered specific for the precipitation of a-fetoprotein by
adsorbing each ml of rabbit antiserum with 0.2 ml of pooled
adult human serum (10).
RESULTS
Cultures of liver consistently
yielded relatively large
amounts of labeled a-fetoprotein (Table 1; Fig. 1), but in none
980
Fig. 1. Left, culture fluid supernatants after immunoelectrophoresis,
developed with rabbit antiserum specific for a-fetoprotein; anode was
to the left in each instance, and a-fetoprotein precipitin line is indicated
by arrows in A. Right, radioautographs of the immunoelectrophoreses
shown on the left. A, C8-68, mince of whole embryo; B, C2-68, liver; C,
C5-68, yolk sac;D, Cl 1-68, yolk sac;£,C3-68, gastrointestinal tract.
of the cultures of lung, thymus, pancreas, skeletal muscle,
amnion, or chorion was any labeled a-fetoprotein detected.
The yolk sacs obtained at 4.5 to 8.5 weeks of gestation
synthesized highly labeled a-fetoprotein (Table 1; Fig. l),but
the protein was found to be much less labeled in the culture of
the yolk sac obtained at 11.5 weeks of gestation; the yolk sacs
of 4.5 to 8.5 weeks of gestation were vesicular, well
vascularized, and filled with fluid, but the yolk sac at 11.5
weeks was solid and atretic, having undergone normal
involution. Radioactive a-fetoprotein was also detected in the
gastrointestinal tract cultures from all but 1 of the 10
conceptuses so studied (Table 1); in each instance, however,
the amount of labeled protein found was very much less than
that observed in the liver cultures (Fig. 1). Where portions of
the stomach and small intestine were cultured separately, i.e.,
CANCER RESEARCH VOL. 32
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1972 American Association for Cancer Research.
Synthesis of a-Fetoprotein in the Human Conceptas
in H9-67 and H3-67, the different areas seemed to produce of the lung, thymus, pancreas, skeletal muscle, amnion, and
similar amounts of a-fetoprotein. Kidney cultures from at least chorion, as well as those of kidneys and placenta, was not
1 conceptus produced a faint but definite a-fetoprotein line on attributable to a simple failure of metabolism in that tissue:
every tissue culture yielded 1 or more labeled plasma proteins
radioautography
(Table 1). Only 1 of the 14 different
placentas cultured synthesized a-fetoprotein (Table 1).
which were readily detected as reported elsewhere (11),
Labeled a-fetoprotein was not detected in either of the 2 whether a-fetoprotein was produced or not. Conversely, the
labeling of a-fetoprotein in tissue culture was not due to
cultures of fetal whole blood or in any of the control
incubation fluids that contained fetal serum rather than tissue. simple binding of labeled amino acids to carrier a-fetoprotein,
None of the tissue cultures prepared in the absence of the since cultures of many tissues did not form labeled
labeled amino acids yielded a detectable a-fetoprotein line on a-fetoprotein, although the incubating medium was the same
as in those that did.
radioautography.
Some patients with hepatoma do not have demonstrable
serum levels of a-fetoprotein. Increasing the sensitivity of the
DISCUSSION
method that is used to detect a-fetoprotein,
however,
apparently increases the percentage of patients with hepatoma
According to the data obtained in this investigation, it who do have the protein in their sera (3); this is in accord with
would seem that the sites of synthesis of a-fetoprotein include
the observation that different individual hepatomas in
primarily the liver and the yolk sac and, to a lesser extent, the monkeys, whether in the same monkey or in different
gastrointestinal
tract. The relative degree of a-fetoprotein
monkeys, may produce different amounts of a-fetoprotein
labeling seen in the liver cultures clearly indicates that (13). Unlike the hepatoma,
however, gastrointestinal
throughout most of gestation the liver is the principal source carcinoma is associated with a-fetoprotein only rarely. It has
of this protein, although the yolk sac is equally active in been suggested that the serum a-fetoprotein found in some
a-fetoprotein synthesis early in development. As the yolk sac
patients with gastric carcinoma metastatic to the liver may
becomes atretic toward the end of the 1st trimester, however, originate in the altered hepatic parenchyma (4, 16). Although
its production of the protein apparently diminishes sharply. this is a distinct possibility, it is also possible that the tumor
The
amount
of a-fetoprotein
synthesized
by the
itself may be the source of the anomalous protein. In rats with
gastrointestinal tract appears to be much less than that transplanted mouse hepatoma only mouse a-fetoprotein is
synthesized by equivalent amounts of either liver or early yolk found (2), and in monkeys with hepatoma, a-fetoprotein can
sac as judged from the relative amounts of labeled be detected with fluorescent antibodies only in the cells of the
a-fetoprotein found in these cultures.
tumor and not in the surrounding parenchyma (M. G. Kelly, P.
That the liver of the human embryo can produce radioactive P. Carbone, and D. Gitlin, unpublished data). As noted in the
a-fetoprotein
in tissue culture when incubated
with present
study,
the relative amount
of a-fetoprotein
14C-labeled amino acids has been reported previously (11).
synthesized by the gastrointestinal tract of the embryo or
Although it was found that a-fetoprotein in the rat can be
fetus is considerably less than that produced by an equivalent
synthesized in the yolk sac (11), the rat yolk sac being a weight of liver. It would be reasonable to expect, therefore,
well-developed and biologically active structure until birth, the
that a carcinoma of the gastrointestinal tract on the average
human yolk sac was not studied in that investigation. would produce much less a-fetoprotein, if any at all, than a
Synthesis of a-fetoprotein by the gastrointestinal tract and hepatoma and that the resulting serum levels of a-fetoprotein,
other organs, although examined, was not detected (11). In therefore, might be below the limit of detection of the
the present inquiry, however, (a) the tissue was incubated 3 to methods used thus far.
10 times longer, (b) the relative amount of culture fluid used
Serum a-fetoprotein is produced by the fetal hepatocyte,
for immunoelectrophoresis
was 2 to 4 times greater, (c) the and tumors arising from hepatocytes may synthesize the
film used for radioautography was more sensitive, and (d) the protein. Small amounts of a-fetoprotein are produced by the
time allowed for radioautography was 2 to 8 times longer than gastrointestinal tract in utero, and an occasional carcinoma
that used in the previous investigation. Hence, in the study arising from this area may be associated with detectable serum
reported here, the technique used permitted the detection of levels of the protein. Synthesis of a-fetoprotein by a tumor
much smaller amounts of labeled protein synthesis than did arising from an organ which earlier had had the capacity to
the technique used in the earlier report.
manufacture the protein may represent a derepression of the
A trace amount of labeled a-fetoprotein was present in cistron responsible for a-fetoprotein production. On this basis,
cultures of kidney from 1 of 9 conceptuses and in cultures of the presence of serum a-fetoprotein in patients with certain
placenta from 1 of 14 conceptuses. Using similar methods, van
Furth and Adinolfi (28) found a-fetoprotein labeling in 2 of 7 gonadal carcinomas might be difficult to explain. However,
Huntington and his colleagues (14, 15, 22) and Bailas (6) have
human placentas cultured. It is possible, of course, that the
recently suggested that some gonadal tumors are of yolk sac
renal and placental cultures from the other conceptuses in the
origin,
and the yolk sac in the present study was found to be a
present study did produce a-fetoprotein, but the amount
major site of a-fetoprotein
production in early human
produced may have been below the limit necessary for
development.
detection by the method used. On the other hand, it is also
possible that these apparently negative cultures did not reveal ACKNOWLEDGMENTS
labeled a-fetoprotein simply because the protein was not
synthesized. The failure to find evidence of a-fetoprotein
The authors are pleased to record their indebtedness and their
gratitude to Dr. Valdemar Madsen at the Amts Sygehuset, Gentofte,
synthesis in any tissue culture in this study, including cultures
MAY
1972
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1972 American Association for Cancer Research.
981
D. Gitlin, A. Perricelli, and G. M. Gitlin
Denmark, for providing the embryos and fetuses reported in this
investigation and for permitting us the use of the facilities of his
department.
13.
14.
REFERENCES
15.
1. Abelev, G. I., Assecritova, I. V., Kraevsky, N. A., Perova, S. D., and
Perevodchikova, N. I. Embryonal Serum a-Globulin in Cancer
Patients: Diagnostic Value. Intern. J. Cancer, 2: 551-558, 1967.
2. Abelev, G. I., Perova, S. D., Khramkova, N. I., Postnikova, Z. A.,
and Irlin, I. S. Production of Embryonal a-Globulin by
Transplantable Mouse Hepatomas. Transplantation, /: 174-180,
1963.
3. Alpert, E., Hershberg, R., Schur, P. H., and Isselbacher, K. J.
a-Fetoprotein in Human Hepatoma: Improved Detection in Serum,
and Quantitative Studies Using a New Sensitive Technique.
Gastroenterology,6/:
137-143, 1971.
4. Alpert, E., Pinn, V. W., and Isselbacher, K. J. Alpha-Fetoprotein in
a Patient with Gastric Carcinoma Metastatic to the Liver. New
Engl. J. Med., 285: 1058-1059,1971.
5. Alpert, M. E., Uriel, J., and de Nechaud, B. Alpha, Fetoglobulin in
the Diagnosis of Human Hepatoma. New Engl. J. Med., 278:
984-986, 1968.
6. Bailas, M. Yolk Sac Carcinoma of the Ovary with a-Fetoprotein in
Serum and Ascitic Fluid Demonstrated by Immuno-Osmosphoresis.
Am. J. Clin. Pathol., in press.
7. Beigstrand, C. G., and Czar, B. Demonstration of a New Protein
Fraction in Serum from the Human Fetus. Scand. J. Clin. Lab.
Invest.,«: 174, 1956.
8. Bourreille, J., Metayer, P., Sauger, F., Matray, F., and Fondimare,
A. Existence d'Alpha Poeto Protéineau Cours d'un Cancer
Secondaire du Foie d'Origine Gastrique. Presse Med., 78:
1277-1278,1970.
9. DeMuralt, G., and Roulet, D. L. A. Etude Immunologique des
ProtéinesSériquesFoetales Humaines. Helv. Paediat. Acta, 16:
517-533,1961.
10. Gitlin, D., and Boesman, M. Serum a-Fetoprotein, Albumin, and
"xG-Globulin in the Human Conceptus. J. Clin. Invest., 45:
1826-1838,1966.
11. Gitlin, D., and Boesman, M. Sites of Serum a-Fetoprotein
Synthesis in the Human and in the Rat. J. Clin. Invest., 46:
1010-1016, 1967.
12. Halbrecht, I., Klibanski, C., Brzoza, H., and Lahav, M. Further
Studies on the Various Hemoglobins and the Serum Protein
982
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
Fractions in Early Embryonic Life. Am. J. Clin. Pathol., 29:
340-344, 1958.
Hull, E. W., Carbone, P. P., Gitlin, D., O'Gara, R. W., and Kelly,
M. G. a-Fetoprotein in Monkeys with Hepatoma. J. Nati. Cancer
Inst.,42; 1035-1044,1969.
Huntington, R. W., Jr., and Bullock, W. K. Yolk Sac Tumors of the
Ovary. Cancer, 25: 1357-1367,1970.
Huntington, R. W., Jr., and Bullock, W. K. Yolk Sac Tumors of
Extragonadal Origin. Cancer, 25: 1368-1376, 1970.
Kozower, M., Fawaz, K. A., Miller, H. M., and Kaplan, M. M.
Positive Alpha-Fetoglobulin in a Case of Gastric Carcinoma. New
Engl. J. Med.,2S5: 1059-1060, 1971.
Masopust, J., Kithier, K., Rà di, J., Koutecky, J., and Kotál,L.
Occurrence of Fetoprotein in Patients with Neoplasma and
Non-Neoplastic Diseases. Intern. J. Cancer, J: 364-373, 1968.
Masopust, J., and Kotál, L. Fetoprotein: Immunochemical
Behavior of an Autonomous Fetal Component in Sera from Human
Fetuses. Ann. Paediat., 204: 138-140, 1965.
Mehlman, D. J., Bulkley, B. H., and Wiernik, P. H. Serum
Alpha,-Fetoglobulin with Gastric and Prostatic Carcinomas. New
Engl. J. Med., 285: 1060-1061, 1971.
O'Conor, G. T., Tatarinov, Yu. S., Abelev, G. I., and Uriel, J. A
Collaborative Study for the Evaluation of a Serologie Test for
Primary Liver Cancer. Cancer, 25: 1091-1098, 1970.
Patten, B. M. Human Embryology. New York, Blakiston Company,
1948.
Pierce, G. B., Bullock, W. K., and Huntington, R. W., Jr. Yolk Sac
Tumors of the Testis. Cancer, 25: 644-658, 1970.
Scheidegger, J. J. Une Micro-Méthode
de l'Immuno-Électrophorèse.
Int. Arch. Allergy Appi. Immunol., 7. 103-110, 1955.
Streeter, G. L. Developmental Horizons in Human Embryos.
Carnegie Inst. Wash. Pubi., 32: 133-204, 1948.
Tatarinov, Y. S. New Data on the Embryo-Specific Antigenic
Components of Human Blood Serum. Vop. Med. Khim., 10:
584-589, 1964 (translated in Federation Proc., 24: T916-T918,
1965).
Tatarinov, Y. S. Content of Embryo-Specific a-Globulin in Fetal
and Neonatal Sera and Sera from Adult Humans with Primary
Carcinoma of the Liver. Vop. Med. Khim., lì: 20-24, 1965
(translated in Federation Proc., 25: T344-T346, 1966).
Uriel, J., de Nechaud, B., Stanislawski-Birencwajg, M., Masseyeff,
R., Leblanc, L., Quénum,C., Loisillier, F., and Grabar, P.
Antigènes Embryonnaires et Cancer du Foie chez l'Homme.
Association de la a, -Foetoproteine Sériqueavec l'Hépatome
Primaire. Compt. Rend. Acad. Sci., 265: 75-78,1967.
van Furth, R., and Adinolfi, M. In Vitro Synthesis of the Foetal
a,-Globulin in Man. Nature, 222: 1296-1299, 1969.
CANCER RESEARCH VOL. 32
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1972 American Association for Cancer Research.
Synthesis of α-Fetoprotein by Liver, Yolk Sac, and
Gastrointestinal Tract of the Human Conceptus
David Gitlin, Anita Perricelli and Geraldine M. Gitlin
Cancer Res 1972;32:979-982.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/32/5/979
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1972 American Association for Cancer Research.