The location and synthesis of transferrin in mouse embryos and teratocarcinoma cells

DEVELOPMENTAL

BIOLOGY

(1982)

‘&22’7-234

The Location and Synthesis of Transferrin in Mouse Embryos and Teratocarcinoma Cells EILEEN Cancer Research

Center,

La Jolla Received

Cancer August

Research

D. ADAMSON

Foundation,

10901 North

21, 1980; accepted

in revised

form

Twrey February

Pines

Road,

La Jolla,

California

92037

I, 1982

In early postimplantation mouse development, transferrin synthesis appears to be a marker of visceral endoderm cell types. Transferrin was identified using immunoperoxidase staining, in the proximal (visceral) endoderm of the sixth-day egg cylinder, in some tissues at later stages, and in the visceral yolk sac (VYS) at all stages examined. Since the location of a plasma protein does not necessarily indicate its site of synthesis, the incorporation of labeled amino acids into transferrin was studied. Synthesis could be detected in egg cylinders on the seventh day of gestation onwards and in the VYS at all stages. However, although endoderm was the likely tissue source, its ability to synthesize transferrin after its isolation from the embryo was either much reduced or absent. The data are suggestive of a modulating influeme by mesoderm and other cell types on transferrin synthesis in visceral endoderm cells. Three types of endoderm-like cells which are produced by teratocarcinoma embryonal carcinoma (EC) cells were analyzed for transferrin synthesis to assess possible parallels with the embryo. Embryoid bodies from PSAl EC cells contained some outer endoderm cells which stained for transferrin and others which did not. The endoderm line PSA5E but not PYS-2 synthesized transferrin. The third type of endoderm-like cell (END cells) synthesized very little (OC15Sl) or no (PC13 clone 5) transferrin. The conclusion that PSASE, OC15 END, and some differentiated PSAl cells have visceral endoderm-like character while PYS-2 reflects parietal endoderm phenotype is in agreement with published data.

generate after the sixth week of gestation. Nevertheless, this organ synthesizes transferrin as well as CYfetoprotein, al-antitrypsin, prealbumin, and albumin (Gitlin and Pericelli, 1970). The visceral yolk sac of the mouse is well developed at the 13th day and persists until parturition. It is a major source of a-fetoprotein (Wilson and Zimmerman, 1976) and it seemed likely that this membrane would be responsible for the early production of fetal mouse transferrin. This has been confirmed in the present study, however, the earliest detectable synthesis was found to be by embryos on the 7th day of gestation, that is, well before the formation of the visceral yolk sac. Immunoperoxidase reactions were also employed to detect the location of transferrin in postimplantation embryos from the fifth day of gestation. Transferrin located in embryonic cells before the formation of the placenta would not necessarily be indicative of its synthesis there, since there is no evidence for a barrier to the maternal supply at these earlier times. Later, it is thought that the placenta is a barrier to the passage of maternal transferrin to the human fetus (Faulk and Galbraith, 1979). The present studies were made to identify the tissues and the earliest times of development in which fetal transferrin is synthesized. Teratocarcinoma stem cells or embryonal carcinoma (EC) cells can be induced to differentiate in vitro to form endoderm-like cells, either by forming floating

INTRODUCTION

Transferrin is one of the major iron-carrying serum proteins and its main function is to transport and supply ferric ions for the cellular production of hemoglobin and cytochromes, etc. In the mouse, hemopoiesis starts at about the eighth day of gestation (Bateman and Cole, 1971) and later the blood islands of the inner mesoderm layer of the visceral yolk sac are probably the source of hemopoietic cells (Moore and Metcalfe, 19’70).Transferrin can be detected in fetal serum and in amniotic fluid as soon as these fluids accumulate; presumably this is the immediate source of iron needed for hemoglobin production in the developing red cells in the blood islands of the visceral yolk sac. The transferrin in mouse amniotic fluid is fetal in origin (Renfree and McLaren, 1974) and it has been shown that transferrin is synthesized by mouse embryos at least as early as the 13th day of gestation, in an undersialylated form which is gradually replaced by the adult-type sialylatetl forms by the 17th day (Gustine and Zimmerman, 1972, 1973). Yeoh and Morgan (1974) demonstrated that rat fetal liver on the 15th day of gestation and all stagev afterwards synthesizes transferrin. These workers also detected the synthesis of transferrin by combined fetal membranes and this started at the 13th day of gestation. In the human fetus, the visceral yolk sac rcemains small and starts to de227

OOlZ-1606/82/060227-08$02.00/O copyright All righta

Q 1982 by Academic Press, Inc. of reproduction in any form reserved

228

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aggregates called embryoid bodies (Martin and Evans, 1975) or by low-density plating (Adamson and Graham, 1980) or by retinoic acid (Jetten and Jetten, 1979; Strickland and Mahdavi, 1978; Rees et al., 1979; Strickland et al., 1980). Endoderm cells formed by each of these three methods were examined for transferrin synthesis. The possibility that EC cells differentiate to give endoderm cells with a phenotypic expression similar to early embryonic endoderm was therefore tested. MATERIALS

Biological

AND

METHODS

Materials

Embryos were obtained from a variety of outbred strains of mice, and they were usually PO (Pathology Department, Oxford), MFl (Olac Ltd., Oxon, U. K.), or Dub (ICR) (Flow Laboratories, Inglewood, Calif.). The day of detection of the copulation plug was designated the 1st day of gestation. For immunoperoxidase studies, early embryos (5th to 9th days of gestation) were fixed in utero; later embryos and tissues were dissected in solution A of Dulbecco and Vogt (1954, PBS) and pooled before being fixed. Inner cell masses were obtained by immunosurgery (Solter and Knowles, 1975) of early5th-day blastocysts and cultured for 2 to 5 days before being fixed (see Adamson et al., 1979). Visceral yolk sacs of embryos from the 12th to the 16th day of gestation were dissected out and also separated into their two component layers, the endoderm and mesoderm, by means of an enzyme mixture (0.5% trypsin and 2.5% pancreatin, Levak-Svajger et al., 1969) for 2 hr at 4°C. In some experiments a nonenzymatic method was employed to loosen the mesoderm from the endoderm. This occurs after 5 to 10 min at room temperature in 1 M glycine-2 mM EDTA, pH 7.3 (McClay and Marchase, 1979). The tissues were then separated in tissue culture medium containing 10% fetal calf serum. Visceral yolk sacs were also incubated with purified collagenase (Adamson et al., 1979) for 90 min at 37°C to remove part of the mesoderm from the remaining portion. This was a gentle way of removing most of the mesoderm while leaving all the endoderm and some of the adhering mesoderm intact. The teratocarcinoma cell line, PSAl, was used to make embryoid bodies (Martin et al., 1977). Two embryonal carcinoma cell lines, OC15Sl (McBurney, 1976) and PC13, clone 5 were the source of differentiated cells called END. Their maintenance and formation have been described by Adamson et al. (1979). Teratocarcinoma endoderm cell lines PSA5E (Adamson et al., 1977) and PYS-2 (Lehman et al., 1974) were cultured in the same medium but in nongelatinized dishes.

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Anti-transferrin

Antiserum

Amniotic fluid of embryos from the 12th to 18th day of gestation was collected and separated on S-mm-thick preparative polyacrylamide gels (Davis, 1964) containing 7.5% acrylamide. A band containing transferrin was cut out, the protein was eluted, concentrated, and used as the immunogen to raise antibodies in a rabbit. The methods used were as described for the preparation of anti-a-fetoprotein (AFP) antiserum (Dziadek and Adamson, 1978). Immunoelectrophoresis (Grabar and Williams, 1953) showed that there was some contamination with antibodies against AFP and albumin. These were removed by adsorption to acrylamide gel bands containing these proteins. The resulting antiserum had no detectable activity in immunoelectrophoresis tests against AFP, albumin, or any other fetal or adult serum protein except transferrin (Figs. Id, e, and g). The antiserum reacted with both adult and fetal mouse transferrin (Figs. lb, c, and f) but did not react in doublediffusion assays against the transferrin in fetal calf serum (Fig. la, well B). The latter observation was important because some tissues were incubated in culture medium containing fetal calf serum before immunoperoxidase tests. The control antiserum used in the studies described below was either preimmune serum or the reactive serum which had been adsorbed with polyacrylamide gel bands containing transferrin (Fig. la, center well on the right-hand side). Control and reactive antisera were precipitated with 40% saturated ammonium sulfate to obtain the immunoglobulin fraction. This was dialyzed against PBS, centrifuged to clarify, and frozen in small aliquots at -70°C before use in immunoperoxidase and immunoprecipitation reactions. Immunoperoxidase

Reactions

Details for the fixation, embedding, sectioning, and reaction with the antibody have been described previously (Dziadek and Adamson, 1978; Dziadek, 1978). Internal

Labeling

of Transfer&

The details for labeling embryos and dissected tissues were described earlier (Dziadek and Adamson, 1978). Confluent monolayers containing from 1 to 5 X lo6 END or EC cells per 50-mm dish were labeled by incubation for 16-24 hr in a medium consisting of l/10 a-medium (supplemented with nucleosides and deoxynucleosides Adamson et al., 1979) and 9/10 leucine-free minimum essential medium, modified Eagle’s with Earle’s salts (Flow Laboratories, Irvine, Scotland) with 20 &i/ ml [L-4, 53H]leucine (146 &i/mmole, Radiochemical Centre, Amersham, U. K.) and 10% dialyzed fetal calf

EILEEN

D. ADAMSON

Transferrin

in

Mouse

Emirryonic

229

Cells

A similar medium containing 50 &i/ml L[35S]methionine (New England Nuclear, 500 Ci/mmole) was constructed in a methionine-reduced background. Radioactive supernatants (0.5-3 ml) were either (a) clarified by centrifugation at 200,OOOg for 30 min and immunoprecipitated by the addition of 10 ~1 of adult mouse serum as carrier (6 mg/ml) and 20 ~1 of antitransferrin immunoglobulin (0.8 mg/ml; these were shown to give maximum precipitation in a separate experiment) or, (b) immunoprecipitated by the addition of 3-5 pg immunoglobulin and 50 ~1 of a suspension of Sepharose linked to protein A of Staphylococcus aureus (Pharmacia, Piscataway, N. J.) as described before (Hogan et al., 1981). The radioactive supernatants were “cleared” by preadsorption with Sepharose-protein A and nonimmune serum. Immunoprecipitates were analyzed by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis on 7.5% gels (Laemmli, 1970). Slab gels were used to show radioactive bands by fluorography (Bonner and Laskey, 1974). Aliquots of radioactive supernatants were monitored for total activity and for trichloroacetic acid (TCA) precipitated activity on glass fiber disks (GF/C, Whatman; W. and R. Balston, U. K.). Radioactive samples were counted in a Beckman LS-230 scintillation counter. serum.

RESULTS

AND

DISCUSSION

Immunoperoxidase Localization of Transfer& Early Postimplantation Embryos

FIG. 1. Double-diffusion and immunoelectrophoretic analysis of rabbit anti-mouse transferrin. (a) Antibody in the left center well; antibody adsorbed with transferrin for control reactions on the right. Outer wells in anti-clockwise direction contain: B, fetal calf serum and a one-fifth dilution; F, fetal mouse serum and a one-fifth dilution; A, adult mouse serum and a one-fifth dilution. (b-g) In the immunoelectrophoretic diffusion plates, the trough in b contains antitransferrin immunoglobulin which had been adsorbed twice with AFP and albumin-containing polyaxrylamide gel bands. The other troughs contain the unadsorbed antiserum and faint traces of aFP and albumin can be discerned. F, fetal mouse serum; A, adult mouse serum;

in

Figures 2a and b show the earliest stage (late-6thday embryos) of development at which transferrin was present at higher concentrations in the embryo than in the surrounding maternal tissues. As was shown for AFP (Dziadek and Adamson, 1978) transferrin was clearly located in the endoderm of the seventh day egg cylinder (Figs. 2c and d). In contrast to AFP, the extraembryonic portion as well as the embryonic portion of the endoderm stained. In 7th-day embryos, increased staining in the parietal endoderm and in the trophectoderm was most noticeable (Figs. 2e and f) and only the ectoderm was largely unstained. Similar results were obtained with 8th- and Sth-day embryos. Most tissues had some staining and Fig. 2g shows that extraembryonic membranes were intensely stained. The staining described above showed the localization in embryos of transferrin that could have been derived either from maternal or embryonic sources. It is known that visceral endoderm and trophectoderm are particularly capable of pinocytosing proteins from the surrounding Ruids. In order to test the developing egg cylTr, transferrin; aFP, (Y fetoprotein; teins used at 0.1-0.5 mg/ml.)

Alb,

albumin.

(The last three

pro-

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DEVELOPMENTAL

FIG. 2. Immunoperoxidase reactions with anti-mouse embryos fixed within the uterus in acidified ethanol. (a) (f) controls. In c and d bar = 50 pm; in e and f bar = embryonic visceral endoderm, EV; and extraembryonic layers are shown from the left: Ae, amniotic ectoderm; trophoblast. Bar = 50 pm.

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transferrin immunoglobulin. Six-micrometer sections of embryos were made from Sixth-day reaction; (b) control bar = 20 Frn. (c) and (e) seventh-day reaction; (d) and 20 pm. Note heavy staining in the trophoblast, T, parietal endoderm, P; and both visceral endoderm, EEV. (g) Section of a ninth-day embryo. The extra-embryonic Ve, visceral yolk sac endoderm; P, parietal endoderm and Reichert’s membrane; T,

inder for transferrin synthesis, the maternal source of transferrin would first have to be eliminated. This was done (three experiments) by isolating the inner cell masses of 5th-day blastocysts and then examining the “embryoid bodies” formed after 2 to 5 days in culture.

Immunoperoxidase reactions on sections of these bodies which consist of a sphere of ectoderm cells surrounded by a layer of endoderm cells were negative for transferrin. (Refer also to work by Dziadek (19’79), who showed that in such bodies only a very few cells stained

EILEEN

D. ADAMSON

Transfertin

for AFP.) In addition, transferrin synthesis was also undetectable after immunoprecipitation of the medium following internal labeling of “embryoid bodies” with tritiated amino acids. It was concluded that under the culture conditions described, transferrin synthesis was not initiated by these primitive embryonal cells even at the equivalent of the 10th day of gestation. However, the section below describes that in vivo, embryos at the 7th day of gestation are capable of synthesizing transferrin.

Synthesis of Transfer&

Synthesis

Embrpmic

TrAFPAlbc-

231

Cells

-

*Tr

by Isolated Embryos

Since immunoperoxidase reactions can only show the location of an antigen and not its tissue source, the incorporation of amino acid precursors into immunoprecipitated transferrin secreted by embryos and isolated tissues was tested as described under Materials and Methods section. The earliest day of detection of synthesis was by seventh day embryos during overnight incubation in radioactive medium. Figure 3, lane 8, shows a band of radioactive transferrin synthesized by late-seventh-day embryos and accumulated in the medium; in comparison, th,e synthesis of AFP occurs at a higher level and produces a stronger band (lane 7). Early seventh day embryos consist of two cell layers, inner ectoderm and out’er endoderm, which can be separated manually and incubated separately. The synthesis and secretion of radioactive transferrin by the separated layers of 36 (embryos were tested in each of two experiments. In neither endoderm nor ectoderm was transferrin synthesis detected. It is possible that the tissues were damaged by the separation technique but a more interesting explanation is that interactive influences between the layers is necessary to initiate synthesis. In view of the lack of synthesis by “embryoid bodies” (above) one may speculate that the mesoderm may be important in the initiation of transferrin synthesis.

Transferrin Tissues

in MolLse

ty Isolated

Extraembryonic

Transferrin (Tr) synthesis was not detectable in isolated lOth-day trophectoderm, lOth- to 16th-day parietal endoderm, and IOth- to 16th-day amnion after internal labeling and immunoprecipitation. Therefore the detection of transferrin by immunoperoxidase staining in these extraembryonic tissues (Fig. 2g) is most likely to be from adsorption of transferrin rather than its synthesis. However, it has not formally been ruled out that synthesis occurs in the trophectoderm, parietal endoderm, and amnion of 5th- to Sth-day embryos. Parietal endoderm (Figs. 4a and b) and amnion (not

1

2

3

4

5

6

7

8

FIG, 3. Polyacrylamide gel ectrophoretic analysis of [aH]leucine or [?S]methionine-labeled immunoprecipitates using rabbit anti-mouse anti-transferrin and anti-AFP. Gels were 7.5% and contained 0.1% SDS; the direction of migration was downwards toward the anode. Lanes 1 and 2 show Coomassie blue-stained proteins; lanes 3 to 8 are fluorographs. Lane 1, proteins which were immunoprecipitated by anti-transferrin from radioactive culture medium of lo-day visceral yolk sacs (VYS); from the top they are transferrin and heavy chain of IgG; lane 2, the three major proteins of fetal mouse serum, transferrin (Tr), a-fetoprotein (AFP), and albumin (alb); lane 3, untreated supernatant from metabolically labeled 13th-day VYS (the major band contains both Tr and AFP); lane 4, immunoprecipitated Tr from the incubation medium of lOth-day VYS; lane 5, radioactive proteins immunoprecipitated with anti-Tr from the incubation medium of 13th-day VYS; lane 6, the same as 4 only precipitated with anti-AFP; lane 7, radioactive proteins immunoprecipitated from [%S]methinoinelabeled overnight cultures of ten-7th-day embryos with anti-AFP; lane 8, the same as 7 only immunoprecipitated with anti-Tr. Lanes 5 to 8 were from a different gel.

shown) tissues were also shown to be negative in immunoperoxidase tests provided that they had been incubated in vitro for 3 to 4 hr to allow adsorbed or ingested material to diffuse out or to be degraded. On the other hand, VYS which had been incubated in PBS or had been incubated in the presence of collagenase (1 pg/ml) for 90 min at 37°C to remove most of the mesoderm, were stained strongly especially in the endoderm cells, while the remaining mesoderm was less well stained (Figs. 4c and d). The synthesis of Tr was also detected by immunoprecipitation from the medium after incubation of lOthday (Fig. 3, lanes 1 and 4) and 13th-day (Fig. 3, lanes 3 and 5) VYS with radioactive amino acids. The total amount of radioactivity which was found in Tr increased with gestational age, but at all times the synthesis of AFP was 5 to 20 times greater than that of Tr (Fig. 3, lanes 6 and 7). This was estimated by using the same radioactive culture medium to immunoprecipitate sequentially with anti-Tr and anti-AFP and

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DEVELOPMENTAL BIOLOGY

FIG. 4. Immunoperoxidase reactions with anti-mouse transferrin on sections of embryonic tissues. (a) Parietal endoderm P and Reichert’s membrane dissected from 13th-day embryos and incubated in PBS before fixation. (b) Same as (a) but using control antiserum. (c) Visceral yolk sacs dissected from 13th-day embryos and incubated with collagenase (see Materials and Methods for details) to remove some of the mesoderm layer, Vm. Ve, visceral endoderm layer, is heavily stained. (d) Control. Bar = 50 pm.

excision of the resulting radioactive gel band to measure the incorporated counts. Finally, it was of interest to test which of the layers of the VYS, endoderm or mesoderm, was synthesizing transferrin. As mentioned above, staining in the endoderm portion after immunoperoxidase reactions is not conclusive. Since both AFP and transferrin are synthesized by embryonic VYS and liver, it was expected that both these glycoproteins would be restricted to endodermally derived cells. The two cell layers were separated by incubation at 4°C in pancreatin-trypsin (see Materials and Methods section) and the medium from metabolically labeled cultures was tested by immunoprecipitation with anti-transferrin and as a comparison, also with anti-AFP antibodies. The radioactive products were identified and quantified after polyacrylamide gel electrophoresis. Three types of samples were tested in eight experiments: (a) isolated mesoderm, (b)

VOLUME 91, 1982

isolated endoderm, and (c) mesoderm and endoderm mixed after isolation. All three samples synthesized transferrin and AFP but the results obtained were too variable to make firm conclusions. There are two main reasons. First, it is difficult to prepare mesoderm entirely free of endoderm. Second, the synthesis of transferrin (but less so AFP) seems to be peculiarly sensitive and easily inhibited during both the enzymatic and the nonenzymatic methods employed to separate the layers, while total protein synthesis apparently remained unaffected. All three samples synthesized labeled transferrin and AFP but mixture (c) invariably gave better incorporation (two- to threefold) into transferrin and AFP compared to (a) and (b). This could have been because of a “feeder” effect of one tissue on the other so that transferrin synthesis was stabilized. Or, if one assumes that only the endoderm synthesizes transferrin and AFP, as the data above indicate and from our earlier work on AFP (Dziadek and Adamson, 1978; Dziadek, 1979), it is also possible that the mesoderm has a stimulatory effect on the endoderm. Further studies are needed to distinguish between these alternative explanations. The data above indicate that interacting tissues (mesoderm or possibly ectoderm) may facilitate the synthesis of transferrin by endoderm cells from three types of embryo cultures. In contrast, Dziadek (1978) found that AFP is synthesized by isolated 7th- and 8thday endoderm (detected by immunoperoxidase reactions). Moreover, in the case of AFP, extraembryonic ectoderm seems to have an inhibitory influence on its synthesis. In these respects, although both AFP and transferrin seem to be markers of endoderm cells they are affected differently by the surrounding tissues. Transfer?% Synthesis as a Marker of Endoderm Type in Teratocarcinma Cell Structures

Cell

Sections of embryoid bodies formed when PSAl EC cells were cultured in suspension showed definite intracellular staining in about 30% of the cells of the outer rind of endoderm-like cells (Fig. 5). In contrast the EC cell cores stained unevenly and very faintly. It was concluded that the synthesis of transferrin probably occurred in the endoderm cells, which therefore assigned a visceral-like character to at least some of the outer cells. Our findings are in agreement with others who have suggested that mixed types of endoderm occur in embryoid bodies (Martin et al., 1977; reviewed by Solter and Damjanov, 1979). These results are in accord with our earlier findings that AFP synthesis also occurs in these cells (Adamson et al., 1977). Some teratocarcinoma cell cultures were examined for the synthesis of transferrin by internal labeling

EILEEN

D. ADAMSON

Transferrin

with [3H]leucine. Undifferentiated stem cells of EC cell lines PC13 and OC15 did not synthesize it at detectable levels. Similarly, immunoprecipitation with anti-mouse transferrin failed to detect radioactive transferrin produced by PYS-2 cells after polyacrylamide gel electrophoresis of the product. Small but reproducible amounts of labeled transferrin were synthesized by PSA5E cells and this is shown Fig. 6, lanes 6 and 7. These results confirmed that PYS-2 cells are similar to parietal endoderm cells (see also Hogan, 1980; Hogan et al., 1980); and that PSA5E cells are similar to embryonal visceral endoderm cells (Adamson et al., 1977; Hogan, 1980), although these cells no longer synthesize detectable amounts of AFP as they did in an earlier study (Adamson et al., 1977). Some EC cell lines differentiate into endoderm-like cells when plated at low density (OC15S1, Burke et al., by retinoic 1978; Adamson et al., 1979), or if stimulated acid (F9, Strickland and Mahdavi, 1978; Solter et al., 1979; Strickland et al., 1980; PC13S1, Adamson et al., 1979; Rees et al., 1979; Adamson and Graham, 1980). Monolayer cultures of large flat endoderm cells (END) formed after differentiation of PC13 and OC15 cell lines were tested for transferrin synthesis. Little or no transferrin could be detected( after immunoprecipitation of radioactive culture media from PC13 END cells, but OC15 END gave small and variable amounts after 4 to 7 days of culture at low density (Fig. 6, lanes 4 and 5). It is clear from these findings that the best culture conditions which are permissive to the production of transferrin by teratocarcinoma cells are uncertain and further studies are in progress to determine them. In conclusion, the synthesis of transferrin occurs in embyronal visceral endoderm cells from the seventh day to the end of gestation. The production of transferrin seems to be modulated by other embryonal cells. The known growth requirement of embryos beyond the blastocyst stage, for serum (Hsu, et al., 1974) and the

FIG. 5. Immunoperoxidase sections of embryoid bodies from PSAl teratocareinoma = 20 pm.

reactions fixed after cell line

to anti-mouse transferrin on 6 days in suspension culture (a) reaction (b) control. Bar

in Mouse

Embryonic

Cells

12

3

233

Tr+ AFP, Alb(=

4

5

6

7

FIG. 6. Electrophoretic analysis of [aH]leucine-labeled proteins secreted by teratocarcinoma cell lines. Conditions were similar to those described in the legend to Fig. 3 except that lanes 6 and 7 were from a 5% gel. Lanes 1,2, and 3 are Coomassie blue-stained proteins; lanes 4 to 7 are autofluorographs. Lane 1, adult mouse serum; lane 2, mouse transferrin; lane 3, fetal mouse serum; lane 4, the radioactive proteins secreted by OC15 END cells; lane 5, the same but immunoprecipitated by anti-transferrin. Lanes 6 and 7 show radioactive proteins which were synthesized by PSABE teratocarcinoma cell line and immunoprecipitated with: lane 6, preimmune serum; lane 7 antitransferrin. Tr is the upper band while the lower band may be a degraded form. AFP, a-fetoprotein, Alb, albumin.

requirement of transferrin for the growth and maintenance of all cells which grow in serum-free defined media (Rizzino and Sato, 1978; Sato et al., 1979; Honegger et al., 1979) suggest that there may be a complex system of production and utilization. Transferrin as a marker of visceral endoderm cell type appears less useful than AFP because its production is sensitive to as yet undefined modulating conditions and because it is synthesized and secreted at a lower rate.

I am grateful for the advice and criticism of Dr. C. F. Graham at all phases of this work. Dr. M. Dziadek provided the embryoid body sections. This work was made possible by grants from the Medical Research Council and the Cancer Research Campaign to Dr. C. F. Graham. These studies were also partly supported by grants from the National Cancer Institute: CA 08293, P30 CA 30199, and PO1 CA 28896.

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REFERENCES ADAMSON, E. D., EVANS, M. J., and MAGRANE, G. G. (1977). Biochemical markers of the progress of differentiation in cloned teratocarcinema cell lines. Eur. J. B&hem. 79, 607-615. ADAMSON, E. D., GAUNT, S. J., and GRAHAM, C. F. (1979). The differentiation of teratocarcinoma stem cells is marked by the types of collagen which are synthesized. Cell 17,469-476. ADAMSON, E. D., and GRAHAM, C. F. (1980). Loss of tumorigenicity and gain of differentiated function by embryonal carcinoma cells. In “Results and Problems in Cell Differentiation” (R. McKinnell, ed.), Vol. 11, pp. 288-295. Springer-Verlag, Berlin. BATEMAN, A. E., and COLE, R. J. (1971). Stimulation of haem-synthesis by erythropoietin in mouse yolk-sac-stage embryonic cells. J. EmbryoL Exp. MmphoL 26,475-480. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. B&hem. 46, 83-88. BURKE, D. C., GRAHAM, C. F., and LEHMAN, J. M. (1978). Appearance of interferon inducibility and sensitivity during differentiation of murine teratocarcinoma cells in vitro. Cell 13, 243-248. DAVIS, B. J. (1964). Disc electrophoresis II. Methods and applications to human serum proteins. Ann. N. Y Acad. Sci. 121, 404-427. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med 99, 167-182. DZIADEK, M. (1978). Modulation of alpha-fetoprotein synthesis in the early postimplantation mouse embryo. J. Embryol. Exp. MorphoL 46, 135-146. DZIADEK, M. (1979) Cell differentiation in isolated inner cell masses of mouse blastocysts in vitro: Onset of specific gene expression. J. EmbryoL Exp. MwphoL 53, 367-379. DZIADEK, M., and ADAMSON, E. D. (1978). Localization and synthesis of alpha-fetoprotein in postimplantation mouse embryos. J. EmLnyoL Exp Morphor 43, 289-313. FAULK, W. P., and GALBRAITH, G. M. P. (1979). Trophoblast transferrin and transferrin receptors in the host-parasite relationship of human pregnancy. Proc. Rwy. Sot. London B 204, 83-97. GITLIN, D., and PERRICELLI, A. (1970). Synthesis of serum albumin, pre-albumin, a-fetoprotein, al-antitrypsin and transferrin by the human yolk sac. Nature (London) 223, 995-997. GRABAR, P., and WILLIAMS, C. A. (1953). Mdthode permettant l’etude conjugee des proprietes electrophoretiques et immunochimique d’un melange de proteines. Application au serum sanguin. B&him. Biophys. Acta 10, 193-194. GUSTINE, D. L., and ZIMMERMAN, E. F. (1972). Amniotic fluid proteins: Evidence for the presence of fetal plasma glycoproteins in mouse amniotic fluid. J. O&et. GynecoL 114, 553-560. GUSTINE, D. L., and ZIMMERMAN, E. F. (1973). Developmental changes in micro-heterogeneity of foetal plasma glycoproteins of mice. B&hem J. 132,541-551. HOGAN, B. L. M. (1980). High molecular weight extracellular proteins synthesized by endoderm cells derived from mouse teratocarcinoma cells and normal extraembryonic membranes. Develop. BioL 76,275285. HOGAN, B. L. M., COOPER, A. R., and KURKINEN, M. (1980). Incorporation in Reichert’s membrane of laminin-like extracellular proteins synthesized by parietal endoderm cells of the mouse embryo. Develop. BioL 80. 289-300. HOGAN, B. L. M., TAYLOR, A., and ADAMSON, E. D. (1981). Cell interactions modulate embryonal carcinoma cell differentiation into parietal or visceral endoderm. Nature (London) 291, 235-237. HONEGGER, P., LENOIR, D., and FAVROD, P. (1979). Growth and dif-

VOLUME

91, 1982

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