Synthesis of serum proteins by cultures of chick embryo yolk sac endodermal cells

DEVELOPMENTAL

BIOLOGY

100,

50-58 (1983)

Synthesis of Serum Proteins by Cultures of Chick Embryo Yolk Sac Endodermal Cells MARIAN Department

F. YOUNG’ AND NORMAN

of Animal Genetics and Genetics and Cell Biology Received November

W. KLEIN’

Section, University

1, 1982; accepted in revised

of Connecticut,

Storrs, Connecticut

06268

form June 24, 1983

Endodermal cells were isolated from yolk sacs of 3-day chick embryos and cultured for 6 days in Eagle’s minimal essential media plus 10% fetal calf serum. During this period cells rapidly lost their ability to synthesize DNA as judged by [‘Hlthymidine incorporation into DNA. In spite of this loss of DNA synthesis serum protein synthesis and secretion remained at a constant 45% of total protein synthesis and secretion. This was determined by immunoprecipitation of culture media using antibodies directed against embryonic chick serum proteins. Media were also analyzed for the synthesis and secretion of specific serum proteins using polyacrylamide gel electrophoresis. The relative synthesis and secretion of the individual serum proteins followed that previously observed in ouo with the exception of cY-globulin-a which became undetectable. When culture media were supplemented with ovalbumin or insulin the relative synthesis and secretion of certin specific serum proteins were altered. However, analysis of these same media samples showed that the total amounts of serum protein synthesis and secretion were unaffected.

nutrient protein breakdown within the yolk sac of the chick embryo (Hassell and Klein, 1971). When yolk sacs Serum proteins have been used extensively to follow were incubated in the presence of radioactive amino changes in the regulation of gene expression. For exacids, an analysis of the incubation medium by polyample, the synthesis of transferrin by cultured mouse acrylamide gel electrophoresis revealed that of the many hepatocytes has been shown to respond to hydrocortiproteins synthesized by the yolk sac only four proteins sone (Brown and Papaconstantinou, 1979) and the synhad been both synthesized and secreted (Kram and thesis of albumin by diabetic mice was found to be modKlein, 1976). Their identification as serum proteins was ulated by treatment with insulin (Peavy et aZ., 1979). based on their ability to comigrate with proteins drawn The’levels of circulating individual serum proteins have from the blood vessel of an embryo and by their mobeen observed to change during embryonic development lecular weights. Serum protein synthesis by yolk sacs for the chick (Sanders and Kline, 197’7; Weller, 1976), was observed as early as the primitive streak stage and rat (Tam and Chan, 1977), and mouse (Renfree et ak, was found to change with development and with changes 1975). In neoplastic transformation the synthesis of (Yin the nutrient milieu (Kram and Klein, 1976). In view fetoprotein has been observed for a variety of hepatoma of these findings it was proposed that changes in serum types (Alpert et al, 1968) as well as in tumors of germ protein synthesis leading to changes in their circulating cell origin (Abelev, 1974). Serum proteins have also relevels could provide stage-specific signals for developceived attention because of their nutritional and regmental events. ulatory activities. For example, they have been shown In a recent study the cells of the endoderm were found to affect the proliferation of cells in culture (Rudland to be the only cell layer of the yolk sac involved in serum et ah, 1978; Temin, 1972) as well as the growth and protein synthesis (Young et ah, 1980). The objective of development of cultured mammalian embryos (Steele the present study was to characterize further yolk sac and New, 1974; Klein et aL, 1978; Hsu, 1980). endodermal cells considering first their ability to synOur attention was drawn initially to serum proteins thesize serum proteins in culture and second their ability in an attempt to identify the products synthesized from to respond to various environmental factors under these conditions. INTRODUCTION

’ Present address: Laboratory of Developmental Biology and Anomolies, National Institute of Dental Research, National Institutes of Health, Bethesda, Md. 20205. * To whom correspondence should be addressed: Department of Animal Genetics and Genetics and Cell Biology Section, University of Connecticut, Storrs, Conn. 06268. 0012-1606/83 $3.00 Copyright All rights

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

MATERIALS

AND METHODS

Culture media Eagle’s minimal essential medium (Gibco) was stored in five times concentrated aliquots 50

YOUNG

AND KLEIN

Synthesis

at -20°C. Fetal calf serum was heat inactivated at 56°C for 30 min and stored at -20°C. Conalbumin and ovalbumin were isolated from fresh egg white as described by Rhodes et aZ. (1958) with modifications of Hassell and Klein (1971). These purified egg proteins were added directly to the culture medium and dilute NaOH was added to adjust the pH to 7.4. A stock solution of insulin was prepared by dissolving 1.0 mg crystalline bovine insulin (Sigma) in 0.3 ml 10 mM HCl (Granick et at., 1975) and adjusting the volume to 1.0 ml with Eagle’s minimal essential medium. All media were filtered through type HA Millipore filters and contained (mg/ ml) 0.66 streptomycin sulfate and 0.006 penicillin Gpotassium. Cell culture. Employing watchmaker’s forceps, the yolk sac endodermal cell layers were isolated as intact sheets from eggs that had been previously incubated for 3 days (Young et al., 1980). Although trypsin dissociation was attempted, brief treatment of the endodermal sheets by Vortex agitation was found sufficient to provide singlecell suspensions. When such suspensions were placed in culture dishes (Falcon No. 3002) containing Eagle’s minimal essential medium with 10% (v/v) fetal calf serum, few cells were found to attach to the culture dish. Because of the reported differences between endodermal cells located proximal (in the area vasculosa) and distal (in the area vitellina) to the sinus terminalis blood vessel (Bellairs, 1963; Mobbs and McMillan, 1979), the abilities of endodermal cells from each region to attach in culture was examined separately. Only endodermal cells isolated from the area vitellina were found to attach and spread in culture. This sharp demarcation at the sinus terminalis (the limit of peripheral mesodermal layer expansion) of ability to attach was dramatically demonstrated when an attempt was made to culture an intact continuous strip of tissue containing endoderm from both regions (Fig. 1). Because of this difference in attachment capability, all subsequent studies were conducted with endodermal cells derived from the area vitellina of the 3-day chick embryo. Endoderm cells were obtained by Vortex agitation of area vitellina tissue for 10 set within a solution of Tris-buffered saline (g/liter: 8.0 NaCl, 0.37 KCl, 0.1 Na2P04, 1.0 glucose, 3.0 Tris, pH 7.4). This mixture was then filtered through a 200-mesh nylon filter cloth (National Filter Media Corp., Hamden, Conn.) which allowed only single endoderm cells to readily pass. Cells were concentrated by centrifugation (1000 rpm for 3 min), resuspended in Eagle’s minimal essential medium with 10% fetal calf serum, counted by hemocytometer, and cultured in 60-mm culture dishes (No. 3002 Falcon) with 2.0 ml of the same medium at a density of 5 X lo5 cells/ ml. Following an initial 3 hr for attachment the medium was replaced. Subsequently the medium was replaced

by Endodemal

Cells

51

at 2-day intervals. Cultures were incubated at 37°C with 95% air plus 5% COz. DNA synthesis. Cells were cultured in the presence of 10 &i/ml [3H]thymidine (New England Nuclear, specific activity 20 Ci/mmole) for 4 hr on each day for 5 days. Zero time was considered the time point following the 3-hr period given for attachment. Aliquots of 100 yl hydroxyurea were added to 2.0 ml medium from a stock solution (2 Min Eagle’s minimal essential medium) to estimate nonmitotic DNA synthesis (Yager and Miller, 1978). Following radioactive thymidine exposure, cultures were extensively washed (Tris-buffered saline) and cells were scraped from plates and stored at least overnight in 95% ethanol at 4°C. DNA was extracted with 5% perchloric acid and aliquots were used for DNA quantitation with diphenylamine (Giles and Myers, 1964) using calf thymus DNA as the standard. The amount of radioactivity within DNA was determined using toluene-Triton-X-100 (0.2 g POPOP, 9.0 g PPO, 1 liter Triton X-100, 2 liters toluene) as scintillation fluor. Serum protein synthesis and secretion, Area vitellina endodermal cells were cultured in the presence of 10 &i/ml [3H]valine (New England Nuclear, specific activity 1.5 Ci/mmole) for 24hr intervals (zero time considered the time point after 3 hr attachment) for a total of 5 days with Eagle’s minimal essential medium. Effects of ovalbumin, conalbumin, or insulin supplementation were evaluated by exposure during O-48 hr culture. At 48 hr media were replaced with Eagle’s minimal essential medium containing 10 &i/ml radioactive valine for an additional 24 hr of culture. Media were centrifuged at 1000 rpm for 10 min and supernatant fluids were dialyzed for 24 hr against a solution containing 10 mMKCl,l.5 mMMgCl,, and 17.0 mM Tris-HCl, at pH 7.4 and 4°C for 24 hr. Samples were concentrated to approximately 1.0 ml with Sephadex G-200 and redialyzed for an additional 24 hr. Protein content of the samples was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. Immunological procedures. Antisera were prepared as previously described (Young et al., 1980). The amount of antiserum required to precipitate the maximum amount of radioactivity from a mixture of radioactive serum proteins was used to establish antigen antibody equivalence. Immunoprecipitations were carried out by mixing 50 ~1 radioactive culture medium plus 5.0 ~1 (20 pg) chick embryo serum protein as “carrier” with an appropriate volume of antisera (volume was usually 0.4 ml). Immunoprecipitation reactions were allowed to proceed for 1 hr at 37°C and then for 24 hr at 4°C. Immunoprecipitates were isolated by centrifugation at 2000 rpm for 10 min, washed three times in chick Ring-

52

DEVELOPMENTALBIOLOGY

VOLUME 100, 1983

Vitellina

\

/

Strip of endoderm isolated and placed in culture

v\ AREA VASCULOSA floats in media FIG. 1. Drawing with photograph of tissue illustrating

AREA VITELLINA will attach and spread in culture

a cultured strip of endoderm containing area vitellina and area vasculosa.

er’s solution, and then finally dissolved in 0.3 ml Protosol. Samples were then counted for radioactivity in 3.7 ml toluene-Omnifluor. Total protein radioactivity was determined by placing 50 ~1 radioactive culture medium plus 1.5 mg bovine serum albumin (Sigma) in 5% trichloroacetic acid overnight at 4°C. Samples were centrifuged at 2000 rpm and precipitates resuspended in fresh 5% trichloroacetic acid, heated to 9O”C, and then rinsed twice with 5% trichloroacetic acid. Precipitates were then dissolved in 0.3 ml Protosol and counted in 3.7 ml toluene-Omnifluor scintillation fluid. The synthesis and secretion of serum protein was then quantified by dividing the amount of immunoprecipitable radioactivity by the amount of trichloroacetic acid-precipitable radioactivity within each experimental sample. Two types of controls were performed to correct for nonspecific immunoprecipitation. First, serum from

nonimmunized rabbits was used to precipitate radioactive media samples. For all samples examined, the radioactivity within such precipitates never exceeded 4% of the total protein radioactivity within the reaction and averaged 2% for all samples examined. The second control involved the immunoprecipitation of a heterologous array of proteins that did not include chick serum protein. Such radioactive proteins were obtained by incubating rat embryos in vitro in the presence of rH]valine for 48 hr, followed by homogenization and centrifugation to obtain soluble protein. Immunoprecipitation of this protein with antisera to chick embryo serum showed only 2% of total protein radioactivity precipitated. The percentage of radioactivity precipitation in the controls was subtracted from each experimental value. Student’s t test was used to evaluate differences with P > 0.05 considered insignificant.

YOUNG

AND KLEIN

53

Synthesis bg Endodermal Cells

Polyacrylamide gel analysis. Culture media were analyzed directly by polyacrylamide gel electrophoresis as described by Davis (1964) with the modification of Kram and Klein (1976). A maximum of 200 pg protein were included in an equal volume of 2.5% acrylamide stacking gel and electrophoresed into a 7.5% cylindrical 85 X 5mm separating gel. Gels were run at 2 mA/gel tube in a running buffer of 7.5 mM Tris-glycine, pH 8.6, at 4°C until the tracker bromophenol blue was within 1 cm of the bottom of the tube. Gels were frozen in solid COP and cut into l.O-mm slices. Individual slices were dissolved in 0.3 ml 30% H202 overnight at 37°C and counted in 4.0 ml toluene-Triton-X-100 scintillation fluor. The relative synthesis of serum proteins was determined as previously described by Kram and Klein (1976). Specifically, radioactivity within each serum protein peak was corrected for differences between proteins in valine content and corrected values were expressed as a percentage of total radioactivity.

1 4

6-

-4

O RESULTS

t

DNA Synthesis

Synthesis

and Secretion

Cells were cultured in the presence of [3H]valine for the 24-hr periods of O-24 hr (Day l), 48-92 hr (Day 3) and 96-120 hr (Day 5) and media aliquots were either immunoprecipitated with antiserum to chick embryo serum proteins or precipitated with trichloroacetic acid. By comparing the amounts of radioactivity in these precipitates, it was possible to estimate the percentage of total protein synthesis and secretion attributed to serum proteins. Throughout the entire culture period, serum protein synthesis and secretion remained a highly active process. Synthesis and secretion ranged from 50.8 + 2.7% of total synthesis and secretion after the first day of culture to 45.8 f 4.7% after Day 3 and 41.5 ? 1.9% after Day 5. These decreases were not significant (P > 0.05).

I

I

I

I

2

3

4

5

J

DAYS IN CULTURE

Although mitosis and DNA synthesis have been observed for endodermal cells of the intact area vitellina (Bennet, 1973), mitotic figures were not observed once these cells were dissociated and placed in culture. Exposures to [3H]thymidine for 24-hr periods showed a rapid decline in DNA radioactivity from 540 dpm/ng DNA on Day 1 to only 60 dpm/ng DNA by Day 3 of culture (Fig. 2). This low level was comparable to that observed with hydroxyurea-treated cultures. Because hydroxyurea inhibits mitotic DNA synthesis only background levels or nonmitotic incorporation was detected in the presence of the drug. Total Serum Protein

I I

FIG. 2. DNA synthesis in the presence of hydroxyurea. Endodermal cell cultures were incubated in the presence of [3H]thymidine at O-4, 24-28, 48-52, 72-76, and 96-100 hr of culture counting 3 hr after the start of culture as zero time. One-half of such cultures received 100 mM hydroxyurea (dotted line) during the incubation period while the other half did not (solid line). DNA was then extracted and analyzed for radioactivity and DNA content for the determination of specific activity. Values represent single determinations.

Specific Serum Protein

Synthesis and Secretion

As previously described, endodermal cells were exposed to radioactive valine for 24-hr periods starting on Days 1, 3, and 5 of culture; but in this study media were analyzed by nondenaturing polyacrylamide gel electrophoresis. The relative synthesis of serum proteins on Day 1 of culture (labeled during the 0- to 24-hr period) was precisely the same as previously reported (Young et al., 1980) for the intact area vitellina (Fig. 3a and Table 1). On Days 3 and 5 of culture, however, changes were observed (Figs. 3b, c). Most striking were the increases in synthesis and secretion for albumin and the complete cessation for a-globulins-a and -b. Although slight, a consistent decrease in the synthesis and secretion of prealbumin was observed. Transferrin synthesis and secretion remained constant relative to the changes observed for the other proteins. These changes paralleled those observed in ova with the exception of a-globulin-a. The synthesis of a-globulin-a was found to increase in the yolk sac until Day 14 of incubation (Weller, 1976).

DEVELOPMENTALBIOLOGYVOLUMEloo,1983

54

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[al

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1

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1

A. I Day of

culture

240 -

200 -

i 0

160-

2 a

120

x

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I b I

t 80

50

60

70

80

90

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GEL SLICE

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240

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3 days of

6.

culture

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1 20

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I 70

SLICE

FIG. 3. Serum protein synthesis with time for area vitellina endodermal cell cultures. Cultures were incubated in the presence of [‘Hjvaline at the start of 1 (a), 3 (b), and 5 (c) days of culture for 24 hr. Incubation media were analyzed by nondenaturing polyacryamide gel electrophoresis. The serum proteins are labeled (a) transferrin, (b) a-globulin-a, (c) o-globulin-b, (d) albumin, (e) prealbumin, and (f) unidentified.

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55

Synthesis by Endodermal Cells

YOUNG AND KLEIN

C. 5 Days of culture

a 4 240 -

T

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200 i T 0

160-

, 1

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,

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120-

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SLICE

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1 70

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80

FIG. a--Continued

+ 1.0%; and for control cultures 59.1 + 1.1%. In the conalbumin studies the values were 5 mg/ml, 47.2 f 6.8%; The effects of ovalbumin and conalbumin on endo- 10 mg/ml, 44.7 + 4.2%; and for control cultures dermal cell cultures were studied because serum protein 42.2 + 4.8%. Differences between values for supplesynthesis by intact yolk sacs of chick embryo explants mented (either ovalbumin or conalbumin) and controls was shown to be altered by these proteins (Kram and were not significant (P > 0.05). Klein, 1976). Supplementing cultures with ovalbumin Media were also analyzed for specific serum protein or conalbumin during 0 to 48 hr did not appreciably synthesis using polyacrylamide gel electrophoresis. It influence total serum protein synthesis and secretion should be noted that although variability was apparent relative to total protein synthesis and secretion. The between experiments, analysis of culture plates in tripvalues for serum proteins relative to total proteins were, licate within an experiment showed differences for inat 10 mg/ml ovalbumin, 51.5 * 3.5%; at 20 mg/ml, 56.8 dividual proteins that did not exceed 4%. In four separate Ovalbumin

and Conalbumin

Supplements

TABLE

1

RELAT~VESYNTHESISANDSECRETIONOFSERUMPROTEINSBYENDODERMALCELLCULTURES Transferrin Day”

1 3 5

* (%)

a-Globulin-a

(%)

a-Globulin-b

(%)

Albumin

(%)

Prealbumin

(%)

Expt. 1

Expt. 2

Expt. 1

Expt. 2

Expt. 1

Expt. 2

Expt. 1

Expt. 2

Expt. 1

Expt. 2

33 44 40

36 48 45

24 0 0

21 0 0

15 0 0

23 0 0

6 34 47

10 42 49

20 22 13

10 11 6

“Endodermal cell cultures were incubated in the presence of [3H]valine for 24 hr on 1, 3, and 5 days of culture. Media were resolved by nondenaturing polyacrylamide gel electrophoresis. *Radioactivity within each serum protein peak was corrected for differences between proteins in valine content and corrected values were expressed as a percentage of total radioactivity.

56

DEVELOPMENTAL BIOLOGY

TABLE 2 SYNTHESIS AND SECRETION OF PREALBUMIN RELATIVE TO OTHER SERUM PROTEINS IN RESPONSE TO OVALBUMIN SUPPLEMENTATION~

Supplementation Ovalbumin (mg/ml) Control 15 Control 20 Control 10 20 Control 12.5 25.0

Expt. I dpm (%I

Expt. II dpm (%)

Expt. III dpm (%)

Expt. IV dpm (%)

Ob(O)C 4207 (16) 0 (0) 451 (20) 2354 (15) 3961 (26) 5640 (31) 0 (0) 3120 (13) 4426 (24)

a Endodermal cell cultures were supplemented with various concentrations of ovalbumin within Eagle’s minimal medium or Eagle’s minimal medium alone as control for 48 hr of culture. Cells were then incubated in the presence of [‘H]valine for 24 hr and media resolved by nondetergent polyacrylamide gel electrophoresis. bValue represents the actual quantity of radioactivity in dpm within the prealbumin region of the gel after electrophoresis of the various culture media. ‘Radioactivity for all serum proteins within a gel was corrected for differences between proteins in valine content before summation. The value in parenthesis represents the percentage of total corrected serum protein radioactivity that was prealbumin.

experiments ovalbumin was observed to increase synthesis of prealbumin in comparison to unsupplemented cultures. Further, it was possible to demonstrate a relationship between ovalbumin concentration and relative prealbumin synthesis (Table 2). The synthesis of transferrin and serum albumin by these ovalbumin-supplemented cultures, on the other hand, did not differ consistently from unsupplemented cultures. Conalbumin did not influence the relative synthesis of prealbumin or the other serum proteins. Insulin

Supplement

Similar to the ovalbumin and conalbumin experiments cultures were supplemented with insulin during O-48 hr and then incubated with Eagle’s minimal essential medium containing [3H]valine for 48-72 hr. Media were analyzed again for total serum protein synthesis and secretion as well as specific serum protein synthesis. At concentrations of insulin ranging from 1 X lo-’ to 1 X 1O-5M neither total nor specific serum protein synthesis and secretion was affected. When the concentration was increased to 1 X 10m4M insulin, total serum protein synthesis and secretion was still unaltered but a change in the relative synthesis of individual proteins was observed. Insulin appeared to increase the relative synthesis and/or secretion of prealbumin and decrease that of albumin (Table 3).

VOLUME 100. 1983 DISCUSSION

In the present investigation area vitellina endodermal cells were cultured under conditions which permitted some temporal changes to occur in expression of serum protein synthesis and secretion which parallel those observed in ouo and to respond to some environmental factors previously shown to affect serum protein synthesis by the intact yolk sac (Kram and Klein, 1976; Grieninger and Granick, 1975). On the other hand, cells of the area vitellina endoderm have high proliferative capacity in ova (Bennet, 1973), but this characteristic was lost when the cells were placed in culture. This conclusion was supported both by a decrease in [3H]thymidine incorporation into DNA to background levels by Day 3 of culture and by the absence of mitotic cells. The synthesis of serum protein was measured quantitatively and qualitatively with time in culture. Throughout a culture period of 6 days, the absolute quantity of serum protein synthesized and secreted into the media did not change appreciably, remaining between 40 and 50% of the total protein synthesized and secreted into the media. The relative synthesis of some TABLE 3 SYNTHESIS AND SECRETION OF ALBUMIN AND PREALBUMIN IN COMPARISON TO OTHER SERUM PROTEINS IN RESPONSE TO INSULIN Albumin Concentration W)” Expt. I Control 1.0 x 1o-5 2.5 X 1O-5 5.0 x 1o-5 1.0 x lo-’

(dpm)

7404s 2711 8919 6743 4073

Expt. II Control 5.0 x 1o-5

12421 5924

Expt. III Control 1.0 x 1o-4

2479 3935

Prealbumin (So)

(dpm)

(%I

(44)c (43)

0 0 1767 9151 2690

(0) (0) (5)

(28) (20)

(32)

0 3272

(0) (18)

(50) (32)

0 1993

(0) (23)

(27) (21) (29)

(6-i’)

a Endoderm cultures were supplemented with various concentrations of insulin with Eagle’s minimal medium or Eagle’s minimal medium alone as control for 48 hr. Cells were then pulsed for 24 hr in the presence of rH]valine in Eagle’s minimal medium and the media finally analyzed by nondetergent polyacrylamide gel electrophoresis. b Values represent actual amount of corrected radioactivity within albumin or prealbumin region of gels after electrophoresis. c Radioactivity within serum protein peaks was corrected for differences in valine content and then summated. Corrected radioactivity within albumin and prealbumin was divided by the total corrected serum protein radioactivity to give the numbers in parentheses.

YOUNG AND KLEIN

Synthesis

but not all serum proteins, however, changed dramatically. Specifically, the synthesis of albumin increased, and prealbumin decreased but transferrin remained constant. For all serum proteins except a-globulin-a, these changes in relative synthesis paralleled precisely those which take place in ova (Weller, 1976; Sanders and Kline, 1978). Although a-globulin-a has been found to be synthesized in increasingly larger quantities with development (Lingren, 1974), its synthesis was not detected after 1 day of culture. Thus, the synthesis of this protein may have required some factors not present within the culture environment. It is also possible that a-globulin-a synthesis by chick extra embryonic endoderm is regulated by some factors requiring interaction with the embryo or interactions with extra embryonic tissues, as has been reported for the yolk sac of the mouse (Dziadek, 1978). Because the egg white proteins ovalbumin and conalbumin had previously been shown to affect serum protein synthesis in the intact yolk sac (Kram and Klein, 1976), these proteins were used to determine whether isolated cells would respond in the same manner. In the previous study, yolk sacs from whole chick embryo explants were cultured in the presence of an ovalbuminconalbumin mixture and found to synthesize more transferrin than those cultured on whole egg medium. When the same combination was added to isolated cultures of endoderm cells no difference in the relative synthesis of serum protein was detected. For this reason, individual supplements of these two nutrient proteins were examined. While no quantitative changes in the percent total synthesis of serum protein could be detected in these cultures, acrylamide gel analysis of the individual proteins synthesized showed that ovalbumin but not conalbumin consistently increased the synthesis of prealbumin. Thus, endodermal cells appear to respond to certain proteins but the precise nature of the response appears influenced by the state of the cells. The effect of insulin on serum protein synthesis and secretion by cultured vitellina cells was examined because this hormone was previously shown to stimulate albumin synthesis in cultures of amphibian liver cells (Stanchfield and Yager, 1977), chick embryo liver cells (Grieninger and Granick, 1976), and in the intact rat (Peavy et ah, 1979). Immunoprecipitation of incubation media from endodermal cell cultures supplemented with high doses of insulin showed that comparable amounts of serum protein per total protein were synthesized and secreted. Acrylamide gel analysis of the incubation media, however, indicated that insulin-supplemented endoderm cells synthesized relatively less albumin and more prealbumin. The vitellina endoderm cell culture system offers a

by Endodermal

57

Cells

useful system in which to study developmental regulatory mechanisms. Large quantities of cells capable of synthesizing and secreting cell-specific products may be easily isolated and cultured. Furthermore, the synthesis and secretion of serum protein by the cultured cells changes in a manner comparable to that which takes place during development in ova and agents exogenously added to the medium alter the synthesis and/or secretion of serum proteins by these cells. Thus, by manipulating the environment of these cells it may be possible to identify a variety of factors that play a role in their differentiation. We thank Ms. Clare Chatot and Ms. Helen Fish for technical assistance and Ms. Mary Jane Spring for illustrations. This report is scientific contribution No. 979, Storrs Agricultural Experiment Station, The University of Connecticut, Storrs, Connecticut 06268. This work was supported by the U. S. Department of Energy Contract EV03139 (Office of Health and Environmental Research).

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58 (1978). Serum protein

DEVELOPMENTAL BIOLOGY depletion

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