DEVELOPMENTAL

Chondroitin

BIOLOGY

X$367-377

(19761

Sulfate Synthesis by Mouse Embryonic, and Teratoma Ceils in Vitro

JEROME CANTOR,"" Institute of Molecular

’ Roche

Nutrition,

STANLEY

S. SHAPIRO,~ AND MICHAEL

Biology, Nutley, Roche Research Accepted

Extraembryonic, I. SHERMAN"~

New Jersey 07110; and 1 Department Center, Nutley, New Jersey 07110 January

of Biochemical

20,1976

Sulfated glycosaminoglycan (GAG) synthesis by primary cultures of embryo, yolk sac, and trophoblast was compared with synthesis by the same tissues in utero. In general, the in uivo and in vitro results were in good agreement. As was the case in uiuo, the three tissues synthesized chondroitin-4-sulfate and chondroitin-6-sulfate (but no dematan sulfate) at characteristic ratios. Cultured embryos are already capable of synthesizing chondroitin sulfates, primarily chondroitin-4-sulfate, before, or at, the 64-cell stage. During the attachment and initiation of outgrowth stages, blastocysts synthesize more chondroitin-6-sulfate than chondroitin-4-sulfate. Thereafter, progressively more chondroitin-4-sulfate is synthesized so that the 4:6 ratio increases, resembling that of tropboblast cells. Blastocyst-derived cell lines and teratoma cell cultures were also studied. One blastocystderived line, MB4, synthesized GAG with a pattern similar to that of yolk sac, which it resembles biochemically in other respects as well. The GAG profile of MBZ, a parietal endoderm-like cell line resembled neither that of embryo, yolk sac, nor trophoblast cells. Embryonal carcinoma (undifferentiated teratomal cells had a chondroitin sulfate pattern different from that of most of the other cultures. INTRODUCTION

phological (Hsu et aZ., 1974), biochemical (see Sherman, 1974, for a review), and functional (Salomon and Sherman, 1975; Sherman and Salomon, 1975) criteria, we undertook to compare in vitro GAG production by embryonic and extraembryonic cell types with the pattern already obtained in vim. We have found that embryo, yolk sac, and trophoblast all have their own characteristic patterns of chondroitin sulfate synthesis, and that there is, in most cases, a good correlation between in vivo and in vitro patterns. With this in mind, we have analyzed GAG production by both primary and established (but not conclusively identified) blastocyst cultures, and compared the results with those obtained with midgestation tissues. Finally, since numerous analogies exist between embryonal carcinoma (undifferentiated teratoma) cells and early embryonic cell types (see Damjanov and Solter, 1974, and Martin, 1975, for reviews), we have also carried out studies to determine

Because relatively little was known about sulfated glycosaminoglycan (GAG) synthesis during mammalian embryogenesis, we recently undertook to characterize patterns of GAG production in midgestation mouse embryonic and extraembryonic tissues (Shapiro and Sherman, 1974). We found that embryo proper, yolk sac, and trophoblast all produced chondroitin-4-sulfate (ch-4-S) and chondroitin-g-sulfate (ch6-S) as well as heparin and/or heparan sulfate (hep-S), but little or no dermatan sulfate. Furthermore, each of the three tissues produced the three species of GAG in different, characteristic ratios. Since mouse embryos cultured from as early as the two-cell stage develop in a manner similar to that in uivo on the basis of mor,’ Present address: Department of Pathology, Columbia College of Physicians and Surgeons, New York, New York 10032. -I Author to whom requests for reprints should be addressed. 367 Copyright All

rights

0 1976 by Academic Press, of reproduction in any form

Inc. reserved.

368

DEVELOPMENTAL BIOLOGY

whether this relationship GAG synthetic profiles. MATERIALS

AND

extends to their METHODS

Enzymes and chemicals. Carrier-free [35S]H,S0, was obtained from New England Nuclear Corp. (Boston, Mass.). Chondroitin-C&fate, chondroitin-6-sulfate, dermatan sulfate, 2-acetamido-2-deoxy-30- [ P-n-gluco-4-enepyranosyluronic acidl4-0-sulfo-Dgalactose (ADi-4s) and 2-acetamido - 2 - deoxy - 3 - 0 - [p - D - gluco - 4 enepyranosyluronic acid]-6-O-sulfo-n-galactose (ADi-6s) were obtained from Miles Laboratories (Kankakee, Ill.). Chondroitinase ABC (chondroitin ABC lyase, EC 4.2.2.4) and AC (chondroitin AC lyase, EC 4.2.2.5) were obtained from Miles Laboratories and from Sigma Chemicals (St. Louis, MO.). Papain and bovine testicular hyaluronidase were obtained from Worthington Biochemicals (Freehold, N.J.). Cetyl pyridinium chloride was obtained from Sigma Chemicals. Culture reagents. NCTC-109 medium and fetal calf serum were purchased from Microbiological Associates (Bethesda, Md.). Phosphate-buffered saline (PBS; solution A of Dulbecco), Dulbecco-modified Eagle’s (DME) medium, sulfate-free (MgCl, substituted for MgSO,) Eagle’s Basal (BME-S) medium, and trypsin (0.05%)-EDTA (0.02%) were purchased from Gibco (Grand Island, N.Y.) Preparation of cells. SWRIJ female and SJL/J male mice were obtained from Jackson Laboratories, Bar Harbor, Me. For midgestation tissues, random-mated mice were checked daily for sperm plugs. The day of observation of the sperm plug is considered the first day of pregnancy. On the tenth or eleventh day, embryo proper, yolk sac, and trophoblast were collected in PBS as described previously (Sherman, 1972). The tissues were washed in PBS and disaggregated with trypsin-EDTA in the presence of DNase at 37°C for 10 min as described for primary trophoblast cultures (Salomon and Sherman, 1975). In all

VOLUME 50, 1976

cases, single cells and small clumps of cells were present. The cells were plated at moderate to high densities in 60-mm tissue culture dishes (Falcon Plastics, Oxnard, Calif.) in NCTC-109 medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics (Sherman, 1975a, 1976). For preimplantation and postblastocyst cultures, SWR/J female mice were superovulated (Runner and Palm, 1953) and mated, and embryos were collected either from oviducts (2-cell embryos) or from uteri (blastocysts). Two-cell embryos were cultured in WB medium (Whitten and Biggers, 1968), and blastocysts were cultured in supplemented NCTC-109 medium as described elsewhere (Sherman, 1975a, 1976). Blastocyst-derived cell lines MB2 and MB4 (Sherman, 1975a,b) were cultured in supplemented NCTC-109 medium. A pluripotent embryonal carcinoma cell line, PCC4.azal (Jakob et al., 1973) was either maintained in an undifferentiated state in DME medium supplemented with 15% fetal calf serum and antibiotics, or induced to differentiate by culture in a bacterial petri dish and then transferred to a tissue culture dish containing supplemented NCTC-109 medium (Sherman, 1975c). Incorporation of 13”SIH,S0,. Prior to incubation with labeled sulfate, cultures were washed to remove debris and fresh medium was added. In some cases, as indicated, BME-S medium was substituted for NCTC-109. Cultures were incubated with lo-20 FCi [““SlH,SO, for various lengths of time. After incubation, media were collected, and each dish was washed three times with PBS. The cells were then resuspended in PBS and dislodged from the culture dish with a rubber policeman. The cells and the medium plus washes were centrifuged at 15OOg for 5 min. The supernates were pooled. The cell pellet was resuspended in PBS and homogenized. Preparation of [35S]GAG. The combined media-wash fractions were dialyzed for 24 hr against 10 liters of 0.1 N ammonium

CANTOR,

SHAPIRO

AND SHERMAN

GAG Synthesis

by Embryonic

Tissues

369

with either chondroitinase ABC or chonsulfate to remove unincorporated radioacdroitinase AC as described above. As a tive sulfate. In some experiments, after control, a sample of 13”S]GAG was treated counting aliquots , the dialyzed medium as above, except that incubation with hyaand the cells were further processed sepaluronidase treatment was omitted. This rately, while in others the two fractions treatment did not alter the susceptibility were pooled. Papain digestion, followed by of the GAG to chondroitinase hydrolysis. trichloroacetic acid precipitation of undigested polypeptides and dialysis against RESULTS water were then carried out as described Cultures previously (Shapiro and Sherman, 1974). Primary Midgestation After the addition of carrier chondroitin Cultures were incubated with radioacsulfates to the dialysate (final concentrative sulfate beginning on the secoizd to tion of 1 mg/ml), GAG material was presixth days in vitro. The duration of the cipitated with 3 vol of ethanol. After 24 hr exposure varied between 24 and 90 hr. Neiat 4”C, GAG were resuspended in water in ther of these factors had any qualitative preparation for analysis. effects upon the pattern of [3”SlGAG synAnalysis of [35S]GAG. Digestion of the thesis. In some cases, BME-S was substi[““S]GAG with chondroitinase ABC and tuted for NCTC-109 during the incubation AC according to the procedure of Saito et period. Due to the higher specific activity al. (1968) was modified from our previous of :WO, when the former medium was method (Shapiro and Sherman, 1974) in used, the number of W counts per minute that a larger incubation volume (260 ~1 vs incorporated per milligram protein in 70 ~1) and twice the amount of enzyme (0.3 BME-S (l-7 x 10” cpm/mg protein) was units instead of 0.15) were used. The incufour- to sixfold higher than in NCTC-109. bation period (18 hr at 37°C) was the same. However, this did not significantly alter Analysis of the products (ADi-4S, ADi-GS, the proportions of different types of P5Sland undigested material) by chromatograGAG synthesized. Consequently, the data phy was exactly as described by Shapiro obtained for each tissue in several experiand Sherman (1974). Nitrous acid degraments have been considered as a single dation of [3”S]GAG and chromatographic class in Tables 1 and 2. analysis of the products were carried out Unlike the in uivo situation, in which as described in the same communication. TABLE 1 For combined hyaluronidase-chondroDISTRIBUTION OF PSlGAG IN CELLS AND MEDIUM itinase analysis, the [3”S]GAG preparafe;;~ Average percentCulture tions were treated with 15 units of bovine age cpm in testicular hyaluronidase for 4 hr at 37°C in deterMeminaCells 0.02 M phosphate buffer pH 5.5, 0.08 M dium tions” -__ NaCl, 0.001 M MgCl, and 0.01% bovine Embryo 5 35.4 64.6 serum albumin. After incubation, 0.5 mg Yolk sac 5 33.5 66.5 of dermatan sulfate was added and undiTrophoblast 5 7.1 92.9 gested GAG was precipitated with 5 mg of MB2 1 6.2 93.8 cetyl pyridinium chloride. Cetyl pyridinMB4 1 12.2 87.8 ium chloride was removed from the GAG by repeated precipitation of the latter in a 65.4 34.6 PCCl.azal (undiffer3 saturated solution of KCNS in alcohol entiated) (Scott, 1960). The precipitated GAG was PCC4.azal 52.5 47.5 (differen2 tiated) then washed with ethanol, resuspended in “enriched” Tris buffer (Saito et al., 19681, ” A different culture was used for each determination. split into two equal portions and treated

370

DEVELOPMENTAL

BIOLOGY TABLE

CHONDROITINASE Cell twe

NUllIber of assayrtl

ABC

DIGESTION

VOLUME

50,

1976

2

OF [Y3lGAG

PREPARATIONS

In Vitro

In vitm Percenta e ADi-4S & s D

Percenta e ADi-6S 2 8 D

AND In Vivo

In vivd Total percentage hydrolyzed f SD

ch-4-S C~-B-S

Percentage ADi-4S

Percentage ADi-6S

Total percent-

Ch-4-S Ch-6-S

h; f :olyzed Embryo Yolk sac Trophoblast

i

33.2 2 5.2 26.9 k 6.0 67.7 k 9.9

17.0 ? 4.3 8.1 * 5.7 7.5 2 4.2

50.2 k 4.7 35.0 * 7.4 75.2 + 9.8

2.0 3.3 9.0

MB2 MB4

2 2

10.5 * 3.4 22.5 k 1.5

13.6 k 7.4 6.3 ? 4.9

24.1 k 10.8 28.8 -+ 6.4

0.8 3.6

(undiffer-

2

12.6 ? 2.1

10.2 * 3.7

22.8 k 1.6

1.2

(differen-

2

16.4 2 3.8

10.4 * 4.7

26.8 +- 8.5

1.6

PCC4.azal entiated) PCCl.azal tiated)

6

a A different culture was used for each determination. calculations for the in vitro values. h Data from chondroitinase ABC digestions in Shapiro

[:‘“SIGAG

31.5 a.7 39.7

26.6 3.1 2.3

58.1 11.8 42.0

from both cells and media have been included

and Sherman

1.2 2.8 17.3

in the

(1974).

the relative amount of :YSO, incorporated AC (see Methods). By determining the into nondialyzable material per milligram amount of material hydrolyzed by chonwet weight for midgestation em- droitinase ABC, but not by chondroitinase AC, it was calculated that less than 5% of bryo:trophoblast:yolk sac was 2.2:1.2:1.0 the [““S]GAG synthesized by any midges(Shapiro and Sherman, 19741, both yolk tation tissue in culture could have been sac and trophoblast cultures incorporate more YSO, per milligram protein than do dermatan sulfate. The same was found to be the case for MB2, MB4, and PCC4.azal embryo cultures (embryo:trophoblast:yolk cultures. These results are, therefore, in sac:: 1.0:3.5:2.3). There is also a difference in the proportion of nondialyzable “5S-con- agreement with the observation in in vivo taining material in cells and medium (Ta- studies that embryonic and extraemble 1): while both embryo and yolk sac bryonic tissues do not synthesize signifirelease two-thirds of the synthesized la- cant amounts of dermatan sulfate. beled material into the medium, more The results of chondroitinase ABC degradation are analyzed in Table 2. For comthan nine-tenths of the [““SIGAG produced by trophoblast cells are secreted. parative purposes, data obtained previWhen the [““SIGAG preparations were ously in in vivo experiments (Shapiro and treated with chondroitinase ABC, in every Sherman, 1974) are included in the same case both ADi-6S and ADi-4S were ob- table. In general, the in vivo and in vitro tained. The former indicates that ch-6-S results are consistent: yolk sac and trophoblast [““Slchondroitin sulfates are dehad been synthesized. The latter product graded mainly into ADi-4S, indicating could have been obtained from degradathat ch-4-S is the primary species synthetion of either ch-4-S or dermatan sulfate. To distinguish between the two, GAG sized. In the embryo preparation, although ch-4-S is predominant, proportionately preparations were exhaustively treated with testicular hyaluronidase. This en- more ch-6-S is present than in yolk sac or zyme degrades ch-4-S and ch-6-S but not trophoblast. This is reflected in the ch-4dermatan sulfate or hep-S (Mathews, 1966; S:ch-6-S ratios: the embryo preparations Meier and Hay, 1973). Undegraded mate- have a relatively low ratio both in vivo and rial was then recovered, split in half, and in vitro, the yolk sac ratio is about 3 in ch-4treated with either chondroitinase ABC or both cases, while the trophoblast

GAG Synthesis

CANTOR, SHAPIRO AND SHERMAN

S:ch-6-S proportions are substantially higher. Both yolk sac and trophoblast cultures appear to synthesize relatively more ch-6S than the same cell types do in uiuo. A possible explanation of this discrepancy is apparent when the L3”SlGAG profiles of cells and media are analyzed separately (Table 3). In the instance cited, the ch-4S:ch-6-S ratios are similar in the cells and in the medium of the embryo preparation. On the other hand, no ch-6-S is detected in the [““S]GAG associated with trophoblast and yolk sac cells; all the ch-6-S synthesized has been secreted. In in uiuo studies, the values obtained reflect only synthesized GAG that is associated with the cells, and do not take released GAG into account. Another discrepancy is that in yolk sac and trophoblast cultures, a substantially higher fraction of the incorporated 3”S0, is in chondroitinase-sensitive material than is the case with in uiuo tissues. This may again be explained by the fact

Culture

Embryo Yolk sac Trophoblast

Preimplantation Embryo Blastocyst Cultures

Initial

stage

and

Primary

Incubation of preimplantation and in uitro postimplantation embryo cultures with l”SlH,SO, revealed that GAG synthesis could be detected during all stages of in vitro development (Table 4, Fig. 1). Twocell embryos cultured in WB medium for 72 hr develop to the blastocyst stage (ca. 64 cells). During this time, GAG synthesis is taking place. Although the extent of incorporation of :YSO, into GAG is very low, analysis of chondroitinase ABC-treated 3

OF [““SIGAG

IN CELM AND MEDIA

Cells

-

Medium

Percentage ADi4s

Percentage ADi6s

Total percentage hydrolyzed

Ch-4-S -Ch-6-S

Percentage ADi4s

Percentage ADi6s

Total percentage hydrolyzed

Ch-4-S Ch-6-S

13.2 26.7 15.3

6.2 0 0

19.8 26.7 15.3

2.1 -

38.9 27.9 73.2

14.7 6.1 4.6

53.6 34.0 77.8

2.6 4.6 15.9

TABLE QUANTITATION

371

Tissues

that the secreted [““SIGAG is proportionately more chondroitinase-sensitive than the cellular [““SIGAG (Table 3). However, this explanation may not be totally adequate, since the in vitro embryo sample, which has about the same proportion of chondroitinase-sensitive [YS]GAG as it does in uiuo (Table 2), nevertheless also secretes [“S]GAG proportionately rich in chondroitinase-sensitive material.

TABLE ABC DIGESTION

CHONDROITINASE

by Embryonic

AND ANALYSIS

Number of embryos

1123 903 380 210 210

OF 1”“SIGAG

tXi,“h” wo, added 1’ 1’ 1’ 5 12

Duration of incu“?l% 72 72 185 72 72

4

SYNTHESIZED CULTURES

2-CelP Blastocystb Blastocyst” Blastocyst! Blastocysti fl Cultured in WB medium. h Cultured in NCTC-109 medium. c Isotope added at the beginning of the culture ” Not determined.

‘% cpm incorporated/hr/ embryo 0.7 - ‘1 6.0 17.7 52.3

period.

BY PREIMPLANTATION

AND POSTBLASTOCYST

From chondroitinase L;‘;;;;

ABC analysis:

4s

Percentage ADi6s

Percentage hydrolyzed

Ch-4-S -Ch-6-S

37.7 11.8 36.5 61.8 58.7

12.6 19.6 18.9 12.0 7.1

50.3 31.4 55.4 73.8 65.8

3.0 0.6 1.9 5.2 8.3

----

372

DEVELOPMENTAL BIOLOGY

VOLUME 50. 1976

During this period, low levels of GAG are being synthesized, although here more ch6-S than ch-4-S is being made (Fig. 1B). A relatively high percentage of 35S0, is incorporated into chondroitinase ABC-resistant material (Table 4). With increasing time in culture, the pattern shifts again (Figs. lC-E) so that between 12 and 15 days in vitro, there is almost no evidence of ch-6-S synthesis. The extent of incorporation of 35S0, increases dramatically with increasing age of the culture. This is to be expected, since there is also a substantial increase in cell number throughout this period. ”

FRACTIONS

r

FIG. 1. Chromatography of chondroitinase ABC digested material from preimplantation and postimplantation embryo GAG preparations. Cultures were incubated with [%lH,SO, for 72 hr except in C, where the incubation period was 185 hr. [%]GAG was purified from pooled cells and medium, digested with chondroitinase ABC, and chromatographed in butanol-acetic acid-ammonia as outlined in Methods. The undigested GAG remains at the origin (O), and is not shown here. In every case, background counts from chromatographs of undigested GAG have been subtracted from the experimental profiles. The position of ADi-6S and ADi-4S markers are shown. F is the solvent front. The number of embryos used and the stage at which labeled sulfate was added are as follows: (al 1123,2-cell (second day) embryos; (b) 903, fourth day blastocysts; (cl 380, fourth day blastocysts; (d) 210, fourth day blastocysts after 4 days in culture; (el 210, fourth day blastocysts after 11 days in culture. Further details are given in Table 4.

preparations clearly reveals the production of ADi-4S (Fig. 1A) and, consequently, the presence originally of ch-4-S. Small amounts of ch-6-S might also have been present, although the number of counts cochromatographing with the ADi-6S marker is very low. When blastocysts are removed from the uterus on the fourth day cf pregnancy and cultured in NCTC-109 medium, during the following 72 hr they hatch from the zona pellucida, attach to the culture dish and the trophoblast cells begin to outgrow (see, e.g., Salomon and Sherman, 1975; and Sherman, 1975a).

Blastocyst-Derived Lines

and

Teratoma

Cell

A number of blastocyst-derived cell lines have been generated and partially characterized (Sherman, 1975a, b). Two such cell lines, an epithelioid type designated MB2 and a fibroblastic type designated MB4 have been analyzed for their patterns of GAG synthesis. In both cases, the vast majority of the [35SlGAG is secreted into the medium by the cells (Table 1). Chondroitinase analysis reveals that MB4 cultures synthesize [35SlGAG with a pattern remarkably similar to that of yolk sac cells (Table 2). MB2 cells, however, synthesize more ch-6-S than ch-4-S, a property in common only with early blastocyst cultures (Table 4) of all the cell types assayed in this study. Teratoma cells can also be derived from blastocysts, although to date, the embryos must be cultured in vivo to yield multipotential teratoma cells (see Damjanov and Solter, 1974, and Sherman, 1975b, for reviews). We have analyzed [““SIGAG synthesis by teratoma cells. The line used, PCC4.aza1, gives rise only to embryonal carcinoma under ordinary culture conditions (Jakob et al., 1973). When cultured in bacterial petri dishes, to which the cells do not adhere, multicellular aggregates are formed. These aggregates, when returned to regular tissue culture dishes,

CANTOR, SHAPIRO AND SHERMAN

give rise to mixed cultures containing both embryonal carcinoma cells and a variety of differentiated cell types. By culture at high densities in NCTC-109 medium, the proportion of embryonal carcinoma cells present can be sharply reduced (Sherman, 1975c). Cultures containing either undifferentiated or primarily differentiated cells were incubated with [“‘SlH,SO,. In both cases, a substantial portion of the [‘{“SIGAG synthesized is retained by the cells (Table 1). This is different from the observations with in vitro blastocyst-derived cells. The pattern of GAG synthesized by teratoma cells (Table 2) also differs from that of MB2 and MB4, particularly the latter; on the other hand, there was not a substantial difference between the GAG patterns in embryonal carcinoma and differentiated teratoma cell cultures. Nature of the Chondroitinase-Resistant 1WIGAG In our previous studies on synthesis of we GAG by embryonic tissues in Go, found that embryo, yolk sac, and trophoblast all synthesized material that was insensitive to chondroitinase ABC digestion (Shapiro and Sherman, 1974). From nitrous acid analysis, we concluded that virtually all of this material was heparin and/or hep-S. As illustrated in Tables 2 and 4, from 25 to 75% of the GAG synthesized by the various cultures was insensitive to chondroitinase. Nitrous acid analyses similar to those carried out previously indicate that all midgestation cultures, as well as blastocyst-derived and teratoma cell lines, synthesize heparin and/or hepS. In these cases, however, the chondroitinase ABC and the nitrous acid digestible material did not together equal the total amount of [:‘“S]GAG, even when increased amounts of chondroitinase were used and when the incubation period with nitrous acid was prolonged. Usually, lo-20% of the total L”“S]GAG was resistant to both treatments. At present, the nature of this resistant material is uncertain. A similar

GAG Synthesis

by Embryonic

Tissues

373

proportion of hyaluronidase-, chondroitinase-, and nitrous acid-resistant material was recently found in GAG synthesized by cornea1 flbroblast cultures by Conrad and Dorfman (1974). This material (which was not keratan sulfate) appeared to be undersulfated. We have noted previously that some of the GAG synthesized by embryonic and extraembryonic tissues in viva was undersulfated (Shapiro and Sherman, 1974); although incompletely sulfated chondroitin sulfates would be expected to be cleaved by chondroitinases, it is probable that undersulfated hep-S would be only partially degraded in nitrous acid. The undegraded segments might be large enough to be retained at the origin during chromatography in the isobutyric acid:ammonia system (see Shapiro and Sherman, 1974), and would thus not be included in the calculation of the amount of hep-S. DISCUSSION

The results of this study indicate that primary embryo, yolk sac, and trophoblast cell cultures continue to synthesize GAG with patterns characteristic of those observed in viva even after 6 or more days in vitro. The variations from one experiment to the next in the proportions of the different GAG species synthesized by each tissue are small, as indicated by the standard deviations in Table 2. This is somewhat surprising, since one might expect a fair amount of heterogeneity in the midgestation cultures, especially in the case of the embryo, which contains many different cell types. In a study of 10 cell lines, Saito and Uzman (1971) reported that all of them synthesized dermatan sulfate. In our studies, however, none of the cultures produced significant amounts of this species of GAG. This is consistent with observations on frog embryos (Kosher and Searls, 1973) and primary rat and mouse embryo cultures (Dietrich and Montes De Oca, 1970). It should be noted that in the latter study, Dietrich and Montes De Oca did not detect

374

DEVELOPMENTALBIOLOGY

dermatan sulfate synthesis by HeLa and L cells as did Saito and Uzman. In our experience, some chondroitinase AC preparations are not as active on ch-4-S as is chondroitinase ABC, leading to the incorrect impression that dermatan sulfate is present in the GAG sample (Shapiro and Sherman, unpublished observations). This may be the reason for the discrepancy between the results of Saito and Uzman (1971) and Dietrich and Montes De Oca (1970), and explains why in our analyses for dermatan sulfate, we have resorted to treating our preparations with testicular hyaluronidase before exposing them to chondroitinases ABC and AC. It is of interest that while the ch-CS:ch6-S synthesis ratios in the midgestation and other cell cultures studied varied from less than 1:l to almost lO:l, and while the proportion of chondroitinase-resistant 13”SlGAG synthesized varied from 2575%, the percentage of ch-6-S synthesized in all of the cultures studied was only between 6 and 20% (Tables 2 and 4). We cannot at present explain this relatively small variation in the ratio of ch-6-S: total GAG synthesized. This phenomenon may be restricted to embryo- and teratoma-derived cultures, since Saito and Uzman (1971) did not find this relationship in a variety of established cell lines. Saito and Uzman (1971) also observed a large variation among the cell lines they studied with respect to the amount of synthesized GAG secreted into the medium. Similarly, in our study we found that while trophoblast and MB2 cultures secreted more than 90% of the synthesized GAG, differentiated teratoma cells secreted only half of their [33S]GAG, while the other cell lines were intermediate (Table 1). The active secretion of GAG by trophoblast, MB2, and, to a slightly lesser extent, MB4, may be related to the production by these cells of specialized extracellular materials. Trophoblast cells are known to secrete an abundance of a mucoprotein called “fibrinoid” by Kirby et al. (1964).

VOLUME 50, 1976

MB2, which conains a large proportion of parietal endoderm-like cells (Sherman, 1975b), secretes a material which resembles Reichert’s membrane (see Pierce et al., 1962), particularly in tumor form (M. I. Sherman, R. A. Miller, and C. Richter, manuscript in preparation). Although early in the derivation of MB4, an extracellular matrix was in evidence in the cultures (Sherman 1975b), the cells at the present time do not produce conspicuous levels of this material. On the other hand, MBCderived tumors do produce significant quantities of a periodic acid-Schiff stain positive, reticulum stain positive, extracellular matrix (Sherman, Miller, and Richter, in preparation). Such material may also be secreted in culture, but below the levels of detection by inverted phase microscopy. Whatever the reason for, and the nature of, the GAG secreted by the cells under study, the data in Table 3 make it clear that the secretion is not a representative sample of the [““SIGAG synthesized. In all the cases cited, little or none of the ch-6-S synthesized remained associated with the cells; conversely, chondroitinase-resistant material, presumably mainly hep-S, constituted a very high proportion (7565%) of the cell-associated [““SIGAG. Kraemer (1971) has reported that hep-S has a number of cellular locations. The two cell lines, MB2 and MB4, although initially derived from the same primary blastocyst culture, differ from each other in a number of respects (Sherman 1975a, b; Miller et al., 1975). Their pattern of GAG synthesis (Table 2) is no exception. As a matter of fact, the marked similarity of the MB4 pattern with that of yolk sac reinforces earlier observations that the two cell types have a number of biochemical properties in common (Sherman, 1975a). MB2, on the other hand, has a ch4-S:ch-6-S ratio quite different from that of any of the primary midgestation cultures. This is to be expected if, as we propose (Sherman, Miller, and Richter, in prepa-

CANTOR,

SHAPIRO

AND

SHERMAN

ration), MB2 consists primarily of parietal endoderm cells; these cells were removed as thoroughly as possible from their location adjacent to trophoblast cells during dissection of midgestation tissues, and were discarded. Further studies will have to be carried out to confirm that primary cultures of parietal endoderm cells share a GAG synthetic profile in common with MB2. The data in Table 4 reveal that the mouse embryo synthesizes GAG from its earliest stages of develoment, i.e., at and probably prior to the 64-cell stage. This might not be surprising when it is recalled that the mouse embryo synthesizes RNA from as early as the two-cell stage (Knowland and Graham, 1972) and expresses paternal genes by the &cell stage (Brinster, 1973; Chapman and Wudl, 1975). Furthermore, by autoradiographic and biochemical analyses, Kosher and Searls (1973) demonstrated that very early frog embryos were also capable of synthesizing GAG. It should be noted that in the case of both the mouse and frog embryo up to the time of blastulation, little or no ch-6-S is synthesized. Thereafter, the patterns differ; most notably, a much higher fraction of the GAG synthesized by the frog embryo is chondroitinase resistant (Kosher and Searls, 1973). Such differences are to be expected, however, since a large fraction of the cells developing in early mouse postblastocyst cultures are extraembryonic and do not have counterparts in the frog embryo. The mouse embryo shows two dramatic reversals in the ch-4-S:ch-6-S synthetic ratios in its in vitro development (Table 4 and Fig. 1). The first change is observed when the blastocyst attaches to the substratum and begins to outgrow: during this phase, there is a sharp drop in both the ch-4-S:ch-6-S ratio and in the proportion of chondroitinase-sensitive GAG. It is quite possible that these differences are merely due to the fact that preblastocyst and postblastocyst cultures require mark-

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edly different media; there have been a large number of studies by others suggesting that GAG synthetic profiles in vitro are very sensitive to culture conditions. On the other hand, one of the earliest cell types to proliferate in the implanting blastocyst in vitro is parietal endoderm (see Sherman and Salomon, 1975). If, as we have speculated above, parietal endoderm cells, like MB2, have a ch-4-S:ch-6-S ratio less than 1:l and produce a large amount of chondroitinase-resistant GAG, and if this cell type were predominant in GAG synthesis at the onset of implantation, then this could explain our observations. The next distinct alteration in the ch-4S:ch-6-S ratio takes place with further postblastocyst development. The ratio rises again and continues to do so until it exceeds 8:l between 12 and 15 days in uitro. Throughout this period, trophoblast cells outgrow and assume giant proportions (Sherman 1975a); at the same time, the inner cell mass proliferates, giving rise to a variety of cells, the predominant one of which resembles yolk sac biochemically (Bell and Sherman, 1973; Sherman, 1975d) and morphologically (Sherman, 1975a). The [“S]GAG profile observed is, therefore, quite consistent with the major constituents of the culture. The observation that a trophoblastlike GAG synthetic profile is still observed after 12 days in culture, at which time the number of trophoblast cells is greatly outnumbered by inner cell mass derivatives, suggests that the former cells have the capacity to produce a relatively large quantity of GAG. Undifferentiated and differentiated teratoma cell cultures produced ch-4-S and ch-6-S in about equal amounts. On the basis of their multipotentiality and surface antigens, Stevens (1958) and Artzt et al. (1972) have compared embryonal carcinoma cells to those of the preimplantation embryo. On the other hand, histochemical and morphological properties of embryonal carcinoma cells in viva and in vitro have led Damjanov et al. (1971) and Sherman

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(1975b) to propose that these cells most closely resemble some of those present in postimplantation embryo. The the [:15S]GAG profile of PCC4.azal is quite different from that of preimplantation embryos. This in itself certainly does not settle the issue, but taken together with preliminary biochemical studies (Mintz et al., 1975; Sherman, M. I., unpublished observations; Bernstine, E., personal communication), the evidence suggests that embryonal carcinoma cells are more closely analogous to certain cell types in the postimplantation, rather than preimplantation, embryo. Finally, Table 2 indicates that there is no substantial difference in the GAG profiles of an undifferentiated teratoma cell culture and a culture containing a majority of its cells in the differentiated state. This result is probably coincidental, reflecting an overall GAG synthetic profile in the differentiated culture contributed to by parietal endoderm-like cells with low ch-CS:ch-6-S ratios and fibroblastic and neuroblastic cell types which probably have higher ratios. Differentiated cultures containing other types of cells or the same types of cells in other proportions might be expected to give different GAG profiles. J. Cantor is grateful to the Roche Institute of Molecular Biology for its support while this work was being carried out. We wish to thank Drs. J. Abbott and K. Gibson for comments on the manuscript. REFERENCES ARTZT, K., DUBOIS, P., BENNETT, D., CONDAMINE, H., BABINET, C., and JACOB, F. (1973). Surface

antigens common to mouse cleavage embryos and primitive teratocarcinoma cells. Proc. Nat. Acad. Sci. USA 70, 2988-2992. BELL, K. E., and SHERMAN, M. I. (1973). Enzyme markers of mouse yolk sac differentiation. Develop. Biol. 33, 38-47. BRINSTER, R. (1973). Parental glucose phosphate isomerase activity in three-day mouse embryos. B&hem. Genet. 9, 187-191. CHAPMAN, V. M., and WUDL, L. (1975). The expression of P-glucuronidase during mouse embryogenesis. In “Isozymes” (C. L. Markert, ed.), Vol. 3, pp. 57-65. Academic Press, New York.

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G. W., and DORFMAN, A. (1974). Synthesis of sulfated mucopolysaccharides by chick cornea1 libroblasts in vitro. Exp. Eye Res. 18, 421-433. DAMJANOV, I., SOLTER, D., and SKREB, N. (1971). Enzyme histochemistry of experimental embryoderived teratocarcinomas. Z. Krebsforsch. 76,249256. DAMJANOV, I., and SOLTER, D. (1974). Experimental teratoma. Curr. Topics Pathol. 59, 69-130. DIETRICH, C. P., and MONTES DE OCA, H. (1970). Production of heparin-related mucopolysaccharides by mammalian cells in culture. Proc. Sot. Exp. Biol. Med. 134, 955-962. Hsu, Y.-C., BASKAR, J., STEVENS, L. C., and RASH, J. E. (1974). Development in vitro of mouse embryos from the two-cell stage to the early somite stage. J. Embryol. Exp. Morphol. 31, 235-245. JAKOB, H., BOON, T., GAILLARD, J., NICOLAS, J.-F., and JACOB, F. (1973). Teratocarcinome de la souris: Isolement, culture et proprietes de cellules a potentialites multiples. Ann. Microbial. Inst. Pasteur 125B. 269-282. KIRBY, D. R. S., BILLINGTON, W. D., BRADBURY, S., and GOLDSTEIN, D. J. (1964). Antigen barrier of the mouse placenta. Nature fLondon) 204, 548549. KNOWLAND, J., and GRAHAM, C. (1972). RNA synthesis at the two-cell stage of mouse development. J. Embryol. Exp. Morphol. 27, 167-176. KOSHER, R. A., and SEARLS, R. L. (1973). Sulfated mucopolysaccharide synthesis during the development of Rana pipiens. Develop. Biol. 32, 50-68. KRAEMER, P. (1971). Heparan sulfates of cultured cells. I. Membrane-associated and cell-sap species in Chinese hamster cells. Biochemistry 10, 14371445. MARTIN, G. (1975). Teratocarcinomas as a model system for the study of embryogenesis and neoplasia. Cell 5, 229-243. MATHEWS, M. (1966). Animal mucopolysaccharidases. In “Methods in Enzymology” (E. F. Neufeld and V. Ginsburg, eds.), Vol. 8, pp. 654-662. Academic Press, New York. MEIER, S., and HAY, E. D. (1973). Synthesis of sulfated glycosaminoglycans by embryonic cornea1 epithelium. Deuelop. Biol. 35, 318-331. MILLER, R. A., RUDDLE, F. H., and SHERMAN, M. I. (1975). Surface antigens of blastocyst-derived cell lines. In “Teratomas and Differentiation” (M. I. Sherman and D. Solter, eds.), pp. 123-136. Academic Press, New York. MINTZ, B., ILLMENSEE, K., and GEARHART, J. D. (1975). Developmental and experimental potentialities of mouse teratocarcinoma cells from embryoid body cores. In “Teratomas and Differentiation” (M. I. Sherman and D. Solter, eds.), pp. 5082. Academic Press, New York. PIERCE, G. B., JR., MIDGLEY, A. R., JR., SRI RAM, J. S., FELDMAN, J. Dr (1962). Parietal yolk sac carciCONRAD,

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noma: Clue to the histogenesis of Reichert’s membrane of the mouse embryo. Amer. J. Path. 41, 549-566. RUNNER, M. N., and PALM, J. (1953). Transplantation and survival of unfertilized ova in relation to postovulatory age. J. Erp. 2001. 124, 303-316. SAITO, H., YAMAGATA, T., and SUZUKI, S. (1968). Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243, 1536-1542. SAITO, H., and UZMAN, B. G. (1971). Production and secretion of chondroitin sulfates and dermatan sulfate by established mammalian cell lines. Biothem. Biophys. Res. Commun. 43, 723-728. SALOMON, D. S., and SHERMAN, M. I. (1975). Implantation and invasiveness of mouse blastocysts on uterine monolayers. Exp. Cell Res. 90, 261-268. SCOTT, J. E. (1960). Aliphatic ammonium salts in the assay of acid polysaccharides for tissues. In “Methods of Biochemical Analyses” (D. Glick, ed.), Vol. 8, pp. 145-197. Wiley (Interscience), New York. SHAPIRO, S. S., and SHERMAN, M. I. (1974). Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse. Arch. Biochem. Biophys. 162, 272-280. SHERMAN, M. I. (1972). The biochemistry of differentiation of mouse trophoblast: Alkaline phosphatase. Develop. Biol. 27, 337-350. SHERMAN, M. I. (1974). In uiuo and in uitro differentiation during early mammalian embryogenesis. Front. Rad. Therapy 9, 28-41.

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SHERMAN, M. I. (1975a). Long term culture of cells derived from mouse blastocysts. Differentiation 3, 51-67. SHERMAN, M. I. (1975b3. The culture of cells derived from mouse blastocysts. Cell 5, 343-349. M. I. (1975c). Differentiation of teratoma cell line PCCl.azaI in uitro. In “Teratomas and Differentiation” (M. I. Sherman and D. Solter, eds.), pp. 189-205. Academic Press, New York. SHERMAN, M. I. (1975d). Esterase isozymes during mouse embryonic development in uiuo and in uitro. In “Isozymes” (C. L. Marker%, ed.), Vol. 3, pp. 83-98. Academic Press, New York. SHERMAN, M. I. (1976). Generation of cell lines from preimplantation mouse embryos. In “Tissue Culture Association Manual. Techniques, Methods and Procedures for Cell, Tissue and Organ Culture” (V. J. Evans, V. P. Perry and M. M. Vincent, eds.), In press. SHERMAN, M. I., and SALOMON, D. S. (1975). The relationship between the early mouse embryo and its environment. Symp. Sot. Develop. Biol. 33, 277309. STEVENS, L. C. (1958). Studies on transplantable testicular teratomas of strain 129 mice. J. Nat. Cancer Inst. 23, 1257-1276. WHITTEN, W. K., and BIGGERS, J. D. (1968). Complete development in vitro of the preimplantation stages of the mouse in a simple chemically defined medium. J. Reprod. Fertil. 17, 399-401.

SHERMAN,