Glycoprotein synthesis and inhibition of glycosylation by tunicamycin in preimplantation mouse embryos: Compaction and trophoblast adhesion

Cell, Vol. 18, 217-227,

September,

1979,

Copyright

0 1979

by MIT

Glycoprotein Synthesis and Inhibition of Glycosylation by Tunicamycin in Preimplantation Mouse Embryos: Compaction and Trophoblast Adhesion M. Azim H. Surani Physiological Laboratory University of Cambridge Cambridge CB2 3EG, England

Summary The synthesis of glycoproteins and inhibition of protein glycosylation by tunicamycin were examined during development of preimplantation mouse embryos and trophoblast adhesion. Tunicamycin specifically inhibits glycosylation of asparaginyl residues of glycoproteins. Tunicamycin, 0.25-5.0 pg/ ml, had no effect on early cleavage or aggregation between embryos, but the embryos remained irreversibly uncompacted when control embryos developed to the blastocyst stage. Trophoblast adhesion and giant cell outgrowth were reversibly inhibited and the binding of Con A was also reduced. Incorporation of 3H-mannose into blastocysts was inhibited by 80%, but that of 3H-glucosamine and 3Hleucine by only 28 and 18%, respectively, in the presence of 1.0 pg/ml tunicamycin. Qualitative analysis showed that the incorporation of the sugars was markedly reduced in the majority of the fractions, but the synthesis of these carbohydratedeficient glycopeptides was essentially normal. However, protein-polysaccharide fractions with nearly 40% of the incorporated glucosamine and only 5% mannose and 1% leucine were insensitive to inhibition by tunicamycin. Membrane-bound Nglycosidically linked glycoproteins therefore evidently play an important role during compaction and in trophoblast adhesion of mouse embryos. Introduction Cell surface components apparently play a crucial role during development of preimplantation mouse embryos (Bennett, Boyse and Old, 1971); for example, in the Ca2+-dependent process of compaction (Ducibella and Anderson, 1975; Kemler et al., 1977), in aggregation between embryos (Burgoyne and Ducibella, 1977) and in trophoblast adhesion (Jenkinson and Wilson, 1973). Changes in the cell surface antigens (Jacob, 1977) and progressive reduction in the agglutination of embryos by Concanavalin A (Con A) during early development (Rowinski, Solter and Koprowski, 1976) are indicative of alterations in the cell surface properties. Cell surface glycoproteins may be especially important in development, since they are implicated in a number of membrane-modulated phenomena such as responsiveness to hormones, agglutination by plant lectins and recognition by antibodies, and in other

behavioral activities such as cell aggregation and adhesion (Cook and Stoddart, 1973; Hughes, 1976; Nicolson, 1976). The cell surface glycoproteins of preimplantation embryos undergo qualitative and quantitative modifications during development (Pinsker and Mintz, 1973) as is also shown by the presence of sialoglycoproteins on blastocysts with the loss of sialic acid before implantation (Jenkinson and Searle, 1977) and the detection of LETS or fibronectin only on the inner cell mass cells of blastocysts (Zetter and Martin, 1978). Active synthesis of a unique fucosyl-glycopeptide in early embryos (Muramatsu et al., 1978) and a nonsulfated polysaccharide by blastocysts (Pike, 1970) suggests that the embryos perhaps synthesize a wide range of developmentally regulated glycoproteins, some of which may be important as cell surface components in morphogenesis and cell differentiation. The oligosaccharide moiety of glycoprotein is thought to be important for cell-cell interactions (Turner and Burger, 1973; Town and Stanford, 1979). Synthesis of carbohydrate-deficient N-glycosidicaliy linked glycoproteins in cells cultured in the presence of tunicamycin (Waechter and Lennarz, 1976; Rothman, Katz and Lodish, 1978) which primarily inhibits synthesis of N-acetylglucosaminyl pyrophosphoryl dolichol phosphate (Takatsuki, Arima and Tamura, 1971; Tkacz and Lampen, 1975; Hemming, 1977; Struck and Lennarz, 1977) causes alterations in cell surface properties (Duskin and Bornstein, 1977a, 1977b; Olden, Pratt and Yamada, 1978; Duskin et al., 1978; Damsky et al., 1979; Pratt et al., 1979). Cellcell interactions and gastrulation are markedly affected in sea urchin embryos cultured in tunicamycin (Lallier, 1978; Heifetz and Lennarz, 1978). In this study, synthesis of glycoproteins by preimplantation mouse embryos is described and the inhibition of protein glycosylation by tunicamycin is shown to prevent compaction and trophoblast adhesion. Results Effects of Tunicamycin on Embryonic Development and Trophoblast Adhesion Mouse embryos were cultured at the 2-cell stage in BMOC3 medium. The initial stages of development and cleavage proceeded normally in the control and experimental groups in the presence of 0.25-5.0 pg/ ml tunicamycin. All embryos reached the 6-8-cell stage after 18-24 hr of culture, and developed to the 16-cell stage after 48 hr of culture. The zona pellucida-denuded embryos cultured from the 2-cell stage were also able to aggregate with other similar embryos after a 24 hr culture in the presence of tunicamycin. After a 48 hr culture, the embryos in control groups underwent compaction. The embryos in experimental

Cell 218

groups also showed initial signs of compaction, but some of the blastomeres remained free. After a further period of 12-16 hr in culture, the embryos in the experimental groups were fully decompacted, as shown in Figure 1. The number of cells per embryo in both groups ranged from 25-32 after 72 hr in culture. While the number of cells in the control embryos continued to increase, to about 77, there was no change in the number of cells in the embryos in experimental groups. When the embryos in the experimental groups were washed and explanted to fresh medium after 24 hr in culture, they remained uncompacted and failed to form blastocysts, and there was no detectable increase in cell number. When the 8cell stage embryos were similarly cultured for 12-24 hr in the presence of tunicamycin, the embryos again showed similar response and remained uncompacted, and failed to form blastocysts even after they were extensively washed and transferred to fresh medium. Hence the effect of the drug was essentially irreversible under these experimental conditions. In the embryos explanted to fresh medium, however, some morphological changes were evident, since the inner group of cells underwent compaction but the peripheral cells remained free and failed to form a blastocoelic cavity. Some of the peripheral cells showed signs of fluid accumulation in a manner suggesting trophectoderm-like cells. Embryos at the blastocyst stage were cultured in the optimal medium with 10% fetal calf serum to study the cell substrate adhesion and trophoblast giant cell outgrowths. Blastocysts were rendered free of the zona pellucida by treatment with the enzyme pronase to allow rapid adhesion and reduce the time of culture in tunicamycin. The zona pellucida-free embryos attached within 24 hr to the petri dish, and the initiation of giant cell outgrowths was observed; substantial trophoblast outgrowths were detected after a 48 hr culture in the control groups. All the cultures in experimental groups were therefore examined after 48 hr in culture. As shown in Table 1 and Figures 2 and 3, a substantial number of blastocysts underwent trophoblast giant cell outgrowths when cultured in 0.25 pg/ml tunicamycin. Generally, however, the outgrowths were considerably smaller than in the control groups. With increasing amounts of tunicamycin, from 0.5-2.0 pg/ml, the giant cell outgrowths were not observed. At I .O and 2.0 pg/ml tunicamycin, no giant cell outgrowths were observed. Approximately half the embryos had attached, however, and could not be dislodged by shaking the dish; the remainder were free-floating in the medium. After 72 hr in culture, the trophoblast outgrowths in the control groups were extensive, but there was no marked change in embryos cultured in the presence of higher concentrations of tunicamycin. Some blastocysts were cultured in 0.025 pg/ml cycloheximide, which is a translational

inhibitor of protein synthesis; the inhibitor caused the same extent of inhibition of protein synthesis as tunicamycin (data not shown). The majority of the embryos in this group attached, however, and underwent extensive giant cell outgrowths. The effect of tunicamycin was found to be reversible to the extent that when blastocysts were washed and explanted to fresh medium after a 24 hr culture in 0.5-l .O pg/ml tunicamycin, the embryos attached to the petri dish and giant cell outgrowths were obtained. Preliminary evidence also shows that when the embryos are transferred to uteri of pseudopregnant mice after a 24 hr culture, the trophectoderm cells are functional, since the blastocysts implant and evoke a decidual response, but the development of the embryo itself appears to be slightly abnormal or absent. Effect of Tunicamycin on Con A Binding Sites on Blastocysts Since tunicamycin has an effect on trophoblast cell surface as shown by the lack of adhesion to substratum, the influence of the drug on Con A binding sites was determined. Binding of the radiolabeled plant lectin could not be used as an assay, since substantial amounts of the lectin enter the blastocoelic cavity. Hence the hemadsorption assay for Con A binding sites was used as described by Sobel and Nebel (1976). As shown in Table 2, there was a reduction in the Con A binding sites in embryos cultured in 0.251 .O pg/ml tunicamycin in comparison with the number of erythrocytes bound to blastocysts in the control group. No binding of erythrocytes was obtained in the presence of a-D-methylmannopyranoside. Effect of Tunicamycin on Incorporation of 3HLeucine, 3H-Glucosamine and 3H-Mannose in Blastocysts The effect of tunicamycin on the incorporation of the labeled amino acid and sugars has so far been determined in detail only at the blastocyst stage, since the incorporation, especially of sugars, was very low during the early cleavage period. Since the number of cells in blastocysts and the total number of embryos that can be used are limited, the medium used in the incorporation studies was modified according to the method of Pinsker and Mintz (1973) but no glucose was added to the medium. The medium contained pyruvate, glutamic acid and aspartic acid, however, which could be utilized as alternative energy substrates. Studies showed that in this medium, development of 2-cell embryos normally occurred to the fully expanded blastocyst stage, with approximately the same rate of cleavage and total number of cells in blastocysts as observed in embryos cultured in BMOC3 medium. The blastocysts were first incubated for 6 hr in the optimal medium containing 10% fetal calf serum and

Protein 219

Glycosylation

Figure

1. Effect

Inhibition

of Tunicamycin

in Mouse

Embryos

on Preimplantation

Mouse embryos were cultured from to the compacted morula stage (A). the embryos in the control group uncompacted and embryos did not

Development

of Mouse

Embryos

After a 48 hr culture, the embryos in the control group de ~veloped the 2-cell stage in 0.25 pg/ml tLmicamycin. with loose blastomeres (CL After 72 hr in culture, The embryos in the experimenta I group were uncompacted. developed to the blastocyst stag le (B). but the blastomeres of embryos in the experimental group n amained develop as blastocysts (D); this i s also shown in (E). Bar = 30 pm.

Cell 220

in the presence of 0.25-2.0 pg/ml tunicamycin. They were then cultured for a further 6 hr in the glucosefree medium in the presence of the precursors and Table 1. Influence of Tunicamycin on Trophoblast Giant Cell Outgrowths after 48 Hr in Vitro % Total

Tunicamycin Q.&ml)

Adhesion

Number

of Blastocysts Attached with Giant Cell Outgrowths

Number of Blastocysts

Free

Control

38

-

7.9

92.1

0.25

31

3.2

25.8

71.0

0.50

34

41.1

52.9

1 .oo

41

53.6

46.3

-

2.00

22

54.5

45.5

-

Cycloheximide (0.025 pg/ml)

26

7.9

-

92.3

Attached

and

5.9

Blastocysts were rendered zona pellucida-free by treatment with 0.5% enzyme pronase prior to culture in an optimal medium with 10% fetal calf serum. The results were recorded after 48 hr culture; no marked change was observed in embryos in experimental groups even after 72 hr. Cycloheximide (0.025 sg/ml) caused the same extent of inhibition of protein synthesis as did tunicamycin, but had little effect on giant cell outgrowths. The embryos that attached without giant cell outgrowths in the presence of tunicamycin were those that could not be dislodged by shaking the petri dish.

Figure

2. Effect

of Tunicamycin

on Blastocyst

Trophoblast

Adhesion

different concentrations of tunicamycin. As shown in Table 3 and Figure 4, the incorporation of 3H-leucine into blastocysts was inhibited by approximately 1630%. The incorporation of 3H-glucosamine at the lower concentration of tunicamycin was inhibited by only about 3%, but increased to nearly 38% with an increase in the concentration of tunicamycin. The incorporation of 3H-mannose was most severely inhibited, since even in the presence of 0.25-0.5 pg/ ml tunicamycin, the incorporation was inhibited by nearly 50%. The inhibition of incorporation rose to nearly 80% when 1 .O-2.0 pg/ml tunicamycin was used. The inhibition of incorporation of the precursors was not due to the effect of tunicamycin on the uptake of the precursors, since similar values were obtained for the acid-soluble counts in the control and experimental groups for all three precursors. In all subsequent experiments, 1 .O pg/ml tunicamycin was used. Time-dependent changes in the incorporation of the precursor were examined in the presence of 1 .O ag/ ml tunicamycin. As demonstrated in Figure 5, the inhibition of the incorporation of the three precursors remained essentially unchanged during continuous labeling of blastocysts for up to 24 hr. Marked inhibition of 3H-mannose incorporation into blastocysts was observed, whereas the incorporation of 3H-leucine

and Giant

Cell Outgrowth

Mouse blastocysts were cultured for 48 hr. In the control group (A), trophoblast adhesion and extensive giant cell outgrowths (T) were obsf ?rved (ICM = inner cell mass). In the experimental group(B), blastocysts were cultured in the presence of 1 pg/ml tunicamycin; no giant cell outgra lwths were observed, although about half the embryos were attached to the dish. Bar = 35 pm.

Protein 221

Glycosylation

Inhibition

in Mouse

Table 2. ConA-Mediated Cultured for 18 Hr

Hemadsorption

Treatment @g/ml Tunicamycin)

Number of Blastocysts

Control 0.25

Embryos

Assay

ConA

100

of Blastocysts

0

A

Pretreatment

90 Blastocysts

Erythrocytes

45

++

++++

31

++

+++

1 .oo

73

+

++

Control + a-D-methylmannopyranoside

27

-

-

No treatment ConA

25

-

-

l $

80

“0 z ;

70

Attached with giant cell outgrowths Attached without giant cell outgrowths Free

i

with

Two types of hemadsorption assays were carried out in which either blastocysts or erythrocytes were pretreated with ConA. In both tests, blastocysts were then co-cultured with erythrocytes, and the number of erythrocytes remaining on the blastocyst surface after washing indicated the extent of hemadsorption mediated by ConA; the results are presented on an arbitrary scale with the highest binding evident in the control group. No binding occurred in the presence of a-Dmethylmannopyranoside or when neither of the two cell types were pretreated with the plant lectin.

and 3H-glucosamine was substantially less inhibited. Preliminary studies have been carried out on the incorporation of precursors at the 6-8-cell stage prior to compaction. These studies show that the incorporation of 3H-leucine is again inhibited by only 16%, whereas the incorporation of both the sugar precursors, 3H-mannose and 3H-glucosamine, is inhibited by as much as 60%. These studies have been carried out in the presence of 1 pg/ml tunicamycin under conditions similar to those described for blastocysts, but using considerably larger numbers of embryos in each group. In the presence of cycloheximide (0.025 pg/ml), a translational inhibitor of protein synthesis, about 20% inhibition of 3H-leucine incorporation in blastocyst proteins was observed; the inhibition of both the sugar precursors was of the same magnitude (data not shown). Effect of Tunicamycin on Qualitative Changes in Protein and Glycoprotein Synthesis Qualitative changes in the proteins and glycoproteins were examined after labeling blastocysts in the presence of the three radioactive precursors. The embryos were preincubated for 6 hr in the presence of 1 .O;ug/ ml tunicamycin in the optimal medium with 10% fetal calf serum. The embryos were then cultured in the glucose-free medium with or without tunicamycin for 6 hr to allow sufficient incorporation of the precursors. The embryos were then washed and incubated once again in the optimal medium with 10% fetal calf serum with or without tunicamycin but without the radioactive precursors for a further period of 12 hr. The embryos were solubilized in SDS and 2-mercaptoethanol and analyzed by disc gel electrophoresis on 6% polyacrylamide gels. The results in Figure 6 demonstrate that apart from a slight reduction in the incorporation of

0.25

0.50 1’0 Tunicamycin

Figure 3. Influence of Tunicamycin Giant Cell Outgrowths Embryos

were observed

2.0 ()g/ml)

on Blastocyst

Attachment

and

after 48 hr in culture

3H-leucine in all the fractions, the patterns of synthesized proteins were essentially similar in both the control and experimental groups. Perhaps somewhat greater inhibition of incorporation of 3H-leucine in the higher molecular weight fractions (fractions 20-30) occurred; these protein fractions are markedly glycosylated, as shown by the incorporation of the sugar precursors. Analyses of glycoproteins after labeling with 3H-glucosamine and 3H-mannose, however, revealed marked inhibition of the incorporation of the sugars in most of the fractions. Substantial incorporation of the sugars, especially 3H-glucosamine, was observed in the first few fractions near the top of the gel, and these fractions were not affected by tunicamycin. When the protein patterns were analyzed at the end of a 6 hr incubation without the pulse-chase experiments, the protein patterns were similar to those described above. In the case of glycoproteins analyzed after labeling with 3H-glucosamine, the fractions migrating near the top of the gel were relatively more prominent. The relative distribution of the precursors in frac-

Cell 222

Table 3. Incorporation

of Leucine

and Sugars

into Trichloroacetic

Acid-Insoluble

Fraction

of Blastocysts

Incorporation (cpm per Blastocyst) Tunicamycin Precursor

(pg/ml)

L-4,5-3H-leucine

0.25

D-6-3H-glucosamine

D-2-3H-mannose

Number of Experiments

Number of Blastocysts

4

67

Control

Experimental

% Inhibition

25,956

21,880

15.70

0.50

4

101

24,867

19,645

21 .Ol

1 .oo

9

252

27,389

22,545

17.69

2.00

4

76

25,013

17,669

29.36

0.25

3

99

4,649

4,519

2.80

0.50

4

78

4,777

3,709

22.35

1 .oo

7

236

5,067

3,645

28.06

2.00

4

112

4,067

2,523

37.96

0.25

4

396

345

191

44.63

0.50

4

415

385

190

50.65

1.00

10

979

367

75

79.56

2.00

3

327

341

73

78.59

Blastocysts were preincubated for 6 hr in the presence of different concentrations of tunicamycin in the optimal medium with 10% fetal calf serum. They were then labeled in the glucose-free medium in the presence of radioactive precursors for 6 hr. Groups of 10 and 50 embryos, solubilized in 0.1% SDS and 0.14 M 2-mercaptoethanol in 100 ~1 samples, were used in each sample. Aliquots of 5 pl were deposited on GFA glass fiber discs and treated with trichloroacetic acid to obtain counts for incorporation of the precursors. All samples were prepared in triplicate.

tions of proteins and glycoproteins was further shown by calculating the percentage of incorporation in individual fractions from the total counts obtained for all the fractions in a gel, as illustrated in Figure 7. The results consistently showed that there was essentially little difference in the incorporation of 3H-leucine in each fraction, whereas there was a significant reduction in the incorporation of the sugars in most fractions when blastocysts were cultured in the presence of tunicamycin. The results also showed substantial incorporation of 3H-glucosamine in the first five fractions, presumably protein polysaccharides, migrating near the top of the gel. These fractions did not migrate significantly into the gel when analyzed on 5% acrylamide gels. Tunicamycin had little effect on the incorporation of sugars in these fractions, which contained only about 1 .O% 3H-leucine, 5.0% 3H-mannose, but nearly 40% 3H-gIucosamine of the total precursors incorporated in blastocysts. The incorporation of 3Hglucosamine in these fractions apparently increased from about 35 to 50% after treatment with tunicamytin. Figure 7 also indicates the groups of proteins which are glycosylated with respect to mannose and glucosamine. Discussion N-glycosidically linked glycoproteins may be important in the early development of mouse embryos, since tunicamycin prevents compaction of blastomeres as well as the adhesion and outgrowth of trophectoderm cells of blastocysts. Tunicamycin inhibits glycosylation of asparaginyl residues of glycoproteins (Wae-

chter and Lennarz, 1976; Rothman et al., 1978) and markedly reduces the incorporation of mannose (but not that of leucine) in mouse blastocysts. The majority of the glycopeptides were substantially carbohydratedeficient after the embryos were treated with tunicamycin. This is likely to result in the modifications of the cell surface properties, as shown in several studies (Duskin and Bornstein, 1977a, 1977b; Duskin et al., 1978; Olden et al., 1978; Damsky et al., 1979; Pratt et al., 1979). The reduction of Con A binding, prevention of compaction and trophoblast adhesion probably reflect altered cell surface properties. Early cleavage divisions, aggregation of embryos and the initial phase of compaction are not affected by tunicamycin. However, the failure of compaction, a Ca’+-dependent process in which the cell surface components may play an important role (Ducibella et al., 1977; Kemler et al., 1977), may occur because of impaired cell-cell interactions, since cleavage is not affected until about the 30-cell stage, and preliminary studies show a marked inhibition of the incorporation of sugars (but not that of leucine) at the 8-cell stage. Gastrulation in sea urchin embryos is similarly affected by tunicamycin (Schneider, Nguyen and Lennarz, 1978), with an inhibition of two ?SO.+- and 3H-gIucosamine-labeled glycopeptides without an effect on protein synthesis (Heifetz and Lennarz, 1978). The reversible inhibition of cell-substratum adhesion of trophectoderm cells and reduction of the binding of Con A is also observed in several other cell types (Duskin et al., 1978; Olden et al., 1978; Damsky et al., 1979; Pratt et al., 1979). Protein-bound cell surface carbohydrates may also be involved in blastocyst

Protein

Glycosylation

Inhibition

in Mouse

Embryos

223

A

3H] Leucine

80

0 Mannose

l

20

3200 2400

10

0

1600 0

3.25

0.50 Tuhicamycin

1.0

2.0 (yglml

/

800

1

Figure 4. Influence of Tunicamycin on Incorporation of Leucine Sugars in Trichloroacetic Acid-Insoluble Fraction of Blastocysts

and

Blastocysts were preincubated for 6 hr in medium with tunicamycin and labeled in glucose-free medium for a further 6 hr. The tunicamycin concentration-dependent effect is expressed as a percentage of maximum counts in the control group.

implantation in vivo (Schlafke and Enders, 1975; Surani, 1977) since significant changes in the cell surface glycoproteins prior to implantation are indicated (Jenkinson and Searle, 1977; Wu and Chang, 1978) and the recognition between embryos and uterine epithelium appears to be species-specific (Surani, 1977). incorporation of D-2-3H-mannose into blastocysts is severely inhibited by tunicamycin, but not that of L4,5-3H-leucine or D-2-3H-glucosamine, although the incorporation of glucosamine also appears to be markedly inhibited in the 8-cell embryos. Some residual incorporation of the sugars in tunicamycin-insensitive steps, such as transfer to terminal sugars (Struck and Lennarz, 1977) and glycosylation of Oglycosidic glycoproteins and glycolipids, may occur. Indeed, nearly 40% of the labeled glucosamine is incorporated in protein-polysaccharide fractions

C

&-,0-;0-; 2

6

Figure 5. Time-Dependent Leucine and Sugars

Effect

10 Time

14 (h)

of Tunicamycin

18

22

on Incorporation

of

Blastocysts were cultured for 6 hr in optimal medium with 10% fetal calf serum in control (W) or experimental groups with 1 .O fig/ml tunicamycin (O- - -0). They were then labeled in the modified BMOC3 medium without glucose in the presence of labeled precursors for up to 24 hr. Groups of blastocysts, between 10 and 50. were removed periodically from the incubation dishes, and the incorporation of the precursors in the trichloroacetic acid-insoluble fraction was determined. Results from two separate experiments are combined.

which are insensitive to inhibition by tunicamycin; incorporation of significant amounts of 3H-glucosamine in a rapidly synthesized nonsulfated polysaccharide by blastocysts has been reported (Pike, 1970). This fraction may be an important major cell surface component of blastocysts. A slight inhibition of protein synthesis by tunicamycin was expected, and a reduction in the number of membrane-bound ribosomes has been demonstrated (Duskin et al., 1978; Damsky et al., 1979). Suboptimal levels of cycloheximide, which cause the same extent of inhibition of protein synthesis, do not, however, affect trophoblast adhesion and

Cell 224

Mr 165 155

68

39

\

r 1)

i 10

165 155

21 5

,

\

-3 68 I BSA

6’=‘t

_

20

30

40

FRACTION

50

60

70

+

NUMBER

Mr 68

r

39

_ 10 Orlgln

20

30

40

50

FRACTION

60

70

NUMBER

X 10e3

21 5

165 155

68

39

21 5 28 0 230 I& 18 0 f 130 z 802 302 3 0 T 2 o”E 25

25

15;

10 O;igl”

giant

cell

quences

camycin, cosylation

20

30

outgrowths. of the and

40

FRACTION

inhibition the

effects

60

70

NUMBER

50

+

10 Lgl”

Nevertheless, of of

protein inhibition

which could subsequenHy

20

40

FRACTION the

synthesis of

30

conseby

protein

on Laof Mouse

Blastocysts were preincubated for 6 hr in the control (-----) or experimental (-) groups in the presence of 1.0 pg/ml tunicamycin. They were then incubated for 6 hr in the glucose-free medium in the presence of labeled leucine or sugars, followed by pulse-chase incubation for 12 hr. The embryos were SOlubilked in SDS and 2-mercaptoethanol, and analyzed on 6% polyacrylamide gels after preparing gel slices. Results are representative of between 8 and 10 analyses for each precursor, with approximately 20 and 100 blastocysts per sample.

B

Glucosamme

lf 55 155

-I

215 T ST1

L3Hl

10 Orlgln

Y 0 2

39 I OL

Figure 6. influence of Tunicamycin beled Proteins and Glycoproteins Blastocysts

tunigly-

affect other cel-

50

60

NUMBER

70

Figure 7. Influence of Tunicamycin on the Relative Distribution of Leucine and Sugars in Different Groups of Proteins and Glycoproteins of Mouse Blastocysts Control groups (A and C) and experimental groups (I3 and D). Note the various groups of ‘H-leucine-labeled proteins (O- - -0) which are glycosylated with respect to ‘H-glucosamine U-A) and ‘H-mannose (W (A and C). In addition, tunicamycin has relatively little effect on protein synthesis, but the incorporation of sugars in the various fractions is substantially reduced (B and D), except for the ‘H-glucosamine in the first few fractions of protein-polysaccharides.

+

lular events, need detailed investigation. Glycosylation of the majority of glycopeptides markedly inhibited after treatment of blastocysts tunicamycin, but the synthesis of carbohydrate-defi-

is by

Protein 225

Glycosylation

Inhibition

in Mouse

Embryos

cient glycopeptides is not substantially affected, nor is the overall protein synthesis. The fate of these unglycosylated proteins was not established. Several membrane-bound glycoproteins of eucaryotes apparently contain asparagine-linked oligosaccharide moieties, as shown in BHK fibroblasts, and the unglycosylated glycopeptides synthesized in the presence of tunicamycin are translocated normally to the cell surface (Damsky et al., 1979). The unglycosylated derivatives of some glycoproteins, such as fibronectin, are less stable and are easily degraded by proteolytic enzymes (Olden et al., 1978), whereas procollagen is secreted normally, but its conversion to a collagen is impaired (Duskin and Borstein, 1977). In any event, the cell surface properties are therefore modified in the cells treated with tunicamycin. Comprehensive studies are needed on developmentally regulated glycoproteins, and especially on the changes in the cell surface glycoproteins of preimplantation mouse embryos, to establish how they are involved in compaction, cell-cell interactions and the differentiation of the blastomeres into ICM or trophectoderm cells of blastocysts. The cell surface glycoproteins of trophoblasts are also of considerable interest for defining the molecular aspects of specific adhesion between embryos and uterine epithelium at the onset of blastocyst implantation. Experimental

Procedures

Animals An outbred strain of albino CFLP mice was obtained from Anglia Laboratories Ltd. They were maintained on a lighting schedule of 05:OO to 19:OO hr. Two or three adult females, approximately 6 weeks old, were caged with each male and checked the following morning for copulation plugs: the day of the vaginal plug was designated as day 1 of pregnancy. Embryos were retrieved from oviducts at the 2cell and 8-cell stage at 16:OO hr on day 2 of pregnancy and at 09:OO hr on day 3 of pregnancy, respectively. Blastocysts were flushed from uterine horns at IO:00 hr on day 4 of pregnancy.

Culture Media Three different culture media were prepared. For the retrieval of embryos from the genital tract and the culture of 2- and 8-cell embryos, an embryo culture medium, BMOC-3 with 4 mg/ml BSA. was prepared (Brinster, 1970). For the culture of embryos at the blastocyst stage, an optimal medium which contained amino acids, vitamins and 10% fetal calf serum (Spindle and Pedersen, 1973) was used. The medium used for the duration of incubation of embryos with the radiolabeled amino acid and sugars was essentially a modification of BMOC-3 medium with 4 mg/ml BSA (Pinsker and Mintz, 1973). This medium contained 0.5 mM pyruvate as an alternative energy substrate, and glucose was omitted. In addition, the medium contained 0.2 mM glycine. 0.33 mM glutamic acid and 0.2 mM aspartic acid, since the last two amino acids can be utilized via the citric acid cycle. This medium was twice concentrated, and the final volume was adjusted afler the addition of radiolabeled precursors, Tunicamycin stock solution (1 mg/ml) was prepared by dissolving in undiluted dimethylsulfoxide. Aliquots of 5-10 gl were made and stored at -2O’C. Between 0.25 and 5.0 pg tunicamycin per ml of medium for experimental groups was prepared, and equivalent quantities of the vehicle alone were added to the control group.

Embryo Culture and Morphological Observations Embryos were cultured in Falcon plastic petri dishes under paraffin oil at 37°C in 5% CO, in air. The 2-and 8-cell embryos were cultured in BMOC-3 medium, and examined during their in vitro culture and development in the presence or absence of 0.25-5.0 pg/ml tunicamycin. The effect on cell proliferation was determined by counting nuclei in air-dried preparations of embryos (Tarkowski. 1966). The adhesion and giant cell outgrowths of trophoblast cells of blastocysts were also examined in the presence of tunicamycin (0.25-2.0 $g/ ml). In these studies, the zona pellucida of blastocysts was first removed by incubating blastocysts in BMOC-3 medium containing 0.5% pronase and 10 mg PVP/ml for 3-5 min at 37°C. They were then cultured in the optimal medium with 10% fetal calf serum (Spindle and Pedersen, 1973) for up to 72 hr. In some experiments, embryos were washed 9 times after 24 hr and cultured in fresh medium without tunicamycin. Manipulations on embryos were carried out under a Wild M5 dissecting microscope, and all detailed examinations and photographs were taken using a Wild M40 inverted phase microscope. incorporation of 3H-Leucine, 3H-Glucosamine and 3H-Mannose in Blastocysts The incorporation of the amino acid and sugars was studied and the dose-dependent effects of between 0.25-2.0 fig/ml tunicamycin were established. In these experiments, freshly obtained blastocysts were first incubated for 6 hr in the optimal medium with 10% fetal calf serum in the presence or absence of tunicamycin. Both the experimental and control embryos were then removed and washed 9 times in the medium without glucose to ensure removal of serum and glucose. The embryos were then cultured for 6 hr in 50 pl of glucosefree medium to which 25 pCi of ‘H-glucosamine (spec. act. 38 Ci/ mmole). 25 pCi of 3H-mannose (spec. act. 2 Ci/mmole) or 5 pCi of 3H-leucine (spec. act. 105 Ci/mmole) were added. The medium in experimental groups also contained appropriate amounts of tunicamycin. Time-dependent changes in the incorporation of the three precursors were then examined in the presence of 1 pg/ml tunicamycin and compared with those cultured in the absence of the drug. Preincubation of embryos for 6 hr in the optimal medium with or without tunicamycin was carried out as above, and labeling of embryos was carried out in the glucose-free medium. Groups of 15-30 blastocysts were removed periodically during a 24 hr culture from each group, except for those embryos cultured in ‘H-mannose, where larger groups of 40-50 embryos were retrieved. The embryos used for qualitative analyses of labeled proteins and glycoproteins were treated similarly. After a 6 hr preincubation in the presence or absence of tunicamycin in the optimal medium, the embryos were transferred to the glucose-free medium with the three radioactive precursors, with or without 1 .O bg/ml tunicamycin. The embryos were cultured for 6 hr and then retransferred to the optimal medium with 10% fetal calf serum without the precursors, but the experimental groups contained 1 .O pg/ml tunicamycin. The pulsechase incubation was carried out for a further 12 hr. In this case, the embryos were cultured in bacteriological plastic culture dishes (Sterilin) to prevent attachment of the embryos to the dish in the control groups. After the completion of the incubation period in each case, the embryos were withdrawn and washed 9 times in 0.1 M phosphatebuffered saline containing 10 mg/ml PVP at 0°C. The embryos were counted and transferred to 100 ~1 of 0.01-M phosphate-buffered saline containing 0.1% SDS and 0.14 M 2-mercaptoethanol and stored at -20°C. Samples used in qualitative analyses of proteins were stored in 200 ~1 of the buffer. The time elapsed between washing and storage of embryos was l-3 min. All the samples were stored for a maximum of 1 week before analysis. Quantitative estimation of the incorporation of labeled amino acid and sugars in blastocysts was carried out after the samples were heated in a water bath for 1 hr at 65OC using 5 nl aliquots. as described by Fishel and Surani (1978). Qualitative analysis of labeled proteins and glycoproteins was carried out on 6% polyacrylamide gels, also as described previously (Surani. 1977). The gel columns

Cell 226

were calibrated by using soybean trypsin inhibitor (molecular weight 21.500), bovine serum albumin (molecular weight 68,000) and RNA polymerase u chain (molecular weight 39.000), /3 chain (molecular weight 155,000) and p’ chain (molecular weight 165,000). Hemadsorption Assay for Con A Binding Sites on Slastocysts The blastocysts were retrieved on day 4 of pregnancy, the zona pellucida was removed by treatment with pronase and the embryos were cultured for 18 hr in the presence or absence of 0.25 or 1 .O @g/ml tunicamycin. The hemadsorption test for Con A binding sites in blastocysts was then carried out according to methods described previously (Furmanski, Phillips and Lubin. 1972; Sobel and Nebel, 1976). Two types of hemadsorption assays were carried out. Either blastocysts or the human type O-positive erythrocytes were first treated with Con A. Blastocysts were washed 9 times in phosphatebuffered saline containing 10 mg/ml PVP. They were then incubated for 10 min at 4°C in phosphate-buffered saline containing 0.1% BSA and 100 pg/ml Con A. The embryos were again washed 9 times and placed individually in microtiter plates. The erythrocytes were washed 3 times in the phosphate buffer containing albumin and suspended at a concentration of 2% (v/v). These erythrocytes were added to the wells containing the blastocysts. Incubation was carried out at 37’C for 1 hr in a shaking water bath. Alternatively, the erythrocytes were treated with 20 pg/ml Con A for 10 min at 37°C. washed 3 times and suspended at a concentration of 2% (v/v). These Con A-treated erythrocytes were then added to individual untreated blastocysts in microtiter plates, and incubation was carried out at 37°C in a shaking water bath for 1 hr. The blastocysts were removed and washed 3 times to remove the nonadhering erythrocytes, fixed in 2.5% PBS-buffered glutaraldehyde and then in methanol, and stained with Giemsa. Control experiments were carried out in the presence of 0.1 M-a-D-methylmannopyranoside. In another control experiment, neither blastocysts nor erythrocytes were treated with Con A prior to their co-incubation. The extent of hemadsorption was scored as described previously (Sobel and Nebel. 1976).

changes in the eight-cell genesis of the blastocyst.

mouse embryos: prerequisites Dev. Biol. 47, 45-58.

for morpho-

Ducibella, T., Ukena, T.. Karnovsky. M. and Anderson, E. (1977). Changes in cell surface and cortical cytoplasmic organisation during early embryogenesis in the preimplantation mouse embryos. J. Cell Biol. 74, 153-167. Duskin, D. and Bornstein, P. (1977a). Changes in surface properties of normal and transformed cells caused by tunicamycin, an inhibitor of protein glycosylation. Proc. Nat. Acad. Sci. USA 74, 3433-3437. Duskin, D. and Bornstein, P. (1977b). Impaired conversion of procollagen to collagen by fibroblasts and bone treated with tunicamycin, an inhibitor of protein glycosylation. J. Biol. Chem. 252, 955-962. Duskin, D., Holbrook. K., Williams, K. and Bornstein, P. (1978). Cell surface morphology and adhesive properties of normal and virally transformed cells treated with tunicamycin, an inhibitor of protein glycosylation. Exp. Cell Res. 7 76, 153-l 65. Furmanski. P., Phillips, P. G. and Lubin, M. (1972). Cell surface interactions with Concanavalin A: determination by microhaemadsorption. Proc. Sot. Exp. Biol. Med. 740. 216-219. Fishel, S. 9. and Surani, ness of preimplantation Morphol. 45, 295-301.

M. A. H. (1978). mouse embryos

Changes in the responsiveto serum. J. EmbryoI. EXP.

Heifetz, A. H. and Lennarz, W. J. (1978). The effect of inhibition of N-glycosidically linked glycoproteins on gastrulation. J. Cell Biol. 79, 167a. Hemming, F. W. (1977). Dolichol glycosylation of animal membrane Sot. Trans. 5 (4), 1221-l 231.

phosphate, a coenzyme in the bound glycoproteins. Biochem.

Hughes, R. C. (1976). Membrane Glycoproteins: ture and Function (London: Butterworth). Jacob, F. (1977). Mouse Immunol. Rev. 33, 3-32. Jenkinson. the mouse

teratocarcinoma

A Review

and embryonic

of Strucantigens.

E. J. and Searle. R. F. (1977). Cell surface changes on blastocyst at implantation. Exp. Cell Res. 106, 386-390.

Jenkinson, E. J. and Wilson, I. 9. (1973). In vitro studies on the control of trophoblast outgrowth in the mouse. J. Embryol. Exp. Morphol. 30, 21-30. I wish to thank Andrea Burling and Sheila Barton for expert assistance; Dr. R. G. Edwards and Mr. S. 9. Fishel for helpful suggestions; and Dr. R. Hamill of Eli Lilly, USA for a generous gift of tunicamycin. This work was supported by an MRC project grant and a grant from the Ford Foundation. The costs of publication of this article were defrayed in part by the payment of page charges, This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received

March

28, 1979;

revised

June 21,1979

Bennett, D., Boyse, E. A. and Old, L. J. (1971). Cell surface nogenetics in the study of morphogenesis. In Cell Interactions, Silvestri, ed. (Amsterdam: North-Holland), pp. 247-263. In vitro culture

of mammalian

immuL. G.

ova. Adv. Biosci.

Burgoyne, P. S. and Ducibella, T. (1977). Changes in the properties of the developing trophoblast of preimplantation mouse embryos as revealed by aggregation studies. J. Embryol. Exp. Morphol. 40, 143157.

Cook, G. M. W. and Stoddart, R. W. (1973). of the Eukaryotic Cell (New York: Academic

Surface Press),

Carbohydrates

Damsky. C. H., Levy-Benshimol, A., Buck, C. A. and Warren, L. (1979). Effect of tunicamycin on the synthesis, intracellular transport and shedding of membrane glycoproteins in BHK cells. Exp. Cell Res. 119,1-13.

Ducibella,

T. and Anderson,

E. (1975).

Cell shape

4452.

Lallier, R. (1978). Effects de deux inhibiteurs de la synthese des glycoproteines, tunicamycine et 2-deoxyglucose, sur le development de I’oeuf de I’oursin. C. R. Acad. Sci. Ser. D. 287, 543-546. Muramatsu. T., Gachelin, G.. Nicolas. J. F.. Condamine, H., Jakob, H. and Jacob, F. (1978). Carbohydrate structure and cell differentiation: unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells. Proc. Nat. Acad. Sci. USA 75, 2315-2319. Nicolson, G. L. (1976). Trans-membrane control of the receptors normal and malignant cells. Biochim. Biophys. Acta 458, l-57.

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