N-linked glycoprotein biosynthesis in the developing mouse embryo

DEVELOPMENTAL

BIOLOGY

113,228-237

(1986)

N-Linked Glycoprotein

Biosynthesis

D. RANDALLARMANT,'HOWARD Department

of Biochemistry and Molecular Tztmnr Institute at Hwstcm,

A.

in the Developing Mouse Embryo KAPLAN,AND

Biology, The University 67(?3 Bertnu Awwue,

WILLIAM

of Texas Houston,

J. LENNARZ' M. D. Anderson Texas 77030

Hospital

and

We have developed microenzymic assays that have, for the first time, enabled analysis of several enzymes in the pathway for N-linked glycoprotein biosynthesis in pre- and peri-implantation mouse embryos. The in vitro activities of the glycosyl transferases responsible for the formation of N-acetylglucosaminylpyrophosphoryldolichol, NJ’-diacetylchitobiosylpyrophosphoryldolichol, mannosylphosphoryldolichol, and glucosylphosphoryldolichol were found to decrease after fertilization before increasing significantly at the blastocyst stage, a stage that was also found to be highly sensitive to the glycosylation inhibitor, tunicamycin. The observed elevation in the activities of these enzymes in blastocysts still occurred when embryos were cultured in n-amanitin, indicating that de novo mRNA synthesis is unnecessary for the observed increase in their activities. Thus, an elevated capacity for N-glycosylation exists at the blastocyst stage, a time when dramatic increases in cell-cell interactions are known to occur. 0 1986 Academic Press. Inc

INTRODUCTION

Nevertheless, indirect evidence has accumulated in support of a role for glycoproteins in mouse embryogenesis through the use of immunological and lectin probes that have demonstrated that certain carbohydrate-bearing molecules change temporally and spatially during embryonic development (Johnson and Calarco, 1980; Shevinski et al., 1982; Marticorena et al., 1983; Fenderson et al., 1983; Chavez et al., 1984; Fox et al., 1984). Perhaps the best evidence suggesting a role for cell surface glycoproteins in mammalian embryogenesis comes from the study of Surani (1979) who used tunicamycin, an inhibitor of N-linked glycoprotein biosynthesis, to block blastocyst formation. However, since protein synthesis inhibitors are present in most preparations of tunicamycin, it has been suggested that these results be interpreted cautiously (Atienza-Samols et al., 1980). In another study preimplantation mouse embryos were treated with compactin (Surani et al., 1983), a potent inhibitor of the synthesis of polyisoprenoids (Brown et al., 19’78) such as dolichol, which serves as the saccharide carrier for the assembly of the oligosaccharide chains of N-linked glycoproteins (Struck and Lennarz, 1980). Although compactin caused the developmental arrest of the embryos and a concomitant decrease in protein glycosylation, its effect could not be reversed by dolichol supplementation as had been done successfully in an analogous study of sea urchin embryo development (Carson and Lennarz, 1979). Thus, the role for N-linked glycoproteins in mouse embryo development remains unclear. In this study we have directly examined the developmental expression of several enzymes responsible for the biosynthesis of N-linked glycoproteins in mouse embryos. Because of the limited availability of embryonic

Cell surface N-linked glycoproteins are thought to play an important role in the cell-cell interactions involved in cellular differentiation and embryonic development. Although considerable progress has been made in understanding the basic mechanism of the synthesis of Nlinked glycoproteins (for review see: Struck and Lennarz, 1980), the processes that regulate the biosynthesis of these macromolecules and their biological function are not well understood. One of the most extensively studied developmental systems in which both the regulation of biosynthesis and the biological function of glycoproteins have been addressed is the sea urchin embryo (for review see: Lennarz, 1983). Biochemical analyses of developing sea urchin embryos have clearly demonstrated that Nlinked glycoprotein biosynthesis is developmentally regulated, increasing during gastrulation when a dynamic series of cell-cell interactions occur (Lennarz, 1983; Welply et al., 1985). The finding that gastrulation is prevented in the presence of the glycosylation inhibitor, tunicamycin (Heifetz and Lennarz, 19’79), indicates a requirement for the synthesis of N-linked glycoproteins at this stage. Extensive biochemical analysis of mammalian embryos similar to that carried out with echinoderm embryos has been precluded by the limited availability of material from mammals. For example, the average yield of viable embryos obtained from a superovulated mouse is 20 to 30, with each embryo containing only 25 ng of total protein (Sellens et al., 1981). i Present address: Department of Obstetrics and Gynecology, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215. a To whom correspondence should be addressed. 0012-1606/86 Copyright All rights

$3.00

0 1986 by Academic Press. Inc. of reproduction in any form reserved.

228

ARMANT,

KAPLAN,

AND

LENNARZ

Protrilr

tissue, we developed microenzymic assays for several important enzymes involved in the dolichol-mediated assembly of the oligosaccharide chains of N-linked glycoproteins (Struck and Lennarz, 1980). The enzymes studied were those involved in the formation of N-acetylglucosaminylpyrophosphoryldolichol (GlcNAc-PP-dolichol), N,N’-diacetylchitobiosylpyrophosphoryldolichol (GlcNAc),-PP-dolichol), mannosylphosphoryldolichol (Man-P-dolichol), and glucosylphosphoryldolichol (Glc-P-dolichol). The results of our study demonstrate that the activities of these enzymes are developmentally regulated in the mouse embryo, initially decreasing after fertilization and then rising significantly at the blastocyst stage. The elevated capacity for assembly of the oligosaccharide chains of N-linked glycoproteins in the mouse blastocyst occurs at a time of dramatically increasing cell-cell interactions, which is analogous to the above cited observations on enhanced glycoprotein synthesis found in the sea urchin embryo at gastrulation. MATERIALS

AND

METHODS

Muterials. CF-1 female and male mice were purchased from Charles River (Kingston, N. Y.). GDP-[3,4-“HIMan, UDP-[l-“H]Glc, UDP-[6-“H]GlcNAc, [5,6-“Hluridine, n[2-“Hlmannose, and L-[4,5-3H]leucine were obtained from NEN Research Products (Boston, Mass.). Gestyl pregnant mare serum gonadotropin (PMSG) was obtained from Diosynth, Inc. (Chicago, Ill.). Human chorionic gonadotropin (hCG) was purchased from CalbiochemBehring (San Diego, Calif.). Tris, adenosine 5’-monophosphate (AMP), uridine, yeast transfer ribonucleic acid (tRNA), a-amanitin, dolichol phosphate, Triton X100, Sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), polyvinyl-pyrrolidinone, ethylenediaminetetraacetate (disodium salt) (EDTA), aprotinin, leupeptin, antipain, and benzamidine were purchased from Sigma Chemical Company (St. Louis, MO.). DEAE-cellulose (DE52) was obtained from Whatman, Inc. (Clifton, N. J.). Silica gel 60 precoated thin-layer chromatography plates were obtained from MCB Manufacturing Chemists, Inc. (E. Merck) (Cincinnati, Ohio). Liquiscint scintillation cocktail was obtained from National Diagnostics (Somerville, N. J.). Hank’s balanced salt solution, fetal calf serum, and CMRL 1066 were purchased from GIBCO (Grand Island, N. Y.). Terasaki tissue culture plates were obtained from Falcon (Oxnard, Calif.). Tunicamycin, purified by high-performance liquid chromatography to remove inhibitors of protein synthesis, was the generous gift of Dr. Alan D. Elbein, The University of Texas Health Science Center, San Antonio, Texas. All other chemicals used were of analytical grade. Embryo production and cultivation. Embryos and eggs were generated from CF-1 mice using females superovulated with 5 IU PMSG followed 40-48 hr later by 5

Glp~x!~lrrf

ion

iv

Mtntw

Errrlrryos

229

IU hCG. During the first 3 days of gestation, the animals were sacrificed and the oviducts removed. Unfertilized eggs were removed by making an incision in the oviduct wall to release the cumulus mass. Eggs were freed from the cumulus cells by incubation with 1 mg/ml hyaluronidase (Worthington) in phosphate-buffered saline containing 4 mg/ml BSA. Embryos at the two-cell to morula stages were flushed from the oviducts with prewarmed Hank’s balanced salt solution. Expanded blastocysts were obtained by flushing the uterine horns removed from pregnant females on the fourth day of gestation. Eggs and embryos were washed free of cumulus and uterine epithelial cells by transfer with a micropipet through several drops of Hank’s solution containing 4 mg/ml BSA. Hatched blastocysts were obtained by culturing expanded blastocysts in CMRL 1066 medium supplemented with 4 mg/ml BSA at 37°C in a 5% COZ plus air atmosphere. Embryo outgrowths were obtained by culturing blastocysts in CMRL supplemented with 10% heat-inactivated fetal calf serum. Microtechxiques *for enxymatic analysis. Microtechniques employed were adapted from those described by Lowry and Passonneau (1972). Embryos and unfertilized eggs were washed in several drops 0.9% NaCl/BSA (2 mg/ml) to remove all media components and/or added drugs, and transferred into either the lo-p1 wells of a Terasaki plate or onto the side wall of a glass vial containing several drops of water. The plate or vial was immediately immersed into liquid nitrogen to freeze the embryos for lyophilization. It was necessary to carry out lyophilization under a vacuum of 200 millitorrs or less in order to retain enzyme activities. Lyophilized tissues in glass vials were stored frozen under vacuum for indefinite periods without loss of enzyme activities. Enzyme assays were carried out in the wells of Terasaki plates either by adding reaction mixture directly to wells in which embryos had been lyophilized or by transferring individual lyophilized embryos with a hair point (an eyelash glued to the end of a glass rod (Lowry and Passonneau, 1972) from the side of the glass storage vial into the wells and then adding reaction mixture. Solutions were added to or removed from wells using handmade quartz constriction pipets that were calibrated with p-nitrophenol standard solutions to deliver volumes in the range of 0.1 to 5 yl (Lowry and Passonneau, 1972). After addition of reaction mixture, the wells were overlayed with mineral oil to prevent evaporation of the small reaction volumes during their incubation at 37°C. Enzyme assays. Preliminary experiments were conducted with mouse embryo homogenates to determine saturating concentrations of substrates for each enzyme assayed. Typical reaction mixtures are given below. The reaction mixture for the assay of Man-P-dolichol synthase activity contained: 65 ,L&ZGDP-[“HIMan (25.1 Ci/mmole), 1.5 mM dolichol phosphate, 0.3 mM MgClz,

230

DEVELOPMENTAL

BIOLOGY

0.3 mMMnClz, 0.15 MNaCl, 4 mMAMP, 0.4% BSA, 0.2% Triton X-100, protease inhibitor cocktail (PIC; 25 KIU/ ml aprotinin, 1 pg/ml leupeptin, 2 pg/ml antipain, 10 pg/ml benzamidine), 50 mM Tris-HCl, pH 7.4. Glc-P-dolichol synthase activity was assayed in a reaction mixture containing: 60 @LMUDP-[3H]Glc (11 Ci/ mmole), 2.5 mM dolichol phosphate, 10 mlM MgCl,, 1 mM MnClz, 0.15 M NaCl, 4 mM AMP, 0.4% BSA, 0.2% Triton X-100, PIC, 50 mM Tris-HCl, pH 7.4. The reaction mixture for the synthesis of (G~cNAc)~.~PP-dolichol contained: 65 PLMUDP-[3H]GlcNAc (20.4 Ci/ mmole), 1.5 mA4 dolichol phosphate, 27 rnM MgClz, 4 mMAMP, 0.4% BSA, 0.2% Triton X-100, PIC, 0.2 MTrisHCl, pH 7.4. Controls consisted of heat denaturing embryos in buffer alone under mineral oil at 95°C for 20 min before assay of enzyme activities. All reactions were terminated by transferring the entire reaction mixture to 3 ml CHC13:CH30H (2:l) and the products extracted as described below. Isolation of reaction products. The labeled products formed during the enzyme assays described above, ManP-dolichol, Glc-P-dolichol, and ( GlcNAc)l-z-PP-dolichol, were isolated according to the extraction method of Lucas et al. (1975). At the end of the incubation periods, reaction mixtures were transferred to tubes containing 0.5 mg of heat-denatured mouse liver microsome fraction protein which served as unlabeled carrier. The reaction products were then extracted three times with 3 ml CHCls:CH30H (2:l). The supernatants, cleared by lowspeed centrifugation, were pooled and washed by mixing vigorously once with 0.25 vol of 0.9% NaCl and five times with 0.2 vol of CH30H:0.9% NaCl(1:l). The upper phase obtained after each wash by low-speed centrifugation was aspirated and discarded. The remaining lower organic phase was subsequently dried under a stream of N2. The dried residue was either dissolved in 5 ml scintillation cocktail for determination of radioactivity or in an appropriate volume of CHC13:CH30H (2:l) for chromatographic analysis. Chromatographic procedures. Enzyme reaction products were analyzed by ion-exchange and thin-layer chromatography. Reaction product in CHC13:CH30H (2: 1) was applied to a 1.5 ml bed volume of DEAE-cellulose acetate (Lucas et ah, 1975) equilibrated with CHC&: CH30H (2:l). The column was eluted first with 12 ml CHC13:CH30H (2:l) followed by 12 ml each of 30 and 100 mM formate in CHC13:CHBOH (2:l). Fractions of 1.5 ml were collected. Thin-layer chromatography was carried out by spotting embryo reaction product and an appropriate radioactive standard prepared from hen oviduct microsomes (Waechter et al., 1973; Lucas et ah, 1975), on silica gel 60-precoated plates and developing in CHC13: CH30H:H20 (60:35:6) or (65:25:4). Segments of 1 cm were

VOLUME

113. 1986

scraped from each lane into scintillation vials for determination of radioactivity. Descending paper chromatography of the carbohydrate moieties of sugar lipids, released by hydrolysis in tetrahydrofuran:0.5 M HCI (4: 1) at 50°C for 90 min (Hanover and Lennarz, 1979), was performed on Whatman No. 3MM paper using butanol: pyridine:water (6:4:3). Strips of 1 cm were cut from each lane for determination of radioactivity; authentic sugar standards were detected by staining with aniline-diphenylamine (Hanover and Lennarz, 1979). Isolation of Poly A+ RNA. The effect of Lu-amanitin on poly A’ RNA synthesis in mouse embryos was determined based on the method of Levey and Brinster (1978). Expanded blastocysts (150) were cultured in the absence or in the presence of n-amanitin (1 pg/ml) for 1.5 hr, then labeled for 4 hr by the addition of 11 pLM[3H]uridine (45 Ci/mmole) to the culture media. Following incubation, embryos were washed by sequential transfer through 10 drops of Hank’s balanced salt solution containing 3 mg/ml polyvinylpyrrolidinone and 4 mg/ml uridine. Embryos were subsequently dissolved in 0.2 ml of binding buffer (0.01 M Tris-HCl, 0.5 M NaCl, 1 mM EDTA, 0.5% SDS, pH 7.5) containing 50 pg of yeast tRNA as carrier and mixed with 100 ~1 of packed wet oligo(dT)cellulose that had been pretreated with total cellular RNA isolated from sea urchin embryos (Lau and Lennarz, 1983) to reduce nonspecific binding. Samples were incubated for 1 hr at 55°C and then centrifuged for 30 see at 5OOOg,,. The supernatant was aspirated and the pellet washed with ten successive 0.5-ml aliquots of binding buffer. The bound poly A+ RNA was eluted in 0.5 ml of 0.01 M Tris-HCl, pH 7.5, containing 1 mM EDTA, and radioactivity determined by liquid scintillation counting. RESULTS

Developmental expression of enzymes involved in the synthesis of oligosaccharide-lipid. Our primary objective was to determine if the in vitro activities of the enzymes involved in the assembly of the oligosaccharide chains of N-linked glycoproteins were altered during development from the unfertilized egg to the peri-implantation stage. Because conventional methodologies for assay of these enzymes are too insensitive for use with the amounts of tissue obtainable from mouse eggs and embryos, we devised new assays based on the pioneering microtechniques first introduced by Lowry and Passonneau (1972). Using these techniques, we assayed the activities of Man-P-dolichol synthase, Glc-P-dolichol synthase, and the enzymes synthesizing GlcNAc-PP-dolichol and ( GlcNAc)z-PP-dolichol (collectively termed chitobiosyl-PP-dolichol synthase) in lyophilized tissues prepared from unfertilized eggs and embryos at various stages of development. It was first demonstrated that

XKMANT,

KAPLAN,

AND

LENNARZ

Protein

lyophilization of the embryos and inclusion of Triton X100 in the reaction mixture afforded active preparations of these microsomal enzymes. All assays were performed at saturating concentrations of the sugar nucleotides and dolichyl phosphate substrates, as shown by the lack of increase in the reaction rates upon further elevation of the substrate levels (data not shown). Linearity of the reactions with time and embryo number (protein concentration) was established for each of the three enzyme activities. The results for Man-P-dolichol synthase, shown in Fig. 1, were similar to those obtained for the other two enzyme activities. Using this sensitive assay system, it was possible to accurately measure Man-Pdolichol synthase activity in a single embryo, although 10 to 20 lyophilized embryos were usually pooled for routine determinations. The validity of these enzyme assays was confirmed by chemical identification of the reaction products. The enzyme reaction products were each found to cochromatograph on silica gel thin-layer chromatograms with authentic standards prepared from hen oviduct microsomes (Fig. 2 and 3A). The reaction products also were found to coelute with authentic standards on DEAEcellulose acetate (data not shown) which differentiates between phosphodiester- and pyrophosphate-containing dolichol-linked saccharides (Lucas et al., 1975). Several studies in other systems have indicated that (GlcNAc),-PP-dolichol synthesis is catalyzed by two enzymes, both of which utilize UDP-GlcNAc as the sugar donor (Struck and Lennarz, 1980). Therefore, the GlcNAc-containing lipid synthesized by mouse embryo homogenates was subjected to mild acid hydrolysis and the released carbohydrate moiety was found by paper

2 I 5 Time (mm)

cl . 0

. 10

I 20

Number

. 30

40

1 5(

of Embryos

FIG. 1. Linearity of the Man-P-dolichol synthase reaction. Man-Pdolichol synthase was assayed using lyophilized hatched blastocysts, varying either the incubation time (A) or the number of embryos assayed (B). The reaction was carried out at 37°C under mineral oil in 0.4 ~1, as described under Materials and Methods. Heat denaturation of the tissue before assay (closed circles) destroyed all activity and was subtracted as background when determining enzyme activities.

Glycosylntion

in

Mmrw

231

En1hryos

c+4

.I D -t

J0

0.5

1.0

Relative

A.0

0.5

1.0

Migration

Frc. 2. Identification of the Man-P-dolichol synthase and Glc-P-dolichol synthase reaction products. Approximately 200 lyophilized hatched blastocgsts were pooled and incubated 30 min in 5 ~1 of the appropriate reaction mixture and the oligosaccharide-lipid was isolated as detailed under Materials and Methods. The isolated Man-P-dolichol (A and B) or Glc-P-dolichol (C and D) were analyzed by thin-layer chromatography in CHC13:CH30H:Hz0 at a ratio of either 60:35:6 (A and C) or 65:25:4 (B and D). The migration of the corresponding standard prepared using hen oviduct microsomes is indicated by the bar and arrow in each chromatogram.

chromatography to consist of a mixture of GlcNAc and (G~cNAc)~ (Fig. 3B). This indicates that the determination of the amount of GlcNAc-containing lipid formed in the embryo homogenates is actually a measure of the sum of enzyme activities catalyzing the formation of both GlcNAc-PP-dolichol and ( GlcNAc)Z-PP-dolichol. As expected (Struck and Lennarz, 1977), the synthesis of the GlcNAc-containing lipid in embryo homogenates was completely inhibited in the presence of tunicamycin (data not shown). Having identified the enzymatic products, we determined the enzymatic activities of chitobiosyl-PP-dolichol, Man-P-dolichol, and Glc-P-dolichol synthases in homogenates over the course of preimplantation development. As shown in Fig. 4, all three enzyme activities showed relatively similar developmental profiles; i.e., a decrease between egg and the early cleavage stages (twocell and eight-cell compacted) followed by a marked increase at the blastocyst stages. The times during development at which the activities began to increase varied somewhat for each enzyme: Chitobiosyl-PP-dolichol synthase elevated following the two-cell stage; Man-P-

232

DEVELOPMENTAL

Relative

Migration

ppE$q-j

iI/, h

,I

0.5

0 Relative

VOLUME 113, 1986

BIOLOGY

1.0

Migration

FIG. 3. Identification of the Chitobiosyl-PP-Dolichol Synthase Reaction Products. The chitohiosyl-PP-dolichol synthase reaction was performed in 10 ~1 using approximately 400 hatched blastocysts and incubating at 37°C for 30 min as detailed under Materials and Methods. The isolated reaction products were analyzed by thin-layer chromatography (A) in CHCla:CH30H:H20 (60:35:6). The glycose released from the reaction products upon mild acid hydrolysis, as described under Materials and Methods, was analyzed by paper chromatography (B) in butanol:pyridine:H,O (6:4:3). The migration of standard chitohiosylPP-dolichol prepared using hen oviduct microsomes is indicated by the bar and arrow in A. Chitobiose and GlcNAc were cochromatographed in separate lanes and detected using aniline-diphenylamine as indicated in B.

dolichol synthase elevated following the eight-cell stage; and Glc-P-dolichol synthase increased following the expanding blastocyst stage. We found that the increased enzyme activities between the early cleavage and blastocyst stages cannot be readily explained by the presence of activators or inhibitors, because a mixture of embryo homogenates from the two stages exhibited levels of activities for all three enzymes which were simply additive. None of the enzyme activities varied significantly whether the embryos were removed from the mother at a particular stage or were cultured in vitro from a prior stage of development. As shown, Man-P-dolichol synthase exhibited the highest and chitobiosyl-PP-dolichol synthase the lowest enzyme activities. The specific activities of the enzymes were within the range observed in a variety of other organisms and tissues (e.g., see Kean, 1980; Clarke et ab, 1983; Welply et al., 1985). Embryo development is blocked in the absence of oligosucchuride-lipid synthesis. To assess the functional importance of the biosynthesis of N-linked glycoproteins during embryogenesis, we cultured mouse embryos in medium containing the glycosylation inhibitor tunicamycin (Tamura, 1982). The tunicamycin preparation used was purified by high-performance liquid chromatography to remove one or more components that inhibit protein synthesis (A. D. Elbein, personal communication). In preliminary experiments, we found that blastocysts cultured in 1 pug/ml of this tunicamycin preparation for 6 to 8 hr incorporated [3H]leucine into protein at the same level as control embryos (100 + 5%),whereas [3H]mannose incorporation into trichloroacetic acid-insoluble material was inhibited 60 to 80%.

B

C

40

6 20

2 I

I

I

I

I

I

I

I

UE

2C

8C

EB

HB

UE

2C

8C

Developmental

FIG. 4. Chitobiosyl-PP-dolichol

I

EB

1

HB

v I

UE

I

2C

,

I

I

8C

E8

HB

Stage

synthase (A), Man-P-dolichol synthase (B), and Glc-P-dolichol synthase (C) activities during preimplantation development. Activities were determined as detailed under Materials and Methods pooling for each determination 50 (A), 10 (B), or 20 (C) unfertilized eggs (UE), two-cell embryos (ZC), eight-cell morulae (8C), expanding blastocysts (EB), or hatched blastocysts (HB). The mean and standard deviation are indicated for each determination, assayed in triplicate. The specific activities were calculated based on a total protein content per embryo of 25 ng which does not significantly change throughout the period of development studied (Seliens et al., 1981).

ARMANT,

KAPLAN,

AND

LENNARZ

Protein

TABLE OF TUNI(:AMYCIN

stage tunicamycin added 4-cell 8-cell morula Blastocyst Hatched

77 142 55

hlastocgst

23

Compaction 73 (95)

Cavitation 0 (0) 47 133)

Mouse

233

Embryos

1

ON EMBRYO

Developmental Total No. of embryos

iv

thesis in mouse embryos within 1 hr without noticeably inhibiting total RNA or protein synthesis, and to block the increase in the activity of n”,3@hydroxysteroid dehydrogenase observed at the late blastocyst stage (Levey and Brinster, 1978; Schindler and Sherman, 1981). Embryos exposed to the drug before blastocyst formation are developmentally arrested (Braude, 1979), but as Schindler and Sherman (1981) have observed, exposure of postcavitation-stage embryos to cr-amanitin for 24 hr does not noticeably affect development. Therefore, it is possible in blastocysts to determine the effect of this drug on the expression of proteins without the potential complication of pleiotropic effects. We incubated early blastocysts for 24 hr in the absence or in the presence of a-amanitin and determined its effect on changes in the expression of Glc-P-dolichol and chitobiosyl-PP-dolichol synthase activities during that period. The developmentally regulated increase in the in vitro activities of these enzymes was not prevented when embryos were cultured in medium containing 1 &ml cY-amanitin (Table 2). Man-P-dolichol synthase activity was also unaffected by the drug, remaining at a constant level throughout the treatment period (data not shown). To be certain that the drug was, in fact, inhibiting mRNA synthesis under these conditions, we [“Hluridine-labeled the RNA of embryos cultured in the presence or absence of a-amanitin and then isolated the poly A+ RNA (see Materials and Methods). Greater than 95% of the poly A+ RNA synthesis was abolished in (Yamanitin treated embryos, as expected. Although it is not clear why actual increases in the activities of chitobiosyl-PP-dolichol and Glc-P-dolichol synthases were observed in the presence of a-amanitin, it seems clear from these results that the developmentally regulated increase in these enzyme activities in control embryos is not the result of increased transcription.

The effect of the purified preparation of tunicamycin on the development of embryos during various periods of in vitro culture is shown in Table 1. Ninety-five percent of the embryos developed normally in the presence of tunicamycin from the four-cell stage to the eight-cell morula stage and then arrested. When examined by light microscopy, the arrested morulae appeared to have compacted normally. All subsequent stages of development were inhibited in the presence of tunicamycin, indicating a requirement for newly synthesized N-linked glycoproteins in blastocyst formation, hatching from the zona pellucida, and in vitro attachment leading to outgrowth of the trophectoderm. The failure of tunicamycin to block early developmental stages could have simply been due to the inability of embryos at those stages to take up the drug. Metabolic labeling with mannose to determine glycoprotein synthetic rates is impossible at these early stages because hexose uptake is very poor prior to the morula stage (Brinster, 1969). Therefore, we cultured embryos for 10 hr from the four- to the eight-cell stage in medium containing 1 pg/ml tunicamycin, washed, and lyophilized them, and determined the activity of chitobiosyl-PPdolichol synthase. The enzyme activity in control embryos was 1.03 pmole/min/mg protein while that in tunicamycin treated embryos was only 0.23 pmole/min/ mg protein. These results directly demonstrate that tunicamycin can enter early embryos. cu-Anmliti~~ does not block the developmental regulutioz qf’sydhase uctivity in blastocysts. To determine whether the observed changes in enzyme activities during preimplantation development could be explained by alterations in the transcriptional activity of the corresponding genes, we examined the effect of a-amanitin on the expression of these enzymes in blastocysts. This drug has been shown to completely inhibit poly A’ RNA syn-

EFFECT

Glycosylation

DEVELOPMENT

stage Expansion

24 (17)

reached

by treated

embryos

Hatching

Attachment

Outgrowth

0 (0) 8 (15)

3 (5)

0 (0)

16 (70)

0 (0)

,Vofc. Embryos were cultured in vitro beginning at the indicated stages in CMRL 1066 medium supplemented as detailed under Materials and Methods. Approximately 85-95’S of embryos cultured in control media reached each developmental stage, while the number of embryos indicated in the table reached the corresponding stages when the media was supplemented with 1 n&ml tunicamycin. Treated embryos were scored as normal on the basis of their morphological appearance when examined by phase microscopy. The values in parentheses are the percentage of embryos reaching each stage.

234

DEVELOPMENTAL TABLE

EFFECT

OF WAMANITIN

Enzyme Chitobiosyl-PPdolichol synthase

Glc-P-dolichol synthase

Stage

EB HB HB

EB HB HB

ON ENZYME

n-Amanitin

-

BIOLOGY

2 ACTIVITIES

IN BLASTOCYSTS

Specific (pmole/min/mg

activity protein)

+

1.12 + 0.08 2.43 f 0.17 2.72 f 0.31

+

3.82 k 0.31 6.40 k 0.53 9.92 f 1.20

Note Embryos collected at the expanded blastocyst (EB) stage were either frozen and lyophilized immediately for enzyme assay or cultured 24 hr to the hatched blastocyst (HB) stage in CMRL 1066 media supplemented with (+) or without (-) 1 pg/ml cu-amanitin. Cultured embryos were then frozen and lyophilized, and all embryos were assayed as described under Materials and Methods. Assays were performed in triplicate using 25 embryos for each determination. The specific activities expressed as the mean k standard deviation of labeled were calculated as described in Fig. 4.

DISCUSSION

In this study we have examined the developmental regulation of N-linked glycoprotein biosynthesis in an effort to better understand the role of these macromolecules during preimplantation mouse embryogenesis. We were interested in establishing a relationship between the level of this biosynthetic activity and the growing functional complexity of cell membranes that occurs during blastocyst formation. Using very sensitive microassays, we determined the stage-specific activities of three key enzymes involved in the assembly of the oligosaccharide chains of N-linked glycoproteins. The results of these experiments suggest that an increased capacity for N-linked glycoprotein synthesis may be associated with blastocyst formation, In vitro culture of mouse embryos, which has opened the way for a molecular analysis of early mammalian development, has been in use for a number of years (Whitten and Biggers, 1968). However, biochemical studies have been greatly impeded by the limited availability of embryological tissue. Each egg or embryo contains only 25 ng of total protein (Sellens et al, 1981) and their production by laboratory mice is extremely costly in comparison with that of echinoderm or amphibian embryos. Therefore, biochemical studies can be performed only when available methods are sensitive enough for use with individual or small numbers of embryos. Our use of such methods has permitted a quantitative analysis in mouse embryos of the in vitro activities of the enzymes involved in N-linked oligosaccharide

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113. 1986

assembly. We have adapted procedures originally developed by Lowry and Passonneau (1972) for working with extremely small amounts of tissue and have extended these approaches, using in this case radioactive substrates, to assay three enzymes involved in the dolichol-linked pathway for N-linked glycoprotein synthesis, namely Man-P-dolichol, Glc-P-dolichol, and chitobiosyl-PP-dolichol synthases. These three enzymes of the dolichol-linked pathway were chosen for two reasons. First, labeled substrates of very high specific radioactivity are available. Second, chitobiosyl-PP-dolichol synthase catalyzes the first steps in the pathway, whereas Man-P-dolichol and Glc-P-dolichol synthases are involved in late stages. Similar methods employing enzymatic cycling and fluorescence detection have been used by other workers to measure enzyme activities and metabolite levels in early mouse embryos (Barbehenn et al., 1974,1978; Biggers and Borland, 1976; Hsieh et al., 1979; Leese et al, 1984). In our case, we were able to scale down the assays for the enzymes involved in N-linked oligosaccharide assembly and increase their sensitivity by using submicroliter volumes and saturating levels of substrates of high specific radioactivity. These conditions allowed for the utilization of relatively small amounts of costly radioactive substrates. Furthermore, although the reaction volumes used were relatively minute (0.2 to 1.0 pl), dilution of the radioactive substrate by endogenous substrates was unlikely since the reaction volume was at least 100 times greater than the cellular volume of the embryos (Lewis and Wright, 1935; Barbehenn et al., 1978). The results of these enzymatic studies demonstrate that the enzymatic apparatus for three steps in the synthesis of N-linked glycoproteins is present throughout murine embryogenesis. The in vitro activities of the enzymes measured, chitobiosyl-PP-dolichol synthase, ManP-dolichol synthase, and Glc-P-dolichol synthase, were all found to be developmentally regulated, generally decreasing between the unfertilized egg and early cleavage stages and then increasing during the blastocyst stage. We were surprised to learn that although the three enzyme activities were low during the stages directly following fertilization, their activities in the unfertilized egg were relatively high. However, it is important to note that the unfertilized egg has just completed a developmental program of oogenesis that may involve elevated glycoprotein synthesis. Indeed, a major product synthesized during oogenesis is the zona pellucida which is composed of three glycoproteins that contain N-linked oligosaccharide chains (Greve et al, 1982). Throughout development the specific activity of chitobiosyl-PP-dolichol synthase was the lowest compared to the other enzymes and exhibited the most dramatic

ARMANT, KAPLAN, AhTnLENNARZ

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developmental changes, increasing over fivefold between the early cleavage and hatched blastocyst stages. Although a number of other enzymes are involved in the assembly of these oligosaccharides, the consistent trend of an increased activity at the blastocyst stage observed for all three enzymes indicates that the capacity of embryos to synthesize N-linked glycoproteins is also elevated in blastocysts. This increase in the biosynthetic capacity for N-linked glycoproteins is presumably related to the increasing complexity of the embryo cell membranes during blastocyst formation. Prior to the eight-cell stage there is little evidence of cell-cell interactions among blastomeres, but thereafter interactions between cells have been shown to induce changes in the organization of their surface membranes (Ziomek and Johnson, 1980). These cellular interactions convey positional information that gives rise to the organization and differentiation of the blastocyst (Johnson and Ziomek, 1981). Our findings are consistent with the idea that N-linked glycoproteins may be involved in these cell-cell interactions. The essential role of N-linked glycoprotein synthesis during blastocyst formation was further demonstrated ilr ~V+Oby the finding that tunicamycin had no effect on development up to compaction, but blocked all subsequent developmental events, including cavitation, blastocyst expansion, hatching from the zona pellucida, and i?r ~ifro attachment leading to trophectodermal outgrowth. Tunicamycin did not strongly inhibit attachment when exposure to the drug was initiated at the hatched blastocyst stage, probably because the glycoproteins required for attachment were already synthesized (Sherman and Atienza-Samols, 1978). However, these attached embryos did not progress to form trophectodermal outgrowths. Inhibition of blastocyst formation and outgrowth by tunicamycin was first reported by Surani (1979) and has been confirmed in a subsequent report (Surani et ccl., 1981), as well as in this one. However, it has been argued that the inhibition observed is due to inhibitors of protein synthesis contaminating the tunicamycin rather than to inhibition of glycosylation (Atienza-Samols et al.. 1980). In this study we have used a purified tunicamycin preparation that is essentially free of inhibitors of protein synthesis and found that the drug blocked all later stages of development. Although we are in agreement with previous investigators that tunicamycin has no effect on the early cleavage stages, a period when Nlinked glycoprotein synthesis is known to occur (Magnuson and Epstein, 1981), we could not confirm the earlier reports that the drug blocks compaction (Surani, 1979; Atienza-Samols et aZ., 1980; Surani et ah, 1981; Pratt ct ol., 1982; Sutherland and Calarco-Gillam, 1983). This

Glycos~/lnt itm in MOUHP Evr/bry/os

235

discrepancy may be due to our use of a purer preparation of tunicamycin. One possible explanation for the differential effect of tunicamycin on early and late preimplantation development is that the newly synthesized glycoproteins do not have an essential function during early development, whereas glycoproteins synthesized at later stages serve essential functions at the time they are made. Alternatively, earlier formed stores of the oligosaccharidelipid may be adequate for glycosylation to occur in the absence of de novo oligosaccharide-lipid synthesis, given the very low level of protein synthesis during early embryogenesis (Brinster, 1971; Epstein and Smith, 1973). Although it is difficult to correlate in vitro activities with in r!iao events, it is interesting that the in vitro activity profiles for two of these enzymes (chitobiosylPP-dolichol synthase and Glc-P-dolichol synthase) in sea urchin embryos were found to correlate well with the profile of oligosaccharide-lipid biosynthesis measured in vi?10 (Welply et al., 1985). As in preimplantation mouse embryos, tunicamycin interferes with sea urchin embryo development only at the later stages when the in vitro activities of these enzymes are elevated (Heifetz and Lennarz, 1979; Welply et ul., 1985). Although echinoderm and mammalian embryos are well separated phylogenetically and confront very different environments, both embryos demonstrate an increased capacity to synthesize N-linked glycoproteins during periods of development characterized by heightened cell interactions and increased membrane complexity. The heightened capacity of mouse embryos to assemble oligosaccharide-lipid occurs at a time of increased protein synthesis (Brinster, 1971; Epstein and Smith, 1973), and most likely represents the coordinated regulation of oligosaccharide-lipid, protein, and glycoprotein synthesis during preimplantation development. Having established that the enzymes chitobiosyl-PPdolichol synthase, Man-P-dolichol synthase, and Glc-Pdolichol synthase are developmentally regulated during mouse embryogenesis, we initiated experiments aimed at characterizing the mechanisms underlying the regulation of these enzymes. Specifically, we asked whether the increased enzyme activity observed at the blastocyst stage was due to increased transcription of the mRNAs encoding these proteins. It has previously been demonstrated that expression of some unidentified proteins (Braude, 1979), as well as two enzymes, P-glucuronidase and plasminogen activator (Schindler and Sherman, 1981) are unaffected by cr-amanitin treatment, whereas the expression of other proteins (Braude, 1979) and the activity of A5,3/%hydroxysteroid dehydrogenase (Schindler and Sherman, 1981) are a-amanitin-sensitive at the blastocyst stage. The present study has demon-

236

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strated the presence of three oc-amanitin insensitive enzymes involved in N-linked glycoprotein synthesis. Because cu-amanitin does not block development at the blastocyst stage (Braude, 1979; Schindler and Sherman, 1981), whereas tunicamycin does (Surani, 1979), it seems likely that the synthesis of N-linked glycoproteins at the blastocyst stage is regulated by a mechanism independent of de novo mRNA synthesis. We are very grateful to Dr. Alan D. Elbein for the generous gift of tunicamycin. The helpful discussions of Dr. Joseph K. Welply, Dr. Stephen R. Grant, Dr. Harold D. Woodward, and Dr. Barry D. Shur, as well as the technical assistance of Ms. Mary Rivera, Fran Tan, and Helen Park, are appreciated. We also thank Ms. Diana Welch for help in preparing the manuscript. This work was supported by National Institutes of Health grants (HD-18600) to W.J.L., and (HD-19977) to D.R.A, a grant from the March of Dimes (MOD l-867) to W.J.L., and a National Research Service Award (GM-07965) from the NIGMS to D.R.A. Dr. William J. Lennarz, who is a Robert A. Welch Professor of Chemistry, gratefully acknowledges the Robert A. Welch Foundation. REFERENCES ATIENZA-SAMOLS, S. B., PINE, P., and SHERMAN, M. I. (1980). Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos. Deu Biol. 79, 19-32. BARBEHENN, E. K., WALES, R. G., and LOWRY, 0. H. (1974). The explanation for the blockade of glycolysis in early mouse embryos. Proc. N&l.

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