THE J O U R N A L OF BIOLOGICAL CHEMISTRY VOI. 258, No. 9, Issue of May 10, pp. 5858-5863, 1983 I’rrntrd In KI S.A.

In Vitro Biosynthesis of Three Sulfated Glycoproteins of Murine Zonae Pellucidae by Oocytes Grown in Follicle Culture*

(Received for publication, November 29, 1982)

Satoru ShimizuS, Masahiro Tsujig, and Jurrien Dean7 From the Laboratory of Chemical Biology, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205

The zona pellucida is an extracellular glycocalyx that surrounds the growing oocyte and mediates spe- cific biological functions essential to early mammalian development. We have isolated by a novel technique intact zonae pellucidae from a murine follicle culture system which mimics in vivo granulosa cell-oocyte in- teractions. Using radioactive precursors we have dem- onstrated that all three zona proteins, ZP-1,ZP-2, and ZP-3, are sulfated glycoproteins. Incubation of the fol- licle culture with tunicamycin (0.5 pg/ml), an antibiotic that blocks N-glycosylation, inhibits new protein dep- osition in the zona. Under these same conditions overall protein synthesis is decreased only 30%. Biosynthetic studies indicate that after 6 days in culture the zona proteins are produced at 0.4 pg/oocyte/h, which rep- resents 2-3% of the total oocyte protein synthesis. This synthesis stops at or before ovulation and the ex- tremely long half-life (>lo0 h) of the zona may be essential to preserve its known biological functions, all of which occur after ovulation.

Preimplantation development in mammals, which begins with oogenesis and proceeds through fertilization to the early blastocyst stage, involves the modulation of gene expression during cell differentiation. Although a number of gene prod- ucts have been identified during early murine development (1, a), only a few appear to be specific for this period of growth (3-6) and still fewer have known biological roles. However, the proteins of the zona pellucida constitute a subset of gene products that are produced during oogenesis, have particular biologic functions, and, furthermore, are produced in suffi- cient quantities to permit analysis (7, 8). We have selected these proteins for more detailed studies in hopes of eventually elucidating the mechanism of the control of their expression in development.

During oogenesis in the murine ovary, the zona pellucida appears as a discrete extracellular structure that surrounds the oocyte and contains three glycoproteins designated ZP-1, ZP-2, and ZP-3 (8,9). This glycocalyx (a) mediates the species specificity of capacitated sperm binding to ovulated eggs, ( b ) is a major block to post fertilization polyspermy, and (c) appears to protect the growing embryo as it passes down the

* 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 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Laboratory of Ultrastructure Research, Aichi Cancer Center Research Institute, Nagoya, Japan.

Present address, Department of Chemistry, Faculty of Science, Nagoya University, Nagoya, Japan.

7 To whom reprint requests should be addressed.

oviduct prior to implantation on the uterine wall (7-9). Al- though the zona plays a crucial role in development, only recently have biochemical analyses of its constituents been performed. The mass of the murine zona from a single oocyte has been estimated to be 5 ng, of which approximately 80% is protein and the rest presumed to be carbohydrate (8). Each of the three zona proteins is glycosylated and each appears to be synthesized by the growing oocyte (10).

In the ovary there is a close cooperative relationship be- tween the oocyte and the surrounding granulosa cells. During oogenesis, prior to the secretion of the zona pellucida, gap junctions are formed between the oocyte and neighboring granulosa cells (11, 12). Greater than 85% of the nutrients taken up by the follicle-enclosed oocyte are first taken up and metabolized by granulosa cells prior to their transfer via gap junctions to the oocytes (13, 14). Without this contact with the surrounding granulosa cells, the metabolism of the oocyte is markedly different (13, 14), and denuded oocytes do not grow at a normal rate (13-15). Thus, we have taken advantage of a follicle culture system (15) which mimics the in vivo granulosa-oocyte interaction to study the production of the zona pellucida. In this report we describe the biosynthesis of three sulfated glycoproteins that make up the murine zona pellucida as well as the effect of tunicamycin on their produc- tion.

EXPERIMENTAL PROCEDURES

fied. Medium was obtained from the National Institutes of Health Materials-All reagents were from Sigma unless otherwise speci-

Media Facility, Bethesda, MD. Radioisotopes were purchased from New England Nuclear.

Radiolabeling of Follicle Cultures-Mouse follicles were isolated from ovaries of 8-day-old DBA mice and cultured in modified Way- mouths MAB 87/3 media for 7 days (15). Fresh medium was added every 2 days. After 6 days in culture, follicles were washed twice with amino acid-free culture medium containing 15% dialyzed and heat- inactivated fetal calf serum (Flow Laboratories, McLean, VA). One ml of the same medium with 125 pCi of ~-’~C-aminoacid mixture (55 mCi/matom of carbon) was added and after 16 h, the cells were washed three times with normal culture medium before analysis. The same procedure was used to label with [35S]sulfate except that MgS0,- free culture media containing 50 pg/ml of gentamicin sulfate (32% sulfate) and 1.25 mCi of carrier-free (35S)sulfuric acid were used. The calculated final concentration of sulfate in the medium was 167 pM. Labeling with [32P]phosphate was carried out in phosphate-free media using 500 pCi/ml of carrier-free (32P)phosphoric acid. The carbohy- drate side chains were labeled using 1.0 mCi/ml of ~-[5,6-~H]fucose (56 Ci/mmol) or 500 pCi/ml of D-[1,6-3H]glucosamine (31.3 Ci/mmol) in normal medium added to the follicle culture on the 5th day. The zonae were isolated after 48 h of incubation. Follicle cultures were also labeled following incubation with tunicamycin (Calbiochem- Behring) introduced in 4 pl of 0.1 M NaOH (final concentration, 0.5 pg/ml). Control samples received the same amount of alkali without tunicamycin. After 24 h, cultures were labeled for 12 h as described above except for the continued presence of tunicamycin (0.5 pg/ml).

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Biosynthesis of the Murine Zona Pellucida 5859

Isolation of Oocytes and Zonae Pellucidae-After a week in culture, oocytes were freed from the surrounding granulosa cells, collected by micropipette and washed three times in HEPES'-modified Brinster's media containing BSA (0.1 mg/ml). Washed oocytes were suspended in 0.3 ml of HEPES-Brinster media containing BSA (0.1 mg/ml), EDTA (10 mM), lima bean trypsin inhibitor (10 pg/ml, P-L Biochem- icals), phenylmethylsulfonyl fluoride (0.1 mM), and 0.2% NP40 (BDH Chemicals, Poole, England). After three cycles of freezing in dry ice- ethanol and thawing a t 20 "C, the suspension was transferred to a culture dish containing 2 ml of the same medium and examined. Intact zonae pellucidae with no apparent cellular debris were isolated by micropipette and washed twice with 2 ml of HEPES-Brinster medium containing BSA (0.1 mg/ml) and NP40 (0.2%), and twice with medium containing BSA alone. Acid-insoluble radioactivity was ascertained by adding BSA (0.2 mg/ml) and making the sample 10% in trichloroacetic acid. After centrifugation, the supernatant was counted by liquid scintillation in 10 ml of Aquasol (New England Nuclear). The pellet was dissolved in 10 e1 of 1 M NaOH, neutralized, and counted.

Lactate dehydrogenase activity was determined (16) after freezing and thawing the isolated oocytes or zonae. ATPase activity of freeze- thawed oocytes or zonae was assayed in a 10O-pl reaction mixture using 0.1 pmol of [y3'P]ATP (5 pCi/pmol) (17). After absorption of ATP by Norit A followed by centrifugation, the amount of inorganic [32P]phosphate in the supernatant was determined.

Isolation of Superouulated Eggs-Six-week-old DBA mice were injected intraperitoneally with 10 IU of pregnant mare serum gonad- otropin followed 48 h later with 10 IU of human chorionic gonadotro- pin (18). The mice were sacrificed by cervical dislocation 16-18 h after the last injection and the eggs isolated from their oviducts. Following a 30-min incubation with hyaluronidase (300 units/ml, Sigma type VI) the eggs were harvested by micropipette. The eggs and zonae pellucidae were labeled, washed, and isolated as described above.

SDS-Polyacrylamide Gel Electrophoresis-Slab SDS-PAGE was performed using a 4.5% stacking gel and a 10% running gel of 0.75- mm thickness as previously reported (19) except that samples were incubated for 15 min at 37 "C in the presence or absence of 40 mM dithiothreitol (Bethesda Research Laboratories). Samples (30 pl) containing 20-100 zonae (500-2500 dpm) or 10-15 oocytes (5,000- 10,000 dpm) were applied to the gel and electrophoresed at 20 mA. Gels were fixed and stained (20), and molecular weights were calcu- lated using the following proteins as markers (Boehringer Man- nheim): Escherichia coli RNA polymerase (a, @, @' subunits, 39,000, 155,000, and 165,000 respectively), phosphorylase b (94,000), BSA (68,000), ovalbumin (43,000), and soybean trypsin inhibitor (21,500). The protein hands in fixed gels were detected by fluorography (21) and densitometer tracings were obtained with a LKB laser densitom- eter.

Gel Filtration of Glycopeptides-Zonae pellucidae labeled with either 13H]glucosamine or [35S]sulfate were isolated as described above, incubated in 0.2 M NaOH at room temperature for 16 h, neutralized, and twice digested with 200 pg of pronase (Calbiochem) for 2 h a t 56 "C. The material was then applied to a Sepharose CL- 6B (Pharmacia) column (100 X 0.8 cm) equilibrated with 0.2 M NaCI. The fractions of the single peak were pooled, lyophilized, and aliquots were treated with endo-@-galactosidase (Pseudomonas sp., Miles Lab- oratories, Inc., Elkhart, IN), chondroitinase ABC (Proteus oulgaris, Miles Laboratories, Inc.,) or 18% (w/w) HONO as previously de- scribed (22). A control aliquot and the three treated samples were each applied to a Bio-Gel P-4 (Bio-Rad) column (40 X 0.5 cm, 200- 400 mesh) equilibrated in 0.3 M pyridine-acetic acid, pH 5.0.

Biosynthesis and Turnover Studies-After the 6th day in culture, 125 pCi of I4C-aminoacid mixture was added to a culture as described above. Oocytes and zonae pellucidae were isolated after 2, 4, and 8 h of labeling. In a parallel experiment, the 125 FCi of 14C-aminoacids used to label the follicle culture were diluted with a $-fold excess of unlabeled amino acids (in the same mass ratio) and oocytes were collected in a similar manner. Acid-insoluble counts were used in an isotope dilution method to determine the amino acid pool size and calculate the amount of de nouo protein synthesis (23).

The turnover rate was ascertained by labeling each of four parallel cultures with 125 pCi of 14C-aminoacids for 16 h following the 4th

The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-2-piper- azineetbanesulfonic acid BSA, bovine serum albumin; NP40, Noni- det P-40; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

day in culture. The cultures were washed six times with modified Waymouth's MAB 87/3 medium containing 15% fetal calf serum and incubated in the same medium (15). Fresh medium was added every 48 h. Trichloroacetic acid-insoluble radioactivity was determined for the isolated zona at appropriate times after the initial labeling period. Each of four cultures was used as an individual time point.

RESULTS

Isolation of Zonae Pellucidae from Follicle Cultures-For a single preparation, 30 DBA female mice (8-day-old) were sacrificed and 25-35 follicles were isolated from each ovary (see Fig. la). Each follicle consisted of a central oocyte in meiotic arrest surrounded by several layers of granulosa cells. The granulosa cells proliferated and anchored the oocytes to the culture dish substratum (see Fig. lb) . After 7 days in culture the zona pellucida was enlarged and the oocytes had diameters of 45-55 Fm. The oocytes with their surrounding zonae pellucidae were freed from the granulosa cells and isolated by micropipette (see Fig. IC). The recovery of oocytes ranged from 15-50% (median 33%) of the number of follicles originally seeded.

Zonae pellucidae obtained by freeze-thawing in the presence of 0.2% NP40 were examined by Nomarski interference con- trast optics. They appeared as wraith-like circles with diam- eters of 50-60 wm (see Fig. Id). There was no evidence of cytoplasmic or nuclear structures and they appeared free of any intracellular debris. Intact zonae were routinely obtained from more than 60% of the harvested oocytes. To test further the purity of these preparations, we measured activities of enzymes present in isolated zonae pellucidae (compared to the intact oocyte) which would be indicative of residual cy- toplasm or cell membranes. The lactate dehydrogenase activ- ity of the zona was 0.8% and ATPase activity was 0.2% of that of the cultured oocyte. (NP40 treatment of lysed oocytes under identical conditions resulted in 98% retention of enzy- matic activity by the oocytes.) Finally, the presence of only three major zona proteins, designated ZP-1, ZP-2, and ZP-3 (8 ) , after labeling with I4C-aminoacid precursors, was used as a criterion for purity (see Fig. le).

Glycoproteins of the Zona Pellucida-Table I shows the incorporation of radioisotope into the oocytes and zonae pellucidae when the follicles in culture were labeled with I4C- aminoacid mixture, [3H]glucosamine, ['H]fucose, or [35S]sul- fate. Most of the radioactivity in the oocytes (60-95%) and all of the radioactivity in the zonae was acid insoluble. Acid- precipitable radioisotope in the zonae pellucidae represented 2-6% of the [14C]amino acid present in the total oocytes, 35- 45% of the [3H]fucose, 37-40% of the [3H]glucosamine, and 15-25% of the [''SS]sulfate.

Using approximately 3000 dpm of I4C activity, all three zona proteins were visualized on SDS-PAGE by fluorography after 10 days at -80 "C. The protein pattern of the zonae pellucidae isolated by freeze-thawing in the presence of deter- gent revealed three diffuse bands (see Figs. le and 2b) with average molecular weights of 185,000 (175,000-195,000), 140,000 (120,000-160,000), and 83,000 (68,000-98,000). In addition, there is a sharp band of 48,000 daltons which has the same apparent molecular weight as mouse actin (8). The majority of the radioactivity appeared in ZP-2. After reduction of disulfide bonds with dithiothreitol, the diffuse band of the highest molecular weight disappeared, although a sharp band with a. molecular weight of 175,000 remained and two other diffuse bands with apparent average molecular weights of 120,000 and 95,000 were visualized (compare Fig. 2, b and c).

Zonae isolated from follicles labeled with ['H]fucose dem- onstrated the same three diffuse bands as were observed after labeling with 14C-aminoacids (see Figs. 2d and le). However,

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5860 Biosynthesis of the Murine Zona Pellucida

containing oocytes with intact nuclei surrounded by granulosa cells. b, follicles , . after 7 days in culture showing prolifer- ating granulosa cells anchoring oocytes to the substratum. c, oocytes with sur- . ,, ~. ~

rounding zonae pellucidae isolated from follicles in culture. Nuclear membranes , are intact and each contains a prominent nucleolus. d, intact zonae pellucidae iso- lated after freezing and thawing in the presence of 0.2% NP40. e, fluorogram of zonae pellucidae proteins separated by SDS-PAGE. Sample contained 1.5 X lo3 dpm.

. , .-

TABLE I Incorporation of "C-arnirwacids, 3H-sugars, and rJ5Slsulfate into

oocytes and isolated zonae pellucidae Isotope concentrations were: "C-aminoacids, 125 pCi/ml; [3H]

glucosamine, 500 pCi/ml; [3H]fucose, 1 mCi/ml; and [35S]sulfate, 1.25 mCi/ml.

Intact oocyte Zona pellucida

Total Acid in- Ti$ Acid in- dpm/egg soluble soluble ". " __

% %

"C-aminoacids 860 70 34 99 [:'H]glucosamine 179 58 67 99 [:'H]fucose 290 67 72 99 [:'"S]sulfate 31 93 6 100

most of the label appeared in ZP-2 and ZP-3 with less appar- ent in ZP-1. After reduction with dithiothreitol, the highest molecular weight band, ZP-1, disappeared completely and two other diffuse bands shifted their apparent molecular weights exactly like those bands labeled with 14C-aminoacids. There was no residual band a t 175,000, in contrast with that seen after labeling with 14C-aminoacids and SDS-PAGE under reducing conditions (compare Fig. 2, c and e). A similar pattern was seen after labeling with [RH]glucosamine (data not shown).

When the follicles were labeled with ["S]sulfate, three diffuse bands (see Fig. 2f) were demonstrated although ZP-1 and ZP-2 appeared equally labeled while ZP-3 contained very little '"S. Labeling with ["*P]phosphate resulted in no radio- activity incorporated in any of the three zona proteins. The ratio of radioactivity incorporated after labeling with I4C- amino acids, "H-sugars, and [:"S]sulfate differed among the three zona proteins, which suggests that each is a distinct sulfate containing glycoprotein. Zonae pellucidae isolated after labeling either with [3H]glucosamine or [R5S]sulfate were treated with alkali and, following exhaustive digestion with pronase, chromatographed on a Sepharose CL-GB column. The same major peak was observed for both radioisotope labels and had a Kay of 0.7-0.85 which contained 90-95% of the radioactivity applied to the column. The peak containing "H material was lyophilized and either digested with a hydro- lase specific for keratan sulfate, dermatan sulfate, chondroitin sulfate A and C, or treated with nitrous acid to degrade heparin or heparan sulfate. The treated samples were rechro- matographed on a P-4 column and all of the labeled material appeared in the void volume suggesting a lack of degradation by these procedures.

a b c d e f "- 165K-

93K- 67K-

39K-

m 3ZP-1 b 4 1 zp-2

] ZP-3

FIG. 2. Fluorograms of SDS-PAGE of oocytes and isolated zonae pellucidae labeled with I4C-aminoacids, [3H]fucose, or [3'S]sulfate. Lune a, protein pattern of the intact oocyte labeled with "C-aminoacids, 2.0 X IO4 dpm. Lane b, zonae pellucidae labeled with "C-aminoacids, 1.0 X lo3 dpm. Lane c, the same as lane b but after reduction with dithiothreitol. Lane d, zonae isolated after label- ing with [''H]fucose, 2.2 X lo' dpm. Lane e, the same as lane d after reduction with dithiothreitol. Lane f, isolated zonae pellucidae after labeling with ["S]sulfate, 1.3 X lo3 dpm.

Effects of Tunicamycin on Radioactive Presursor Incorpo- ration-Incubation of the follicle culture with tunicamycin (0.5 pg/ml) dramatically altered the incorporation of radioiso- topes into the zonae pellucidae (see Table 11). Under our experimental conditions, the amount of I4C-aminoacid radio- activity in the oocyte protein was 70% of normal and ["HI glucosamine was 40% of normal. [''HlFucose incorporation was unchanged from the control culture incubated without tunicamycin, which suggests that in the oocyte fucose is not primarily associated with N-linked oligosaccharides. Less than 3% of the radioactivity seen in [3H]glucosamine or ['HI fucose labeled control zonae was seen in tunicamycin-treated zonae. Because zonae pellucidae labeled with I4C-aminoacids in the presence of tunicamycin still had radioactivity associ- ated with them (see Table II), I4C-aminoacid labeled zonae isolated after culturing in the presence and absence of tuni- camycin were compared by SDS-polyacrylamide gel electro- phoresis (see Fig. 3). Both had similar amounts of the 48,000- dalton (presumed actin) band, but the tunicamycin-treated

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Biosynthesis of the Murine Zona Pellucida 5861

zona had essentially no labeled zona proteins. The remaining “‘C-aminoacids associated with the zona appeared as large macromolecules that did not enter even the stacking gel. Similar large molecular weight material was seen both in the presence or absence of tunicamycin. As yet our data have not permitted us to determine the nature of this material.

Biosynthesis and Turnover Rates-We have labeled the follicle culture for 16 h with “C-aminoacids after 1, 2, 4, and 6 days in culture and determined the amount of radioactivity in the zonae pellucidae as a percentage of the total acid- insoluble radioactivity of the intact oocyte. Following 1 day in culture 7.5% of the “‘C-aminoacids was found in the zona, and this percentage decreased progressively to 3% after 6 days in culture (see Fig. 4). Radioisotope incorporation studies of superovulated eggs freed from their granulosa cells demon-

TABLE II Effect of tunicamycin on protein synthesis of oocytes and zonae

pellucidae OCKyte Zona pellucida

Con- Tunica- (5% Con- Con- Tunica- (96 con- trol mycin” trol) trol mycin” trol)

dpn b.loocyte dpn ~./zono

“‘C-aminoacids 958 666 (TO) 39 (41) [“Hjglucosamine 106 42 (40) 32 :; (<3) [“Hjfucose 144 162 (112) 38 1 (<3)

” Tunicamycin, 0.5 pa/ml.

a b

ZP-1 r

zp-2[ o+ ZP-3 1

FIG. 3. Effect of tunicamycin on radioisotope incorporation into the zona pellucida. a, SDS-PAGE of zonae pellucidae isolated from “C-aminoacid-labeled follicle cultures. b, same as a but labeled in the presence of tunicamycin (0.5 &ml). c, densitometry tracing of a. d. densitometry tracing of b.

I 1 I I

2 4 6 8

DAYS IN CULTURE

FIG. 4. Percentage of the total acid-insoluble W-amino- acids associated with the zona pellucida during culture.

TABLE III

Determination of ammo acid pool size and rate of protein synthesis Amino acids dI/dt I, SA- (;

5’1 260

52.5 181 13.2 62

dpm/h/oocvte dpmlwcyte dpml;;i;mino pg amino ocids/oocyte

5.0 36.2 0.9 68.9

500tA

d 400 -

:

8300- /11-14 . E 0"

200-

loo- / L-I

2 4 6 8 Time (hours)

20 8

16 t

w Time (hours)

10 20 30 40 50 60 70 80 Time lhoursl

FIG. 5. Biosynthesis and degradation of the oocyte and zona pellucida. A, “C-aminoacid incorporation into acid-insoluble mate- rial from isolated oocytes grown in follicle culture. W, undiluted “C- aminoacids; 0, “C-aminoacids diluted 1:5 in the same mass ratio with unlabeled amino acids. R, undiluted “C-aminoacid incorporation into zonae pellucidae isolated from oocytes grown in follicle culture (m). C, follicle cultures labeled with ‘“C-aminoacids, washed and incubated with unlabeled amino acids. Radioactivity of zonae pellucidae isolated at various times after initial labeling (0).

strated a substantial amount of radioactivity incorporated into acid-insoluble material obtained from the eggs, but no radioactivity was found in the zonae proteins after labeling either with “‘C-aminoacids or with [“Hlglucosamine.

Using an isotope dilution method (23), we have measured the rate of protein synthesis in intact oocytes and the appear- ance of label in isolated zonae pellucidae during the 6th day of follicle culture. Two different sets of experimental condi- tions were used that differed only in the specific activity of the precursor amino acids (see Table III). We have measured the apparent rates of radioactive amino acid incorporation (dZ/dt) into total oocyte protein (see Fig. 5A). Under both sets of experimental conditions, the expanded amino acid pool is at a steady state between the oocyte and the media within the first hour of incubation and the intra-oocyte, acid-soluble radioactivity, L, can be measured. From this, and knowing the specific activity of amino acids in the media, SAmed, the amino acid concentration, G, can be calculated (see Table III). Assuming that the absolute rate of protein synthesis remains constant under both sets of experimental conditions, we have determined that the size of the amino acid precursor pool is 52.6 pg/oocyte. From this we calculate that during the 6th day in culture, the oocytes incorporate 25.9 pg of amino acid per h into acid-insoluble macromolecules. Because the mix- ture of amino acids that we have added represents 98% of the endogenous available acids of the oocyte (24), we have deter-

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mined that protein synthesis occurs in oocytes in culture a t a rate of 26.4 pg/h/oocyte.

The apparent rate of synthesis of the zona pellucida was determined in a parallel experiment from the siope of the appearance of 14C activity in the zona over time (see Fig. 5B). The dI/dt of the zonae pellucidae was 1.4 dpm/zona/h. Using the already ascertained amino acid pool size of the oocyte, we have calculated that the zona pellucida is synthesized at 0.7 pg/h/oocyte. However, our studies with tunicamycin suggest that only 59% of the 14C-aminoacids associated with the zona pellucida is incorporated into the zona proteins, the rest being found in large molecular weight aggregates that do not enter the stacking gel. Reflecting this observation, we have deter- mined that the amount of protein synthesized as ZP-l,ZP-2, and ZP-3 is 0.4 pg/h/oocyte or 2% of the total oocyte protein production.

Following the 4th day in culture four separate follicle CUI- tures were labeled for 16 h with '*C-aminoacids. All of the cultures were washed repeatedly with medium containing a 20-fold excess of unlabeled amino acids in order to minimize the reutilization of radioactive precursors. Oocytes were bar- vested from one of the cultures and the amount of radioactiv- ity incorporated into the zona pellucida was determined. A similar determination was made after in cub at in^ the other three cultures with media containing unlabeled amino acids for periods of 16,36, or 82 h (see Fig. 5C). The apparent half- life of the zona pellucida in our culture system is quite long and 70% of the 14C-aminoacids remains associated with the zona after 82 h.

DISCUSSION

Using 8-day-old mice as a biological source for our follicle cultures, we have obtained for our biosynthetic studies a fairly homogenous cell population in meiotic arrest with nuclear membranes intact. The follicle culture system (15) has per- mitted labeling with a variety of amino acids, sugars, and inorganic compounds and allowed us to analyze the kinetic rates of synthesis and degradation of the three sulfate-con- taining zona glycoproteins during the 7-day culture period. By internally labeling the proteins of the oocytes and the surrounding zona pellucida, we have avoided potential arti- facts introduced by external labeling. We have routinely ob- tained 1800-2200 follicles from 30 mice. After a week in culture, the oocytes enclosed in the follicle have increased their diameter by 30% and the zona is a readily apparent structure.

A wide variety of mechanical, physical, and chemical meth- ods have been used to solubilize or obtain intact murine zonae pellucidae (8,10,25-28). In the novel procedure reported here, we disrupt the oocyte plasma membrane by freezing and thawing in the presence of a nonionic detergent. Thus, the cell components inside of the zona pellucida are washed out and the zona pellucida is left as an intact structure. We have used morphological appearance, absence of cytoplasmic and plasma membrane enzymatic activity, and the protein pattern on SDS-PAGE gels to ascertain the purity of our preparation. We have found that zonae obtained by this procedure contain fewer con~minating protein bands than those obtained by mechanical disruption by homogenization followed by wash- ing with NP40 (data not shown). They are also comparable to the results reported in the literature for disruption by micropipetting and solubilization with low pH (8). Our method is fast, requires no special equipment, and yields reproducible results. Zonae from oocytes at every stage and of any size can be isolated because, unlike the mechanical methods used, no particular clearance of the homogenizer pestle nor diameter of the micropipette need be considered.

Our studies in culture confirm earlier reports (8) that the zonae is comprised of three proteins, ZP-1, ZP-2, and ZP-3, that are noncovlanetly linked to form the zona pellucida. Our incorporation studies with radioactive presursors indicate for the first time that all three are sulfate-containing glycocon- jugates. The co-migration of [35S]sulfate and [3H]glucosa- mine-labeled material during gel filtration indicates that the sulfate is attached to the carbohydrate and the lack of diges- tion of the isolated side chains with chondroitinase ABC or endo-@-galactosidase and resistance to treatment with nitrous acid suggest that all three zonae proteins are sulfated glyco- proteins. However, definitive determination of the composi- tion carbohydrate side chains of the zona proteins awaits further analysis.

Tunicamycin, an antibiotic which blocks the transfer of UDP-N-acetylglucosamine to dolichol phosphate (29, 30), inhibits N-glycosylation of newly synthesized proteins (30, 31). Most preparations of tunicamycin consist of a family of h o m o l o ~ e s which vary in their ability to inhibit glycosylation and the extent to which they inhibit protein synthesis (32). However, the complete inhibition of protein synthesis has not been reported even with tunicamycin doses as high as 2 pg/ ml (32-41). Our results demonstrate a clear-cut absence of newly synthesized protein in the zona pellucida in the pres- ence of tunicamycin (0.5 pg/ml). Under these conditions less than 3% of the radioactive sugar precursors are found asso- ciated with the zona pellucida and, after gel electrophoresis no I4C-amino acid labeled zona proteins can be detected in isolated zonea pellucidae. Tunicamycin can affect glycopro- teins by altering the rate of their degradation causing intra- cellular aggregation by blocking assembly of subunits. All of these mechanisms can lead to an inhibition of protein secre- tion, but as yet, our data do not permit discrimination among these possibilities.

We have calculated that the oocyte after 6 days in culture synthesizes 26 pg/h/ooc~e of protein which is comparab~e to that observed in a follicle culture system using f35S]methio- nine alone assuming an average methionine composition of 2% of the total protein (23). Under the present culture con- ditions, 0.4 pg/h/oocyte or 2% of the total protein synthesis was secreted as ZP-1, ZP-2, and ZP-3 to form the zona pellucidae. These kinetic data for the zona are in accord with the data presented in Table I showing that after 16 h of labeling 2-6% of the 14C-amino acids were found in the zonae. When the follicle culture was labeled for 16 h during the first day in culture, the zona petlucida contained 7-8% of the total radioactivity incorporated into protein synthesized by the oocyte. By the 6th day in culture this percentage had de- creased to 2-3%. During this time period the total oocyte protein synthesis has approximately doubled (23). Further- more, ovulated eggs do not incorporate radioactive precursors into their zona pellucida a t all. Thus, it would appear that most of the zona pellucida proteins are produced relatively early in oogenesis and that during the culture time period a significant, albeit decreasing, amount of metabolic activity is occupied in the synthesis of the zona proteins. Although the biological functions of the zona pellucida are manifested after ovulation by which time its biosynthesis is complete, the long half-life (>lo0 h) of the zona proteins appears to assure their presence during early preimplantation development.

Acknowledgments-We are grateful to Dr. John J. Eppig for intro- ducing us to the follicle culture system and to Drs. E. Neufeld and V. Hascafl for critical reading of the manuscript. We thank Laura Barry for typing the manuscript.

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S Shimizu, M Tsuji and J Deanoocytes grown in follicle culture.

In vitro biosynthesis of three sulfated glycoproteins of murine zonae pellucidae by

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