T H E JOURNAL OF BIOLOGICAL C H E M I S T R Y Vol. 264, No. 23, Issue of August 15, pp. 13840-13847, 1989 Printed cn U. S. A.

Synthesis and Accumulation of Hyaluronic Acid and Proteoglycans in the Mouse Cumulus Cell-Oocyte Complex during Follicle-stimulating Hormone-induced Mucification*

(Received for publication, March 22, 1989)

Antonietta SalustriS, Masaki Yanagishita, and Vincent C. Hascall From the Bone Research Branch. National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892

In most mammalian ovaries, the cumulus cell-oocyte complex (COC) expands at the time of ovulation by depositing an extensive extracellular matrix between the cumulus cells. This phenomenon can be reproduced in vitro by culturing COCs with follicle-stimulating hormone (FSH) and serum. Biosynthesis of hyaluronic acid (HA) and proteoglycans by mouse COCs in vitro was studied using [3H]glucosamine and [36S]sulfate as metabolic precursors. Radiolabeled complex carbohy- drates were analyzed by ion exchange chromatogra- phy, specific enzyme digestion followed by high per- formance liquid chromatography, and gel filtration. The specific activities of [SH]hexosamines in the la- beled molecules were determined by measuring the incorporation of 3H and 35S into chondroitin 4-sulfate disaccharides. When COCs were stimulated with FSH, HA biosynthesis increased 20-30-fold between 3-12 h later when expansion occurs, reaching a maximum rate of -780 pmol (as glucosamine)/COC/h compared with the unstimulated rate of -26 pmol/COC/h. The final concentration of HA in the expanded COC was calcu- lated to be -250 pg/ml. The effects of dibutyryl cyclic AMP (Bt2cAMP) on COC expansion and HA synthesis were similar to those of FSH, suggesting that the ef- fects of FSH are mediated by CAMP. However, FSH significantly decreased the specific activity of the in- corporated hexosamines while BtscAMP did not. Serum is necessary for the accumulation of HA in the COC matrix. HA synthesis in FSH-stimulated COCs was as high or higher in the absence of serum, but most was recovered in the medium and not in the COC matrix. The molecular size of the HA was >2 million dalton in either case, suggesting that the serum did not alter physical properties of HA. Stimulation of proteo- glycan biosynthesis by either FSH or BtzcAMP was less pronounced (three to four times control) than for HA and was sustained throughout an 18-h culture period. A reduction of 80% in the deposition of newly synthesized PGs in the COC matrix by 0.5 mM 8-xylo- side treatment did not affect the expansion of the cu- mulus.

* This work was supported in part by a grant from the Consiglio Nazionale delle Ricerche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Visiting Associate in the Bone Research Branch, 1988. Present address: Department of Sanita Pubblica e Biologia Cellulare, Faculty of Medicine, 2nd University of Rome, via 0. Raimondo, 00173 Rome, Italy.

In most mammals, oocytes in fully grown follicles are sur- rounded by compact layers of follicle cells to form the cumulus cell-oocyte complex (COC).’ When the circulating level of gonadotropins increases during the preovulatory period, the compact organization of the COC expands with the deposition of a mucoid material around and between the cumulus cells, a process referred to as expansion or mucification (1, 2). At ovulation, the expanded COC is released from the follicle as a viscous and elastic cell mass. Extracellular matrix compo- nents of the mucified COC appear to facilitate the pick-up of the COC by oviductal fimbria (3) and to induce changes in spermatozoa preceding the fertilization process (4). Presently, it is not known if mucification is induced directly in uiuo by the gonadotropins or is mediated indirectly by the mural granulosa cells (5-7). However, gonadotropins can induce expansion of the COC in vitro if serum is present in the culture medium (8, 9). The expansion process appears to be mediated by CAMP. For example, cAMP analogues, adenylate cyclase activators, and phosphodiesterase inhibitors all stim- ulate mucification in vitro (10, ll), and FSH stimulates an increase in intracellular cAMP in the mouse COC in vitro (12, 13).

The ultrastructure of the extracellular matrix of the muci- fied COC contains a fibrillar network with a homogeneous, regular distribution that extends into the outer zona pellucida, the external coat of the oocyte (14). Specific hyaluronidases destroy this network (15) and dissociate the COC into indi- vidual cells (16). All the agents, including FSH, that induce mucification in vitro stimulate the incorporation of 3H, from [3H]glucosamine as a precursor, into newly synthesized mac- romolecules (10). The stimulation is primarily into hyaluronic acid (HA) as determined by precipitation with cetylpyridi- nium chloride and susceptibility to hyaluronidase (10). Thus, HA is an important component of the expanded matrix of the COC. However, little is known about the characteristics of proteoglycans synthesized by COCs nor about their possible function in mucification. These macromolecules contain gly- cosaminoglycan chains covalently bound to core proteins, and

The abbreviations used are: COC, cumulus cell-oocyte complex; FSH, follicle-stimulating hormone; Bt,cAMP, dibutyryl cyclic aden- osine monophosphate; HEPES, N-hydroxyethylpiperazine-N’-eth- anesulfonic acid; FCS, fetal calf serum; Adi-HA, 2-acetamido-2- deoxy-3-~-[(3-~-gluco-4-ene-pyranosyluronic acid]-D-glucose; Adi-4- S, 2-acetamido-2-deoxy-3-0-[(3-D-gluco-4-ene-pyranosy~uronic acid]- 4-0-sulfo-D-galactose; Adi-GS, 2-acetamido-2-deoxy-3-0-[(3-~-gluco- 4-ene-pyranosyluronic acid]-6-O-sulfo-D-galactose; Adi-OS, 2-aceta- mido-2-deoxy-3-O-[~-~-gluco-4-ene-pyranosyluronic acid]-~-galac- tose; hexN, hexosamine; PG, proteoglycan; HA, hyaluronic acid HS, heparan sulfate; DS, dermatan sulfate; CS, chondroitin sulfate; GP, glycoproteins; HPLC, high performance liquid chromatography.

13840

Hyaluronic Acid Synthesis in Cumulus Cell-Oocyte Complexes 13841

different types, such as those in cartilage which specifically bind to HA, contribute directly to the structure, organization, and physical properties of extracellular matrices (17). Further, PGs may influence fertilization processes directly. Addition of sulfated glycosaminoglycans to culture medium prevents physical changes in the zona pellucida which occur in isolated oocytes and which inhibit in vitro fertilization (18, 19). Ex- ogenous glycosaminoglycans also stimulate the acrosome re- action i n vitro for spermatozoa from several species (4, 20).

In the study described in this report, we define the temporal changes in synthesis of HA and PGs during mucification of the mouse COC following stimulation i n uitro with FSH or Bt,cAMP. We show that FSH and Bt2cAMP induce an in- crease of both HA and PG biosynthesis by COC cultured i n uitro, and that increased net synthesis of HA, but not of PGs, correlates closely with the expansion process. A model for mucification is proposed in which cumulus cells require FSH and a factor(s) derived from the oocyte to increase HA syn- thesis and require a factor(s) in serum to accumulate this HA in the COC matrix.

EXPERIMENTAL PROCEDURES

Materials-Guanidine HC1 and urea were purchased from Be- thesda Research Laboratories; [35S]sulfate (-1.0 mCi/mmol) and D- [6-3H]glucosamine (29.5 Ci/mmol) from Du Pont/New England Nu- clear; Sephadex G-50, Q-Sepharose, prepacked Superose 6 , and Seph- acryl S-1000 from Pharmacia LKB Biotechnology Inc.; Partisil 5 PAC (0.4 X 25 cm) from Whatman. The scintillation mixture, Ready Safe, was obtained from Beckman; Triton X-100 from Pierce Chem- ical Co.; chondroitinase ABC (Proteus uulgaris), heparitinase (Fla- vobacterium heparinum), and chondroitin sulfate disaccharides from Seikagaku Kogyo (Tokyo) through ICN Biochemicals; hyaluronidase (Streptomyces hyalurolyticus nou. sp.) from Behring Diagnostics; preg- nant mare's serum gonadotropin, hyaluronic acid (from human um- bilical cord), Bt,cAMP and dimethyl polysiloxane (type 5X) from Sigma; p-nitrophenyl-P-D-xyloside from Koch-Light Laboratories (United Kingdom); Eagle's minimum essential medium with Earle's salt and HEPES from GIBCO; FCS from Flow Laboratories. Highly purified follicle stimulating hormone (NIDDK-rat-FSH-1-7) was kindly provided by the National Institute of Diabetes and Digestive and Kidney Diseases, and the National Hormone and Pituitary Program, University of Maryland School of Medicine. Female Swiss CD-1 mice (7-8-week old) were purchased from Charles River. The unsaturated hyaluronic acid disaccharide, Adi-HA, was prepared by digesting 10 mg of HA with 1 IU of chondroitinase ABC in 1 ml of 0.1 M Tris, 0.1 M acetate, pH 7.3, at 37 "C for 3 h. Digests were stored at -80 "C until further use.

Metabolic Labeling of COCs and Extraction of Radiolabeled Mole- cules-Adult, 2-month old mice were injected with 5 IU of pregnant mare's serum gonadotropin in 0.1 ml of physiological saline and killed by cervical dislocation 44-48 h later. Ovaries were removed and placed in minimal essential medium containing 1 mg/ml bovine serum albumin and buffered at pH 7.2 with 20 mM HEPES. COCs were released into the medium by puncturing large follicles. They were collected with a micropipette and transferred into droplets of culture medium (50-100 pl) covered with dimethyl polysiloxane to prevent evaporation of medium; between 25 and 100 COCs were used for each treatment, and at least three different experiments were done for each protocol. Unless specified in the text, COCs were cultured in minimal essential medium supplemented with 5% FCS as the basal control condition and with 5% FCS plus 1 pg/ml FSH or plus 2 mM BtZcAMP as the mucification induction condition (9). The cultures were incubated at 37 "C in a humidified atmosphere of 5% CO, in air for 18 h, a sufficient time for mucification to occur (9). For radiola- beling, [35S]sulfate (30 pCi/ml) and [3H]glucosamine (100 pCi/ml) were included in the medium. All manipulation steps of extraction of the COC were done under the dimethyl polysiloxane. After labeling, media samples were carefully removed from COCs with a micropi- pette. The COCs were washed twice with 25 pl of fresh medium and then extracted with 50 pl of 4 M guanidine HC1, 50 mM sodium acetate, pH 6.0, containing 2% (w/v) Triton X-100 and protease inhibitors (21, 22) at 37 "C for 5 h, then the extract was transferred

to a tube and brought to appropriate volume with 4 M guanidine HC1. Media samples were diluted with 500 p1(-5 volumes) of 4 M guanidine HC1 buffer containing protease inhibitors. All extracts were stored frozen until further analyses.

Evaluation of Mucification Process-COCs were examined for cu- mulus mucification with a stereo microscope. Expansion, elasticity, and resistance to mechanical disruption of the COcs were criteria for a positive response.

Isolation of Proteoglycans-Guanidine HCl extracts of COCs and media samples were eluted on Sephadex G-50 columns (8 ml of bed volume for 2 ml of sample) equilibrated with 8 M urea, 50 mM sodium acetate, 0.15 M NaC1,0.5% Triton X-100, pH 6.0, to remove unincor- porated isotopes and guanidine HC1 (23). Excluded fractions with labeled macromolecules were applied onto Q-Sepharose columns (0.7 X 4 cm) equilibrated with the same urea buffer. After sample appli- cation, each column was washed with 5 ml of equilibrating buffer and then eluted with a NaCl gradient (0.15-1.2 M) in the 8 M urea buffer (total volume of 46 ml) with a flow rate of 15 ml/h. Fractions of 1 ml were collected and aliquots were measured for radioactivity and conductivity.

Gel Filtration-A prepacked Superose 6 column (1 X 30 cm) was eluted with 4 M guanidine HC1, 50 mM sodium acetate, 50 mM Tris, 0.5% Triton X-100, pH 7.0, at room temperature at a flow rate of 0.4 ml/min. Fractions of 0.4 ml were collected for analysis. A Sephacryl S-1000 column (0.7 X 95 cm) was eluted with 4 M guanidine HCl, 50 mM sodium acetate, 50 mM Tris, 0.5% Triton X-100, pH 7.0, at a flow rate of 3 ml/h. Fractions of 0.5 ml were collected for analysis.

Enzyme Treatments-Hyaluronidase digestion (30 turbidity reduc- ing units/ml) was done in 0.1 M NaC1, 0.1 M acetate, pH 5.0, for 2 h at 37 "C. Chondroitinase ABC (0.1 unit/ml) or heparitinase (10 mIU/ ml) digestion of samples was done in 0.1 M Tris, 0.1 M acetate, pH 7.3, for 2 h a t 37 "C.

Quantitation of HA, PGs, and Glycoproteins-After adding 200 pg of HA and 100 pg of bovine serum albumin to each sample, aliquots of COC extracts and media samples were eluted on Sephadex G-50 columns (2 ml of bed volume for 0.5 ml of sample), equilibrated in 0.1 M Tris, 0.1 M acetate, 0.5% Triton X-100, pH 7.3, to remove unincorporated isotopes and guanidine HCl. Macromolecules in the excluded fraction were then digested with chondroitinase ABC as described above. A portion of each digest was applied to a Sephadex G-50 column (4 ml of bed volume) equilibrated in 8 M urea, 0.15 M NaC1, 50 mM sodium acetate, 0.5% Triton X-100, pH 6.0, and eluent fractions were analyzed to determine proportions of radioactivity that was enzyme susceptible (HA and dermatan sulfate disaccharides in included volume) and enzyme resistant (heparan sulfate and glyco- proteins in excluded volume). Another portion of each digest was analyzed for the proportions of the disaccharide digestion products by an HPLC procedure using Partisil 5 PAC (24) after adding Adi- 4S, Adi-6S, and Adi-OS disaccharide standards (5 pg each) as internal standards. The Adi-HA in each sample was derived from the enzyme digestion of the carrier HA.

For all experiments, radioactivities of samples were determined with a Beckman LS 5801. Differentiation of 35S and 3H activity was done by calculating 35S spill-over into the 3H channel using 36S standards prepared for each set of samples.

Statistical Analysis-Differences between two groups of data were analyzed by Student's t test. p < 0.05 was considered significant.

RESULTS

Cumulus Cell-Oocyte Complex Mucification in Vitro- Freshly isolated mouse cumulus cell-oocyte complexes (COCs) contain 1,400 f 200 cumulus cells compactly associated with the oocyte, Fig. la.' This structure undergoes little change when the COCs are incubated in basal conditions (medium plus FCS) for 18 h, Fig. l b , but undergoes extensive expansion when FSH is added to the basal medium, Fig. k 3

Procedures to Identify and Quuntify HA and PGs-Isolated COCs were stimulated with FSH in the presence of FCS and incubated with [3H]glucosamine and [35S]sulfate for 18 h,

A. Salustri, unpublished data. We are very grateful to Gary Best, National Institutes of Health

Graphics Department, for preparing the photomicrographs.

Hyaluronic Acid Synthesis in Cumulus Cell-Oocyte Complexes 13842

/. '. -I

FIG. 1. Photomicrographs of COCs using differential inter- ference contrast optics. Freshly isolated COCs (diameter of COC -130 pm) (a ) ; ( 6 ) COCs incubated for 18 h in medium with 5% FCS but without FSH (note that the cumulus is not expanded, diameter of COC -130 pm) ( 6 ) ; and COCs incubated for 18 h in medium with 1 pg/ml FSH and 5% FCS (c) (note that the cumulus is expanded, diameter of COC -250 pm). The bar in panel c is 50 pm.

sufficient time for expansion to occur (9). A COC extract and medium sample were prepared and eluted on Sephadex G-50 columns with an 8 M urea solvent. Labeled macromolecules in the excluded fractions were then analyzed by ion-exchange chromatography on Q-Sepharose. For the COC extract, Fig. 2a, -38% of "H-labeled macromolecules did not bind (peak l ) , -21% eluted as a narrow peak during the gradient at 0.28 M NaCl (peak 2), and -40% eluted as a broad peak between 0.55-0.85 M NaCl (peak 3 ) . Almost all of the incorporated "S activity eluted in peak 3. In a number of different experiments, 60-80% of the labeled molecules in peak 2 were digested to small fragments by Streptomyces hyaluronidase (data not shown). Further, authentic HA added to a similar sample eluted with peak 2. Therefore, this peak contained primarily labeled HA. For peak 3, -65% of the 'H (-70% of the "S) was susceptible to chondroitinase ABC and -25% of the 'H (-30% of the %) to heparitinase. Other experiments showed that most of the heparan sulfate eluted in the earlier fractions of peak 3 (data not shown). Peak 3, then, contained almost exclusively labeled dermatan sulfate and heparan sulfate PGs.

The medium sample gave a similar profile on Q-Sepharose, Fig. 2b. Peaks 1, 2, and 3 contained -54, -11, and -35% of the 'H label respectively; -70% of peak 2 was digested with Streptomyces hyaluronidase, and -55 and -25% of the 'H- labeled molecules in peak 3 were digested with chondroitinase

Q-Sepharose

COC Extract

12

FRACTION NUMBER

FIG. 2. Q-Sepharose elution profiles of extract (a) and me- dium (b) samples from COCs cultured with FSH and FCS. See Table I and text for details.

ABC and heparitinase, respectively. For the 18-h labeling time, -70% of the total incorporated 'H and -60% of the 35S were recovered in the COC extract.

While recoveries of "S-labeled macromolecules from Q- Sepharose were nearly quantitative, averaging between 90- 98%, recoveries of 'H-labeled macromolecules were poorer, 85-95% for media samples and only 45-70% for COC extracts. Most of the missing 'H in the COC extracts bound at the top of the Q-Sepharose columns. The ion-exchange procedure, then, is useful for purifying PGs synthesized by the COC; but the low recoveries of 3H, especially in the COC extracts, indicated that it gives poor yields of either HA (peak 2), or glycoproteins (peak 1) or both. Another procedure was there- fore developed to address this problem.

Bovine serum albumin and HA were added to samples to facilitate recoveries, and the mixtures were eluted on Sepha- dex G-50 columns equilibrated with 0.1 M Tris, 0.1 M sodium acetate, 0.5% Triton X-100, pH 7.3. The recoveries of mac- romolecular 'H and "S were quantitative. Aliquots of the excluded fractions were digested directly with chondroitinase ABC under conditions sufficient to digest all of the carrier and labeled HA as well as the labeled dermatan sulfate. Undigested macromolecules were removed by centrifugation after precipitation with 3 volumes of ethanol. Standard chon- droitin sulfate disaccharides were added to the soluble diges- tion products, and the samples were then analyzed by HPLC on Partisil 5 PAC (24). For the COC extract, two 'H peaks were observed eluting with Adi-HA and Adi-4S, respectively (Fig. 3). The former contained 80-90% of the 'H in the analysis while the latter contained 10-20% as well as all of the '73.

The percent of 'H in heparan sulfate in the each sample was evaluated independently by digesting a separate aliquot with heparitinase followed by elution on Sephadex (3-50 to quantify the digestion products. For the COC extract, -5% of the total 'H and -30% of the 3sS was in heparan sulfate, the latter value in close agreement with the data described above for peak 3. The 3H and ?3 distributions for both the COC extracts and medium samples are summarized in Table I along

Hyaluronic Acid Synthesis in Cumulus Cell-Oocyte Complexes 13843

TABLE I Comparison of ion-exchange chromatography and enzyme digestion plus HPLCprocedures

for quantitative determination of 3H-labeled macromolecules Relative amount of 3H incorporation"

Analytical method COC extract Medium

GP HA PG GP HA PG

Ion exchange Peak 1 Peak 2 Peak 3 Peak 1 Peak 2 Peak 3 % of 3H applied

to column 19 11 20 49 10 31

Enzyme digestion + Partisil 5 PAC

% of 3H in sample 19 60' 21d 51 19' 30d (1.0Y (5.5) (1.1) (1.1) (1.9) (1.0)

- COCs cultured in labeling medium for 18 h with FCS and FSH; total 3H incorporation: -1200 cpm/COC/18

h, with -70% in the COC extract. * 3H resistant to chondroitinase and heparitinase digestion.

3H in Adi-HA in chondroitinase digests. 3H in Adi-4S in chondroitinase digests plus heparitinase digestion products.

e Values in parentheses are ratios of values determined by the enzyme method to those determined by the ion- exchange method.

Partisil 5 PAC

ADI~HA

ELUTION TIME (Minutes)

FIG. 3. Partisil 5 PAC elution profile of disaccharides de- rived from chondroitinase ABC digests. The peaks for disaccha- ride standards are shown at the top, and the radiolabeled products derived from the extract of COCs cultured with FSH and FCS are shown at the bottom. The large UV absorbing peak at the break- through is from the Triton X-100 in the sample.

with the results from the Q-Sepharose analyses. It is clear that HA accounts for virtually all of the 3H-labeled material which did not elute from Q-Sepharose; for example, labeled HA in peak 2 of the COC extract accounted for less than 20% of the labeled HA in the original extract. The distributions of radiolabel in HA, PGs, and glycoprotein fractions in all the following experiments were determined by the enzyme diges- tion and HPLC procedure.

Specific Activity Corrections-When [3H]glucosamine is used as a metabolic precursor, it is diluted in the cytoplasm by endogenous pathways for synthesis of hexosamines. Thus, the specific activity of the UDP-N-acetylhexosamine precur- sors for glycosaminoglycan synthesis is much lower than that of the [3H]glucosamine in the medium, and it often varies depending upon the experimental conditions (25-28). For this reason, an indirect method was used to estimate the specific activity of the UDP-N-acetylhexosamine pool (28). The method uses the specific activity of [35S]sulfate in the medium,

Partisil 5 PAC

5 P I t 3 1 .o

0.5 1 -

0

E

6 x 5 1.5 ;; - 2 3 1.0 8

6

- - I

0 0.5 x 3

- 1 F

I

5 1.5

3 1 .o 0.5

1

0 5 10 15 20 ELUTION TIME (Minutes)

FIG. 4. Partisil 5 PAC elution profiles of disaccharides de- rived from chondroitinase ABC digests of extracts of COCs incubated in labeling medium for 18 h with FCS alone (a), in FCS plus FSH (b ) , and in FCS plus BtZcAMP. The 3H/35S ratios for the Adi-4S peaks indicated in the figure were used to correct for changes in the specific activities for the calculations in Table 11.

sa(S), combined with the 3H/35S-labeling ratio, Ir, in the monosulfated Adi-4S to calculate the specific activity of the galactosamine, sa(H), in this disaccharide derived from the dermatan sulfate synthesized during the labeling p e r i ~ d . ~ The formula is (28):

Because UDP-galNAc and UDP-glcNAc are in a rapid equilibrium (29), the specific activity of glucosamine in the newly synthesized HA is assumed to be equivalent to sa(H). In the experiments reported in this paper the specific activity

This is based on the fact that the specific activity of [36S]sulfate in the medium is not significantly diluted by endogenous sulfate sources in the intracellular phosphoadenosinephosphosulfate pool, the immediate metabolic precursor for the sulfate ester in glycosa- minoglycan synthesis (28). For rat granulosa cells, less than 2% of the sulfate esters on the proteoglycans is derived from cysteine or methionine when environmental sulfate in the medium is not limiting, i.e., above 0.1 mM (M. Yanagishita, J. Kimura, V. Hascall; unpub- lished data).

13844 Hyaluronic Acid Synthesis in Cumulus Cell-Oocyte Complexes

TABLE I1 Effects of FSH and BtZcAMP on synthesis and accumulation of HA, PGs, and G P in COC extracts

COCs were incubated in labeling medium for 18 h with FCS (basal), with FCS plus FSH (FSH) or with FCS plus Bt,cAMP (Bt,cAMP). Values are for the COC extract compartment, and values in parentheses are ratios to basal values.

Treatment 3H incorporation sa(hexN)" Net synthesis

HA DS-PG HS-PG GP HA DS-PG HS-PG GP cprn/COC ( ~ 1 0 - 3 , pCi/nrnol pmol hexN/COC

Basal 0.1Ob 0.03 0.02 0.14 0.16 0.56 FSH 0.61 0.05

0.15 0.03

0.10 0.79 0.16 0.10 5.54 0.46 0.23

(6.1) (1.7) (1.5) (1.1) (9.9) 1.46

0.72 (3.1)

0.10 (2.3)

0.06 (1.8)

0.25 0.18 (7.2) (3.6)

3.64 0.51 0.28 (3.3)

1.25 (1.8) (6.5) (3.4) (2.9) (1.6)

BtzcAMP

a Calculated from equation 1 as described in the text. * Values represent averages of three experiments. Coefficients of variation were less than 25%.

b.

Medium

0 C0""Ol

I FSH F3 Bt. CAMP

HA DSPG HSPG GP

3H-LABELED MACROMOLECULES

FIG. 5. Changes in the synthesis of 3H-labeled components in COC extracts (a) and medium samples ( b ) for COC cultures treated as described in Fig. 4 legend. Error bars represent 1 S.D.

of the [35S]sulfate in the medium was determined to be -37.5 pCi/pmol initially (30 pCi of [35S]sulfate/800 nmol sulfate/ ml), and corrections for radioisotope decay were made there- after. The values for sa(H) and the total incorporation, then, can be used to calculate the masses of HA and PGs synthe- sized during the labeling period.

Changes in Net Synthesis-An example of such an analysis is shown in Fig. 4 for samples derived from COC extracts after incubation in labeling medium for 18 h without (control), or with stimulation of mucification by FSH or Bt2cAMP. The calculated results are given in Table I1 and summarized in Fig. 5. It is clear that FSH decreased the specific activity of the UDP-N-acetylhexosamine precursors relative to the con- trol, whereas BtzcAMP did not. Thus, the values for the 3H- labeled macromolecules must be corrected for these differ- ences in specific activity. Both FSH and BtzcAMP increased net synthesis of all of the glycosylated components, but the largest increases occurred for HA. Even though Bt2cAMP stimulated a larger incorporation of 3H into HA (7.2 x control) than FSH (6.1 X control), the correction for specific activity differences shows that FSH increased net HA synthesis (9.9 x control) more than did Bt2cAMP (6.5 x control).

The amounts of the newly synthesized glycosylated com- ponents released into the culture medium in this experiment were also determined by the same procedure. There were no significant changes between control and treated samples for any of the glycosylated components except the small increase of DS-PG by FSH treatment (Fig. 5). For this reason, in the following experiments only data for the COC extracts are presented.

a.

9-

7 -

L ? , I ' . . l ' l s l a c l l l l c l "

0 0.01 0.1 1 .o Et, CAMP, mM

FIG. 6. Changes in synthesis of HA and PG in COC extracts of cultures incubated in labeling medium for 18 h with FCS and different doses of FSH (a) or BtzcAMP (b ) . The insets show the specific activity of the hexosamines (Sa) calculated from the 3H/ 3S ratios of the Adi-4S disaccharides for the different samples as defined by equation 1. Error bars represent 1 S.D.

The effects of different concentrations of FSH and Bt2cAMP on the specific activity of the UDP-N-acetylhexo- samines and the net synthesis of HA and PGs in COC extracts for 18-h incubations are shown in Fig. 6. Maximal levels of stimulation of synthesis were obtained with 5 ng/ml FSH and 0.5 mM Bt2cAMP, with EDso values of -1 ng/ml and -0.2 mM, respectively. As noted above, HA synthesis increased much more than for PG synthesis. Both types of PGs, heparan sulfate and dermatan sulfate, showed about the same degree of stimulation. The specific activities of the Adi-4s galactos- amine decreased for FSH but remained unchanged for Bt2cAMP (insets, Fig. 6).

Time Course of HA and PG Synthesis-HA and PG syn-

Hyaluronic Acid Synthesis in Cumulus Cell-Oocyte Complexes 13845

30

20

-I g 10

s 8

t Q

LT 20

10

1

a. n FSH II b.

U HA DSPG

El HSPG GP

Btz cAMF

n

0-3 3 6 6-12 12-18

LABELING TIME (h)

FIG. 7. Changes in synthesis of HA and PG during different time intervals following stimulation of COC cultures with FSH (a) or BtzcAMP (b ) . Values were calculated based on averages of two observations for each time period and expressed as the ratio to control COC cultures incubated with FCS alone. For this experi- ment basal control levels were constant at 26, 5, and 7 pmol/h (as hexosamine) for HA, HS-PG, and DS-PG, respectively.

7 Superose 6 Peak 3

1 FSH

a

2 COC Extract

FSH + Xyloside / I c

i

1

Kd

FIG. 8. Superose 6 elution profiles for the PG fractions (peak 3, Fig. 2) of COC cultures incubated in labeling medium for 18 h with FCS and FSH. Panels a and b show samples derived from the COC extract and medium compartments, respectively. Panel c shows a sample from the medium compartment of a COC culture incubated additionally with P-xyloside as described in the text.

thetic activities by COCs were investigated at different time intervals during the 18-h culture period, Fig. 7. The control COCs maintained constant rates of synthesis for each glyco- sylated component for all time periods (values given in Fig. 7 legend). Relative to control values, both FSH and Bt2cAMP stimulated HA synthesis transiently. A small stimulation was observed in the first 3 h. Between 3-6 h, a dramatic increase (20-30 times control) occurred which was sustained between

TABLE 111 Effects of FCS and xylosides on synthesis and distribution of

HA and PGs COCs were cultured in labeling medium for 18 h under the treat-

ments indicated. Values in parentheses are ratios to FCS treatment alone; values in brackets are percentages in COC extracts and respec- tive medium samples. Values are means f S.D.

COC extract Medium Treatment

PG HA PG HA prnol hexN/COC

FCS 0.33 f 0.12 0.70 f 0.32 FCS + FSH 0.18 f 0.12 4.33 f 1.50

+ xyloside (0.5) (6.2) FCS + FSH 0.83 f 0.30 6.39 f 2.10 1.01 f 0.20 1.40 f 0.50

BSA + FSH 0.63 f 0.25 2.39 & 0.70 1.27 f 0.23 10.57 f 3.00 (2.5),[451 (9.1),[821 [551 [181

(1.9),[331 (3.4),[191 [671 [811

Sephacryl S-loo0

COC Extract /\ COC Extract

10

5 -

v)

Y

0:

101 5 t

0 0.5 1.0 0 0.5 1.0

Kd Kd

FIG. 9. Sephacryl S-1000 elution profiles for radiolabeled macromolecules from COC cultures incubated in labeling me- dium for 18 h with FSH, and either with (a and b) or without (c and d ) FCS. COC extracts (a and c) and medium samples ( b and d ) are shown. The dashed lines in panels a and d are the elution profiles of separate samples after digestion with Streptomyces hyalu- ronidase.

6-12 h. The rate then returned toward control levels during 12-18 h. The maximal increases in both dermatan sulfate and heparan sulfate PG synthesis (three to four times control) were statistically significant but were much less than for HA. Additionally, the time course of stimulation was different, with elevated synthesis of PGs sustained through the 12-18- h time period.

Characterization of PGs Synthesized by the COG-PG frac- tions (peak 3) were isolated from COC extracts and media samples of control and FSH stimulated COCs by chromatog- raphy on Q-Sepharose (see above). After concentration, ali- quots were eluted on Superose 6 in 4 M guanidine HC1 with 0.5% Triton X-100. The elution profiles for the COC extract and medium PGs of the FSH-stimulated COCs are shown in Fig. 8. The medium sample showed two major 35S peaks, one excluded (Mr greater than 300 kDa) and the second centered at a K d of 0.21 (M, -150 kDa). Since dermatan sulfate is predominant in the medium, these two PG peaks probably correspond to DS-PG-I and DS-PG-I1 previously described as synthetic products of rat granulosa cells (30,31). The COC extract contained PG peaks with similar elution positions

13846 Hyaluronic Acid Synthesis in Cumulus Cell-Oocyte Complexes

followed by a broad peak centered between Kd 0.2-0.6. This latter peak probably contains intracellular glycosaminoglycan degradation products similar to those described for the rat granulosa cells (32, 33). The elution profiles for samples from the basal, unstimulated COCs were essentially identical. More complete analyses of the PGs synthesized by the COCs are underway. These preliminary results, however, indicate that there are no obvious qualitative changes in the PGs produced by COCs stimulated to mucify compared with unstimulated COCs.

Effects of Nitrophenyl-P-D-xyloside on HA and PG Synthesis and on COC Expansion-/3-Xylosides act as exogenous initi- ators for glycosaminoglycan synthesis, thereby competing with the endogenous core protein acceptors and decreasing mature PG synthesis, particularly for chondroitin sulfate/ dermatan sulfate PGs (34, 35). FSH-stimulated COCs were continuously exposed to 0.5 mM nitrophenyl-P-D-xyloside during 18 h of culture to determine if inhibition of PG synthesis affects expansion. The xyloside treatment increased total 35S incorporation 9-10-fold. Almost all of the 35S-labeled molecules were recovered in the medium and eluted as free glycosaminoglycan chains on Superose 6, Fig. 8c (compare with 8b). The xyloside also inhibited the 3H incorporation into PGs accumulated in the COC matrix to -20% of FSH- treated COC, Table 111. In agreement with previous observa- tions (36), HA synthesis was slightly inhibited (-75% of FSH- treated COC) by the xyloside treatment, Table 111. The xylo- side treatment did not alter the FSH-stimulated COC muci- fication in any obvious way as assessed by light microscopy (data not shown). Thus, a significant reduction in the depo- sition of newly synthesized PGs in the COC matrix by the xyloside treatment did not affect the expansion of the cumu- lus, suggesting that PGs have little or no role in the mucifi- cation process.

Effect of the FCS on HA and PG Synthesis and Distribu- tion-In the presence of FSH, but the absence of serum, cumulus expansion does not occur in vitro and the proportion of 3H-labeled macromolecules ( [3H]glucosamine as a precur- sor) in the medium increases (37). For this reason, the effects of FCS on net synthesis of HA and PG and on their distri- bution were studied in FSH stimulated COCs (Table 111). In the absence of FCS (BSA + FSH), net synthesis of PGs was unchanged while net synthesis of HA increased -60% com- pared to cultures with FCS (FCS + FSH). Further, the dis- tribution of the PGs between the COC extract and the medium was unchanged. However, there was a dramatic redistribution of HA from the COC matrix into the medium, Table 111 and Fig. 9. After 18 h in the presence of FCS, -80% of the total labeled HA accumulated in the COC matrix. Conversely, in the absence of FCS, only -20% of the total labeled HA accumulated in the COC matrix with the remainder released into the medium, Table 111. Morphological observations con- firmed that cumulus expansion did not occur for COCs incu- bated without FCS (data not shown).

Labeled macromolecules in the cell and medium fractions for both treatments were analyzed by Sephacryl S-1000 chro- matography in 4 M guanidine HC1, 0.5% Triton X-100, Fig. 9. 3H-Labeled HA synthesized by the COCs in the presence of FCS and extracted from the COC matrix eluted in the excluded column volume, as indicated by the sensitivity of this peak to Streptomyces hyaluronidase digestion, Fig. 9a. Similarly, the labeled HA in the medium for COCs incubated without FCS was also excluded from the column, Fig. 9d. Thus, in both conditions, the hydrodynamic size of the labeled HA molecules indicates that they have M, values greater than

2 million (38), and it is unlikely that changes in their physical properties per se account for the observed redistribution.

DISCUSSION

The experiments described in this report use [3H]glucosa- mine and [35S]sulfate to study the synthesis and distribution of hyaluronic acid and proteoglycans in mouse cumulus oocyte complexes stimulated to mucify in vitro. While extraction of labeled macromolecules from the COC matrix with 4 M gua- nidine HC1 and detergent was efficient, there were significant losses of labeled HA in the subsequent ion-exchange step. Thus, this macromolecule was measured by an alternative procedure which involved chondroitinase digestion and analy- sis of the disaccharides derived from both the HA and the DS-CS PGs. The known specific activity of the radiosulfate in the medium and the ratio of 3H/35S in the chondroitin 4- sulfate disaccharide were used to correct for changes in spe- cific activity of hexosamine pools so that the amounts of HA and PGs synthesized/time could be estimated.

COCs stimulated to mucify by FSH in the presence of FCS synthesized HA at -10 times the rate for unstimulated COCs during 18 h of treatment, and most of this newly labeled HA accumulated in the COC matrix (Fig. 5 ) . The time course for the stimulation of HA synthesis and its accumulation corre- lates with COC expansion. After FSH stimulation, HA syn- thesis and accumulation was four to five times the control in the first 3 h, increased to 20-30 times the control during 3- 12 h, and decreased back to four to six times the control during 12-18 h (Fig. 7 ) . COC expansion in these conditions becomes apparent 4-5 h after FSH stimulation and appears to reach completeness by morphological criteria by 12-14 h. The net increase in HA in the COC matrix during the 18 h was -2.0 ng/COC which would contribute a concentration of 250 pg of HA/ml in the matrix based on the estimated volume of the expanded COC (8 x mm3). COCs stimulated by FSH in the absence of FCS increased net HA synthesis as much or more as for COCs stimulated by FSH in the presence of FCS. However, the HA accumulates in the medium and not the COC matrix (Fig. 9), and these COCs do not expand. These results confirm and extend those reported by Eppig (37) who used cetylpyridinium chloride precipitation of 3H- labeled macromolecules and treatment with Streptomyces hy- aluronidase to show that HA was present in the COC matrix for cultures stimulated to mucify in the presence of FCS and in the medium when FCS was absent.

PGs bind to HA in some connective tissue matrices such as cartilage (17) and are important structural components in the organization of such matrices. PG synthesis and accumulation in the COC matrix did increase during mucification to levels two to three times the control values during the 18 h of treatment (Fig. 5 ) . However, this increase does not appear to contribute significantly to the expansion or organization of the COC matrix since xyloside treatment effectively pre- vented the accumulation of PGs in the COC matrix (Table 111) but did not prevent expansion. The PGs synthesized by the COC have molecular properties similar to those synthe- sized by rat ovarian granulosa cells. One HS PG, partially included on Superose 6, and two CS/DS PGs, one excluded and one partially included, were identified (Fig. 8). If the PGs are homologous, the observation that the PGs synthesized by mural granulosa cells do not bind to HA (39) would be consistent with the lack of their direct involvement in the organization of the HA matrix. However, PGs synthesized by cumulus cells may be an important component of the envi- ronment of the oocyte and for the fertilization process. It has been reported that the zonae pellucidae of isolated mouse

Hyaluronic Acid Synthesis in

oocytes cultured i n vitro become increasingly resistant to solubilization by chymotrypsin (40) thereby decreasing the success of i n vitro fertilization (19). These physicochemical changes in the zonae pellucidae are prevented by adding sulfated glycosaminoglycans (heparin and DS) to the culture medium (18). Moreover, these changes do not occur during oocyte maturation i n vivo nor when isolated oocytes are incubated with intact COC (40).

While our results and those of Eppig (37) have shown that factors in serum (or in follicular fluid) are required for accu- mulation of HA and expansion of COC matrix, their mecha- nism of action remains uncertain. The Sephacryl S-1000 analyses (Fig. 9) suggest that the absence of serum does not cause a decrease in the molecular size of the newly synthesized HA molecules, at least not below 2 million, which could have altered their physical properties. Serum contains HA-binding proteins (41), and these may be necessary for organizing the newly synthesized HA in the COC matrix. Protease digestion, for example, can dissociate the COC as can treatment with a specific hyaluronida~e.~ Alternatively, the serum factor(s) may be required for the cumulus cells to synthesize a critical component involved in organization of the HA in the matrix.

Cyclic AMP analogues have been shown to stimulate HA synthesis and mucification of COC i n vitro (9, lo), and it is thought that FSH exerts its influence on this process via CAMP pathways. Like FSH, BtzcAMP stimulates 3H incor- poration from [3H]glucosamine as a precursor into all the glycosylated components of the COC matrix (Table 11). Dose response curves showed that FSH and Bt2cAMP were half- maximally effective at 1 ng/ml and 0.2 mM, respectively (Fig. 6), which, for FSH at least, is within physiological range. The COCs respond differently to these two factors in one respect. The specific activity of the UDP-hexNAc pools decreased significantly in FSH-stimulated cultures (-60% at maximal stimulation) but remained unchanged in BtzcAMP-stimulated cultures (Fig. 6). After correcting for the changes in specific activity, we found that BtzcAMP and FSH showed the same temporal patterns for stimulation of HA, PG, and GP synthe- sis. However, while levels of synthesis of PGs and GPs in- creased to the same extent with both treatments, FSH-stim- ulated cultures showed higher net synthesis of HA than obtained with Bt2cAMP. These observations are consistent with a direct role for CAMP in mediating FSH action, but they also suggest that other factors may be involved. In separate experiments, we have shown that FSH does not increase HA synthesis in cumulus cells separated from the oocyte unless medium conditioned by isolated oocytes is used during the hormonal stimulation.'j Thus, minimally, a soluble factor produced by the oocyte is essential in combination with FSH to stimulate HA production by cumulus cells.

In summary, we have shown that HA and PG synthesis by COCs are increased during the FSH stimulation but that only HA synthesis and accumulation are directly involved in the mucification process. Studies are in progress to define roles of the oocyte and of serum on synthesis and organization of HA in the extracellular matrix of COC.

Acknowledgment-We wish to thank Dr. Ronald J. Midura for his valuable advice during these studies.

A. Salustri, M. Yanagishita, and V. C. Hascall, unpublished

A. Salustri, M. Yanagishita, and V. C. Hascall, manuscript in observations.

preparation.

hmulus Cell-Oocyte Complexes 13847

REFERENCES 1. Boling, J. L., Blandau, R. J., Soderwald, A. L., and Young, W . C.

2. Dekel, N., and Phillips, D. M. (1979) Biol. Reprod. 21,9-18 3. Mahi-Brown, C. A., and Yanagimachi, R. (1983) Gamete Res. 8,

4. Meizel, S. (1985) Am. J. Anat. 17, 285-302 5. Eppig, J. J. (1980) Biol. Reprod. 23, 545-552 6. Eppig, J. J. (1981) Biol. Reprod. 25, 191-195 7. Salustri, A., Petrungaro, S., and Siracusa, G . (1985) Biol. Reprod.

8. Dekel, N., and Kraicer, P. F. (1978) Endocrinology 102, 1797-

9. Eppig, J. J. (1979) J. Exp. Zool. 208, 111-120

(1941) Anat. Rec. 79, 313-331

1-10

33,229-234

1802

10. Eppig, J. J. (1979) Nature 281,483-484 11. Dekel, N., and Phillips, D. M. (1980) Biol. Reprod. 22, 289-296 12. Schultz, R. M., Montgomery, R. R., Ward-Bailey, P. F., and

13. Salustri, A., Petrungaro, S., DeFelici, M., Conti, M., and Siracusa,

14. Yudin, A. I., Cherr, G. N., and Katz, D. F. (1988) Cell Tissue Res.

15. Talbot, P. (1984) J. Exp. Zool. 229, 309-316 16. McClean, D., and Rowlands, I. W . (1942) Nature 150, 627-628 17. Hascall, V. C. (1988) ZSZ Atlas of Science: Biochemistry 1, 189-

18. DeFelici, M., Salustri, A., and Siracusa, G. (1985) Gamete Res.

19. Downs, S. M., Schroeder, A. C., and Eppig, J. J. (1986) Gamete

20. Meizel, S., and Turner, K. 0. (1986) J. Exp. Zool. 237, 137-139 21. Oike, Y., Kimata, K., Shinomura, T., Nakazawa, K., and Suzuki,

22. Kato, M., Oike, Y., Suzuki, S., and Kimata, K. (1985) Anal.

23. Yanagishita, M., Midura, R. J., and Hascall, V. C. (1987) Methods

24. Zebrower, M. E., Kieras, F., and Brown, W. T. (1986) Anal.

25. Yanagishita, M., and Hascall, V. C. (1985) J. Biol. Chem. 260,

26. Yanagishita, M. (1987) Arch. Biochem. Biophys. 251,287-298 27. Silbert, C . K., Palmer, M. E., Humphries, D. E., and Silbert, J.

28. Yanagishita, M., Salustri, A., and Hascall, V. C. (1989) Methods

29. Kim, J. J., and Conrad, H. E. (1974) J. Biol. Chem. 249, 3091-

30. Yanagishita, M., and Hascall, V. C. (1979) J. Biol. Chem. 254,

31. Yanagishita, M., and Hascall, V. C. (1983) J. Biol. Chem. 258,

32. Yanagishita, M., and Hascall, V. C. (1984) J. Biol. Chem. 259, 10260-10269

33. Yanagishita, M., and Hascall, V. C. (1984) J. Bid . Chem. 259, 10270-10283

34. Okayama, M., Kimata, K., and Suzuki, S. (1973) J . Biochem. (Tokyo) 74,1069-1073

35. Sobue, M., Habuchi, H., Ito, K., Yonekura, H., Oguri, K., Sakurai, K., Kamohara, S., Keno, Y., Noyori, R., and Suzuki, S. (1987) Biochen. J . 241, 591-601

36. Galligani, L., Hopwood, J., Schwartz, N. B., and Dorfman, A. (1975) J. Bid. Chem. 250,5400-5406

37. Eppig, J. J. (1980) Biol. Reprod. 22, 629-634 38. Tengblad, A., Laurent, U. B., Lilja, K., Cahill, R. N., Engstrom-

Laurent, A., Fraser, J. R., Hansson, H. E., and Laurent, T. C. (1986) Biochem. J. 236, 521-525

39. Yanagishita, M., Rodbard, D., and Hascall, V. C. (1979) J . Biol. Chem. 254, 911-920

40. DeFelici, M., and Siracusa, G. (1982) Gamete Res. 6, 107-113 41. LeBoeuf, R. D., Raja, R. H., Fuller, G. M., and Weigel, P. W .

Eppig, J. J. (1983) Deu. Biol. 95,294-304

G . (1985) Biol. Reprod. 33, 797-802

251,555-564

198

12,227-235

Res. 15, 115-122

S. (1980) Biochem. J. 191, 193-207

Biochem. 148, 479-484

Enzymol. 138, 279-289

Biochem. 157, 93-99

5445-5455

E. (1989) Arch. Biochen. Biophys. 268,393-397

Enzymol., in press

3097

12355-12364

12847-12856

(1986) J. Biol. Chem. 261, 12586-12592