Effect of Oocytectomy on Glycosaminoglycan Composition during Cumulus Expansion of Porcine Cumulus-Oocyte Complexes Cultured In Vitro'

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1 BIOLOGY OF REPRODUCTION 55, (1996) Effect of Oocytectomy on Glycosaminoglycan Composition during Cumulus Expansion of Porcine Cumulus-Oocyte Complexes Cultured In Vitro' Taisuke Nakayama, 2 Maki Inoue, and Eimei Sato Department of Reproductive and Developmental Biology, Institute of Medical Science, The University of Tokyo, Tokyo 18, Japan ABSTRACT To investigate the role of oocytes in the accumulation of glycosaminoglycans during cumulus expansion, we analyzed the amount and composition of glycosaminoglycans in porcine intact and oocytectomized cumulus-oocyte complexes. Follicular fluid induced cumulus expansion in intact and oocytectomized cumulus-oocyte complexes cultured for over 24 h, but the degree of expansion in oocytectomized complexes reached only 76% of that in intact complexes after 24 h in culture. There were no differences in the amounts of [3H]glucosamine incorporated into each type of complex. Metabolic labeling studies on glycosaminoglycans with [3H]glucosamine and [35S]sulfate showed that the amounts of 3 H- and 35S-labeled glycosaminoglycans increased, and rapidly so, from 16 h in culture. The 3 H- labeled glycosaminoglycans at these high levels were digested by Streptomyces hyaluronidase or chondroitinase ABC, and the rate of increase in 3 H-labeled glycosaminoglycans was reduced by oocytectomy. In contrast, the increased levels of 35S-labeled glycosaminoglycans were affected only by chondroitinase ABC, and oocytectomy was ineffective. In conclusion, follicular fluid promotes cumulus expansion, and the oocyte plays an essential role in the acute synthesis of hyaluronic acid, but not chondroitin sulfate, during cumulus expansion stimulated in vitro. INTRODUCTION In most mammals, fully grown oocytes are surrounded by compact layers of follicle cells to form the cumulusoocyte complex (COC). During the preovulatory period, the cumulus cells synthesize and deposit an intercellular matrix enriched in hyaluronic acid (HA) [1]. This phenomenon, referred to as cumulus expansion, facilitates detachment of the COC from the follicle wall, its extrusion at ovulation, and its capture by oviductal fimbria. Furthermore, cumulus expansion may also influence a variety of fundamental developmental changes in both the oocyte and the spermatozoan required for successful fertilization and development of the zygote [2, 3]. Compact COCs can expand in vitro if the culture medium contains an appropriate hormone, such as FSH [1], via a mechanism that appears to be mediated by camp [4, 5] in mice and other species. To determine whether the oocyte plays a role in the regulation of cumulus expansion in vitro, the oocyte was removed from isolated COCs by a microsurgical procedure termed oocytectomy [6-8]. The oocytectomized COCs, consisting of the cumulus cells at- Accepted July 24, Received December 1, 'This study was supported in part by grants (numbers and 7565) from the Ministry of Education, Science, Sports and Culture, and a grant for Pioneering Research Projects in Biotechnology from the Ministry of Agriculture, Forestry and Fisheries, Japan. 2Correspondence: Taisuke Nakayama, Department of Reproductive and Developmental Biology, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 18, Japan. FAX: ; tai-suke@ims.u-tokyo.ac.jp 99 tached to the zona pellucida, did not respond to FSH by undergoing cumulus expansion and HA synthesis. In contrast, intercellular camp levels in the oocytectomized COCs were increased by FSH stimulation to the same extent as in intact complexes [6]. The oocytectomized COCs produce HA and undergo cumulus expansion in response to FSH if the COCs are cultured in medium conditioned by fully grown oocytes. These findings suggest that the oocytes secrete a cumulus expansion-enabling factor(s) [5, 6]. Follicular fluid in the medium improves the rate of cumulus expansion and oocyte maturation [9, 1]. Porcine follicular fluid contains a heat-stable factor(s) that promotes cumulus expansion, with a molecular mass below 6.5 kda measured by HPLC [11]. Although the nature of this active factor(s) has not been identified, follicular fluid is useful as a medium for oocyte maturation and other purposes. We examined the effect of oocytectomy on the morphology of COCs and glycosaminoglycan (GAG) synthesis to determine whether or not an oocyte factor(s) is involved in the cumulus expansion or GAG synthesis of porcine COCs cultured in follicular fluid. MATERIALS AND METHODS Materials The following were obtained as indicated: Streptomyces hyaluronidase and heparitinase I from Seikagaku Kogyo (Tokyo, Japan); chondroitinase ABC, hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and papain from Sigma Chemical Company (St. Louis, MO); D-[6-3 H]glucosamine hydrochloride (25 Ci/mmol) and [ 35 S]sulfuric acid (1 M solution, 2-1 mci/mmol) from Amersham (Buckinghamshire, UK); penicillin G potassium and streptomycin sulfate from Meiji Seika Company (Tokyo). The other reagents were obtained from Wako Pure Chemical Industries (Tokyo). Preparation of Porcine COCs and Follicular Fluid Ovaries were obtained from prepubertal gilts at a slaughterhouse and transported to the laboratory within 3 min in saline, maintained at 37 C, containing 1 IU/ml of penicillin and.1 mg/ml of streptomycin. The ovaries were immediately freed from the hilus and connective tissues and washed five times in sterile saline at 37 C. Medium-sized nonatretic follicles with a diameter of 2-5 mm were punctured with a needle directly in a sterile dish, and the COCs were collected and washed in modified Krebs-Ringer bicarbonate solution containing 4 mg/ml BSA (mkrb). Porcine follicular fluid (pff) was prepared as follows. Ovaries were collected as described above and transported to the laboratory in chilled saline. The pff was aspirated from medium-sized follicles and centrifuged at 15 x g at 1 C for 2 min. The supernatant was stored at -2 C. Downloaded from by guest on 19 January 219

2 13 NAKAYAMA ET AL. ci, Cm >Z (63) (4) I ) (55) FIG. 1. GVBD rates of porcine oocytes. Porcine COCs were cultured with pff at 37 C in 5% CO 2 in air. Oocytes showing signs of dissolving the germinal vesicle and having a nuclear phase of prometaphase I or later were considered to have reached GVBD. The number of oocytes examined is shown in parentheses at each point. *Significant differences as compared with the -h point. Oocytectomy This was accomplished by using two micromanipulators according to the method of Buccione et al. [6]. Each COC was attached to a holding pipette. A glass needle was then introduced through the cumulus cells and oocyte into the holding pipette. After the needle was withdrawn, the ooplasm, but not the zona pellucida, was aspirated into the holding pipette by a burst of negative pressure. Evacuated zonae usually collapsed after this procedure, but within a few minutes they returned to their normal spherical shape. The medium for micromanipulation was mkrb. Oocytectomized COCs were washed with mkrb several times, then placed in culture. Evaluation of Cumulus Expansion Each group of ten porcine COCs was cultured in vitro in 1,al of mkrb or pff for 24 h at 37 C under 5% CO 2 in air. The media contained 1 IU/ml ecg. Photographs of the oocytes were taken every 2 h during culture through a phase contrast microscope (x25). Cumulus expansion was evaluated as described by Daen et al. [11]. Briefly, the area occupied by a COC in the photographs was measured with a ruler and calculated with the formula: area = length x width x The length and the width were the distances between the two most widely separated points and between the two closest points, respectively. Metabolic Labeling of GAGs To determine the total incorporation of the HA precursor, COCs were cultured in medium containing [ 3 H]glucosamine (25 [.Ci/ml). The culture conditions were the same as described above. Every 2 h after culture was initiated, COCs were collected and washed three times with mkrb; then 1 of them were transferred to a tube with 15 R1 of mkrb. The COCs were lysed with 5 pi1 of.1 N NaOH, and the amount of incorporated label was examined by liquid scintillation counting. Metabolic labeling, extraction, and enzymatic digestion of GAGs proceeded as described by Tirone et al. [] and Breen et al. [13] with slight modifications. For dual GAG radiolabeling, [ 3 H]glucosamine (25 Ci/ml) and [ 35 S]sulfate (1,uCi/ml) were added to the medium, and * 64) the COCs were cultured for 3 h at 37 C under 5% CO 2 in air. After every 4-h interval during culture, complexes were transferred to radioisotope-free mkrb with a largebore micropipette and washed three times with 5 pxl of fresh mkrb. At the final wash, 4 COCs were transferred to a tube with 15 l of mkrb. Thirty-five microliters of papain (1 IU/ml) in.1 M sodium acetate containing 5 mm EDTA, ph 7.3, was then added to the tube. After a -h incubation at 65C, 5 l of 8 M guanidine-hci containing 2% CHAPS was added and left overnight at 4C. Each extract was brought to 3 jil with.5 M sodium acetate containing as a carrier 1% CHAPS, 1 mg/ml HA, and.5 mg/ml chondroitin sulfate (CS) A and C. Ethanol ( ml) containing 2% sodium acetate was added and mixed for 1 h at 4 C. The GAGs were pelleted by centrifugation at x g for 1 min at 4 C, and the precipitate was washed with ethanol. After air-drying, the GAG sample, dissolved in 2 pl1 of water, was divided into four equal aliquots, each of which was digested with enzymes with a different substrate specificity. Streptomyces hyaluronidase, chondroitinase, and heparitinase digestion proceeded in.1 M acetate buffer (containing.1 M NaCl, ph 5., at 1 turbidity reducing units/ml),.1 M Tris-acetate buffer (containing.1 M NaCl, ph 8., at.1 U/ml), and.5 M Tris-HCI buffer (ph 7.2, at.1 U/ml), respectively, for 24 h. The reaction temperature was 65 C for hyaluronidase and 37 C for the others. One aliquot was not treated. After digestion was completed, the mixtures were boiled for 5 min, then mixed with ethanol and centrifuged. The precipitate was dissolved in 1 L1 of water for liquid scintillation counting. Statistical Analysis The germinal vesicle breakdown (GVBD) rate was statistically analyzed by means of the chi-square test at various times after the beginning of culture and at h in culture. Each of the other experiments was repeated three times. The experimental validity of multiple treatments with four culture groups was analyzed by two-way analysis of variance; then differences among the four culture groups at various times after the beginning of culture were analyzed by Bonferonni's multiple range test. A p value less than.5 was considered significant. RESULTS GVBD Rates Porcine COCs dissected from ovaries were cultured in pff. Although GVBD was not induced during the first 18 h in the culture of COCs (Fig. 1), at 24 and 3 h in culture, 64.3% and 82.8% of oocytes in COCs, respectively, underwent GVBD. Cumulus Expansion The cumulus expansion area (in mm 2 ) of porcine COCs cultured in vitro was determined every 2 h (Fig. 2). Oocytectomy, per se, did not influence the cumulus expansion area; thus, the area of the intact COCs (21.3 ±.9 mm 2 ) was not different from that of oocytectomized COCs (2.6 ± 1.1 mm 2 ) at the beginning of the experiment. There were no differences in the degree of cumulus expansion induced between the two media until 6 h after the start of the culture. In culture with pff, the degree of cumulus expansion gradually increased until 2 h. After 24 h in culture, the cumulus expansion induced in pff was mm 2. Downloaded from by guest on 19 January 219

3 GLYCOSAMINOGLYCAN SYNTHESIS DURING CUMULUS EXPANSION 131 Cumulus expansion of the oocytectomized COCs was induced in pff. The curve of cumulus expansion of the oocytectomized group resembled that of the intact group, but the response was smaller. After 24 h in culture, the degree of cumulus expansion of oocytectomized COCs induced in pff (the area was 6.7 ± 6.1 mm 2 ) was much smaller than that for the intact group at the same time. A morphological study revealed differences between intact and oocytectomized COCs in the degree of expanded cumulus masses (Fig. 3). Cumulus expansion was not stimulated with mkrb in either the intact or the oocytectomized group. Incorporation of PH]Glucosamine The porcine COCs were cultured with medium containing [ 3 H]glucosamine alone to determine the net incorporation of [ 3 H]glucosamine into COCs. Incorporation into intact COCs cultured in pff immediately increased after the start of the culture, and linearly increased with small variations (Fig. 4). The incorporation of [ 3 H]glucosamine was maximal at 22 h, being about 8.2 times that at 2 h. In oocytectomized COCs, [ 3 H]glucosamine incorporation was also stimulated by pff Net incorporation of [ 3 H]glucosamine acutely increased from 8 h, and there was a maximal incorporation at 18 h of culture that was 7.6 times the amount at 2 h. The incorporation of [ 3 H]glucosamine was not stimulated with mkrb in either the intact or the oocytectomized group. Profiles of 3 H-Labeled GAGs The COCs were metabolically labeled simultaneously with [ 3 H]glucosamine and [ 35 S]sulfate. To judge the nature of the GAGs into which the label was incorporated, the extracted GAGs were digested with Streptomyces hyaluronidase, chondroitinase ABC, or heparitinase I. In COCs cultured in pff, total 3 H-labeled GAGs in the undigested aliquot dramatically increased linearly in the first h in culture, at 16 h they had decreased by 27% of the amount at h but then increased acutely from h in culture (Fig. 5A). This total was significantly reduced by digestion with Streptomyces hyaluronidase or chondroitinase ABC (Fig. 5, C and E). At 24 h, the radioactivity of total 3 H- labeled GAGs was 37.2% (by hyaluronidase) and 19.8% (by chondroitinase) of that in the undigested aliquot. Digestion with heparitinase I marginally reduced the profile of the total 3 H-labeled GAGs (Fig. 5G). There was no acute synthesis of GAGs in oocytectomized COCs cultured in pff (Fig. 5B). The radioactivity level of intact complexes at 24 h in culture was reduced to 34.2% by oocytectomy. The sensitivity to each enzyme of total 3 H-labeled GAGs was similar to that of intact COCs (Fig. 5, D, F, and H); differences were not significant. As a control, mkrb did not induce the synthesis of 3 H-labeled GAGs in the COCs. Profiles of 3-S-Labeled GAGs Total 3 5 S-labeled GAGs in the undigested aliquot showed an increasing curve similar to the profile for total 3 H-labeled GAGs throughout the culture period (Fig. 6A), with an acute increase from h. These levels were significantly reduced only by digestion with chondroitinase ABC (Fig. 6E), by which the radioactivity level at 24 h was decreased to 13.7%. Streptomyces hyaluronidase or heparitinase I weakly digested the 35 S-labeled GAGs or had no effect (Fig. 6, C and G). As compared with 3 H-labeled GAGs, oocytectomy had only a weak effect or none at all _.2 r_ q co Q E x E u O Z 2! E FIG. 2. Time-course changes in the degree of cumulus expansion. Intact COCs (solid circles) or oocytectomized COCs (open circles) were cultured with pff (solid line) or mkrb (dotted line). (The profile of intact COCs cultured with mkrb is not shown because the data corresponds with oocytectomized COCs cultured in mkrb.) Details of the calculation of the cumulus expansion area are given in Materials and Methods. Data are shown as the mean SEM for 28-3 complexes. *Significant differences when the intact and oocytectomized groups were compared. in reducing the synthesis of 35 S-labeled GAGs (Fig. 6B). The total of 3 5 S-labeled GAGs from oocytectomized COCs reached 91.3% of that in intact COCs at 24 h in culture. As with 3 H-labeled GAGs, mkrb did not induce the synthesis of 35 S-labeled GAGs in the COCs. DISCUSSION nu I' L We investigated the role of oocytes in the accumulation of GAGs during cumulus expansion. We found that cumulus expansion induces a dramatic rise in HA synthesis, which is oocyte-dependent in porcine COCs cultured with pff. Daen et al. [11] have reported that pff contains an active factor(s) that promotes cumulus expansion. They eluted the active factor(s) for cumulus expansion as a single peak by HPLC, then showed that it was heat-stable at 1 C for 15 min and was not affected by a repeated freeze-thaw cycle. The molecular mass of the active factor(s), estimated by chromatography, was below 6.5 kda. In contrast, cumulus expansion is a paracrine phenomenon regulated by the cumulus expansion-enabling factor secreted from the oocytes in mice [6, 8, 14]. Eppig et al. [15] demonstrated that mouse COCs cultured on the surface of a matrix in medium conditioned by oocytes grown in vitro secreted an active cumulus expansion-enabling factor. This factor possessed protein-like properties or depended on a protein for its activity and had a molecular mass between 3 and 1 kda. This macromolecule has not been detected in pff [11]. These findings suggest that the active pff and oocyte factors are different. That the oocytectomized COCs do not expand in response to FSH stimulation in mice [6] is also evidence of an oocyte factor. However, we induced cumulus expansion in oocytectomized COCs by culture in pff as shown in Figure 2, although to a lesser extent than that in intact COCs. These results suggest that pff induces cumulus expansion independent of the paracrine factor from the oocyte. A serum factor(s) induces cumulus expansion. Daen et al. [16] have estimated the properties of this factor. They showed that the serum factor(s) was heat-stable, with a molecular mass between 1. and 6.5 kda. These properties are Downloaded from by guest on 19 January 219

4 132 NAKAYAMA ET AL. FIG. 3. Photomicrographs of cumulus expansion of porcine COCs. Fresh (A) and oocytectomized COCs (B), and intact (C) and oocytectomized COCs (D) cultured in mkrb for 24 h showed no expansion of the cumulus mass. E) Intact COCs cultured in pff for 24 h showed a fully expanded cumulus layer, and (F) oocytectomized COCs cultured in pff for 24 h also showed cumulus expansion with a glistening appearance. Scale bar = 1 m. very similar to the active factor(s) derived from pff as described above. These findings suggest that the follicular fluid factor(s) is of serum origin. Although it is unknown whether these factors derived from two sources are the same, serum can be used as a starting material with which to purify the active factor(s). Serum also contains a protein that stabilizes the cumulus matrix through direct binding with HA [17]. This protein, referred to as cumulus extracellular matrix stabilizing factor, has been identified with the inter-a-trypsin inhibitor family [2]. To achieve mouse cumulus expansion in vitro, the medium must contain gonadotropin and serum [5]. In our porcine system, however, medium containing pff induced cumulus expansion without serum. Perhaps pff contains a serum-derived factor. It has been thought that the cumulus cells synthesize GAGs and proteoglycans to organize the cumulus matrices during cumulus expansion, and that glucosamine is utilized to produce their components, especially HA and CS [5]. To determine whether the changes in cumulus expansion are reflected in GAG synthesis, we measured [ 3 H]glucosamine incorporation into intact and oocytectomized COCs. Figure 4 shows that [ 3 H]glucosamine incorporation did not reflect the differences between intact and oocytectomized COCs shown in the degrees of cumulus expansion. Thus, although the incorporation of [ 3 H]glucosamine into COCs was minimally affected by oocytectomy, this treatment induced a major change in incorporation of this precursor into GAGs. The differences in cumulus expansion may reflect utilization of this substrate for HA or CS synthesis. We therefore postulated that there are differences between the GAG compositions in the cumulus matrix synthesized through cumulus expansion of intact versus oocytectomized COCs. We examined the changes in GAG composition of cumulus matrix during cumulus expansion using a combination of GAG lyases and metabolic labeling of GAGs with [ 3 H]glucosamine and [ 3 5 S]sulfate. In this experimental system, to determine the nature of the GAGs into which the label was incorporated, the extracted GAGs were digested with Streptomyces hyaluronidase, chondroitinase ABC, or heparitinase I. Chondroitinase ABC digests CS and HA. Downloaded from by guest on 19 January 219

5 GLYCOSAMINOGLYCAN SYNTHESIS DURING CUMULUS EXPANSION 133 V) a_ o Iu, x13 4 r FIG. 4. Time-course changes in [H]glucosamine incorporation into COCs. Ten intact (solid circles) or oocytectomized COCs (open circles) were cultured with pff (solid line) or mkrb (dotted line) containing p1h]glucosamine. Each point represents the mean SEM of three experiments. *Significant differences when the intact and oocytectomized groups were compared. n O T X 1 3 ao) z 3 O Inttact 19 C 2 6 a3 26 o r i6 a 2a- :3- E G L P~E.....I * * _ a Oocytectomized D F H FIG. 5. Time-course changes in 3H-labeled GAGs digested with various enzymes (Streptomyces hyaluronidase, chrondroitinase ABC, or heparitinase I) after extraction from COCs. Ten porcine COCs were cultured with pff (solid circles) or mkrb (open circles) containing [Hl]glucosamine and p[5s]sulfate. The changes in GAGs from intact and from oocytectomized COCs are shown in the left and right panels, respectively. Each point represents the mean SEM of three experiments. *Significant differences when the digested and total GAGs were compared; points with different superscripts are significantly different. b -9 L 7-. -,. I. 2 a U, no S 2 - C M C I E = o Intact 2 G A l ri Oocytectomized D F H FIG. 6. Time-course changes in 35 S-labeled GAGs digested with various enzymes (Streptomyces hyaluronidase, chondroitinase ABC, or heparitinase I) after extraction from COCs. Ten COCs were cultured with pff (solid circles) or mkrb (open circles) containing [3H]glucosamine and p[5s]sulfate. The changes in GAGs from intact and from oocytectomized COCs are shown in the left and right panels, respectively. Each point represents the mean + SEM of three experiments. *Significant differences between the digested and total GAGs. The radioactivity levels after digestion are approximate indicators of the content of HA, CS+HA, and heparan sulfate, respectively. As shown in Figure 5A, total 3 H-labeled GAGs of intact COCs dramatically increased at h in culture. Furthermore, some of the GAGs were digested with Streptomyces hyaluronidase or chondroitinase ABC, which eliminated the prominent increase of total GAGs (Fig. 5, C and E). Most of the eliminated GAG is HA, and the difference between the hyaluronidase- and the chondroitinasedigested groups was attributable to CS. Total 35 S-labeled GAGs also had a similar profile to 3 H-labeled GAGs (Fig. 6A). Oocytectomy eliminated the dramatic rise in the total 3 H-labeled GAGs of intact COCs (Fig. 5B), whereas total 35 S-labeled GAGs derived from oocytectomized COCs were synthesized to the same level as that derived from intact COCs at 24 h in culture (Fig. 6B). These results indicated that oocytectomy abolishes the synthesis of 3 H- labeled GAGs while it is either not effective, or marginally effective, in synthesizing 35 S-labeled GAGs. That is, the oocyte regulates the synthesis of HA, but not of CS, during cumulus expansion. Our findings are partly confirmed by other reports showing that cumulus expansion is independent of the oocyte in pigs [18] and cattle [19]. However, it seems that the cu- r Downloaded from by guest on 19 January 219

6 134 NAKAYAMA ET AL. mulus expansion shown by oocytectomized COCs is attributable to the synthesis and accumulation of CS. We speculate that HA synthesis of cumulus cells during cumulus expansion is dependent on the oocyte, and that CS synthesis is a basic response of cumulus cells. In conclusion, we demonstrated that 1) cumulus expansion of porcine COCs was reduced by oocytectomy, 2) GAG synthesis had a time-dependent profile with a dramatic rise after 16 h, 3) the profile of total GAG synthesis was attributed to HA rather than CS in intact COCs, and 4) oocytectomy reduced this HA synthesis. These results suggest that HA synthesis by porcine COCs is regulated by oocyte-dependent mechanisms. Furthermore, it is likely that cumulus expansion is not abolished completely by oocytectomy, because CS is synthesized independently of the oocyte. REFERENCES I. Eppig JJ. FSH stimulates hyaluronic acid synthesis by oocyte-cumulus cell complexes from mouse preovulatory follicles. Nature 1979; 281: Chen L, Russell PT, Larsen WJ. Functional significance of cumulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass. Mol Reprod Dev 1993; 34: Vanderhyden BC, Armstrong DT Role of cumulus cells and serum on the in vitro maturation, fertilization, and subsequent development of rat oocytes. Biol Reprod 1989; 4: Dekel N, Kraicer PE Induction in vitro of mucification of rat cumulus oophorus by gonadotrophins and adenosine 3',5'-monophosphate. Endocrinology 1978; : Salustri A, Yanagishita M, Hascall VC. Synthesis and accumulation of hyaluronic acid and proteoglycans in the mouse cumulus cell-oocyte complex during follicle-stimulating hormone-induced mucification. J Biol Chem 1989; 264: Buccione R, Vanderhyden BC, Caron PJ, Eppig JJ. FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte. Dev Biol 199; 138: Prochazka R, Nagyova E, Rimkevicova Z, Nagai T, Kikuchi K, Motlik J. Lack of effect of oocytectomy on expansion of the porcine cumulus. J Reprod Fertil 1991; 93: Vanderhyden BC. Species differences in the regulation of cumulus expansion by an oocyte-secreted factor(s). J Reprod Fertil 1993; 98: Naito K, Fukuda Y, Toyoda Y. Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro. Gamete Res 1988; 21: Yoshida M, Ishizaki Y, Kawagishi H, Bamba K, Kojima Y. Effects of pig follicular fluid on maturation of pig oocytes in vitro and on their subsequent fertilizing and developmental capacity in vitro. J Reprod Fertil 1992; 95: Daen FP, Sato E, Naito K, Toyoda Y. The effect of pig follicular fluid fractions on cumulus expansion and male pronucleus formation in porcine oocytes matured and fertilized in vitro. J Reprod Fertil 1994; 11: Tirone E, Siracusa G, Hascall VC, Frajese G, Salustri A. Oocytes preserve the ability of mouse cumulus cells in culture to synthesize hyaluronic acid and dermatan sulfate. Dev Biol 1993; 16: Breen M, Knepper PA, Weinstein HG, Blacik LJ, Lewandowski DG, Baltrus BM. Microanalysis of glycosaminoglycans. Anal Biochem 1981; 113: Vanderhyden BC, Caron PJ, Buccione R, Eppig JJ. Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev Biol 199; 14: Eppig JJ, Peters AHFM, Telfer EE, Wigglesworth K. Production of cumulus expansion enabling factor by mouse oocytes grown in vitro: preliminary characterization of the factor. Mol Reprod Dev 1993; 34: Daen FP Sato E, Nakayama T. Toyoda Y. Serum factor(s) stimulating cumulus expansion in porcine oocyte-cumulus complexes matured and fertilized in vitro. Cell Struct Funct 1995; 2: Chen L, Mao SJT, McLean LR, Powers RW, Larsen WJ. Proteins of the inter-oa-trypsin inhibitor family stabilize the cumulus extracellular matrix through their direct binding with hyaluronic acid. J Biol Chem 1994; 269: Singh B, Zhang X, Armstrong DT Porcine oocytes release cumulus expansion-enabling activity even though porcine cumulus expansion in vitro is independent of the oocyte. Endocrinology 1993; 132: Ralph JH, Telfer EE, Wilmut I. Bovine cumulus cell expansion does not depend on the presence of an oocyte secreted factor. Mol Reprod Dev 1995; 42: Downloaded from by guest on 19 January 219