Secretion of Paracrine Factors Enabling Expansion of Cumulus Cells Is Developmentally Regulated in Pig Oocytes 1

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1 BIOLOGY OF REPRODUCTION 63, (2) Secretion of Paracrine Factors Enabling Expansion of Cumulus Cells Is Developmentally Regulated in Pig Oocytes 1 Eva Nagyová, 2,3,4 Barbara C. Vanderhyden, 4 and Radek Procházka 3 Academy of Sciences of the Czech Republic, 3 Institute of Animal Physiology and Genetics, Liběchov, Czech Republic Ottawa Regional Cancer Centre, 4 Cancer Research Group, Ottawa, Ontario, Canada ABSTRACT To demonstrate secretion of cumulus expansion-enabling factor (CEEF) by porcine oocytes, we used an interspecies testing system. Porcine oocytes were used to condition culture medium, and the presence of CEEF was tested using mouse oocytectomized complexes (), which require CEEF for expansion. Follicle-stimulating hormone-stimulated expansion and synthesis of hyaluronic acid (HA) by mouse were assessed after 18 h of culture in media conditioned by porcine oocytes: 1) at different stages of maturation and 2) in which maturation was inhibited with a specific inhibitor of cdk-kinases, butyrolactone I. Fully grown (GV-germinal vesicle), late-diakinesis (LD), metaphase I (MI), and metaphase II (MII) oocytes were prepared by culture of oocyte-cumulus complexes (OCC) for, 22, 27, and 42 h, respectively. To block GV breakdown, porcine oocytes were cultured for 27 h in medium supplemented with butyrolactone I (5 M). Medium conditioned by oocytes in GV, LD, and after butyrolactone I block allowed full expansion of 9% of mouse, whereas oocytes in MI and MII caused disintegration of mouse without cumulus mucification. To measure synthesis of HA by cumulus cells, 25 mouse were cultured in the conditioned media in the presence of 2.5 Ci of D-[6-3 H]glucosamine hydrochloride. After 18 h, incorporation of the [ 3 H]glucosamine into HA was determined either in complexes (retained HA) or in medium plus complexes (total HA). Total HA accumulation by mouse was not different from that of intact OCC. However, oocytes in GV, LD, and after butyrolactone I treatment enabled mouse to retain significantly more HA within the complex than oocytes in MI and MII. The results indicate that secretion of factors that promote the retention of HA within the complex is developmentally regulated during oocyte maturation. cumulus cells, developmental biology, follicle, FSH, granulosa cells, growth factors, meiosis, oocyte development, ovary, ovum INTRODUCTION In most mammals, as the Graafian follicle progresses to ovulation in response to a gonadotropin surge, the oocytecumulus cell complex (OCC) expands dramatically [1]. This expansion is the result of the synthesis and organization of an extensive extracellular matrix around the cumulus cells [2]. Follicle-stimulating hormone stimulates the 1 This research was supported by collaboration via a fellowship (to E.N.) under OECD Cooperative Research Programme: Biological Resource Management for Sustainable Agricultural Systems, as well as by grant 524/ 98/231 from the Grant Agency of the Czech Republic. 2 Correspondence: FAX: ; nagyova@iapg.cas.cz Received: 7 February 2. First decision: 1 March 2. Accepted: 29 May 2. 2 by the Society for the Study of Reproduction, Inc. ISSN: expansion of cumuli oophori isolated from mice, rats and pigs in vitro [2 4]. The major structural macromolecule in the matrix of the expanded OCC is hyaluronic acid (HA), which is synthesized by the cumulus cells. Net synthesis of HA during FSH-stimulated expansion of the OCC correlates directly with the accumulation of this glycosaminoglycan in the matrix and with the morphological change of the OCC [5, 6]. In addition to FSH, the oocyte also plays an important role in regulation of cumulus expansion. It was demonstrated in experiments in which mouse cumulus cells and oocytes were separated either by dissection or by removal of the oocytes from OCC by oocytectomy that oocytes release a soluble factor(s) that is necessary to induce synthesis of HA by the cumulus cells [6, 7]. Removal of the oocyte from OCC also inhibits FSH and EGF-induced expansion of the cumulus. However, conditioning of the culture medium with denuded oocytes reverses the inhibitory effect of oocytectomy on expansion of cumulus cells [7], indicating that mouse oocytes produce a soluble cumulus expansion-enabling factor (CEEF). Mouse oocytes acquire the ability to secrete CEEF during the growth period at the time of acquisition of competence to undergo germinal vesicle breakdown (GVBD). The ability of the oocyte to secrete CEEF is lost after fertilization [8]. In contrast to the mouse model, we have shown [9, 1] that under in vitro conditions, expansion of porcine OCC does not depend on an oocyte-produced factor. Moreover, our studies have demonstrated that the synthesis of HA by pig cumulus cells in vitro is stimulated by FSH and that oocytectomy does not change this synthesis [11]. However, using an interspecies testing system, it was demonstrated that pig, bovine, and rat oocytes all produce CEEF because they enable the FSH-stimulated expansion of mouse complexes [12 15]. Nevertheless, the pattern of CEEF production differs from that observed in the mouse, where CEEF secretion continues throughout the period of growth and maturation [8]. In in vitro-matured pig oocytes, secretion of CEEF occurs only in oocytes in GV stage and during to MI transition. The metaphase I, metaphase II, activated, and pronuclear pig oocytes also enabled expansion of the mouse ; however, the cumuli were disintegrated and not mucified [15]. The activity of mouse CEEF was found to be heat labile (65C for 15 sec), lost by proteinase K digestion, and retained by 1-kDa, but not by 3-kDa, membranes [16], suggesting that the factor is a protein or depends upon a protein for its activity. However, frozen-thawed bovine oocyte-conditioned media still permitted mouse complexes to undergo cumulus cell expansion [14]. The substantial species differences mentioned above enabled us to use an interspecies testing system to demonstrate production of CEEF by porcine oocytes during their maturation. In addition, we examined the production of CEEF by porcine oocytes after they were blocked in GV

2 115 NAGYOVÁ ET AL. stage for 27 h with the inhibitor butyrolactone I. Butyrolactone I has been described as a potent and specific inhibitor of cdk-kinases, acting as a competitor to ATP [17, 18]. We have shown previously the effect of butyrolactone I, a specific inhibitor of cdc-kinase, on GVBD and chromosome condensation during the first meiotic division of pig oocytes [19]. The 5 M concentration of butyrolactone I was sufficient to block GVBD in pig oocytes, and after washing off the inhibitor, oocytes resumed and completed the first meiotic division at a rate comparable with that of controls. Pig oocytes arrested in the germinal vesicle stage with butyrolactone I possessed no histone H1 activity and MBP kinase activity, indicating that maturation-specific rise of p34 cdc2 /cyclin B and MAP kinase activation was completely blocked by the drug. As has been mentioned above, mouse cumulus cells must interact with two distinctly different factors to synthesize the maximum amount of HA, an unknown soluble factor released by oocytes and FSH [6]. To investigate the expansion-enabling activity produced by porcine oocytes, we assessed FSH-stimulated synthesis of HA by mouse cumulus cells () after 18 h of culture in medium conditioned by porcine oocytes: 1) at different stages of maturation and 2) in which maturation was inhibited with a specific inhibitor of cdk-kinases, butyrolactone I. MATERIALS AND METHODS Isolation and Culture of Porcine Oocytes Porcine ovaries were obtained from crossbred gilts (Landrace, Large White) at a local abattoir and transferred to the laboratory in a thermos at 3C. OCC were isolated from follicular fluid that was aspirated from 2 5 mm antral follicles. The following groups of porcine oocytes were used for assessment of CEEF secretion: fully grown (GVgerminal vesicle) oocytes and oocytes at late diakinesis, metaphase I, and metaphase II prepared by culture of complexes for, 22, 27, or 42 h, respectively. The culture medium was M-199 (Sevac, Prague, Czech Republic), buffered with 2 mm NaHCO 3 and 6.25 mm Hepes and supplemented with 5% fetal calf serum (FCS),.91 mm sodium pyruvate, 1.62 mm calcium lactate, and antibiotics [2]. This culture medium was supplemented with.1 g/ ml FSH (Biogenesis, Poole, England). Oocytes were blocked at the GV stage for 27 h by culture with a specific inhibitor of cyclin-dependent kinases, butyrolactone I (5 M, Sigma-Aldrich, Prague, Czech Republic). Isolation of Mouse Oocyte-Cumulus Complexes and Oocytectomy Mouse OCC were isolated from ovaries of 4- to 6-wkold ICR mice, stimulated h previously with 1 IU ecg (Bioveta, Ivanovice na Hané, Czech Republic). Complexes were released from large antral follicles by puncture of the follicular wall with fine needles in M-199 medium (above), and the oocytes were removed as described elsewhere [7]. The resulting, composed of evacuated zone and surrounding cumulus cells, were washed twice and immediately placed into culture. Preparation of Conditioned Media and Assessment of the Cumulus Expansion Because expansion of porcine cumulus does not depend on an oocyte-secreted factor or factors, we used an interspecies testing system [12 15] to prove secretion of CEEF by porcine oocytes. Porcine oocytes were allowed to condition culture medium, and then the presence of CEEF in the conditioned media was tested by adding mouse, which require CEEF for expansion. Conditioned media were prepared by the culture of 5 denuded porcine oocytes in 5 l of the culture medium for 24 h. Subsequently, 1 to 15 mouse and.1 g/ml FSH were added to the drop of conditioned medium, and expansion was assessed 18 h later, according to a subjective scoring system ( to 4) described previously [8]. Briefly, indicates no detectable response, whereas 1 indicates the minimum observable response: peripheral cumulus cells have a glistening appearance, 2 indicates expansion of the peripheral layers of the cumulus; 3 indicates expansion of all layers of the cumulus except corona radiata cells, and 4 indicates expansion of the cumulus including corona radiata cells. The extent of mucification of the expanded cumuli was evaluated by morphological and physical criteria using a stereomicroscope [6]. Positive response (): the expanded cumuli were mucified and very sticky and were difficult to remove from oocytes by repeated pipetting through a fine-bore pipette. Negative response (): the expanded cumuli were not mucified and not sticky and were very easy to remove from oocytes by pipetting. To test the ability of porcine CEEF to survive freezing, fully grown oocytes and oocytes in late diakinesis were isolated as described above, and conditioned media were prepared by culturing 5 denuded porcine oocytes in 5 l of culture medium for 24 h. At the end of the incubation period, the conditioned media were collected and stored frozen at 2C, along with aliquots of unconditioned media. Media were frozen for up to 2 mo before thawing and testing for the ability to enable expansion of mouse as described above. Once proven that CEEF activity in conditioned media was retained after freezing, frozen-thawed porcine oocyte-conditioned media were used for HA synthesis measurements (below). Hyaluronic Acid Synthesis Groups of 25 mouse were cultured for 18 h in 1 l media conditioned by porcine oocytes (at different stages or after treatment with butyrolactone I) at 37C inanatmosphere of 5% CO 2 in air and in the presence of 2.5 Ci of D-[6-3 H]glucosamine hydrochloride (Amersham Pharmacia Biotech, Buckinghamshire, UK) and 1 g/ml FSH. Hyaluronic acid synthesis was measured by a procedure that was described elsewhere [3, 21], with slight modifications. Briefly, the cultures were terminated by adding 1 l of a solution containing 5 mg/ml pronase (Sigma-Aldrich) and 1% Triton X-1 in.2 M Tris buffer, ph 7.8. The samples were incubated for 2 h at 37C and then transferred to Whatman 3MM filter paper circles. The circles were air dried and then washed through three changes of solution containing.5% cetylpyridinium chloride and 1 mm nonradioactive glucosamine hydrochloride (Sigma-Aldrich) for 45 min each. The circles were dried once again, and radioactivity was measured in 5 ml scintillation fluid (Biodegradable Counting Scintillant, Amersham Canada Ltd., Oakville, ON, Canada) using a liquid scintillation counter. Synthesis of HA was measured either in medium plus OCC or complexes (total HA) or within the complexes alone (retained HA); this was achieved by simply transferring the complexes through three changes of culture medium without labeled precursor before addition of the pronase-triton X-1 solution. The specificity of incorpo-

3 SECRETION OF PARACRINE FACTORS ENABLING EXPANSION OF CUMULUS CELLS 1151 TABLE 1. Effect of freezing CEEF on the expansion of OCCs and s. Culture conditions* Number of complexes Type of complex Degree of expansion Fresh medium Fresh medium Frozen medium Frozen medium Frozen medium, GV oocyte conditioned Frozen medium, LD oocyte conditioned 6 6 OCC OCC 3 to4 3 to4 3 to4 3 to4 * For evaluation of expansion of mouse complexes cultured in frozen-thawed oocyte conditioned media, there were six replicates (n 6). Total number of complexes examined in at least three replicates. Subjective evaluation of the degree of expansion (see Materials and Methods). ration of radioactivity into HA was determined by sensitivity to highly specific Streptomyces hyaluronidase (Sigma- Aldrich). After the overnight culture period, some samples were treated with 1 IU of Streptomyces hyaluronidase for 2hat37C before addition of the pronase-triton X-1 solution. Assessment of GVBD For all experiments, samples from each pool of oocytes used to condition the media were assessed for their stage of meiotic maturation. To prepare the oocytes for assessment of GVBD, cumulus cells were mechanically removed. Denuded oocytes were mounted on slides, fixed in an acetic acid/alcohol (1:3) mixture for 24 h, stained with 1% orcein, and examined under a phase-contrast microscope. Statistical Analysis The differences between responses to the various treatments were identified by analysis of variance (ANOVA), followed by use of Bonferroni s method to determine significance between specific treatment groups or unpaired, two-tailed t-tests when only two treatments were being compared. For all figures, error bars indicate the standard error of the mean from at least three independent experiments. RESULTS Effect of Freezing Porcine CEEF on Expansion of OCCs and s The aim of this experiment was to determine whether media conditioned by porcine oocytes would retain CEEF activity if stored frozen. Both fresh and frozen-thawed media were able to support FSH-induced expansion of mouse OCC, whereas were unable to expand (Table 1). When media conditioned by porcine oocytes in either GV stage or LD stage were frozen and thawed before culture with mouse and FSH, both media were able to support the expansion of the mouse to levels comparable to that of OCC in frozen-thawed media. These results indicate that porcine oocyte-conditioned media can be stored frozen without a change in its expansion promotion activity. For all subsequent experiments, conditioned media were stored frozen and thawed before adding and FSH. Production of CEEF by Porcine Oocytes During Their Maturation Intact mouse OCC underwent 3 to4 expansion when cultured in medium supplemented with FSH and FCS; however, expansion did not occur in cultured under the same conditions (Table 2). The ability to expand was restored when were cultured in medium conditioned by pig oocytes that were fully grown or in late diakinesis. In both situations, the expanded cumuli were mucified and very sticky (Table 2, Fig. 1A). The conditioned media from metaphase I and metaphase II porcine oocytes caused disintegration of the mouse. The cumuli were fragile, not mucified and not sticky (Table 2, Fig. 1B). Nevertheless, these cumuli were substantially different from those cultured in unconditioned medium that were compact and firmly attached to the bottom of the culture dish (Fig. 1C). Total HA Synthesis by Mouse OCC and after Stimulation by FSH Mouse OCC or were cultured in the presence of FSH for 18 h, a period of time required for full expansion in vitro and for maximal HA accumulation in the OCC matrix. In initial experiments, total HA accumulation was measured, which included the accumulation in both cells and media. In association with the morphological changes TABLE 2. Expansion of intact and oocytectomized mouse cumulus complexes cultured with medium conditioned by porcine oocytes at different stages of maturation.* Type of oocytes used for conditioning Type of complexes Degree of expansion Mucification Number of complexes None (M-199) None (M-199FSH) Fully grown oocytes Late diakinesis oocytes Metaphase I oocytes Metaphase II oocytes Oocytes butyrolactone I Intact Intact 3 to4 3 to4 3 to4 3 to4 * Intact oocyte-cumulus complexes cultured in conditioned media displayed a uniform response: expansion, 3 to4; mucification,. More than 92% of complexes exhibited the degree of expansion and mucification shown in the table. In those treatment groups that enabled expansion, there was no difference in the proportion of complexes showing 3 to4 expansion (P.1; chi-square analysis). No expansion; cumulus cells disintegrated

4 1152 NAGYOVÁ ET AL. FIG. 1. Expansion of mouse oocytectomized complexes () in medium conditioned by porcine GV (A) or MII oocytes (B) or in unconditioned medium (C). in OCC that occurred in response to FSH, FSH caused an increase in the accumulation of HA (Fig. 2). FSH was unable to induce HA synthesis by. Treatment of the expanded OCC with the highly specific Streptomyces hyaluronidase resulted in loss of association of the majority of the cumulus cells with each other and with the oocyte. This morphological observation was supported by quantitative assessment of HA content; of the total [ 3 H]-glucosamine incorporation into OCC stimulated by FSH, 9% was sensitive to Streptomyces hyaluronidase FIG. 2. Total HA synthesis by mouse OCC and after stimulation by FSH. Tritium-labeled HA was measured in medium plus complexes (OCC or ) after 18 h of culture in fresh or frozen medium with or without FSH. Sensitivity to Streptomyces hyaluronidase (Sh) was also measured. Data represent the mean SEM from four replicates for a total of 1 complexes (OCC or ) per treatment. Bars with different superscripts are significantly different (P.1). (Fig. 2), indicating that HA is the predominant glycosaminoglycan product of the [ 3 H]-glucosamine. FSH-Stimulated HA Synthesis by Mouse after Culture in Media Conditioned by Porcine Oocytes at Different Stages of Maturation The effect of porcine oocyte-conditioned media (GV, LD, MI, or MII) on synthesis of HA by mouse after FSH stimulation was tested. Mouse cultured in the presence of porcine oocyte-conditioned media (GV, LD, MI, or MII) were capable of synthesizing HA (measured in medium ) to levels equivalent to those of OCC (Fig. 3), but four- to sixfold greater than those of cultured alone (Fig. 2). The total accumulation of HA by mouse complexes cultured in media conditioned by oocytes at different stages of development was not different from that of intact OCC after FSH stimulation (P.5). The amount of HA retained within the complexes was found to be similar between OCC and cultured in the presence of media conditioned by GV or LD stage oocytes (P.5). However, a significant difference (P.1) was observed in the amount of HA retained in intact OCC vs. cultured in media conditioned with MI or MII stage oocytes. When media were conditioned with MI or MII oocytes, the ability for HA to be retained within the complexes was lost, and almost all HA was released into the culture media. FSH-Stimulated Expansion and HA Synthesis by Mouse after Culture in Medium Conditioned by Porcine Oocytes Treated with Butyrolactone I To block the breakdown of the germinal vesicle, porcine oocytes were cultured for 27 h in medium supplemented with butyrolactone I. After removal from the inhibitor, the

5 SECRETION OF PARACRINE FACTORS ENABLING EXPANSION OF CUMULUS CELLS 1153 FIG. 3. FSH-stimulated HA synthesis by mouse after culture in media conditioned by porcine oocytes at GV, LD, MI, and MII stages of maturation. HA retained within the complexes and total (complexes plus medium) were measured. Data represent the mean SEM from three replicates for a total of 75 OCC or per treatment. Bars with different superscripts are significantly different (P.5). oocytes were used to condition media for 22 h, and then expansion and HA synthesis by mouse cultured in this conditioned media was assessed. Mouse cultured in conditioned media from butyrolactone I-treated oocytes underwent both full cumulus expansion (3 to4) and mucification (Table 2). When complexes were washed before processing to measure HA content retained within the complexes, the amount retained was found to be similar that of to intact OCC (P.5). Total HA synthesis was also found to be not significantly different between these two groups (P.5; Fig. 4). Meiotic Maturation of Porcine Oocytes Used to Condition the Media Oocytes from each pool of OCC (GV, LD, MI, and MII) that were denuded and used to condition the media were concomitantly fixed and assessed for their stage of meiotic maturation. The results indicate that although there is some degree of overlap, the timed cultures (i.e.,, 22, 27, and 42 h) were reasonably effective in enabling the collection of the majority of oocytes at specific stages of development (Table 3). For porcine OCC cultured for 27 h in the presence of butyrolactone I, the stage of meiotic maturation was FIG. 4. FSH-stimulated HA synthesis by mouse after culture in media conditioned by porcine oocytes blocked in GV stage with butyrolactone I (BT). HA retained within the complex or total amount of HA (complexes plus medium) was measured. Data represent the mean SEM from three replicates for a total of 75 OCC or per treatment. Bars with different superscripts are significantly different (P.5). assessed at the end of culture (1% in GV stage), as well as 22 h after they were released from the butyrolactone I block (96% GVBD). Results are summarized in Table 4. DISCUSSION These experiments provide evidence that porcine oocytes produce at least two paracrine factors during meiotic maturation in vitro that affect expansion of mouse and that production of these is regulated developmentally. The first factor affects total production of HA by cumulus cells, and the second affects retention of HA in the extracellular matrix of expanding cumulus cells. We demonstrated for the first time that both factors produced by porcine oocytes could be stored frozen while maintaining both their expansion-promoting activity and ability to retain HA within the cumulus cell mass. These data are in agreement with the results of Ralph et al. [14], who demonstrated that frozen-thawed media conditioned by bovine oocytes permitted mouse complexes to undergo cumulus expansion. Using an interspecies testing system, we have found differences in production of expansion and retention promoting factors by porcine oocytes at different stages of maturation [15]. In in vitro-matured porcine oocytes, secretion of both factors occurred only in GV stage oocytes and dur- TABLE 3. Meiotic stage of porcine oocytes used for conditioning medium. Culture period (h) Number of oocytes GV (%) LD (%) MI (%) MII (%) GVBD (%) (1) 2 () 9 (11) 5 (7) 24 (48) 1 (13) 4 (6) 6 (12) 42 (53) 4 (6) 18 (23) 55 (81) 3 (6) 7 (89) 63 (93)

6 1154 NAGYOVÁ ET AL. TABLE 4. Meiotic stage of porcine oocytes used for conditioning medium after treatment with butyrolactone I. Culture period (h) 27 BT* BT 78 Number of oocytes GV (%) LD (%) MI (%) MII (%) GVBD (%) 57 (1) 3 (4) 7 (9) 2 (26) 48 (62) 75 (96) * Oocyte-cumulus complexes after culture in medium supplemented with butyrolactone I (BT) for 27 h. After washing off the inhibitor, denuded oocytes were cultured 22 h to condition the medium. ing the GV to MI transition. Oocytes that progressed to and beyond MI appeared to produce a factor or factors that enabled production of HA after stimulation of cumulus cells with FSH but not its retention within expanded cumuli. Quantification of HA accumulation in mouse showed that oocytes in GV and LD stages as well as after butyrolactone I treatment enabled mouse to retain significantly more HA within the complex than oocytes in MI and MII. Although oocytes in MI and MII stimulated the production of HA at levels comparable to earlier stage oocytes, almost all of the HA was released into the culture medium, and as a result, the complexes disintegrated. It has previously been shown in mouse cumulus complexes stimulated with FSH and cultured in vitro that serum is required for incorporation of HA into the extracellular matrix [3, 22]. The serum factor responsible for retaining HA in the expanding cumulus was subsequently identified as a member of inter--trypsin inhibitor family [23]. Thus, one can speculate that pig oocytes beyond MI, in contrast to oocytes in GV and LD stages, do not support the function of this factor. Moreover, inter--trypsin inhibitor may have a role as a protease inhibitor. It is likely that proteases either synthesized by somatic cells or activated locally (such as by the conversion of plasminogen to plasmin) participate in basement membrane breakdown or degradation of follicular fluid components at the time of ovulation [24 27]. Because the OCC extracellular matrix is susceptible to proteolytic degradation in vitro [28, 29], the presence of inter--trypsin inhibitor in the matrix may help protect it from degradation by endogenous proteases prior to ovulation. It has been demonstrated that isolated mouse cumulus cells cultured with FSH or a camp analogue are stimulated to synthesize urokinase plasminogen activator (upa), a serine protease that can activate plasminogen to plasmin [3]. When oocytes (or oocyte-conditioned medium) are present, however, upa synthesis is suppressed to basal levels. This suggests that the oocyte may contribute to matrix stability by inhibiting the local synthesis of proteases [3]. The ability of mouse oocytes to produce factors that control extracellular matrix production and retention is dependent upon their stage of development. Fully grown oocytes, but not growing oocytes or two-cell embryos, are able to secrete the CEEF that supports HA synthesis [38] and to inhibit upa synthesis [3]. Thus, mouse oocytes appear to promote preovulatory matrix accumulation by modulating the gonadotropin action on both the synthesis and the degradation of specific matrix components [3]. The results from this study would indicate that at least in porcine oocytes, regulation of the preovulatory matrix is achieved using at least two oocyte-secreted factors, that the secretion of the factors is not linked because one can be secreted without the other, and finally that the secretion of at least one (the HA retention factor) is finely connected to the stage of meiotic maturation. Alternatively, there may be only one factor that has differential activities based on different concentration thresholds. The observation that porcine oocytes beyond MI stage enable the production of HA but do not support its retention in the complex would suggest that these oocytes produce the CEEF but can no longer suppress upa production. However, upa activity has not been detected in porcine OCC before or after maturation. Porcine complexes may preferentially utilize tissue-type plasminogen activator (tpa) because both tpa and tpa-pa inhibitor complex activity increase during maturation in vitro [31]. Also, Huart et al. [32] have suggested that spontaneous increases in oocyte tpa activity correlated with germinal vesicle breakdown in rats and mice. Immunohistochemical staining revealed tpa antigen only in those oocytes that had undergone apparent meiotic maturation as confirmed by GVBD. Thus, oocytes contain tpa mrna and synthesize the active protease under a variety of stimuli that result in GVBD [33]. It seems likely that porcine oocytes in MI and MII, in contrast to oocytes in GV and LD, synthesize active proteases (e.g., tpa), which promote proteolytic degradation of the extracellular matrix of expanded cumulus cells. Consequently, expansion of mouse cultured with the maturing oocytes could not be observed because the matrix had disintegrated. Although there is no direct evidence for production of upa or tpa in the present study and the precise role of plasminogen activators in OCC has not yet been elucidated, their possible involvement in the final stages of oocyte maturation and ovulation [34 36], and cumulus expansion or dispersion [34, 37] has been suggested. One clear distinction between mouse and porcine oocytes regarding the production of expansion-promoting factors arises from this study. Mouse oocytes have been shown to support cumulus expansion and retention of HA in mouse in serum-containing medium [7, 8]. In contrast, pig oocytes supported cumulus expansion, but only early-stage oocytes supported retention of HA in mouse in serum-containing medium (this study). Therefore, pig, but not mouse, oocytes appear to lose or have decreased secretion of the factors involved in the retention of HA within the complex during meiotic maturation. Because porcine complexes in vivo do not lose the expanded cumulus cells until after ovulation, it is clear that there must be some other follicular factor (not present in serum) and/ or some porcine cumulus cell product (CEEF produced by porcine cumulus cells; [1], not present in mouse cumulus cells) that play important roles in retaining the HA in expanded porcine complexes until after ovulation. One possibility to explain the increased fragility of the expanded mouse cumuli cultured with mature porcine oocytes may be a decreased content of glycoproteins (laminin, type IV collagen, fibronectin), which have been identified in cumulus extracellular matrix [38]. Using the inhibitor of meiotic maturation, butyrolactone, the production of both paracrine factors by porcine oocytes after they were blocked in GV stage for 27 h could be demonstrated. It has been shown previously that porcine oocyte-cumulus complexes isolated from antral follicles possess a large GV with decondensed chromatin [39]. After h of culture, GVBD is observed, and after 24 h, the first metaphase spindle is formed. Inhibition of GVBD and chromosome condensation for 27 h with butyrolactone I, a specific inhibitor of cdk-kinases, followed by removal of the inhibitor, results in the resumption of oocyte maturation and completion of the first meiotic division at a rate com-

7 SECRETION OF PARACRINE FACTORS ENABLING EXPANSION OF CUMULUS CELLS 1155 parable with controls ([19]; our present results). These results indicate that simply lengthening the period of culture is not sufficient to alter the ability of the oocytes to produce the HA retention factor and that the production of this factor is tightly linked to the meiotic stage of the oocytes. Although all stages of porcine oocytes tested in this study were able to produce the factor that enables synthesis of HA, the results indicate that secretion of factors that cause the retention of HA within the complex is developmentally regulated during oocyte maturation. These data also suggest that porcine oocytes produce at least two factors that contribute to the production and stability of the preovulatory matrix and that secretion of these oocyte factors is differentially controlled. Although the identity of these factors is currently unknown, recent evidence would suggest that growth differentiation factor-9 might be a candidate for the CEEF []. Interestingly, GDF-9, like mouse oocytes, enabled cumulus expansion of mouse in a manner that suggested both the production and retention of HA, suggesting that in mice, one oocyte-secreted factor can serve both functions. As a result, the interspecies assay described here has revealed its ability to detect the differential secretion of two porcine oocyte factors that appear to control HA production and retention independently. This assay should prove valuable for investigating the function and identity of the oocyte-derived factors. ACKNOWLEDGMENTS We thank our colleagues at the Institute of Animal Physiology and Genetics, especially Prof. Jan Motlík for useful discussions. We also thank Dr. Milan Tománek (Research Institute of Animal Production, Prague, Czech Republic) for use of the liquid scintillation counter. 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