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a Academy of Sciences of the Czech Republic, Institute of Animal Physiology and Genetics, Lib
chov, 277 21 Czech Republic
b Ottawa Regional Cancer Centre, Cancer Research Group, Ottawa, Ontario, Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cumulus cells, developmental biology, follicle, FSH, granulosa cells, growth factors, meiosis, oocyte development, ovary, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast to the mouse model, we have shown [9, 10] 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 OOX 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 OOX; however, the cumuli were disintegrated and not mucified [15].
The activity of mouse CEEF was found to be heat labile (65°C for 15 sec), lost by proteinase K digestion, and retained by 100-kDa, but not by 300-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 OOX 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 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 50 µM 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 p34cdc2/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 (OOX) 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Porcine ovaries were obtained from crossbred gilts (Landrace, Large White) at a local abattoir and transferred to the laboratory in a thermos at 30°C. 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 (GV-germinal vesicle) oocytes and oocytes at late diakinesis, metaphase I, and metaphase II prepared by culture of complexes for 0, 22, 27, or 42 h, respectively. The culture medium was M-199 (Sevac, Prague, Czech Republic), buffered with 20 mM NaHCO3 and 6.25 mM Hepes and supplemented with 5% fetal calf serum (FCS), 0.91 mM sodium pyruvate, 1.62 mM calcium lactate, and antibiotics [20]. This culture medium was supplemented with 0.1 µg/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 (50 µM, Sigma-Aldrich, Prague, Czech Republic).
Isolation of Mouse Oocyte-Cumulus Complexes and Oocytectomy
Mouse OCC were isolated from ovaries of 4- to 6-wk-old ICR mice, stimulated 44–46 h previously with 10 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 OOX, 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 OOX, which require CEEF for expansion. Conditioned media were prepared by the culture of 50 denuded porcine oocytes in 50 µl of the culture medium for 24 h. Subsequently, 10 to 15 mouse OOX and 0.1 µg/ml FSH were added to the drop of conditioned medium, and expansion was assessed 18 h later, according to a subjective scoring system (0 to +4) described previously [8]. Briefly, 0 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 50 denuded porcine oocytes in 50 µl of culture medium for 24 h. At the end of the incubation period, the conditioned media were collected and stored frozen at -20°C, 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 OOX 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 OOX were cultured for 18 h in 100 µl media conditioned by porcine oocytes (at different stages or after treatment with butyrolactone I) at 37°C in an atmosphere of 5% CO2 in air and in the presence of 2.5 µCi of D-[6-3H]glucosamine hydrochloride (Amersham Pharmacia Biotech, Buckinghamshire, UK) and 1 µg/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 10 µl of a solution containing 50 mg/ml pronase (Sigma-Aldrich) and 10% Triton X-100 in 0.2 M Tris buffer, pH 7.8. The samples were incubated for 2 h at 37°C and then transferred to Whatman 3MM filter paper circles. The circles were air dried and then washed through three changes of solution containing 0.5% cetylpyridinium chloride and 10 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 OOX 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-100 solution. The specificity of incorporation 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 10 IU of Streptomyces hyaluronidase for 2 h at 37°C before addition of the pronase-Triton X-100 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 OOX 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 OOX and FSH, both media were able to support the expansion of the mouse OOX 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 OOX and FSH.
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Production of CEEF by Porcine Oocytes During Their Maturation
Intact mouse OCC underwent +3 to +4 expansion when cultured in medium supplemented with FSH and FCS; however, expansion did not occur in OOX cultured under the same conditions (Table 2). The ability to expand was restored when OOX 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 OOX. 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).
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Total HA Synthesis by Mouse OCC and OOX after Stimulation by FSH
Mouse OCC or OOX 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 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 OOX.
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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 [3H]-glucosamine incorporation into OCC stimulated by FSH, 90% was sensitive to Streptomyces hyaluronidase (Fig. 2), indicating that HA is the predominant glycosaminoglycan product of the [3H]-glucosamine.
FSH-Stimulated HA Synthesis by Mouse OOX 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 OOX after FSH stimulation was tested. Mouse OOX cultured in the presence of porcine oocyte-conditioned media (GV, LD, MI, or MII) were capable of synthesizing HA (measured in medium + OOX) to levels equivalent to those of OCC (Fig. 3), but four- to sixfold greater than those of OOX cultured alone (Fig. 2). The total accumulation of HA by mouse OOX complexes cultured in media conditioned by oocytes at different stages of development was not different from that of intact OCC after FSH stimulation (P > 0.05). The amount of HA retained within the complexes was found to be similar between OCC and OOX cultured in the presence of media conditioned by GV or LD stage oocytes (P > 0.05). However, a significant difference (P < 0.01) was observed in the amount of HA retained in intact OCC vs. OOX 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.
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FSH-Stimulated Expansion and HA Synthesis by Mouse OOX 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 oocytes were used to condition media for 22 h, and then expansion and HA synthesis by mouse OOX cultured in this conditioned media was assessed. Mouse OOX cultured in conditioned media from butyrolactone I-treated oocytes underwent both full cumulus expansion (+3 to +4) 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 > 0.05). Total HA synthesis was also found to be not significantly different between these two groups (P > 0.05; Fig. 4).
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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., 0, 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 assessed at the end of culture (100% 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 during 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 OOX showed that oocytes in GV and LD stages as well as after butyrolactone I treatment enabled mouse OOX 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 [30]. 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 [30].
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 [30]. 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 [30]. 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 OOX 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 OOX in serum-containing medium [7, 8]. In contrast, pig oocytes supported cumulus expansion, but only early-stage oocytes supported retention of HA in mouse OOX 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; [10], 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 16–17 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 comparable 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 [40]. Interestingly, GDF-9, like mouse oocytes, enabled cumulus expansion of mouse OOX 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 524/98/0231 from the Grant Agency of the Czech Republic. 
2 Correspondence: FAX: 420 206 697 186; nagyova{at}iapg.cas.cz
Accepted: May 29, 2000.
Received: February 7, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ová Z, Nagai T, Kikuchi K, Motlík J. Lack of effect of oocytectomy on expansion of the porcine cumulus. J Reprod Fertil 1991; 93:569–576[Abstract]-trypsin inhibitor family. J Biol Chem 1992; 267:12380–123862-antiplasmin. Endocrinology 1989; 124:415–421[Abstract]
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P, Flechon JE, Pavlok A. Microfilaments, microtubules and intermediate filaments fulfill differential roles in gonadotropin induced expansion of bovine cumulus oophorus. Reprod Nutr Dev 1994; 34:415–425This article has been cited by other articles:
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 R. B. Gilchrist, M. Lane, and J. G. Thompson Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality Hum. Reprod. Update, March 1, 2008; 14(2): 159 - 177. [Abstract] [Full Text] [PDF]
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L. Nemcova, E. Nagyova, M. Petlach, M. Tomanek, and R. Prochazka Molecular Mechanisms of Insulin-Like Growth Factor 1 Promoted Synthesis and Retention of Hyaluronic Acid in Porcine Oocyte-Cumulus Complexes Biol Reprod, June 1, 2007; 76(6): 1016 - 1024. [Abstract] [Full Text] [PDF]
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M. Stojkovic, O. Krebs, S. Kolle, K. Prelle, V. Assmann, V. Zakhartchenko, F. Sinowatz, and E. Wolf Developmental Regulation of Hyaluronan-Binding Protein (RHAMM/IHABP) Expression in Early Bovine Embryos Biol Reprod, January 1, 2003; 68(1): 60 - 66. [Abstract] [Full Text] [PDF]
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C. D'Alessandris, R. Canipari, M. Di Giacomo, O. Epifano, A. Camaioni, G. Siracusa, and A. Salustri Control of Mouse Cumulus Cell-Oocyte Complex Integrity before and after Ovulation: Plasminogen Activator Synthesis and Matrix Degradation Endocrinology, July 1, 2001; 142(7): 3033 - 3040. [Abstract] [Full Text] [PDF]
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