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Endocrinology Vol. 142, No. 7 3033-3040
Copyright © 2001 by The Endocrine Society

ARTICLES

Control of Mouse Cumulus Cell-Oocyte Complex Integrity before and after Ovulation: Plasminogen Activator Synthesis and Matrix Degradation1

Cristina D’Alessandris, Rita Canipari, Monica Di Giacomo, Olga Epifano2, Antonella Camaioni, Gregorio Siracusa and Antonietta Salustri

Department of Public Health and Cell Biology (C.D., M.D., A.C., G.S., A.S.), Histology Section, Faculty of Medicine, University of Rome Tor Vergata, 00173 Rome, Italy; and Department of Histology and Medical Embryology (R.C., O.E.), Faculty of Medicine, University of Rome La Sapienza, 00161 Rome, Italy

Address all correspondence and requests for reprints to: Antonietta Salustri, Ph.D., Department of Public Health and Cell Biology, Faculty of Medicine, University of Rome Tor Vergata, Via Orazio Raimondo, 00173 Rome, Italy. E-mail: salustri{at}med.uniroma2.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During the preovulatory period, cumulus cells (CCs) form a hyaluronan-protein extracellular matrix (cumulus expansion) that positively influences oocyte fertilization. Degradation of this matrix and CC-oocyte complex (COC) dissociation occurs within a few hours of ovulation and parallels the aging of oocytes. Modulation of CC proteolytic activity by gonadotropins and oocyte soluble factors has been hypothesized to determine such cumulus matrix changes. In the present study, we investigated plasminogen activator (PA) synthesis by COCs during the expansion and disassembly processes. Our results show that the secretion of tissue type PA and urokinase type PA (uPA) by oocytes and CCs, respectively, does not change significantly during expansion but dramatically increases thereafter. Compact COCs were isolated from immature mice, primed 48 h earlier with 5 IU PMSGs, and were induced to expand in vitro with 100 ng/ml FSH in the presence of 1% FCS. Full expansion was achieved at 16 h, when hyaluronan synthesis ceased. Release of hyaluronan and CCs from the COC matrix began between 18 and 20 h of culture, which indicates that matrix degradation started at this time. PA activities in culture media were determined by SDS-PAGE, followed by a zymography at various time intervals between 4 and 32 h of culture. Secreted tissue type PA and uPA activity abruptly increased between 16 and 20 h after FSH stimulation. Slot blot hybridization of CC messenger RNA showed that uPA messenger RNA levels correlated with the increase in uPA activity. Similar temporal patterns of PA synthesis and matrix degradation were found in COCs induced to expand in vivo by injection of 5 IU human CG into PMSG-primed mice. Cultures of CCs, both in the presence and absence of oocytes, revealed that uPA synthesis is repressed in FSH-stimulated CCs by an oocyte-soluble factor for the first 16 h of culture, whereas CC responsiveness to this factor is lost thereafter. In conclusion, the data show that a sophisticated interplay between oocyte and CCs causes the two cell types to simultaneously secrete PA activity after ovulation. The fact that matrix degradation parallels PA production strongly supports the hypothesis that these enzymes may destabilize the expanded COC matrix.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, the oocyte in the Graafian follicle is surrounded by tightly packed layers of follicle cells, referred to as cumulus cells (CCs). During the preovulatory period, CCs lose contact with each other and with the oocyte, but are held together by the deposition of a mucoelastic extracellular matrix. Hyaluronan is an essential component of this matrix, in which it forms a homogeneous intercellular mesh-like network (1). Proteins derived from the serum and synthesized by CCs are required to organize and maintain hyaluronan strands in such a highly structured gel (2, 3, 4, 5, 6, 7). The formation of this matrix promotes a significant increase in the CC-oocyte complex (COC) volume, which has led to this process being named cumulus expansion. In nearly all mammalian species, the oocyte reaches the oviductal ampulla still surrounded by the CC-matrix mass. The extracellular matrix seems to provide an essential vehicle for oocyte extrusion from the follicle and pickup by the oviductal fimbriae, because when its synthesis is prevented, a significantly lower number of oocytes are released from the follicles and transferred to the oviduct (8). The expanded cumulus also provides the proper environment for successful fertilization. A close correlation between the degree of cumulus expansion and oocyte fertilization rate has been found in in vitro matured mouse COCs (8). In addition, reduced cumulus expansion in PG E receptor EP2 subtype knockout mice severely impairs fertility (9). Gradual shedding of CCs occurs after ovulation and is correlated with a decline in oocyte fertilizability and capacity to develop in a normal embryo (10, 11). Like COCs expanded in vivo, COCs expanded in vitro by FSH in the presence of serum undergo progressive oocyte denudation, with CCs individually settling on the bottom of the dish (5). This result suggests that COCs closely control the mechanism leading to oocyte denudation, though a contribution in vivo of the oviductal environment cannot be excluded. During this process, most hyaluronan is released from the matrix to the culture medium without any apparent variation in molecular size, suggesting that the disassembly of the matrix does not depend on extensive cleavage of this polymer. Thus, it has been hypothesized that proteases produced by COCs may destabilize cumulus matrix by degrading proteins required for hyaluronan organization (5).

Plasminogen activators (PAs) are serine proteases that cleave plasminogen to form the active protease plasmin. These enzymes are involved in matrix remodeling in several tissues. In mouse ovary, an ovulatory dose of gonadotropins induces rapid and transient synthesis of urokinase PA (uPA) by mural granulosa cells (12). However, it has been observed that when cumulus matrix deposition begins, 4 h after hormone stimulation, CCs synthesize much less uPA than mural granulosa cells (13, 14). In vitro studies have shown that CCs, dissected from mouse COCs and cultured for 4 h with FSH, synthesize similar amounts of uPA as mural granulosa cells but secrete lower levels of enzyme in the presence of oocytes or oocyte-conditioned medium (13). These results show that the oocyte exerts a paracrine control on uPA production by CCs by attenuating cell response to gonadotropin stimulus. In addition, it has been shown that the oocyte accumulates tissue PA (tPA) messenger RNA (mRNA) during the growth phase and that translation of this mRNA is triggered upon resumption of meiotic maturation (15).

In the present study, we analyzed PA synthesis by COCs during the expansion and disassembly processes, to determine whether changes in production and secretion of these proteases are correlated with matrix degradation. Changes in the responsiveness of CCs to the inhibitory action of the oocyte were also investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Immature 22- to 24-day-old female Swiss CD-1 mice were ip-injected with 5 IU PMSG in 0.1 ml physiological saline and, 48 h later, were either killed or injected with 5 IU human CG (hCG) to induce ovulation. Animals were maintained in accordance with the NIH Guidelines for Care and Use of Laboratory Animals. Experimental protocols have been approved by the University Committee on Animal Care and Use.

Isolation and culture of intact COCs, CCs, and mural granulosa cells
For in vitro studies, cultures of COCs and CCs were prepared as described elsewhere (16). Briefly, ovaries were isolated from mice, 48 h after PMSG injection, and placed in MEM with Earle’s salt, buffered at pH 7.3 with 25 mM HEPES containing 1 mg/ml BSA. Compact COCs were released by puncturing large ovarian follicles. Groups of 20 COCs were transferred to 20-µl drops of basal culture medium (MEM with Earle’s salt supplemented with 0.25 mM pyruvate, 50 µg/ml gentamycin, 3 mM L-glutamine, and 1% FCS), supplemented with 100 ng/ml FSH (highly purified rat-FSH, kindly provided by the NIDDK and The National Hormone and Pituitary program, NIH, Bethesda, MD). The drops were covered with dimethylpolysiloxane (Sigma, St. Louis, MO) to prevent evaporation. In some experiments, groups of 20 COCs were transferred to a 20-µl drop of basal culture medium supplemented with 100 ng/ml FSH and mechanically dissociated into CCs and oocytes by repeated passage through a micropipette. Isolated CCs were either cultured alone, by removing the oocytes, or cocultured with their own released oocytes plus 20 additional oocytes obtained from other compact COCs. Such supplementary oocytes were added to CC cultures to maximally stimulate matrix synthesis by CCs (17). The cultures were incubated at 37 C with 5% CO2 in humidified air for the times specified in the text.

For in vivo studies, PMSG-primed mice were injected with hCG, and ovaries and oviducts were excised (respectively, from 0–9 h and from 13–28 h after hCG injection) and placed in MEM with Earle’s salt, buffered at pH 7.3 with 25 mM HEPES, containing 1 mg/ml BSA. COCs were released by puncturing the ovarian follicles or by cutting and gently squeezing the oviducts. They were then treated with testicular hyaluronidase (30 U/ml; Sigma) for 5 min at room temperature, and isolated CCs were collected by centrifugation. The pellet was washed, and the cells were suspended in basal culture medium at a concentration of 106 cells/ml. Aliquots of the cell suspension were cultured for 1 h at 37 C in a humidified atmosphere of 5% CO2 in air.

To obtain mural granulosa cell cultures, after follicle puncture, COCs were removed and granulosa cells were centrifuged at 300 x g for 10 min. After the wash, the cells were resuspended in basal culture medium at a final density of 106 cells/ml and cultured for 1 h.

Determination of cell number for COC
At different times after in vivo and in vitro hormone stimulation, between 20 and 50 COCs were transferred to a 50-µl drop of PBS containing 1 mg/ml BSA and 30 U/ml testicular hyaluronidase, and were incubated for 5 min at room temperature. Cell aggregates were dispersed by pipetting the cells several times under the microscope. Cell suspensions were then transferred to 1.5 ml tubes, and the number of cells in each suspension was determined by counting with a hemocytometer.

Quantitation of hyaluronan
The amount of hyaluronan synthesized in the cultures was determined by metabolic labeling as described elsewhere (5). Briefly, 20 COCs were cultured in a 20-µl drop of basal culture medium supplemented with 100 ng/ml FSH in the presence of 35S sulfate (60 µCi/ml) and 3H glucosamine (100 µCi/ml; NEN Life Science Products, Zaveten, Belgium) for the times indicated in the text. At the end of each culture, the incubation medium was aspirated. The medium and the cell-matrix fraction were then treated with 20 µl of a papain solution (750 mIU final activity) for 1 h at 65 C. The extraction was completed by adding 1 vol of 8-M guanidine HCl containing 4% (wt/vol) Triton X-100. Each extract was eluted on a column of Sephadex G-50 (2 ml bed vol) equilibrated with 0.1 M Tris, 0.1 M sodium acetate, and 0.5% Triton X-100, pH 7.3. Aliquots from the excluded volume for each extract were digested with chondroitinase ABC (0.1 U/ml) (Seikagaku Corp., Tokyo, Japan) for 2 h at 37 C. An aliquot of each digest was chromatographed on a column of Sephadex G-50 (4 ml bed vol) to determine the proportion of radiolabeled macromolecules digested by the enzyme. The remaining portion of each sample was analyzed for its relative proportion of hyaluronan and dermatan sulfate disaccharides, by HPLC, on Partisil 5 PAC (0.4 x 25 cm; Whatman, Fairfield, NJ). The mass of hyaluronan was determined by calculating the specific activity of the UDP-N-acetylhexosamine pools from the ratio of 3H to 35S in monosulfated chondroitin 4-S disaccharide derived from the dermatan sulfate proteoglycans (18).

Gel electrophoresis and zymography
For zymography of PA, culture media were separated by electrophoresis in 8% polyacrylamide slab gels in the presence of SDS (SDS-PAGE) under nonreducing conditions (19). The PA was then visualized by placing the Triton X-100-washed gel on a casein-agar-plasminogen underlay as previously described (20). Molecular weights were calculated from the position of prestained molecular weight markers, subjected to electrophoresis in parallel lanes. Casein underlays were photographed with dark-field illumination. Densitometric scanning of zymographies was performed to obtain semiquantitative estimation of protease activities.

RNA preparation and analysis
Total RNA was prepared according to the method of Chomczynski and Sacchi (21). In vitro- or in vivo-expanded COCs were treated with testicular hyaluronidase to dissolve hyaluronan, and oocytes were removed. Northern and/or slot-blot hybridization were performed as previously described (22). 32P-radiolabeled uPA (pDB4501; kindly provided by Dr. Dominique Belin, University of Geneva, Switzerland) (23) antisense probe was generated by transcription with SP6 polymerase according to the protocol enclosed in the kit (Promega Corp., Madison, WI). Filters were prehybridized, hybridized, and washed as previously described (24). After autoradiography, the filters were probed with a random primed complementary DNA for mouse 18S RNA to adjust for any variability in the amount of RNA present in the filters.

Statistical analysis
Statistical analysis was performed by one-way ANOVA followed by a Tukey-Kramer test for comparison of multiple groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Temporal pattern of disassembly of in vitro- and in vivo-expanded COCs
Experiments were performed to determine the time course of COC disassembly after in vitro and in vivo cumulus expansion. For in vitro studies, COCs were isolated from ovaries of mice primed 48 h earlier with PMSG and stimulated to expand in vitro by treatment with 100 ng/ml FSH in the presence of 1% FCS. Radioactive metabolic precursors were added to a set of COC cultures to label the newly synthesized hyaluronan. Previous studies have shown that hyaluronan synthesis ceases at 16 h, when maximum COC expansion is achieved (18). After this time, hyaluronan accumulated in the matrix is progressively released into the culture medium, and CCs dissociate from the oocyte (5). Therefore, to determine the time at which cell-matrix disassembly begins, matrix-hyaluronan content and cell number per COC were evaluated at different times after the first 16 h of culture. As shown in Fig. 1AGo, hyaluronan content did not significantly change between 16 and 18 h of culture, whereas a decrease of approximately 30% was observed at 20 h. Progressively lower hyaluronan amounts were found in COCs at later times, with a loss of approximately 70% of the hyaluronan originally present in the matrix occurring at 28 h. Similarly, the number of cells/COC remained constant up to 18 h. A loss of approximately 40% of the original cell number occurred after 20 h of culture, and almost complete oocyte denudation was achieved at 28 h (Fig. 1BGo).



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  		Figure 1. Changes in matrix-hyaluronan content and cell number in COCs after in vivo and in vitro expansion. A, Compact COCs were stimulated to expand in vitro, with 100 ng/ml FSH and 1% FCS in the presence of glycosaminoglycan radiolabeled precursors. Net amount of hyaluronan (HA) retained in the matrix was determined at 16 h (full expansion) and at later times. B, COCs were enzymatically dissociated at different times from in vitro FSH stimulation and in vivo hCG injection, and the number of cells per COC was determined. The values represent the mean ± SEM of four independent experiments. SEM values have not been indicated when smaller than the symbols.

 
To study the time course of in vivo COC matrix disassembly, PMSG-primed mice were injected with hCG, and COCs were collected from the oviducts at different intervals, starting 13 h after hCG injection, the time at which ovulation occurs and full expansion is achieved. Considering the striking correlation found between the decrease in hyaluronan-matrix content and the decrease in cell number/COC during in vitro COC disassembly, only the latter method was used for in vivo COC disassembly studies. As shown in Fig. 1BGo, the number of CCs/COC did not significantly change during the first 3 h after ovulation but decreased by 30% at 18 h. Almost complete oocyte denudation occurred 28 h after hCG injection.

These results indicate that destabilization of the COC matrix begins between 18–20 h and 16–18 h after hormone stimulus, in vitro and in vivo, respectively. Although the number of cells in in vivo-expanded COCs was three time as high as that in in vitro-expanded COCs (Fig. 1BGo; see also 25), denudation of the oocytes required approximately the same time.

PA synthesis by CCs in vitro and in vivo
The rate of PA synthesis was investigated during in vitro expansion and disassembly of COCs. Because the number of cells per COC does not change during in vitro expansion (25), CCs were maintained in physiological association with the oocyte and cultured as intact COC. CC-oocyte complexes were stimulated in vitro with FSH in the presence of FCS. Cell cultures were stopped at different times, and PA activity accumulated in the media was analyzed by zymography (Fig. 2AGo). Densitometric analysis of tPA and uPA bands is shown in Fig. 2Go, B and C.



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  		Figure 2. PA activity in COC cultures. COCs were cultured in the presence of 100 ng/ml FSH and 1% FCS. Conditioned medium was collected at the indicated times. Aliquots (15 µl) of conditioned media were analyzed by SDS-PAGE, followed by zymography. A, Representative zymography; B, tPA; C, uPA activity quantified by densitometer scanning of zymographies. Values represent mean ± SEM of five independent experiments. The inset shows the rate of uPA accumulation per hour during the indicated time periods, indirectly obtained from the densitometric values.

 
In accordance with data on polyadenylation and translation of tPA mRNA by oocytes that have resumed meiosis (26), tPA production was found in COC cultures. tPA greatly increased between 16 and 20 h of culture, reaching a plateau at 24 h (Fig. 2BGo). Likewise, uPA enzymatic activity present in the medium increased slightly between 12 and 16 h of culture and rose abruptly between 16 and 20 h, reaching a plateau between 28 and 32 h (Fig. 2CGo). The rate of uPA synthesis per hour was indirectly calculated from the values reported in Fig. 2CGo and shown in the inset. The results show that uPA rate drastically increased between 16 and 20 and remained at maximal values between 20 and 24 h. It declined thereafter, returning to approximately basal levels between 28 and 32 h. We also examined uPA mRNA levels in CCs isolated at different times of culture. Total mRNA from each sample was subjected to slot blot analysis and hybridized with the uPA probe and subsequently with 18S ribosomal RNA (rRNA) probe (Fig. 3Go, A and B). The results showed a transient increase in uPA mRNA levels, which was temporally correlated with the increase in uPA secreted by CCs.



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  		Figure 3. Total RNA was extracted by CCs cultured for the indicated times in the conditions described in Fig. 2Go. RNA (10 µg) was subjected to slot blot analysis and hybridized with uPA and 18s rRNA probes. A, Autoradiography of a representative slot blot; B, averaged data from densitometry of slot blots from three independent experiments (mean ± SEM). Optical density values of uPA mRNA in each lane were normalized by the respective optical density values of 18s rRNA signals.

 
The rate of uPA synthesis was investigated during in vivo expansion and disassembly of COCs. Because the number of cells per COC varies during in vivo expansion (25), COCs isolated at different times from hCG were dissociated by hyaluronidase treatment, and CCs were cultured at 106-cells/ml density for 1 h. For comparison, mural granulosa cells obtained from the same follicles were cultured at the same density and for the same time as CCs. At the end of incubation, the culture medium from each sample was collected, and PA activity was assayed by SDS-PAGE, followed by visualization of the enzyme by zymography (Fig. 4Go, A and B). Densitometric analysis of uPA bands was then performed (Fig. 4CGo). In agreement with previous observations (12), uPA activity produced by mural granulosa cells rapidly and transiently increased during the preovulatory period, reaching maximal levels 4 h after hCG stimulus (Fig. 4Go, A and C). uPA production by CCs showed a different pattern, slowly increasing during the preovulatory period, to reach the maximal level after ovulation, approximately 17 h after hCG injection. Enzymatic activity thereafter decreased toward basal levels (Fig. 4Go, B and C). We also examined uPA mRNA levels in CCs isolated before and after ovulation. A transient increase in the uPA mRNA level occurred after ovulation; this increase correlated well with that in the uPA activity of CCs (Fig. 5Go, A and B).



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  		Figure 4. PA activity secreted by mural granulosa and CCs after in vivo hCG stimulation. Mural granulosa cells and CCs were isolated from ovaries or oviducts at different times after hCG injection and cultured at a density of 103 cells/µl for 1 h. Aliquots (15 µl) of conditioned media were analyzed by SDS-PAGE, followed by zymography. A, Mural granulosa cells (MGCs); B, CCs (representative zymographies); C, PA activity was quantified by densitometer scanning of zymographies from two independent experiments (mean ± SEM).

 


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  		Figure 5. Levels of uPA mRNA in CCs at different times after hCG injection. Total RNA was extracted by CCs at the times indicated and subjected to Northern blot analysis. The filters were hybridized as described in Fig. 3Go. A, Representative autoradiography of Northern blot; B, averaged data from densitometry of Northern blots from three independent experiments (mean ± SEM). The values were normalized as described in Fig. 3Go.

 
Influence of oocytes on uPA synthesized by CCs
We previously reported that a soluble factor produced by mouse oocytes inhibits in vitro uPA synthesis by CCs during the first 4 h of FSH treatment (13). The results reported in Fig. 6Go show that the level of uPA secreted during the first 16 h of FSH-stimulation by CCs cultured in the presence of oocytes (either as intact COC or CC-oocyte coculture) was significantly lower than that secreted by CCs cultured in the absence of oocytes, which suggests that the oocyte inhibitory action was exerted up to this time. However, at 32 h of culture, no significant difference was found between uPA secreted by CCs cultured either in the presence or absence of oocytes. This observation suggests that a significant increase in uPA synthesis by CCs occurred after 16 h of culture in the presence of oocytes. It could be explained by an insufficient amount of inhibitory factor being produced by long-time-cultured oocytes or, alternatively, by a loss of CC responsiveness to the oocyte soluble factor. To discriminate between these two hypotheses, freshly isolated fully-grown oocytes at the germinal vesicle (GV) stage were added to CC-oocyte cocultures at 16 h of culture. The results show that the late increase in uPA synthesis could not be prevented by freshly isolated oocytes, which suggests that, at later times, CCs no longer respond to the oocyte inhibitory factor (Fig. 6Go).



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  		Figure 6. Influence of oocytes on uPA activity secreted by CCs. CCs were cultured in the presence of FSH (100 ng/ml) for the indicated times, alone (solid bars), or in the presence of oocytes, either as intact COCs (empty bars) or as cocultures (hatched bars), as specified in Materials and Methods. In a set of experiments, 40 supplementary oocytes, freshly isolated from compact COCs, were added to cocultures at 16 h of culture (cross-hatched bars). Aliquots (15 µl) of media were analyzed by zymography. PA activity was quantified by densitometer scanning of zymographies from four independent experiments (mean ± SEM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Ovulation requires tight control of follicle extracellular matrix modification in the follicle. Hyaluronan synthesis by COCs begins 3–5 h after the peak of circulating gonadotropins and ceases between 12 and 14 h, when ovulation occurs (25, 27). This glycosaminoglycan is organized in a highly structured extracellular matrix through interactions with specific proteins (3, 4, 5, 6, 7). Several studies have provided evidence indicating that the preovulatory gonadotropin surge induces mural granulosa cells to increase the synthesis of proteases, such as PAs and matrix metalloproteases, which may act in conjunction to promote matrix degradation involved in follicle remodeling and wall rupture at ovulation (28). The results reported in the present paper show that CCs, unlike mural granulosa cells, do not significantly increase uPA mRNA expression and uPA synthesis during the preovulatory period, when COC extracellular matrix deposition occurs. This seems to be attributable mainly to the paracrine action of the oocyte on the surrounding cells. We have previously shown that a soluble oocyte factor(s) prevents FSH-induced uPA synthesis by CCs for up to 4 h of culture (13), which is when hyaluronan synthesis begins (18). We now show that the oocyte inhibitory action on uPA production is exerted for up to 16 h of culture, when maximum hyaluronan accumulation in the matrix and full expansion are achieved. uPA synthesis by CCs abruptly rises thereafter and is soon followed by matrix degradation. Similarly, in vivo, uPA synthesis increases in COCs after ovulation, just before cumulus dispersion. Interestingly, it has been shown that a mouse oocyte-soluble factor stimulates CCs to synthesize hyaluronan in response to FSH (16, 29) and that its action is exerted for up to 16 h of culture (30), a timing that parallels that described in this study for inhibition of uPA production. All these findings support a model in which the oocyte exerts a double control on cumulus matrix formation during the preovulatory period, contributing both to promoting its synthesis and to preventing its degradation.

Regarding the identity of this paracrine activity, recent findings attribute these effects to one of the three members of the TGF-ß superfamily secreted by the mouse oocyte, GDF-9 (31). In fact, GDF-9, but not BMP-15 and BMP-6, is able to inhibit in vitro uPA-mRNA expression and to stimulate hyaluronan synthase-2 transcription by mural granulosa cell cultures, and FSH-stimulated oocytectomyzed COCs are able to expand in the presence of the recombinant GDF-9 protein.

Desensitization of CCs to GDF-9, rather than a decreased ability of oocytes to produce this factor, seems to underlie hyaluronan down-regulation and uPA up-regulation synthesis by CCs after ovulation. In fact, ovulated mouse oocytes, at the metaphase II stage, express GDF-9 mRNA (32) and are still able to stimulate hyaluronan and inhibit uPA synthesis in vitro by CCs isolated from PMSG-primed mice, albeit at a lower rate than fully-grown oocytes at the GV stage (13, 17, 33). Furthermore, we show that the addition of freshly isolated GV oocytes to CC-oocyte cocultures after the first 16 h of culture cannot prevent the rise in uPA activity by CCs that occurs after this time. It is noteworthy that an increase in uPA mRNA and a decrease in hyaluronan synthase-2 mRNA have also been detected in GDF-9-treated mural granulosa cells after 24 h of culture, even though recombinant factor was present throughout this time (31). All these findings suggest that inactivation of GDF-9 receptor or of the intracellular signal pathway takes place as a result of CC differentiation.

The removal of GDF-9 inhibition might not be the sole cause of the uPA rise in expanded COCs. In fact, it should be noted that significantly less uPA activity was found in CC-oocyte than in CC cultures at 16 h, and no difference was found between the two culture conditions at 32 h. Thus, between 16 and 32 h of culture, uPA is produced at a higher rate by CCs cultured in the presence of oocytes. This suggests that the oocyte, though repressing uPA synthesis stimulated by FSH during cumulus expansion, may promote CC differentiation, which, in turn, may lead to up-regulation of uPA synthesis at later times. An analogous situation seems to occur for progesterone synthesis. Mouse oocytes inhibit progesterone synthesis by CCs during the preovulatory period; however, after ovulation, CCs produce significant amounts of this steroid, even though oocytes continue to secrete progesterone-inhibitory factor (34). Elvin and colleagues (35) have recently shown that progesterone synthesis induction is actually a late GDF-9 effect mediated by PGs. Experiments are in progress to determine whether the increase in uPA synthesis after ovulation is caused by metabolic changes set in motion by the oocyte during the preovulatory period.

In addition to uPA secretion, we have observed a significant increase in tPA production by COCs, starting after 16 h of culture. It is well known that mouse oocytes accumulate tPA mRNA during the growth phase and that the translation of this dormant mRNA is triggered upon meiotic resumption (15). It has also been hypothesized that the bulk of the enzyme is released in the medium after fertilization (15, 36). However, we observed a rapid increase in this enzyme in the culture medium that was independent of fertilization and was time-correlated with uPA production. Therefore, it seems possible that, after the COC expansion process is completed, the oocyte promotes matrix degradation by secreting tPA and by allowing and/or stimulating CCs to synthesize uPA. A possible role for oocytes in matrix destabilization has been suggested also for porcine oocytes. In fact, it has been shown that pig oocytes that have resumed meiosis secrete a factor that does not allow hyaluronan organization in the mouse cumulus matrix (37).

Besides the control of production of proteolytic enzymes, COC matrix protection before ovulation is also achieved through a direct inhibition of the residual proteolytic activity. Although CCs do not synthesize specific PA inhibitors (PAI-1 and PAI-2), they do express high levels of nexin-1 mRNA, a broad spectrum protease inhibitor, constitutively (14). Other molecules may also be involved in protecting the COC matrix from premature degradation. During COC expansion, CCs up-regulate the expression of tumor necrosis factor-stimulated gene-6 (TSG-6) (7, 38). This protein is able to complex with hyaluronan and inter-{alpha}-trypsin inhibitor (I{alpha}I), a serum component with serine protease inhibitor activity (39, 40). It is noteworthy that circulating proteins belonging to the I{alpha}I family rapidly enter the follicle after an ovulatory gonadotropin stimulus and colocalize with hyaluronan (3). These molecules seem to be implicated in organizing hyaluronan strands and in stabilizing the cumulus matrix (3, 4, 41). Moreover, the interaction between I{alpha}I and TSG-6 enhances I{alpha}I antiplasmin activity (42). It seems, therefore, that TSG-6/I{alpha}I complexes may specifically localize highly active serin protease inhibitors along hyaluronan strands and protect proteins involved in matrix organization from degradation. After ovulation, COCs increase uPA and tPA secretion, shifting the ratio between proteolytic enzyme and protease inhibitors. This change would lead to a net increase in PA activity and to matrix/CC dispersion and oocyte denudation.

In summary, the results reported in the present paper show that mouse COCs synthesize low levels of PAs throughout the COC expansion process, when matrix deposition occurs, but rapidly increase PA synthesis thereafter, when matrix disassembly and cumulus dispersion begin. We also provide evidence indicating that modulation of uPA activity by CCs mainly depends on changes in CC-oocyte interaction. It is also noteworthy that there is a correlation between the beginning of matrix degradation, 16–20 h after hCG injection, and the time when mouse oocytes undergo changes that make them more likely to produce abnormal embryos when fertilized (43, 44). Thus, it is possible that this sophisticated interplay between CCs and oocytes, which controls the timing of both the stability and the degradation of the matrix, is designed to promote fertilization of freshly-ovulated oocytes and to prevent that of aged oocytes.


   Footnotes
 
1 This work was supported by grants from Ministero per l’Università e la Ricerca Scientifica e Tecnologica (National Project Development and Differentiation of Germ Cells to A.S., and intramural 60% project to R.C.) and from Consiglio Nazionale delle Ricerche (Grant 98.00512.CT04 to A.S.). Back

2 Present address: Olga Epifano, Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 6 Center Drive, Bethesda, Maryland 20892. Back

Received January 8, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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