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a Dipartimento di Istologia ed Embriologia Medica, University of Rome, "La Sapienza", 00161 Rome, Italy
b Departement de Pathologie, University of Geneva Medical School, Geneva, Switzerland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence show that induction of PA synthesis accompanies and participates in gonadotropin-induced ovulation [10, 11]. The type of PA secreted by granulosa cells is species-specific. While rat and pig granulosa cells secrete tPA in response to gonadotropins [12, 13], the same hormonal stimulation induces uPA secretion in mouse granulosa cells [14]. Since the two enzymes share the same substrate, plasminogen, this heterogeneity could reflect a redundancy in the PA/plasmin proteolytic cascade at the time of ovulation.
In the rat, both types of PAs are synthesized in the ovary, and gonadotropins stimulate in vivo and in vitro production of these enzymes by granulosa and theca cells [12, 15–17]. The increase in PA activity is time correlated with ovulation, and the enzymes are induced only in follicles nearing ovulation [7, 18]. Finally, gonadotropin-induced ovulation in rats is reduced by intrabursal injection of serine protease inhibitors [16, 19].
Although both forms of PA have been demonstrated in the rat ovary, several studies identified tPA as the follicular PA induced by the preovulatory surge of gonadotropins. Tissue-type PA was therefore thought to be the major form of PA contributing to ovulation in the rat [17, 19–21]. However, we have previously observed an increased production of uPA by whole rat follicles in response to hCG injection [15]. In contrast, Li et al. [22] recently observed a marked decrease of uPA transcripts and protein at the expected time of ovulation. These discrepant results prompted us to investigate in more detail the hormonal regulation of uPA synthesis in rat preovulatory follicles.
We document here that, with regard to uPA regulation, the same hormonal stimulation has opposite effects in the two follicular compartments. Gonadotropins up-regulated uPA mRNA level in theca-interstitial (TI) cells, whereas in granulosa cells they down-regulated the same uPA transcript. Despite the decrease of uPA mRNA in the granulosa cells, an increase in cell-associated uPA activity was observed in the same cells. We show evidence that this apparent paradox is due to an interesting redistribution of the uPA molecule from the secreted to the cell-associated fraction. These observations can have implications in differentiating the tPA and uPA functions in the preovulatory follicles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature female Wistar rats were obtained from Charles River, Como, Italy. In the morning of their 25th day, they were injected i.p. with 10 IU of eCG (eCG-primed animals). Forty-eight hours later the animals were either killed or injected i.p. with 10 IU of hCG. Animals were maintained in accordance with the NIH Guide to the Care and Use of Laboratory Animals.
Materials
Ovine FSH (oFSH-19-SIAFP) was supplied by the NIDDK rat Pituitary Hormone Distribution Program (NIH, Bethesda, MD). Equine CG and hCG were purchased from Intervet (Livorno, Italy), minimum essential Eagle's medium (MEM) was obtained from Gibco (Grand Island, NJ). The chromogenic plasmin substrate S-2251 (D-val-L-leu-L-lys, p-nitroanilide·2HCl) was purchased from Bachem Feinchemilkalien AG (Basel, Switzerland). All other reagents were of analytical grade and obtained from BDH (London, UK) or from Sigma Chemical Co. (St. Louis, MO). Plasmid pDB4501, containing the 658-base pair (bp) PstI-HindIII fragment of the mouse uPA cDNA clone, and plasmid pDB4701, containing the 726-bp PvuII–SpeI fragment of the mouse tPA cDNA clone, have been described previously [23].
Cell Preparation
For studies involving in vivo stimulation, ovaries were collected at various times after hCG stimulation. Granulosa cells were obtained by puncturing preovulatory follicles with a needle in Hepes-buffered medium M2 [24] supplemented with 0.1% BSA. Viable cells were either plated at a density of 1.5 x 105 cells in 100 µl of medium supplemented with 0.1% BSA and cultured for 2 h at 37°C in a 5% CO2 atmosphere or were solubilized in Triton X-100 in 0.1 M Tris-HCl, pH 8.1, at a density of 105 cells/50 µl. Cell viability was assessed by trypan blue exclusion. At the end of the culture, conditioned medium was collected, and the cells were solubilized in 0.5% Triton X-100 at a density of 105 cells/50 µl. TI cells were obtained from preovulatory follicles dissected from ovaries as previously described [15]. In brief, after dissection the follicles were hemisected using a scalpel blade; they were then gently scraped to eliminate adhering granulosa cells, transferred to fresh medium, and washed vigorously by pipetting up and down through a large tip of a Pipetman P1000 (Gibson, Middleton, WI). The tissue was again placed in fresh medium and washed as before; this procedure removes most but not all of the granulosa cells. The final tissue, mainly TI cells, the equivalent of 3 follicles, was either cultured for 2 h in 100 µl of medium as described for granulosa cells or solubilized immediately after collection in 75 µl of 0.5% Triton X-100. At the end of the culture period, conditioned medium was collected and the tissue was solubilized in 75 µl of 0.5% Triton X-100.
For in vitro studies, animals were killed 48 h after eCG injection and granulosa cells obtained as described above. Granulosa cells were either plated at a density of 1.5 x 105 cells in 100 µl of medium and cultured for 6 h or immediately solubilized in Triton X-100 at a density of 105 cells/50 µl.
Gel Electrophoresis and Zymography
For zymography of PA, culture fluids or cell homogenates were separated by electrophoresis in 8% polyacrylamide slab gels in the presence of SDS (SDS-PAGE) under nonreducing conditions according to the procedure of Laemmli [25]. PA was then visualized by placing the Triton X-100-washed gel on a casein-agar-plasminogen underlay as previously described [26]. Molecular weights were calculated from the position of prestained markers that were subjected to electrophoresis in parallel lines. The lytic zones were plasminogen dependent. For granulosa cells a volume corresponding to 3 x 104 cells, and for TI cells a volume corresponding to 5 µg of protein, were loaded into the gel. Protein content was measured by the method of Lowry et al. [27] with BSA used as standard. Densitometric scanning of zymographies was performed to derive semiquantitative estimation of protease activities.
Phase Separation in Triton X-114 Solutions
Triton X-114 was precondensed as described by Bordier [28] and stored as a 13% (w:v) stock solution. Granulosa cells obtained from eCG-primed rats 10 h after hCG injection were solubilized in 3% Triton X-114. Phase separation was achieved after 3 min at 37°C, and the lysates were centrifuged for 15 min at 12 000 x g. Aliquots of the total cell lysate before phase separation (Total) of the upper (Aqueous) and the lower (Detergent) phases were analyzed by SDS-PAGE and zymography.
Acid Treatment of Granulosa Cells
Granulosa cells obtained from eCG-primed rats 10 h after hCG or saline injection, as a control, were treated for 3 min at room temperature with 50 mm glycine-HCl buffer, pH 3.0, containing 0.1 M NaCl. Then cells were quickly neutralized with 0.5 M Hepes, pH 7.5, and washed three times with MEM supplemented with 0.1% BSA. Viable acid-treated cells were plated at a density of 1.5 x 105 cells in 200 µl of medium and incubated at 37°C for 2 h. At the end of the culture period, conditioned media were collected and cells lysed with 0.5% Triton X-100 at a density of 105 cells/50 µl.
Binding of uPA In Vitro
Granulosa cells obtained from eCG-primed rats were plated as described above and cultured for 10 h at 37°C in the presence or absence of 100 ng/ml FSH. At the end of the culture, the medium was collected, and cells were washed twice with fresh medium. The cells were further incubated for 1 h at 4°C in the presence or absence of exogenous rat uPA. At the end of incubation the cells were extensively washed and lysed in Triton X-100 at a density of 105 cells/50 µl. Medium conditioned by rat granulosa cells cultured for 10 h under basal conditions was used as a source of exogenous uPA.
Isolation and Analysis of Total Cellular RNA
Total RNA prepared from granulosa and TI cells [29] was analyzed for relative abundance of specific mRNAs by Northern and/or slot-blot hybridization. For Northern blot analysis, RNAs (40 µg/lane) were denatured with formaldehyde, electrophoresed on a 1.2% agarose gel containing 6% formaldehyde, and transferred to Hybond Nylon membrane (Amersham) by capillary blotting with 20-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) for 24 h [30]. For slot-blot analysis, RNAs (10 µg/slot) were resuspended in 100 µl of a solution containing 10% formaldehyde and 50% formamide. The mixture was incubated 30 min at 60°C, placed into ice, and then applied with suction to a nylon membrane by means of an IBI (Eastman Kodak, Rochester, NY) slot-blotter apparatus. Each blot was washed twice with 20-strength SSC. Each specific mRNA was quantified by densitometry of the films after autoradiography and was normalized to the amount of rRNA.
Probe Synthesis and Northern Blots Hybridization Conditions
32P-Radiolabeled tPA (pDB4701) and uPA (pDB4501) antisense probes were generated by transcription with SP6 polymerase according to the protocol for the Promega (Madison, WI) kit. Filters were prehybridized, hybridized, and washed as previously described [31]. After autoradiography the filters were probed with a random-primed cDNA for the mouse 18s rRNA [32] to account for any variability in the amount of RNA present in the filters.
Intrabursal Injection of Antibodies
Immature female rats treated 48 h earlier with eCG were anesthetized with sodium pentobarbital (35 mg/kg). One of the ovaries was exteriorized via a small incision. Rabbit anti-human tPA or anti-mouse uPA [33] IgG or preimmune IgG (150 µg) dissolved in 50 µl of PBS was injected using a 30-gauge needle in the ovarian bursa. Both antisera had previously been shown to be specific in inhibiting either tPA or uPA [15, 33]. Distension of the bursa was indicative of successful injection. After the injection, the ovary was replaced into the peritoneal cavity, and skin was sutured. The contralateral ovary served as an uninjected control. The intrabursal injection was performed immediately before induction of ovulation by hCG or 6 h after hormone administration. The animals were killed 15 h after hCG injection, and the number of ova present in the oviducts was counted.
Statistical Analysis
Statistical analysis was performed by one-way ANOVA followed by Tukey-Kramer test for comparisons of multiple groups.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the effect of in vivo hCG injection upon PA production in the different follicular compartments, granulosa and TI cells obtained from immature eCG-treated rats at 0, 5, and 10 h after hCG injection were treated as described in Materials and Methods. Aliquots of media conditioned by cells cultured for 2 h or cell homogenates (prepared at cell isolation time or after the 2 h of culture) were processed for SDS-PAGE followed by zymography. The results showed that, in untreated rats (Time [t] 0), a band with apparent molecular mass of about 45 kDa, corresponding to rat uPA, was the predominant enzyme present in the conditioned medium and cell-associated fraction in both granulosa and TI cells. After hCG stimulation, its activity increased with time in both cell types and in both the secreted (medium) and the cell-associated (cell lysate) fractions (Fig. 1). Urokinase-type PA activity was quantified by densitometric scanning of zymographies, and values were expressed as fold induction with respect to values of the controls set equal to 1 (Table 1). It should be noticed that when granulosa cells were cultured for 2 h, a reduction in cell-associated uPA occurred (Fig. 1B), indicating that the uPA molecule, under these conditions, was probably released from its binding to the uPAR. This phenomenon can also explain the apparent uPA increase in granulosa cell-conditioned media (Fig. 1A).
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After hCG treatment, tPA activity, represented by a lytic zone of about 70 kDa, increased in a time-dependent manner in both cell types (Fig. 1). The induction of tPA was particularly dramatic in the granulosa cell-conditioned medium, as previously demonstrated [12].
Hormonal Regulation of tPA and uPA mRNA Levels
To determine whether hCG stimulatory effects on PA activity were associated with a parallel increase in mRNA levels, Northern blot analysis of total RNA from TI and granulosa cells obtained 0, 5, and 10 h after hCG was performed. In TI cells, the results showed an up-regulation of the steady state levels of both tPA and uPA mRNA after hormonal stimulation, with both messages reaching their maximum levels already at 5 h after hCG (Fig. 2, A and B). Thus, in TI cells the observed RNA levels mirrored the enzymatic activity of both tPA and uPA. In contrast, in granulosa cells, the results showed an induction in the steady state level of tPA mRNA but a rapid decrease in the levels of uPA transcript (Fig. 3, A and B). Therefore, a discrepancy between enzyme activity and corresponding uPA mRNA levels was observed in these cells.
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Analysis of Cell-Associated uPA
The uPAR has been demonstrated to be present in rat follicles and its expression to increase with follicular maturation in both granulosa and TI cells [22]. Therefore, we wanted to test the hypothesis that after uPAR induction in preovulatory follicles, an increase in receptor occupancy could account for the observed discrepancy between the increase in granulosa cell-associated uPA activity and the decrease of uPA mRNA levels. Granulosa cells were obtained from eCG-primed rats 10 h after hCG injection, and the cell-associated PAs were analyzed by detergent phase partitioning, based on the heat-induced separation of Triton X-114 solutions in two phases [28]. Both aqueous and detergent phases were analyzed by SDS-PAGE and zymography (Fig. 4). Tissue-type PA partitioned almost exclusively in the aqueous phase, as expected for a secreted protein. Conversely, two thirds of uPA behaved as an amphiphilic protein (detergent), while the rest was hydrophilic (aqueous), typical for a membrane-associated protein. Moreover, when granulosa cells were treated with an acidic buffer under conditions that have been shown to dissociate the uPA from its receptor [34], the amount of uPA remaining bound to the cells decreased significantly in both control and hCG-treated cells (data not shown). These results suggest that the uPA molecules in granulosa cells of late preovulatory follicles are present mainly bound to their receptors.
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Effect of Gonadotropins and Dibutyryl cAMP on PA Production in Cultured Granulosa Cells
To establish whether the uPA activity modulation and repartitioning observed in granulosa cells after hCG stimulation could be mimicked by hormonal stimulation in vitro, PA production was examined in cultures of isolated granulosa cells.
Cells, obtained from eCG-primed rats, were stimulated for 6 h with 100 ng/ml FSH or with 1 mM dibutyryl cAMP (dbcAMP). Conditioned media and cell lysates were analyzed by zymography. As already observed in vivo, tPA activity increased in response to both molecules either in the medium or in cell lysates. Conversely, the secreted uPA decreased in response to both FSH and dbcAMP, and no increase in the cell-associated enzyme with time was observed (Fig. 5, A and B). Slot-blot analysis of uPA and tPA mRNA levels in granulosa cells after in vitro treatment (Fig. 5C) gave results that mirrored those obtained in vivo, where a down-regulation of the message for uPA was observed (Fig. 3). Moreover, the results indicated that the divergent effects on tPA and uPA mRNA levels produced by FSH stimulation were both mediated by cAMP.
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Effects of FSH on uPAR Binding
In order to determine whether gonadotropin stimulation could modulate uPA binding to uPAR on the granulosa cell surface, we performed a functional assay to test the ability of granulosa cells to bind exogenous uPA. Granulosa cells obtained from eCG-primed rats were stimulated with 100 ng/ml FSH. After 10 h, conditioned medium was collected and replaced for 1 h with fresh medium with or without exogenous uPA. At the end of incubation, the cells were extensively washed and solubilized in 0.5% Triton X-100. Conditioned media and cell lysates were analyzed by SDS-PAGE and zymography. As shown in Figure 6, secretion of uPA by granulosa cells decreased after FSH stimulation, and no increase in cell-associated enzyme was observed. However, when exogenous uPA was added to the cells, the amount of cell-associated enzyme increased significantly in the FSH-treated samples. These results confirm the data of Li et al. [22] on modulation of uPAR expression by gonadotropins and suggest a consequent presence of the available uPA molecules in preovulatory follicles as receptor bound.
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Effect of Antibodies to tPA and uPA on Ovulation
To test when, after gonadotropin stimulation, a block of PA activity would be more effective in inhibiting ovulation, antibodies anti-tPA or anti-uPA or preimmune IgGs were administered intrabursa. When the antibodies were injected immediately before hCG, ovulation was significantly inhibited in the treated ovary compared to that from the untreated control side (57% and 56% for anti-tPA and anti-uPA, respectively) (Table 2). In contrast, when the administration of antibodies was performed 6 h after hCG, only a weak inhibition was observed (Table 2). Both antibodies injected simultaneously did not give any further inhibition (data not shown). Interestingly, though tPA has been considered the most important of the PAs contributing to ovulation, the two antibodies had similar strength in inhibiting ovulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While a decrease in uPA RNA in granulosa cells was observed also by Li et al. [22], our data on uPA expression in TI cells are in contrast with those obtained by that group. They found a decrease in uPA production by TI cells in response to hCG while we observed stimulation. One explanation for these different results could be that the previous researchers analyzed residual ovarian tissues after granulosa cell collection; therefore their results are an average of uPA presence in follicles at all developing stages. In contrast, we selected individual follicles approaching ovulation; thus our data refer strictly to preovulatory follicles. Moreover, these data are in agreement with the results obtained by Tsafriri et al. [40] showing an increase of uPA mRNA in rat thecal tissue 6 h after hCG injection.
The complex regulation of tPA and uPA production by TI and granulosa cells in response to the same hormonal stimulus reinforces the notion that the two PAs might have different functions. It has been proposed that tPA is involved in the rupture of the follicle during ovulation [16, 19] while uPA is associated with tissue remodeling during follicular development [41]. Nevertheless, our data on uPA production by TI cells and the binding of uPA to granulosa cells, together with the inhibition of ovulation by anti-uPA antibodies, suggest that this enzyme could also contribute to the ovulatory process. Moreover, we show here that only when the anti-tPA and anti-uPA antibodies (alone or in combination) are injected immediately before hCG is a significant inhibition observed (> 50%), confirming that the main contribution of both PAs to ovulation occurs in the initial steps of the process.
In this paper we also report an unanticipated relocation of the uPA activity in preovulatory granulosa cells, from the fluid phase to the cell membrane compartment. We found that when granulosa cells from stimulated animals were lysed in Triton X-114, 60–70% of the uPA behaved as a membrane-associated protein (Fig. 4). We also observed that acid treatment of in vivo-stimulated granulosa cells, which disrupts the uPA-uPAR interaction, abolishes the detection of uPA in cell lysates. These results suggest that in preovulatory follicles, uPA accumulates on the granulosa cell surface through binding to its specific receptor, the uPAR. In line with these data, uPAR has been demonstrated to be present in granulosa cells and to increase with follicular development [22]. Moreover, this phenomenon occurs in the absence of novel uPA molecules being produced by granulosa cells, since we show that the uPA messenger RNA is down-regulated after hCG stimulation (Fig. 3).
While passage of uPA in the ovarian follicle from the TI compartment to the granulosa cell compartment across the basement membrane is theoretically possible but has not been reported, the most likely origin for the granulosa cell-associated uPA is the enzyme secreted by the same cells at earlier stages of development and accumulated in the follicular fluid. This hypothesis is supported by our observations that granulosa cells cultured in the presence of FSH increase their ability to bind uPA. Nevertheless, in vitro FSH stimulation does not lead to an increase in receptor-bound uPA unless a high concentration of enzyme is achieved by adding exogenous uPA to the culture medium (Fig. 6). Presumably, a high uPA concentration is what occurs in vivo inside the preovulatory follicle, creating favorable conditions for the uPA-uPAR interaction.
Thus, in rat granulosa cells the uPAR level increases with follicular maturation, reaching its maximum expression in preovulatory follicles. Yet the physiological meaning of the uPAR presence at this stage has not been explained. On the basis of the results presented here, we can speculate about a possible novel functional role for the uPA-uPAR complex in preovulatory follicles. In the hours immediately before ovulation, it is necessary for the expanded cumulus-oocyte complex to breach through the tightly associated mural granulosa cells. While tPA is active in solution and could be involved in the follicular fluid fluidification, an increased uPA binding to its receptor would fit the localized requirement of extracellular matrix degradation between the mural granulosa cells. Novel information has recently expanded uPA-uPAR functions, independently of the proteolytic activity. Binding of uPA to its receptor appears to mediate a variety of cellular events, including proliferation, transcriptional activation, cell migration, and adhesion (reviewed in [42]). However, these pleiotropic properties of the uPAR molecule have not yet been studied in ovarian cells.
Although these findings and other evidence support a role for the PA/plasmin system in follicular rupture, recent gene inactivation studies of main components of this proteolytic system indicated a redundant role of tPA and uPA in ovulation. Mice with a single PA deficiency, lacking either tPA or uPA, have the same ovulation efficiency as wild-type animals [43], while the combined deficiency of the two proteases results in a 26% reduction of ovulation [43]. Furthermore, mice lacking plasminogen are also able to reproduce [44]. There are growing indications that the PA/plasmin system cooperates with the matrix metalloproteinases (MMPs) system to activate a proteolytic cascade leading to the degradation of the follicle wall and to ovulation [40, 45, 46]. Ovulation is a very complex event that involves localized digestion of tissues composed of several diverse proteins: laminin, proteoglycans, and type IV collagen in the basal lamina around follicles and different types of collagen in the connective tissue. As envisioned by Ossowski's group [47], it is possible that the different components of the extracellular matrix and basement membrane need to be sequentially degraded. In addition, recent evidence indicates that pro-MMP-2 and MMP-9 can be activated by PA/plasmin components [48, 49], unraveling a network between the two protease systems. Yet interfering with only the PA/plasmin system is not sufficient to completely block follicular rupture. Taken together, these results lead to the following conclusions: 1) one type of PA can compensate for the loss of the other; 2) plasmin generated in mice with single PA deficiencies is sufficient to ensure normal ovulation efficiency; 3) plasmin action takes place early after the LH peak; 4) plasmin contributes to an optimal efficiency of ovulation, together with metalloproteases with which it shares some substrate specificity. Further studies in which both proteolytic systems are disrupted will help to elucidate whether other unknown pathways are involved. The engagement of several, at least partially redundant, proteolytic systems has probably evolved to ensure follicular rupture, an event essential for reproduction.
In conclusion, our results demonstrate a different distribution and regulation by gonadotropins of the two PAs in the different compartments of the rat ovary. In particular, we report a relocalization of the uPA in granulosa cells of preovulatory follicles from the fluid phase to the cell membrane as a result of binding to its specific receptor. The complex regulation of tPA and uPA production by granulosa and TI cells in response to the same hormonal stimulus reinforces the notion that the two PAs might perform different functions. In this paper we suggest a possible novel role for the uPA-uPAR complex in preovulatory follicles, in which the receptor-bound uPA provides a localized extracellular matrix degradation between the tightly associated mural granulosa cells in order for the cumulus-oocyte complex to escape from the follicle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from MURST (60%) to R.C., from MURST (40%) and CNR 95.02941.CT14 to M.S., and from Swiss National Science Foundation to D.B. 
2 Correspondence: Rita Canipari, Dipartimento di Istologia ed Embriologia Medica, University of Rome "La Sapienza" Via A. Scarpa 14, 00161 Rome, Italy. FAX: 39 6 4462854; canipari{at}uniroma1.it
3 Current address: Olga Epifano, Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, 6 Center Drive, Bethesda, MD 20892.
4 These authors contributed equally to this manuscript.
Accepted: November 15, 1999.
Received: October 8, 1998.
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