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Biology of Reproduction 66, 830-835 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Plasminogen Activator Expression in Rat Granulosa Cell1

Rosanna Apaa, Antonio Lanzonea, Fiorella Micelia, Sergio Vaccarib, Elisabetta Macchioneb, Mario Stefaninib, and Rita Canipari2,b

a Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore, and b Department of Histology and Medical Embryology, University ‘La Sapienza,’ 00161 Rome, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamus. It has been demonstrated to be transiently expressed in preovulatory follicles and to positively affect several parameters correlated with the ovulatory process. The aim of the present study was to investigate whether PACAP influences the plasminogen/plasmin system in rat ovary. Plasminogen activators (PAs) are serine proteases, modulated by gonadotropins and several peptides in preovulatory follicles, that appear to be involved in ovulation. Granulosa cells obtained from immature eCG-treated rats were cultured for 24 h in the presence of increasing concentrations of PACAP and vasoactive intestinal peptide (VIP). A significant, dose-dependent increase in tissue-type PA (tPA) activity and decrease in urokinase-type (uPA) PA activity were observed in PACAP-treated cells. These effects were exerted at the mRNA level. The use of cycloheximide, a protein synthesis inhibitor, suggested that PACAP requires an intermediary protein to decrease uPA-mRNA, but not to induce tPA-mRNA. However, no significant modulation of PAs was observed in the presence of VIP. When granulosa cells were stimulated within the intact follicle (i.e., maintaining the three-dimensional structure and in the presence of the theca cell layers), both PACAP and VIP dose-dependently stimulated tPA. These data suggest that, in addition to the PACAP type I receptor present on granulosa cells, different subtypes of PACAP receptors are present in the different ovarian compartments.

granulosa cells, ovary, ovulation, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pituitary adenylate cyclase-activating polypeptide (PACAP), originally isolated from ovine hypothalamus, exists in two forms; PACAP-38, and PACAP-27 [1]. Their names reflect their potent action in stimulating cAMP production in anterior pituitary cells [2]. On the basis of sequence similarity, PACAPs were included in the vasoactive intestinal polypeptide (VIP)/glucagon/secretin family of peptides. In fact, they share a 68% homology with VIP and a 41% homology with growth hormone releasing factor, two other members of the same family [3].

Three different PACAP receptors have been identified: PACAP type I receptor, which binds specifically to both PACAPs as well as to VIP, though with very low affinity; and two others, called VIP1 [4] and VIP2 [5], which bind to PACAP and VIP with equal affinity. The PACAP type I receptor is coupled to adenylate cyclase and/or phospholipase C pathways [6], whereas VIP1 and VIP2 are coupled only to the adenylate cyclase pathway.

In addition to the central nervous system, both PACAPs and their receptors have been found in various organs and peripheral tissues, such as the lung, testis, adrenal, and ovarian tissues [7–9], which suggests that such peptides may not have an exclusively neuroendocrine role. More specifically, the presence of PACAP and its receptors in the ovary and the fact that this peptide stimulates several ovarian functions, including steroidogenesis and cAMP accumulation in rat granulosa cells [10, 11], and also accelerates meiotic maturation in rat oocytes [12] indicate that PACAP may play a role in the female reproductive system. Moreover, PACAP contributes to the survival of granulosa cells by inhibiting apoptosis in preovulatory follicles [13].

Plasminogen activators (PAs) are proteolytic enzymes involved in numerous biological processes [14, 15]. Two forms of PA have been detected in the ovary: the tissue-type (tPA) and the urokinase-type (uPA), both of which seem to be involved in gonadotropin-induced ovulation [16]. Their synthesis is, in fact, regulated by gonadotropins [17, 18], and in the rat, gonadotropin-induced ovulation is inhibited by injection of serine protease inhibitors in the periovarian bursa [18–20].

Although gonadotropins play a key role in the regulation of PA production in the ovary [18, 19, 21, 22], a number of other factors have been reported to affect PA biosynthesis in this organ [17]. Considering the observed positive effects of PACAP on ovarian physiology at the time of ovulation and the presence of this peptide and its receptors in granulosa cells of preovulatory follicles, we decided to investigate the possible role of this peptide in regulation of the plasminogen/plasmin system in the rat ovary.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Immature female Sprague-Dawley rats (Charles River, Como, Italy) were used for this study. At the age of 25 days, they were either killed (untreated rats) or injected s.c. with 10 IU of eCG and then killed 48 h later (eCG-treated rats). Animals were maintained in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Experimental protocols were approved by the University Committee on Animal Care and Use.

Materials

The PACAP-38 and VIP were purchased from Calbiochem (San Diego, CA). The chromogenic plasmin substrate D-val-leu-lys-p-nitroanilide 2HCl (S2251) was obtained from Bachem Feinchemikalien AG (Basel, Switzerland). The eCG and hCG were purchased from Intervet (Livorno, Italy). The mouse uPA and tPA cDNA clones were kindly provided by Dr. D. Belin (Gen;ageve, Switzerland). All other reagents were obtained from Sigma Chemical (St. Louis, MO).

Preparation of Granulosa Cell Cultures

Granulosa cell cultures were prepared from eCG-treated rats as previously described [21]. Briefly, the contents of individual follicles were expressed in Hepes-buffered medium M2 [23] supplemented with 0.1% (w/v) BSA. Viable granulosa cells were cultured at a density of 1.5 x 105 cells per 200 µl of minimum essential Eagle medium (MEM) supplemented with 0.1% BSA and 1 g/L of glutamine. Viability was estimated by trypan blue dye exclusion. All incubations were carried out at 37°C in a 5% CO2 atmosphere.

Cultures of isolated follicles were prepared from eCG-primed rats. Graafian follicles were dissected from the ovaries and cultured at a density of 6 follicles per 16-mm well in 0.4 ml of MEM supplemented with 0.1% BSA and the concentrations of the peptides indicated. After 8 h of culture, follicles were carefully washed and incised, and the contents were expressed in the medium. Granulosa cells were cultured, as described above, in the absence of peptides for an additional 4 h.

Gel Electrophoresis and Zymography

For the zymography of PA, aliquots of conditioned media 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 [24]. The PA was then visualized by placing the Triton X-100-washed gel on a casein-agar-plasminogen underlay as previously described [25]. That no lytic zones formed in the absence of plasminogen indicates that the lytic zones were plasminogen-dependent. Molecular weights were calculated from the position of prestained markers subjected to electrophoresis in parallel lines. Densitometric scanning of zymographies was performed to derive a semiquantitative estimation of protease activities.

Assay for PA

Enzymatic activity of tPA was assessed according to the method of Shimada et al. [26] using a chromogenic substrate (S2251) assay. Samples were incubated with plasminogen in the presence or absence of 100 µM amiloride to inhibit uPA activity [27]. In this assay, the absorbance generated at 405 nm is related to PA activity. The PA activity is expressed in terms of international units (IU) with reference to a standard preparation of uPA prepared from human urine (Sigma).

Isolation and Analysis of Total Cellular RNA

Total RNA prepared from granulosa cells [28] was analyzed for relative abundance of specific mRNAs by Northern blot hybridization. Total RNAs (40 µg/lane) were denatured with formaldehyde, electrophoresed on a 1.2% agarose gel containing 6% formaldehyde, and transferred to a Hybond Nylon membrane (Amersham Pharmacia Biotech Italia, Cologno Monzese, Italy) by capillary blotting with 20x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate) for 24 h [29]. Each blot was washed twice with 20x SSC. Each specific mRNA was quantified by the densitometry of the films after autoradiography and was normalized to the amount of 18S rRNA.

Probe Synthesis and Northern Blot Hybridization Conditions

The 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 [30]. After autoradiography, the filters were probed with a random-primed cDNA for the mouse 18S rRNA [31] to adjust for any variability in the amount of RNA present in the filters.

Statistical Analysis

Statistical analysis was performed using ANOVA followed by the Tukey-Kramer test for comparisons of multiple groups. The dose-response curve was analyzed and the mean effective dose (ED50) values calculated using the Allfit program [32]. A P value of less than 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of PACAP and VIP on PA Activity in Cultured Granulosa Cells

To examine the influence of PACAP and VIP on granulosa cell PA production, cells obtained from immature eCG-primed rats were cultured for 24 h in medium alone (control), with 100 ng/ml of FSH, or with increasing concentrations of the two peptides (ranging from 10-10 to 10-6 M). The PACAP-38 was used because of its predominant distribution on peripheral tissues [33]. To characterize the type of PA present in the medium at the end of incubation, aliquots of conditioned media at two different peptide concentrations were processed for SDS-PAGE, followed by zymography. As described previously [18], FSH stimulation of rat granulosa cells induced tPA secretion and decreased uPA production. Similarly, the addition of PACAP to the culture medium induced tPA production and reduced uPA activity. The addition of VIP was ineffective (Fig. 1A). When cell lysates were analyzed by zymography, we observed similar effects of PACAP on tPA but no decrease in uPA activity (data not shown).



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  		FIG. 1. Effect of FSH, PACAP, or VIP on PA secreted by granulosa cells. Rat granulosa cells were cultured in medium alone (control [C]), with FSH (100 ng/ml), or with increasing concentrations of PACAP-38 and VIP. After 24 h, conditioned media were collected and analyzed by casein-agar underlay. A) The zymography shown is representative of the three performed. B) Averaged data of uPA activity from densitometry of zymographies from three independent experiments. Values represent the mean ± SEM and are expressed as a percentage of the control value (arbitrarily set at 100). *, P < 0.05; **, P < 0.01 vs. control

The decrease in uPA activity in the medium was subsequently quantified by densitometric scanning of the zymographies, and the values were expressed as a percentage of the value of the control (arbitrarily set at 100). As shown in Figure 1B, PACAP caused a dose-dependent reduction in uPA activity (30% and 43% reduction at 10-7 and 10-6 M PACAP, respectively), whereas no statistically significant decrease in the enzyme was observed after VIP stimulation.

Given the greater efficacy of tPA in the chromogenic substrate assay, we adopted this method to quantify the levels of tPA activity in the samples used for the previous zymographies. The assay was performed in the presence of amiloride to inhibit uPA activity. We found that PACAP induced tPA in a dose-dependent manner and reached a plateau at a concentration of 10-7 M, with a 3.5-fold increase compared to the control (ED50 = 2 x 10-9 M; Fig. 2). The VIP was less potent than PACAP in inducing tPA activity and was effective only at the highest concentration (10-6 M; Fig.2).



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  		FIG. 2. Dose-dependent stimulation of tPA secretion by granulosa cells treated with FSH, PACAP, or VIP. Cells were cultured in medium alone (control [C]), FSH (100 ng/ml), or increasing concentrations (ranging from 10-10 to 10-6 M) of PACAP or VIP. After 24 h, conditioned medium was collected and analyzed for tPA activity by chromogenic substrate assay in the presence of amiloride. Values represent the mean ± SEM of four separate experiments, each performed in duplicate, and are expressed as the fold-induction compared to untreated (control) cells (value arbitrarily set at 1). *, P < 0.05; **, P < 0.01 vs. control

Time-Dependent Stimulation of tPA Secretion by FSH and PACAP

Granulosa cells were cultured in medium alone (control) or with saturating concentrations of FSH (100 ng/ml) or PACAP (10-7 M) for 2, 4, 6, 12, or 24 h. Only a minimal increase in PA activity was found in the control cultures over the 24-h culture period, whereas both FSH- and PACAP-treated cells showed a time-dependent increase in tPA production, which became statistically evident at 6 h (P < 0.005 vs. control; Fig. 3).



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  		FIG. 3. Time course of tPA production by granulosa cells in response to FSH or PACAP. Granulosa cells were cultured in medium alone (control [C]), with FSH (100 ng/ml), or PACAP-38 (10-7 M). At the times indicated, conditioned media were collected and assayed for tPA activity by chromogenic substrate assay in the presence of amiloride. Values represent the mean ± SEM of three separate experiments, each performed in duplicate. *, P < 0.05; **, P < 0.01 vs. control

Effect of FSH and PACAP on Steady-State Levels of tPA and uPA mRNAs

To determine whether the effects of PACAP on PA activity were associated with a parallel modulation of mRNA levels, total RNA was extracted from granulosa cells cultured for 24 h in medium alone (control), with FSH (100 ng/ml), or with PACAP (10-7 M) and analyzed by Northern blot. The filters were hybridized with specific murine tPA and uPA cRNA probes. In accordance with earlier reports, FSH induced tPA mRNA and inhibited uPA mRNA. Similar, though less striking, results were obtained after PACAP stimulation (Fig. 4). Thus, these results closely mirrored those obtained at the protein level by enzymatic assay and zymography.



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  		FIG. 4. Northern blot analysis of PA mRNA levels in granulosa cells treated with either FSH or PACAP and cycloheximide (CHX). Cells were cultured for 12 h in medium alone (control [C]), FSH (100 ng/ml) or PACAP (10-7 M), with and without CHX (50 µg/ml). Total RNA (30 µg) was subjected to electrophoresis on a denaturing agarose gel and then transferred to a nylon membrane, followed by hybridization with murine tPA and uPA cRNA probes. A) Autoradiography of a representative Northern blot. B) Averaged data from densitometry of Northern blots from three independent experiments. Optical density values of mRNA in each lane were normalized by respective optical density values of 18S rRNA signals. The results shown are relative to the control values (arbitrarily set at 100 for uPA and at 1 for tPA)

Effect of Cycloheximide on PACAP Modulation of Steady-State Levels of tPA and uPA mRNAs

To investigate whether protein synthesis was required for control of the two PAs by PACAP, we used the protein synthesis-inhibitor cycloheximide (50 µg/ml) either alone or combined with PACAP (10-7 M). Cells were collected 12 h after stimulation, and RNA was extracted and analyzed by Northern blot. At the concentration used, cycloheximide inhibited protein synthesis by more than 95%, thereby preventing the detection of PA activity by zymography (data not shown). As shown in Figure 4, cycloheximide alone had no effect on the steady-state level of tPA mRNA but enhanced PACAP-stimulated tPA. The uPA mRNA levels were also affected by the presence of cycloheximide, which in PACAP-treated cells blocked the decrease in uPA mRNA.

Effect of PACAP on PA Secreted by Granulosa Cells Stimulated in Whole Follicles

To investigate whether other ovarian compartments may influence the action of PACAP and VIP on PA production by granulosa cells, whole follicles were cultured for 8 h in the presence of LH (100 ng/ml) or increasing concentrations of PACAP or VIP (ranging from 10-8 to 10-6 M). At the end of the incubation period, the follicles were carefully washed in medium without peptides, and granulosa cells were isolated and cultured for an additional 4 h. Zymographic analysis of PA activity in conditioned media showed that both peptides induced tPA production with a similar potency. The induction of tPA activity was quantified by chromogenic substrate assay in the presence of amiloride to inhibit uPA synthesis, with the values being expressed as the fold induction compared to the control value (arbitrarily set at 1) (Fig. 5). The potencies of the two peptides were comparable to that of LH.



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  		FIG. 5. Secretion of tPA by granulosa cells stimulated in whole follicles. Follicles were treated with LH (100 ng/ml) or increasing concentrations of PACAP and VIP for 8 h. Granulosa cells were then isolated from the cultured follicles and cultured for an additional 4 h at a density of 1.5 x 105 cells in 200 µl of MEM. Conditioned media were collected and assayed for tPA activity by chromogenic substrate assay in the presence of amiloride. Values represent the mean ± SEM of three separate experiments, each performed in duplicate. The results are expressed as the fold-induction compared to the control value (arbitrarily set at 1). *, P < 0.05; **, P < 0.01 vs. control

As regards uPA, we observed a slight increase in uPA both in the conditioned media and in the cell lysates (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously demonstrated that gonadotropins differentially regulate uPA and tPA in rat granulosa cells, stimulating production of tPA and inhibiting that of uPA [18]. The present study demonstrates that PACAP significantly modulates production of both PAs in rat granulosa cells. Its action is similar to that obtained by gonadotropin stimulation. In fact, we observed a dose-dependent increase in tPA stimulation and an inhibition of uPA production. We also have previously demonstrated that the FSH-positive and -negative modulation of PA is mediated by the intracellular levels of cAMP [17]. Because the concentration range of PACAP used in this study is the same as that shown to be effective in stimulating cAMP production in rat granulosa cells [10, 12], we may hypothesize that cAMP mediates the action of PACAP on PA production by granulosa cells. The action of PACAP was exerted at the steady-state levels of tPA and uPA mRNAs, and it appeared to be mediated by different mechanisms. Whereas tPA induction occurred in the absence of protein synthesis, suggesting a direct effect on gene expression, transcriptional regulation of uPA required ongoing protein synthesis. In fact, in the presence of cycloheximide, a protein synthesis inhibitor, the effect of PACAP on tPA induction was fully expressed and actually amplified, whereas that on uPA inhibition was abolished. The superinduction of both tPA and uPA mRNA observed in the presence of cycloheximide may involve mechanisms such as stabilization of mRNAs. In fact, it as been shown that in FSH-stimulated rat granulosa cells, the absence of protein synthesis prevents the rapid down-regulation of tPA transcripts [34], and that in FSH-stimulated rat Sertoli cells, it prevents the down-regulation of uPA transcripts [35].

Interestingly, whereas only PACAP stimulated isolated granulosa cells, both PACAP and VIP modulated PA production when granulosa cells were stimulated in whole follicles. Moreover, in accordance with previous data regarding the induction of cAMP and progesterone [10], we have shown that PACAP was 100-fold more potent than VIP in the stimulation of tPA in isolated granulosa cells [11, 36], whereas PACAP and VIP were equally effective in stimulating tPA production when the two peptides were added to whole-follicle cultures.

These different effects of PACAP and VIP on the stimulation of isolated granulosa cells or cells cultured within the follicle can be explained by the presence of different subtypes of PACAP receptors in the different ovarian compartments. In fact, PACAP is known to bind to at least three types of receptors: type I receptor, which binds to both PACAPs as well as to VIP, though with very low affinity; and two others, VIP1 and VIP2, which bind to PACAP and VIP with similar affinities. Reports in the literature regarding what type of PACAP receptor is found in the ovary are, however, contrasting. Binding experiments using whole rat ovary have shown a similar affinity for PACAP and VIP, thereby pointing to the presence of VIP1 receptors [9]. In addition, the presence of mRNA for PACAP type I-receptor transcripts has been observed in whole rat ovary [37], with the mRNA for this receptor having subsequently been localized in granulosa cells of growing and preovulatory follicles [38]. More recently, Gras et al. [36] demonstrated the presence of VIP2 receptors on a small proportion of granulosa cells, thus supporting previous studies showing VIP stimulation of progesterone in a subpopulation of granulosa cells that were unresponsive to FSH [39]. Our data are in accordance with the presence of PACAP type I receptors on granulosa cells and, in addition, suggest the presence of VIP receptors in whole follicles, probably in theca cells, even though neither PACAP nor VIP receptors have been observed in the theca layer [40].

The LH surge is the physiological stimulus that leads to the follicle rupture and luteinization processes by inducing several genes that have been shown to play a role at the time of ovulation [41]. In our previous work, we showed that tPA is regulated by LH, that its production is temporally correlated with ovulation [17], and that uPA is also modulated by gonadotropins. However, unlike tPA, uPA decreases after hormone stimulation [18]. Here, we have shown that PACAP and VIP mimic the effect of gonadotropins in the modulation of PAs. In addition, the levels of tPA induction by PACAP and VIP are similar to those obtained with LH. Taken together, these data strongly support the hypothesis of a role for these two peptides in the preovulatory follicles.

It has been recently shown that PACAP may act as a mediator of LH action during ovulation. In fact, granulosa cells respond to PACAP by increasing progesterone accumulation [42] and decreasing apoptotic cellular death [13]. The fact that, in both cases, the effects of LH can be partially blocked by inhibiting PACAP action with PACAP antagonists or specific antibodies suggests that LH stimulation is also mediated by endogenously produced PACAP. It would be interesting to investigate whether the action of LH is also mediated by PACAP or VIP in PA stimulation.

It should be remembered that the paracrine and autocrine functions of these two peptides in the ovary are also supported by the fact that both peptides are synthesized locally in the ovary. The VIP-containing nerve fibers are present in rat ovarian follicles in close proximity to the theca cell layers during all stages of development [43], whereas PACAP [13] and PACAP receptor [38] are transiently expressed in granulosa cells of preovulatory follicles following hCG and LH stimulation.

In conclusion, our results, which show PACAP and VIP modulation of proteolytic enzymes linked to ovulation in preovulatory follicles, provide further evidence that these two peptides as well as VIP are both deeply involved in the complex regulation of ovarian physiology.


   ACKNOWLEDGMENTS
 
We thank Mr. S. Greci for his excellent technical assistance, Dr. A.F. Parlow and the National Hormone and Pituitary Distribution Program of the NIDDK for providing the ovine FSH and ovine LH, and Mr. Lewis Baker for reviewing the English in the manuscript.


   FOOTNOTES
 
First decision: 29 August 2001.

1 Supported by a grant from MURST (60% to R.C. and 40% to M.S.). Back

2 Correspondence: Rita Canipari, Dipartimento di Istologia ed Embriologia Medica, via A. Scarpa 14, 00161 Roma, Italy. FAX: 39 06 4462854; rita.canipari{at}uniroma1.it Back

Accepted: October 30, 2001.

Received: August 7, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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