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Endocrinology Vol. 148, No. 4 1858-1867
Copyright © 2007 by The Endocrine Society

3'5'-Cyclic Adenosine Monophosphate-Dependent Up-Regulation of Phosphodiesterase Type 3A in Porcine Cumulus Cells

Maxime Sasseville, Nancy Côté, Christian Vigneault, Christine Guillemette and François J. Richard

Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Québec, Canada G1K 7P4

Address all correspondence and requests for reprints to: François J. Richard, Centre de Recherche en Biologie de la Reproduction, Département de Sciences Animales, Université Laval, Québec, Canada G1K 7P4. E-mail: francois.richard{at}crbr.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The means by which cumulus cells react to gonadotropin stimulation and regulate the subsequent production and degradation of cAMP are largely unknown. In this article, we report that cyclic nucleotide phosphodiesterase (PDE) type 3A (Pde3a) is transcriptionally regulated in porcine cumulus cells by a cAMP-dependent pathway during in vitro maturation (IVM). cAMP-PDE activity was increased in the cumulus-oocyte complex (COC) after 10 h of IVM, and 78% of this increase was sensitive to a Pde3-specific inhibitor, cilostamide. Although no variation was observed in the oocyte, cilostamide-sensitive cAMP-PDE activity increased in the cumulus cells after IVM. This was supported by Western blotting, which showed that the intensity of a 135-kDa anti-Pde3a immunoreactive band was increased in COC after IVM. The Pde3a mRNA level was up-regulated 28-fold in the COC after 4 h of IVM and remained high up to 12 h. The mRNA up-regulation and increased activity were inhibited by an RNA synthesis inhibitor, {alpha}-amanitin. The cilostamide-sensitive increase in PDE activity was inhibited by a protein synthesis inhibitor, cycloheximide. Pregnant mare serum gonadotropin (PMSG) caused dose-dependent activation of Pde3. The PMSG-dependent increase in Pde3 activity and Pde3a mRNA were mimicked by the adenylyl cyclase activator forskolin or prostaglandin E2. PMSG-dependent Pde3 activation was inhibited by the protein kinase A-specific inhibitor H89. Collectively, our results show for the first time that degradation of the intracellular cyclic nucleotide by Pde3a is transcriptionally up-regulated via a cAMP-dependent pathway in cumulus cells, suggesting that it has a functional role during the ovulatory gonadotropin surge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN FOLLICLE DEVELOPMENT is stimulated by many paracrine growth factors. FSH and LH are major players in follicular cell functions (1). The FSH and LH receptors are members of the vast family of rhodopsin-like G protein-coupled receptors (GPRs). FSH predominantly stimulates an increase in intracellular cAMP and regulates several physiological functions of somatic granulosa cells, such as proliferation, steroidogenesis, and gene expression (2). Several other known GPRs are present in the ovarian follicle, e.g. leucine-rich repeat-containing GPR 8 (LGR8), GPR3, GPR12, serotonin, and prostaglandin (PG) E receptor subtype EP2 (3, 4, 5, 6). It has recently been proposed that LH stimulates epidermal growth factor-like factors in granulosa cells, and the latter act in an autocrine or paracrine fashion on cumulus cells to stimulate PGE2 production, cumulus expansion, and oocyte maturation in the ovulating follicle (7, 8). The PGE2 receptors EP2 and EP4 activate Gs-protein and stimulate cAMP production (9).

Classically, cAMP signaling activates cAMP-dependent protein kinase A (PKA), leading to phosphorylation of transcription factors such as cAMP response element-binding protein (CREB) (10). cAMP-activated guanine nucleotide exchange factors (cAMP-GEF, also known as EPAC) and cyclic nucleotide-gated Ca2+ channels are alternative cAMP-activated pathways in granulosa (11, 12). Currently proposed models also agree about the central role of cAMP in ovarian function (11, 13, 14, 15, 16, 17, 18). Intracellular cAMP levels are modulated by the equilibrium between synthesis and degradation. The degradation is carried out by the members of the phosphodiesterase (PDE) family. PDEs are known to be important elements in ovarian physiology. The Pde3 and Pde4 subtypes are the most intensively studied of the five known PDE families expressed in rodent ovary; Pde3a is present only in the oocyte, Pde4d in granulosa cells, and Pde4b in theca cells (17, 19, 20). Pde3a-deficient mice are infertile owing to the incapacity of the oocyte to resume meiosis (16). Pde4d-deficient mice have low fertility caused by an apparent defect in the ovulation process (15). Their ovaries and granulosa cells show an altered cAMP response to gonadotropins (17). Pde4d-deficient mice granulosa cells also have an altered gene expression profile for ovulation-related genes (Cox-2, progesterone receptor, PACAP, and cathepsin-L) (17). Pde4d mRNA is up-regulated in rat ovary in response to pregnant mare serum gonadotropin (PMSG) (17). Human cultured luteal granulosa cells show up-regulated Pde4d transcript in response to LH and forskolin (21). These reports suggest that follicular somatic cells up-regulate cAMP-PDE in response to gonadotropin. No report has yet explored the regulation of PDE in response to gonadotropin stimulation in porcine cumulus cells.

A large literature describes the role of cAMP-generating factors in granulosa and cumulus cell physiology. However, only a limited number of studies have described the presence of the PDEs responsible for modulating the cAMP signal in follicular tissues, and an even more limited number have functionally characterized the cAMP-PDE specifically in cumulus cells. The goal of the present study is to describe the presence and regulation of the cAMP-degrading PDE activity in cumulus cells using a well-characterized in vitro cumulus-oocyte complex (COC) maturation system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Ovary collection
Ovaries were collected by the method described previously (22). Briefly, they were recovered from a local slaughterhouse and placed in saline (0.9% NaCl) containing antibiotics and antimycotics (100,000 IU/liter penicillin G, 100 mg/liter streptomycin, and 250 mg/liter amphotericin B) and maintained at 34 C. Upon arrival in the laboratory, they were rinsed with saline containing antibiotics and antimycotics at 34 C.

Culture of COCs
Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Media, COC collection, selection, and culture conditions have been described previously (23). Briefly, COCs were recovered from 2- to 6-mm ovarian follicles, selected, and washed three times with HEPES-buffered Tyrode medium containing 0.01% (wt/vol) polyvinyl alcohol (24). Groups of 30–50 were then cultured in Nunclon {Delta} four-well dishes in 500 µl standard porcine in vitro maturation (IVM) culture medium: BSA-free NCSU 23 medium (25) containing 25 µM 2-mercaptoethanol, 0.1 mg/ml cysteine, 10% (vol/vol) filtered porcine follicular fluid, and gonadotropin supplements at final concentrations of 2.5 IU/ml human chorionic gonadotropin (hCG) and 2.5 IU/ml PMSG (Intervet, Whitby, Ontario, Canada), unless otherwise indicated. Where indicated, cycloheximide and {alpha}-amanitin were used at final concentrations of 2 and 25 µg/ml, respectively. Forskolin and H89 were dissolved in dimethylsulfoxide (DMSO) as 12 and 48.1 mM stock solutions and used as indicated. PGE2 was dissolved in ethanol as a 5 mg/ml stock.

PDE assay
Tissues were suspended in hypotonic buffer [20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.2 mM EGTA, 50 mM NaF, 50 mM benzamidine, 10 mM sodium pyrophosphate, 4 µg/ml aprotinin, 0.7 µg/ml pepstatin, 10 µg/ml soybean trypsin inhibitor, 0.5 µg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride] and homogenized by nine freezing-thawing cycles accompanied by vortex agitation. In all experiments, the hypotonic buffer contained 0.5% Triton X-100 as detergent. Tris-HCl was purchased from Fisher Scientific Limited (Nepean, Ontario, Canada). The homogenate was centrifuged for 20 min at 13,000 x g to obtain the supernatant. PDE activity was assayed at 34 C in 200 µl final volume with 1 µM cAMP as substrate, following the method of Thompson et al. (26) with minor modifications (22). The solution consisted of 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM 2-mercaptoethanol, 0.75 mg/ml BSA (fraction V), 1 µM cold cAMP, and 15 nM [3H]cAMP (GE Healthcare, Baie d’Urfé, Quebec, Canada) (1 x 105 cpm/tube; 30 Ci/mmol). The measurements were performed in the presence of PDE inhibitors 3-isobutyl-methylxanthine (IBMX) (1 mM, nonspecific), cilostamide (10 µM, Pde3-specific), and rolipram (10 µM, Pde4-specific). IBMX-sensitive, cilostamide-sensitive (Pde3), and rolipram-sensitive (Pde4) PDE activities were obtained by subtracting the PDE activity measured in the presence of the respective inhibitors from the total activity. The PDE inhibitors cilostamide and rolipram were purchased from Biomol (Plymouth Meeting, PA).

Western blotting
Tissues were loaded in sample buffer [60 mM Tris-HCl (pH 6.8), 10.5% (vol/vol) glycerol, 2% (wt/vol) SDS, 0.005% (wt/vol) bromophenol blue, and 5% (vol/vol) 2-mercaptoethanol] on to a 10% polyacrylamide gel for electrophoresis. The samples were then transferred on to Hybond-P membrane (GE Healthcare, Baie d’Urfé, Quebec, Canada) using a Mini Protean 3 Cell apparatus (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada). Membranes were blocked for 60 min with Tris-buffered saline (TBS) [150 mM NaCl, 10 mM Tris-HCl (pH 7.4)] containing 0.1% (vol/vol) Tween 20 and 2% (vol/vol) blocking reagent (GE Healthcare). The first hybridization was performed overnight at 4 C in the blocking buffer containing the primary antibody, anti-Pde3a [no. Pde3a (A-19) sc-11828; Santa Cruz Biotechnology, Inc., Santa Cruz, CA] diluted 1:400. The membranes were then washed three times in TBS containing 0.1% (vol/vol) Tween 20 and hybridized with the secondary antibody, horseradish-peroxidase-conjugated antigoat IgG (Jackson Immunoreasearch Laboratories Inc., Bar Harbor, ME) diluted 1:30,000 in TBS containing 0.1% (vol/vol) Tween 20 for 45 min at room temperature. Proteins were detected using an ECL kit (GE Healthcare) and exposed on autoradiographic films (GE Healthcare). Images were analyzed using Quantity One software (Bio-Rad). Where indicated, Restore Western blot stripping buffer (Pierce Biotechnology, Rockford, IL) was used to remove the hybridized antibody to allow subsequent hybridization. The peptide used for antibody preincubation experiments mapped near the N terminus of human Pde3a protein (no. sc-11828P; Santa Cruz Biotechnology).

Quantitative PCR quantification of Pde3a transcript during oocyte nuclear maturation
Ten picograms of an exogenous RNA containing a poly-A tail were added to each pool of COCs before RNA extraction. This exogenous RNA was transcribed from a partial green fluorescent protein (GFP) sequence cloned into pGEM-T Easy (Promega, San Luis Obispo, CA), and a short poly-A tail of 21 bp was added. The GFP fragment that we cloned into pGEM-T Easy was isolated from the phGFP-S65T vector (Clontech, Palo Alto, CA) and corresponds to the sequence of the fragment between bases 892-1598 of GenBank sequence accession number U43284. The exogenous RNA was produced by in vitro transcription of this construct using the AmpliScribe T7 High Yield Transcription Kit (Epicenter, Madison, WI). The RNA extractions from COC pools containing 10 pg GFP RNA were performed using an Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. The RNA solutions were precipitated with 6 µl of 3 M sodium acetate (pH 5.2), 1.5 µl of 1 mg/ml linear acrylamide (Ambion, Austin, TX), and 105 µl of 100% isopropanol. The pellets were washed with cold 75% ethanol. The air-dried pellets were dissolved in 14.75 µl of 1.36 µM oligodT(18) from Ambion (Austin, TX) (to obtain a 1 µM final concentration after the addition of the reverse transcriptase mixture). The RT reaction was performed using OmniScript RT Kit from QIAGEN (Valencia, CA). To each tube, a mixture containing 2 µl Omniscript 5x buffer (QIAGEN, Mississauga, Ontario, Canada), 2 µl of 50 µM dNTPs (QIAGEN), 0.25 µl of 40 U/µl RNASIN (Promega, Madison, WI), and 1 µl Omniscript reverse transcriptase (QIAGEN) was added. The tubes were then incubated at 42 C for 2 h.

The primers for the GFP, ß-actin (Actb), and Pde3a genes were derived from human and mouse sequences from NCBI. The primer sequences for GFP, Actb, Pde3a, and Pde3b were as follows: GFP forward primer, 5'-GTAAACGGCCACAAGTTCAG-3'; GFP reverse primer, 5'-TCACACCACAGAAGTAAGGTTCC-3'; Actb forward primer, 5'-CGTGACATTAAGGAGAAGCTGTGC-3'; Actb reverse primer, 5'-CTCAGGAGGA-GCAATGATCTTGAT-3'; Pde3a forward primer, 5'-GAACAGATGA- CACTGCTCAAGTT-3'; Pde3a reverse primer, 5'-GAGCAAGAATTGGTTTGTCCAG-3'; Pde3b forward primer, 5'-CTGTGTAACTCCTATGATGCTGCTGG-3'; and Pde3b reverse primer, 5'-CTTGTGGTTTTCAGTGAGGTGGTG-3'. They were purchased from Integrated DNA Technologies (Skokie, IL). These primers generate 708-, 242-, 180-, and 216-nucleotide-long PCR products, respectively, which were sequenced and found to be homologous to published sequences (22). For each gene examined, a standard curve was generated in the same run from standard PCR products purified using a QIAquick PCR Purification Kit (QIAGEN) and quantified spectrophotometrically. Pde3a and Actb were amplified from porcine oocyte cDNA and Pde3b from porcine testis cDNA. The standard curve was constructed using five dilutions of the purified PCR products ranging from 1 pg to 0.1 fg. Quantitative PCR was executed on a Lightcycler apparatus (Roche Diagnostics, Laval, Quebec, Canada) using SYBR green incorporation, with the reactions occurring in glass capillaries (Roche). To each capillary we added cDNA corresponding to one COC and a mixture containing 0.5 µl of 10 µM of each primer, 1.6 µl of 25 mM MgCl2 to a final concentration of 3 mM, 2 µl of the SYBR green mix containing dNTPs, FastStart DNA polymerase enzyme and its buffer (Roche), and water to obtain a final volume of 20 µl. The PCR conditions used for all genes comprised a denaturing cycle of 10 min at 95 C, 45 cycles of PCR (denaturing, 95 C for 1 sec; annealing, 58 C for 5 sec; extension, 72 C for 20 sec), a melting cycle consisting of 95 C for 0 sec and 70 C for 30 sec, a step cycle starting from 70 C up to 95 C with a 0.2 C/sec transition rate, and finally a cooling cycle of 40 C for 30 sec. For the GFP, 58 C was used instead of 57 C as annealing temperature in the PCR cycles. cDNA was quantified using Lightcycler Software version 3.5 (Roche) by comparison with the standard curve, and the specificities of the PCR products were confirmed by analysis of the melting curves. The quantity of GFP and Actb obtained for each pool was used to correct the experimental errors caused by the techniques and the materials used for RNA extraction and RT.

Statistical analyses
Statistical analyses were performed using Prism 4.00 GraphPad for Windows (GraphPad Software, San Diego, CA). Statistical significance was assessed using ANOVA analysis followed by Dunnett’s or Bonferroni’s multiple comparison post hoc tests to identify individual differences between means. Probabilities of P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increase of PDE activity in the cumulus-oocyte complex during IVM
cAMP-PDE activity was measured in COC homogenates at different time points from 0–22 h of IVM. Figure 1AGo shows a significant increase of total cAMP-PDE activity in the COC after 10 h of IVM. After 10 h, the total cAMP-PDE activity was maintained at a high level up to 22 h of IVM. Most interestingly, from 10–22 h, about 50% of the cAMP-PDE activity was sensitive to cilostamide (Pde3-specific inhibitor), whereas only 23% was cilostamide sensitive before IVM (0 h, Fig. 1AGo). Seventy-eight percent of the increase in total cAMP-PDE activity observed after 19 h of IVM was sensitive to cilostamide. As observed in Fig. 1AGo, cilostamide-sensitive PDE activity followed roughly the same pattern as total cAMP-PDE activity, suggesting that the activity of type 3 PDEs predominates. There was no significant variation of the cilostamide-insensitive PDE activity from 0–22 h of IVM (data not shown), suggesting that no major changes occurred in other cAMP-degrading PDE subtype activities during this time period.


Figure 1
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  		FIG. 1. A, PDE activity during IVM. Total activity (solid line) and cilostamide-sensitive activity (Pde3-specific, 10 µM) (dashed line) of cAMP-PDE measured in the porcine COC during IVM in the presence of PMSG and hCG (2.5 IU/ml each). PDE activities were measured at 0 and 4 h and thereafter every 3 h until 22 h of IVM. More than 3820 oocytes were used. B, cAMP-PDE activity measured in COCs freshly recovered from the ovary and after 19 h of culture. Total and IBMX-sensitive (1 mM), cilostamide-sensitive (Pde3-specific, 10 µM), and rolipram-sensitive (Pde4-specific, 10 µM) PDE activities in the COC are shown. To determine the inhibitor-sensitive contributions to the overall PDE activity, the activity measured in the presence of the respective inhibitors was subtracted from the total PDE activity. A minimum of 1500 COCs were used. C, Cilostamide-sensitive PDE activity in the denuded oocyte and cumulus cells at 0 h (black bars) or after 19 h of IVM (white bars). cAMP degradation measurements were conducted separately on denuded oocytes and cumulus cells cultured as COCs for 19 h and dissociated after IVM by gentle pipetting. More than 700 oocytes were used. D, cAMP-PDE activity measured in COC-cultured cumulus cells during IVM. Cilostamide- and rolipram-sensitive PDE activities in the cumulus cells are shown. A minimum of 2100 oocytes were used. Each data point represents the mean ± SEM of a minimum of three replicates. Statistical analyses were performed by ANOVA (P < 0.05). Asterisks indicate significant differences from the control according to Dunnett’s multiple-comparison (A and D) or a Bonferroni (B and C) post hoc test (P < 0.05). The control is PDE activity in COCs at 0 h of IVM. E, Expression pattern of Pde3a protein in aorta and denuded oocytes by Western blotting. Numbers on the right indicate migration of protein markers. Lane 1, 40 µg aorta protein extract; lane 2, 200 denuded oocytes. F, Expression of Pde3a protein in COCs after 0 and 13 h of IVM. Lane 1, 50 COCs after 0 h of IVM; lane 2, 50 COCs after 13 h of IVM in the presence of PMSG. In the lower panel is the detection of {alpha}-tubulin in each sample, used as control for the quantification of Pde3a. Representative results from three replicates are shown.

 
cAMP-PDE activities measured in the COC at 0 and after 19 h of IVM were almost completely inhibited by the presence of the nonspecific PDE inhibitor IBMX, as seen on the bar graph by the close similarity between the total and IBMX-sensitive PDE activities (Fig. 1BGo). Although no significant change in rolipram-sensitive PDE activity (Pde4) was observed after 19 h of IVM, total, IBMX-sensitive and cilostamide-sensitive PDE activities increased significantly (P < 0.05) (Fig. 1BGo). The next step was to measure cAMP-PDE activity in cumulus cells and denuded oocytes after they were cultured as COC. At 0 h, cilostamide-sensitive PDE activity was measurable in the oocyte and very low in the cumulus cells (Fig. 1CGo). After 19 h of IVM, no significant change in cilostamide-sensitive PDE activity was observed in the oocyte. However, a robust increase (P < 0.05) was observed in the cumulus cells (Fig. 1CGo). These results suggest that the increase in Pde3 activity occurred in the cumulus cells.

To confirm further the up-regulation of Pde3 in the cumulus cells during IVM, cAMP-PDE activity was measured in cumulus cells that had been cultured as COC for different times. Cilostamide-sensitive and rolipram-sensitive PDE activities were measured for each time point (Fig. 1DGo). Cilostamide-sensitive cAMP-PDE activity was significantly increased in the cumulus cells from 11–19 h of IVM (P < 0.05) (Fig. 1DGo). Rolipram-sensitive activity was stable throughout IVM in the cumulus cells (Fig. 1DGo).

Protein extracts from pig aorta, 200 denuded oocytes, or 50 COC after 0 or 13 h of IVM were separated on SDS-PAGE gels, and Pde3a protein was detected (Fig. 1Go, E and 1FGo). In oocytes, a band was detected at a molecular weight similar to that in aortic protein extract (Fig. 1EGo), consistent with the band of 135 kDa detected in porcine oocytes by Liang and co-workers (27). The same band was detected in COC after 0 h of IVM (Fig. 1FGo). Densitometric analysis of the 135-kDa Pde3a band, compared with {alpha}-tubulin, revealed a 2-fold increase in COC after 13 h of IVM (Fig. 1FGo). To confirm the identity of the 135-kDa immunoreactive band, the membranes were stripped and a second hybridization was conducted. This hybridization was performed under identical conditions, except that the anti-Pde3a antibody was preincubated with the Pde3a peptide used to raise the antibody. This second hybridization displayed no immunoreactive band (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Finally, to demonstrate that the absence of the immunoreactive band was not due to the stripping method, the membranes were stripped and hybridized a third time with the anti-Pde3a antibody. Immunoreactive bands at 135 kDa were detected on the membrane after this third hybridization (supplemental Fig. 1). This result supports the inference that Pde3a is present in oocytes and is increased in cumulus cells in the presence of gonadotropins during IVM, consistent with the increase in Pde3 activity reported in Fig. 1Go.

Pde3a transcript regulation in the COC
Because the toxin {alpha}-amanitin binds irreversibly and inhibits RNA polymerase II (28), COCs were cultured for 19 h, and {alpha}-amanitin was added to the culture medium after 0, 4, 8, or 12 h of IVM. COCs were homogenized, and cAMP-PDE activity was measured. The results in Fig. 2Go, A and B, show that {alpha}-amanitin completely inhibited the increases in total and cilostamide-sensitive PDE activities in the COC if it was added at the beginning of IVM. The earlier {alpha}-amanitin was added during IVM, the more the increase in both activities was inhibited, again supporting the inference that the increase in Pde3 activity during IVM was transcriptionally regulated.


Figure 2
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  		FIG. 2. PDE activity during IVM is regulated. cAMP-PDE activity was measured in the COC during IVM in presence of an RNA synthesis inhibitor, {alpha}-amanitin. Total (A) and cilostamide-sensitive (B) cAMP-PDE activities were measured in COCs after 0 and 19 h of IVM. COCs were cultured for 19 h, and {alpha}-amanitin (25 µg/ml) was added at 0, 4, 8, or 12 h of IVM. A minimum of 1800 oocytes were used. Total (C) and cilostamide-sensitive (D) cAMP-PDE activities were measured in COCs after 0, 10, and 19 h of culture without (black bars) and with (white bars) a protein synthesis inhibitor, cycloheximide (2 µg/ml). A minimum of 1500 oocytes were used. Each data point represents the mean ± SEM of a minimum of three replicates. Statistical analyses were performed by ANOVA (P < 0.05). Asterisks indicate significant differences from the control according to Dunnett’s multiple-comparison post hoc test (P < 0.05). The control is PDE activity in COCs at 0 h of IVM.

 
Cycloheximide is a peptidyl transferase inhibitor that blocks translation on ribosomes, leading to inhibition of peptide elongation (29). Figure 2Go, C and D, shows that cycloheximide significantly prevented the increase of cAMP-PDE activity. Because cycloheximide inhibited the cilostamide-sensitive PDE activity increase after both 10 and 19 h of IVM (Fig. 2DGo), it follows that translation is needed for Pde3 activation in the COC during IVM.

Pde3a mRNA was quantified at different time points during IVM and corrected with an external GFP mRNA spike and by using endogenous Actb mRNA, as previously reported (30). Similar results were obtained with both corrections, and the GFP-corrected Pde3a mRNA measurements are presented in Fig. 3Go. The Pde3a transcript (black bars) was dramatically up-regulated between 0 and 4 h of IVM (28-fold, P < 0.05) (Fig. 3Go). The Pde3a transcript also appeared at an intermediate level after 8 and 12 h, but this was still 18-fold greater than its value at 0 h (P < 0.05) (Fig. 3Go). However, after 20 h of IVM, the levels of Pde3a transcripts were no longer significantly different from those at the start of the culture. This up-regulation was abolished when COCs were cultured in the presence of {alpha}-amanitin, an RNA synthesis inhibitor, supporting the inference that de novo RNA synthesis of Pde3a is up-regulated in the COC during IVM (white bars) (Fig. 3Go).


Figure 3
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  		FIG. 3. Pde3a mRNA quantification by quantitative RT-PCR in the COC during IVM. The signal was corrected using a GFP RNA spike as control. Quantities of Pde3a mRNA are presented as relative proportions of the quantity at 0 h of IVM. COCs were treated either without (black bars) or with (white bars) the RNA synthesis inhibitor {alpha}-amanitin during IVM, and then the Pde3a mRNA levels were measured. About 750 oocytes were used. Each data point represents the mean ± SEM of a minimum of three replicates. Statistical analyses were performed by ANOVA (P < 0.05). Asterisks indicate significant differences from the control according to Dunnett’s multiple-comparison post hoc test (P < 0.05). The control is Pde3a mRNA signal in COCs at 0 h of IVM.

 
Pde3 is activated by gonadotropin
The previous experiments have shown that Pde3a is up-regulated under typical IVM conditions (Figs. 1–3GoGoGo). To determine whether this Pde3a up-regulation and activation is caused by gonadotropin stimulation, COCs were cultured in either the absence or the presence of PMSG and/or hCG, and PDE activity was measured after 19 h of IVM. Figure 4AGo shows that no significant increase in PDE activity occurred between 0 and 19 h of IVM when no gonadotropins were added. Interestingly, COCs treated with both gonadotropins (P < 0.05) or with PMSG alone (P < 0.05) exhibited PDE activation (Fig. 4AGo). However, COCs treated with hCG alone showed no increase in PDE activity (Fig. 4AGo). These results suggest that Pde3a activation is dependent on gonadotropin stimulation.


Figure 4
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  		FIG. 4. The effect of gonadotropins on cAMP-PDE activity measured in COCs after 19 h of IVM. A, Total, IBMX-sensitive, and cilostamide-sensitive cAMP-PDE activities measured in the COC at 0 h (black bars) and after 19 h (dark gray bars) of IVM without gonadotropins, with PMSG and hCG (2.5 IU/ml each) (pale gray bars), with PMSG alone (2.5 IU/ml) (open bars), and with hCG alone (2.5 IU/ml) (hatched bars). More than 1500 COCs were used. B and C, The dose-dependent effect of PMSG on the cAMP-PDE activity in COCs after 19 h of IVM. Total (B) and cilostamide-sensitive (C) PDE activities in the COC are shown. cAMP-PDE activity in COCs cultured without PMSG was arbitrarily set two log units below the lowest PMSG concentration. More than 2700 oocytes were used. Each point represents the mean ± SEM of a minimum of three replicates. Statistical analysis was performed by ANOVA (P < 0.05). Asterisks indicate significant differences from the control according to Dunnett’s multiple-comparison post hoc test (P < 0.05). The control is COC at 0 h of IVM (A) or COC at 19 h of IVM without PMSG (B and C).

 
COCs were stimulated by increasing doses of PMSG, and PDE activity was measured after 19 h of IVM. The doses of PMSG varied from 25 IU/ml down to 2.5 µIU/ml. PDE was activated when COCs were cultured in the presence of 0.25, 2.5, and 25 IU/ml PMSG (Fig. 4BGo). Pde3 activation (cilostamide-sensitive) was also observed at the same concentrations of PMSG (Fig. 4CGo). These results indicate a dose-dependent stimulation of PDE activity by gonadotropin during IVM.

Pde3 activation in COC is cAMP dependent
It is generally accepted that PMSG elevates the intracellular cAMP level in granulosa/cumulus cells (2). To complete the demonstration of the efficacy of the cAMP pathway in stimulating Pde3 activity, alternative cAMP-elevating agents were used to mimic the PMSG effect. Different doses of the adenylyl cyclase stimulator forskolin were added to the culture media, and PDE activity was measured after 19 h IVM. The results show that 10 µM forskolin significantly increased PDE activity compared with the control (Fig. 5Go, A and B). The forskolin-stimulated COCs showed increases in both PDE activities (total and cilostamide-sensitive) to the same level as PMSG-stimulated COCs (2.5 IU/ml). Because PGE2 has been used to stimulate cAMP production in the COC (31), PGE2 was applied for 19 h of IVM. The results show that PGE2 (50 µg/ml) generated a significant increase in PDE activity compared with the control (Fig. 5Go, C and D). PGE2-stimulated COCs showed increases in both PDE activities (total and cilostamide-sensitive).


Figure 5
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  		FIG. 5. The effect of cAMP-producing agents on cAMP-PDE activity during IVM. Total (A) and cilostamide-sensitive (B) cAMP-PDE activities were measured in the COC after 19 h of IVM in the presence of different doses of the adenylyl cyclase activator forskolin. COCs were also cultured in the presence of DMSO (0.1%) and PMSG (2.5 IU/ml). A minimum of 2400 COCs were used. Total (C) and cilostamide-sensitive (D) cAMP-PDE activities were measured in the COC after 19 h of IVM in the presence of different doses of PGE2. COCs were also cultured in the presence of ethanol (0.1%) and PMSG (2.5 IU/ml). A minimum of 1800 COCs were used. Total (E) and cilostamide-sensitive (F) cAMP-PDE activities were measured in the COC after 19 h of IVM in presence of PMSG (2.5 IU/ml), and different doses of a PKA inhibitor, H89. COCs were also cultured in presence of DMSO (0.1%), H89 (50 µM), and PMSG alone (2.5 IU/ml). More than 2100 COCs were used. Each column represents the mean ± SEM of a minimum of three replicates. Statistical analysis performed by ANOVA showed the significant effects of treatments (P < 0.05). Asterisks indicate significant differences from the control according to Dunnett’s multiple-comparison post hoc test (P < 0.05). The control is COC at 19 h of IVM without PMSG.

 
The next question was whether high intracellular cAMP activates PKA leading to the activation of Pde3. PMSG-stimulated COCs were treated with the PKA-specific inhibitor H89 for 19 h of IVM, and cAMP-PDE activity was measured. The results show that the PMSG-stimulated increase in PDE activity was significantly inhibited by 50 µM or higher concentrations of H89, down to the level of the control (Fig. 5Go, E and F). This result further supports the view that the cAMP pathway through PKA efficiently controls the activation of Pde3a.

To support the previous results fully, quantitative RT-PCR measurements of the two type 3 PDEs (Pde3a and Pde3b) were carried out on COCs treated with the above-mentioned cAMP-elevating agents. Pde3a and Pde3b mRNA were quantified after 4 h of IVM, at which point the maximal value was reached, as shown by the previous quantitative RT-PCR experiments (Fig. 3Go). The results show that PMSG, forskolin, and PGE2 significantly increased the quantity of Pde3a mRNA compared with untreated COCs after 4 h of IVM (Fig. 6Go, black bars). Under the same conditions, Pde3b mRNA underwent no significant change (Fig. 6Go, white bars). These results show that cAMP clearly increased Pde3a activity and that Pde3a could serve as a cAMP regulator in cumulus cells for a physiologically relevant cAMP-stimulating agent.


Figure 6
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  		FIG. 6. Quantification of Pde3a (black bars) and Pde3b (white bars) mRNAs by quantitative RT-PCR in COCs after 4 h of IVM. The signal was corrected with Actb mRNA. The quantities of Pde3a mRNA are presented as relative proportions of the quantity measured in untreated COCs after 4 h of IVM (control). Pde3a mRNA was significantly increased after 4 h of IVM in COCs treated with PMSG (2.5 IU/ml), forskolin (10 µM), or PGE2 (50 µg/ml) compared with the control, which is the Pde3a or Pde3b mRNA signal in untreated COCs after 4 h of IVM. No significant variation was observed for Pde3b mRNA. Each point represents the mean ± SEM of a minimum of three replicates. About 240 oocytes were used. Statistical analysis by ANOVA showed a significant effect of treatment for Pde3a mRNA (P < 0.05). Asterisks indicate significant differences from the control according to Dunnett’s multiple-comparison post hoc test (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that Pde3a is transcriptionally regulated in porcine cumulus cells by gonadotropin in a cAMP-dependent manner during IVM. Several lines of evidence support this conclusion. In response to PMSG during IVM, 1) Pde3a mRNA is increased, and this increase is sensitive to {alpha}-amanitin; 2) the intensity of a 135-kDa band detected by an anti-Pde3a antibody is increased; 3) cilostamide-sensitive PDE activity, relating to Pde3, is increased; 4) the increase in Pde3 activity is prevented by {alpha}-amanitin and cycloheximide; 5) both Pde3 activity and Pde3a mRNA are increased by the adenylyl cyclase activator forskolin and by PGE2; and 6) PMSG stimulation of Pde3 activity is prevented by a PKA-specific inhibitor, H89. cAMP-dependent regulation of PDE in cumulus cells is supported by previous studies in rodents showing that gonadotropins up-regulate PDE in the ovary (15, 17).

The pattern of Pde3 activity reported in this study (Fig. 1Go) differs from the currently accepted cellular distribution of PDEs in rodent ovarian follicle, which restricts Pde3a expression to the oocyte (11, 19). These studies described the presence of Pde3a mRNA in rat oocytes (32), and expression was subsequently demonstrated in mouse (20) and human (33) oocytes. Furthermore, Pde3 activity has been demonstrated in mouse and rat oocytes (20, 34), where it was shown to be modulated during the resumption of meiosis in rat oocytes (34). Our team has recently reported the activity of Pde3a in porcine oocytes (22, 23). However, the present study is the first to establish the functional presence of Pde3a in somatic cumulus cells. Liang and co-workers (27) detected Pde3a in oocytes by immunoblotting, consistent with our results showing cilostamide-sensitive PDE activity in denuded oocytes (Fig. 1CGo). However, they did not measure the evolution of Pde3a expression during IVM. They also reported the presence of PDE4A1 in cumulus cells, consistent with our report, which shows that rolipram-sensitive PDE activity in the cumulus cells does not vary during IVM (Fig. 1DGo).

Figure 1Go shows that Pde3 activity was modulated in cumulus cells during IVM. During the same period, Pde4 activity remained unchanged. This result correlates well with the up-regulation of Pde3a transcripts observed in the quantitative RT-PCR experiments (Figs. 3Go and 6Go). In mice, both PMSG and hCG stimulate PDE up-regulation and activity in the ovary, mainly associated with Pde4 (17). Human granulosa cells have been shown to up-regulate Pde4d mRNA in response to LH and forskolin (21). Because the granulosa cells from in vitro fertilization patients were recovered 34–35 h after hCG administration and cultured for 6 d in the presence of serum, they are largely luteinized (21). FSH did not increase PDE4D mRNA, indicating that a different mechanism is involved in regulating cAMP in human luteal granulosa cells (21). The present Pde3a activation was dependent on transcription (Figs. 2Go, A and B, and 3Go) and translation (Fig. 2Go, C and D). Pde3 has also been reported to be up-regulated in response to cAMP stimulation in other cell types (35, 36). This study suggests that a different PDE is up-regulated in porcine cumulus cells in response to cAMP from that in rodent models.

In the present study, a mixture of PMSG (used as an FSH-like hormone) and hCG (used as an LH-like factor) was used to stimulate COCs during IVM. Our results show that only PMSG, not hCG, exerts a dose-dependent effect on the up-regulation of Pde3a (Fig. 3Go, A–C), indicating FSH-like stimulation. PMSG is known to act as a folliculogenesis stimulator by activating the FSH receptor; it has also been shown to have a certain affinity for the LH receptor (37). One could argue that no LH receptors are present in the porcine cumulus cells, which would prevent hCG from being able to up-regulate Pde3a. In porcine cumulus cells, LH begin to bind to the plasma membrane after 12 h of IVM, and LH-stimulated cAMP production is apparent at 20 h, but not at 10 h, of IVM, but Pde3a mRNA is up-regulated at 4 h (Fig. 3Go) (38, 39). This suggests that porcine cumulus cells do not respond to LH early enough for hCG to be involved in Pde3a up-regulation, further supporting the requirement for FSH-like action.

Because Pde3 activity and the Pde3a transcript level were also increased by forskolin, an adenylyl cyclase stimulator, Pde3a up-regulation appears to be a direct response to cAMP stimulation. The forskolin concentration sufficient to increase Pde3a activity (Figs. 5Go, A and B, and 6Go) was similar to the dose required to prevent meiotic maturation of porcine COCs and below the concentration used to activate MAPK phosphorylation in porcine cumulus cells (23, 27). FSH and forskolin both stimulate cAMP-dependent CREB phosphorylation in porcine granulosa cells (40). PGE2-stimulated Pde3a up-regulation further pinpoints the importance of Pde3a as a regulation loop for cAMP-mediated signaling (Fig. 5Go, C and D, and 6Go). EP2 is detected in porcine granulosa cells (data not shown). PGE2 and forskolin similarly stimulated a dose-dependent increase in cAMP in cultured human granulosa cells (31). However, the concentration required for activation was above the reported PGE2 content of primate and bovine follicular fluid (41, 42). The physiological meaning of PGE2-stimulated Pde3a activation in cumulus cells remains to be determined. Nevertheless, PGE2 has been proposed as a major paracrine and autocrine regulator of cumulus cell functions in the porcine periovulatory follicle, such as steroidogenesis, proliferation, and gene expression (7, 43, 44, 45). Shimada et al. (7) included two cAMP-stimulating factors in their model, namely gonadotropins and PGE2, indicating an important role for the cAMP pathway in the cumulus cells of the ovulatory follicle. The role of a negative modulator of the cAMP pathway such as Pde3a provides a new working model of the signaling that takes place after gonadotropin stimulation. At this point, the possibility that other signaling pathways act synergistically with the cAMP pathway cannot be excluded, because FSH stimulates various phosphorylation pathways (12). However, the results using the PKA-specific inhibitor H89 strongly suggest that PKA is the primary mediator of Pde3a activation (Fig. 5Go, E and F). The H89 concentration that inhibited PMSG-stimulated Pde3a activation effectively was equivalent to that found in a previous study on porcine granulosa cells (46). One of the downstream transcription factors activated by cAMP-dependent PKA is CREB, which has been shown to activate the Pde3b promoter and to up-regulate Pde3b mRNA in murine preadipocytes (47). Pde4-specific inhibition extends the temporal pattern of forskolin-stimulated CREB phosphorylation in rat granulosa cells, demonstrating the intimate relationship between cAMP-PDE activity and subsequent transcriptional events (48). This pathway of CREB activation by type 3 PDE has now to be investigated in porcine cumulus cells. Interestingly, these cells express at least one Pde4 subtype (27). Specific inhibition of Pde3 or Pde4 led to different temporal patterns of ERK1/2 phosphorylation during IVM in porcine cumulus cells, suggesting that PDEs modulate discrete compartmentalized pools of intracellular cAMP that affect distinct downstream signaling processes (27). Numerous cell types display divergent responses upon inhibition of Pde3 or Pde4 (49, 50). The progressive change reported here in the balance between PDE subtypes from 0–10 h of IVM in cumulus cells is likely to change the regulation of compartmentalized cAMP signaling and downstream activated kinases.

Whether this Pde3 activation in cumulus cells is unique to the porcine species remains to be determined. Pde4-specific inhibition has been reported to affect human granulosa cell morphology, whereas Pde3 inhibition was ineffective (51). Moreover, no Pde3 activity was detected in cumulus cells from PMSG-stimulated rats (34). Nogueira and collaborators (33) have recently reported improved human oocyte maturation and embryonic development after prematuration culture with a Pde3 inhibitor, which potently delays oocyte nuclear maturation. No evidence of PDE activity in human cumulus cells has yet been reported.

In summary, the present investigation has revealed a new insight into cAMP regulation in cumulus cells. It demonstrates that Pde3a is transcriptionally regulated in porcine cumulus cells by a cAMP-dependent pathway. Additional experiments will provide a better understanding of the key role played by Pde3a in controlling cAMP levels in cumulus cells in response to FSH.


   Acknowledgments
 
We thank Élizabeth Gobeil-Tremblay and Gabrielle Darou for their valuable experimental assistance. Zuzana Bécotte Capova is particularly acknowledged for providing porcine testis cDNA, Pde3b primers, and molecular biology support. Special thanks go to Isabelle Laflamme for professional technical assistance. We also thank Richard Prince and O’Neil Fecteau for collecting ovaries at the slaughterhouse.


   Footnotes
 
M.S. is supported by a Natural Science and Engineering Research Council and Fonds Québécois de la Recherche sur la Nature et les Technologies Ph.D. fellowship. This project was supported by the Natural Science and Engineering Research Council of Canada and by the Canadian Institutes for Health Research in the Program for Oocyte Health granted to F.J.R.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 11, 2007

Abbreviations: COC, Cumulus-oocyte complex; CREB, cAMP response element-binding protein; DMSO, dimethylsulfoxide; GFP, green fluorescent protein; GPR, G protein-coupled receptor; hCG, human chorionic gonadotropin; IBMX, 3-isobutyl-methylxanthine; IVM, in vitro maturation; PDE, phosphodiesterase; PG, prostaglandin; PKA, protein kinase A; PMSG, pregnant mare serum gonadotropin; TBS, Tris-buffered saline.

Received September 13, 2006.

Accepted for publication January 2, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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