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Regular Article |
a Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
b Department of Pharmacology, N.V. Organon, 5340 BH Oss, The Netherlands
ABSTRACT
In the preovulatory follicle, oocyte meiotic resumption occurs soon after the LH surge and is associated with a decrease in cAMP. Inhibition of cAMP degradation blocks germinal vesicle breakdown as well as activation of meiotic promoting factor, both hallmarks of reentry into the cell cycle. In situ and pharmacological analysis of rodent ovaries suggested the presence of a phosphodiesterase 3 (PDE3) in the germ cell but not the somatic cell compartment. Here we have investigated the structure and properties of the PDE form expressed in mouse oocytes. Polymerase chain reactions using a mouse oocyte cDNA library as a template, and primers based on the conserved sequence of rat and human PDE3As, yielded partial fragments corresponding to mouse PDE3A. Further screening of the mouse oocyte cDNA library and subsequent ligation of individual cDNA clones yielded PDE3A cDNA containing the entire coding region of mouse PDE3A. To determine the kinetic properties of this PDE, the cDNAs encoding the full-length PDE3A and NH2-truncation forms Delta 1 (
346aa) and Delta 2 (608aa) were expressed in mouse Leydig tumor cells. Whereas the full-length recombinant protein was always found in the particulate fraction, the Delta 1 and Delta 2 truncated PDE3As were recovered mostly in the soluble fraction. The Michaelis constant values for hydrolysis of cAMP of PDE3A Delta 1 and PDE3A Delta 2 were similar to those of intact full-length PDE3A or oocyte PDE (0.2–0.5 µM). More importantly, there was good correlation between the rank of potency of selective and nonselective compounds in inhibiting recombinant PDE3A or PDE activity derived from cumulus-oocyte complexes and in blocking resumption of meiosis. These data provide evidence that the PDE expressed in the oocyte is a soluble form of PDE3A and that activity of this enzyme is involved in the control of resumption of meiosis.
cyclic adenosine monophosphate, meiosis, oocyte development, ovulatory cycle, phosphodiesterases
INTRODUCTION
The meiotic cell cycle in mammalian oocytes commences in the fetal ovary, proceeds up to the diplotene stage of the first prophase, and is arrested at a G2-like phase [1, 2]. During the growth phase before the follicle antral phase, the oocyte remains arrested at prophase I and is incompetent to complete meiosis.
In vitro and in vivo, rodent oocytes acquire the ability to resume meiosis when they approach their final size [1, 2]. In vivo, meiosis is triggered by the preovulatory surge of LH during each reproductive cycle [3]. Resumption of meiosis and progression to metaphase II involves dissolution of the nuclear membrane, usually referred to as germinal vesicle breakdown (GVBD), chromatin condensation, chromosome segregation, and the extrusion of the first polar body shortly before follicle rupture.
The second messenger cAMP plays an important role in controlling meiotic arrest of oocytes in both mammals and amphibians [1, 4, 5]. Elevated cAMP concentration in the oocyte has been demonstrated to maintain meiotic arrest [1, 6–8]. A transient decrease in cAMP levels in the oocyte is thought to be a primary trigger for resumption of meiosis in most species studied [9, 10]. Consistent with this view, introduction of the regulatory subunit of protein kinase A (PKA) in Xenopus oocytes stimulates resumption of meiosis, whereas injection of the catalytic PKA subunit blocks meiosis [11]. Progesterone and insulin-like growth factor-1, both of which promote meiotic resumption in Xenopus eggs, signal through a decrease in cAMP [12]. The spontaneous maturation of rodent oocytes in vitro can be prevented by the addition of phosphodiesterase (PDE) inhibitors or derivatives of cAMP, both of which cause an increase in intraoocyte cAMP levels. The signaling pathway controlling meiotic resumption is therefore dependent on switching off the PKA activity that is required to maintain meiotic arrest. This inactivation of PKA follows a decrease in cAMP probably caused by PDE-mediated degradation.
From the work of our and other laboratories, it has become clear that at least 11 different families of genes encoding cyclic nucleotide PDEs are present in mammals [13]. Incubation of denuded oocytes in media containing nonspecific or PDE3-specific inhibitors blocks spontaneous resumption of meiosis [14]. Two genes and corresponding isoforms of PDE3, PDE3A, and PDE3B have been described in human and rat [15–17]. In situ hybridization studies have indicated that PDE3A mRNA is expressed in rat oocyte [14, 18]. The PDE3 expressed in heart and ovary was initially named PDE3B [14, 18]. Following a change in nomenclature, they are now termed PDE3As [19]. PDE3A, as well as PDE3B, is characterized by its high affinity for both cAMP and cGMP, and cGMP binding inhibits cAMP hydrolysis by this PDE [19]. Both PDE3 isoforms are structurally similar, containing an NH2-terminal domain important for localization of the enzyme to the particulate fraction and catalytic domain at the carboxyl terminus end [20, 21].
Here we have sought to determine the structure as well as biochemical and pharmacological properties of the PDE3 expressed in mouse oocytes. In addition, we have investigated the correlation between spontaneous resumption of meiosis in the presence of different PDE3 inhibitors and the IC50s of these inhibitors for the activities both of oocytes and of the recombinant PDE3A protein expressed in MA-10 cells. Our data demonstrate that a PDE homologous to PDE3A described in other species is present and necessary for meiotic progression in rodent oocytes.
MATERIALS AND METHODS
Materials
[3H]Cyclic AMP (37.2 Ci/mmol) and [
-32P]dCTP were obtained from NEN Life Science Products, Inc. (Boston, MA). AG 1-X8 resin was from Bio-Rad Laboratories (Hercules, CA), and ECL Western blot detection kits were from Amersham Pharmacia Biotech (Piscataway, NJ). Waymouth MB752/1 medium, gentamycin, and horse serum were purchased from Gibco-BRL (Grand Island, NY). Immobilon was from Millipore Corp. (Bedford, MA). Unless otherwise noted, all chemicals were the purest grade available from Sigma (St. Louis, MO). Cilostamide (N-cyclohexyl-N-methyl-4-[1,2,dihydro-2-oxo-6-quinolyloxy]butyramide) was a gift from Dr. H. Hidaka, University of Nagoya, Nagoya, Japan. Milrinone, 1,6-dihydro-2-methyl-6-oxo-3,4-bipyridine-5-carbonitrile; trequinsin (HL725), 9,10-dimethoxy-2-mesitylimino-3-methyl-3,4,6,7-tetrahydro-2H-pyrimido(6,1-)isoquinolin-4; pimobendan UK-CG115, 4,5,dihydro-6-(2-[4-methoxyphenyl]-1H-benzimadazol-5-yl)-5-methyl-3(2H)pyridazinone; and Org 9935, 4,5,-dihydro-6-(5,6-dimethyoxybenzo[6]thien-2-yl)-5-methyl-3(2H)pyridazinone, were provided by Organon (Oss, The Netherlands).
Reverse Transcription-Polymerase Chain Reaction
Immature female mice (female C57BL/6) were injected s.c. with 5 IU eCG (Calbiochem, La Jolla, CA) to stimulate follicular development. Oocytes were collected, denuded, and stored at -80°C as described [14]. Poly(A)+ RNAs from mouse heart were extracted using a Quickprep micro mRNA purification kit (Amersham Pharmacia Biotech) following the manufacturer's instructions. Poly(A)+ RNAs from mouse oocytes or granulosa cells were extracted with modification of the above procedure using 1:5 elution buffer diluted with RNase-free water and then concentrated five times by Speed Vac (Savant, St. Paul, MN). First-strand cDNAs were generated using the First-Strand cDNA synthesis kit (Pharmacia Biotech) with random hexanucleotides as primers. One reverse transcription-polymerase chain reaction (RT-PCR) contained 5 ng of mouse heart mRNA (200 µg of mouse heart tissue), mRNA from seven oocytes, or mRNA from 30 granulosa cells. Bulk First-Strand cDNA Reaction Mix (containing reverse transcriptase) was replaced by RNase-free water in some reactions to monitor genomic or cDNA contamination. RNase-free water was used to detect contamination of cDNA in Bulk First-Strand cDNA Reaction Mix.
Polymerase chain reactions were performed directly on 1 µl of the first-strand cDNA. For these experiments oligonucleotides employed as primers are listed in Table 1. The PCR incubation mixture was composed of 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 1.5 mM (or 0.8 mM for fragment 415–1055) MgCl2, 200 µM of dNTP, 0.5 µM of each primer, and 2.5 U of Taq DNA Polymerase (Gibco-BRL). The PCR conditions were as follows: 1) denaturation at 94°C for 5 min; followed by 2) 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min; and 3) final extension at 72°C for 10 min. At the end of PCR amplification, products were analyzed on 1% agarose gels stained with ethidium bromide and visualized with UV light.
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Screening of the Library
Two fragments (675 base pairs [bp] and 1.3 kilobases [kb]) of PDE3A were used in the screening. The products were isolated using a PCR-based strategy with the cDNA library as a template (see below). A 675-bp PCR product was isolated using primers designed for regions most conserved in rat and human PDE3A sequences. The PCR amplification of the 1.3-kb fragment was carried out with T7 primer and mouse PDE3A-specific primer corresponding to nucleotides (nt) 2144–2124 (designed on the basis of the 675-bp product), followed by nested PCR with T7 inside primer and a primer corresponding to nt 2078–2058 of mouse PDE3A using the first PCR product as a template. Several PCRs were performed to confirm the sequence. All PCRs were designed to yield products that would overlap each other.
A mouse pSPORT1 oocyte cDNA library was used (a gift from Dr. John Eppig, Jackson Laboratory, Bar Harbor, ME). The library was plated on LB/ampicillin (50 µg/ml) agarose plates at a density of about 30 000 colonies per 140-mm plate. Replica filters on Hybond-N 132 mm nylon membranes (Amersham Life Science, Buckinghamshire, UK) were denatured, baked for 2 h at 80°C and prehybridized in 50% formamide, 5x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA [pH 7.7]), 5x Denhardt solution, 0.1% SDS, and 100 µg/ml sonicated herring sperm DNA at 42°C for 2 h. Hybridization was carried out for 16 h at 42°C in the same buffer containing about 1 x 106 cpm/ml of each of the amplified cDNA fragments (corresponding to bases 851–2144 and 1941–2615, respectively) randomly labeled with [-32P]dCTP (NEN). The filters were washed in buffers of 0.2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% SDS at 65°C, and autoradiographed overnight. Positive clones were purified and subjected to secondary screening.
Construction of PDE3A and Sequencing
The scheme reported in Figure 1 shows the structure of the full-length rat PDE3A, as well as the assembly of the different fragments obtained with different cloning strategies. The PCR fragments and the two clones isolated by hybridization that were used to generate a full-length cDNA (fragments with double-headed arrows) are shown in Figure 1. They were excised by their specific restriction enzymes shown in Figure 1 and subcloned into pBluescript II (SK+) (Stratagene, La Jolla, CA) between XbaI and HindIII. The 5' segment of the sequence is derived from NheI to SacII of 5' clone A, and the 3' segment is derived from SpeI to HindIII of clone B. The middle region was assembled from three PCR products: SacII to BamHI of PCR product nt 415–1055, BamHI to NdeI of PCR product nt 851–2144, and NdeI to SpeI of PCR product nt 1497–2581. To obtain expression of the full-length open reading frame (ORF), the pBluescript construct was digested by NotI and SalI and subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) between NotI and XhoI. Delta 1 truncation of PDE3A was made by removing a BamHI to BamHI fragment from the 5'-end of the full-length PDE3A insert. This creates a 346-amino acid (aa) deletion of the ORF. Delta 2 truncation of PDE3A was made by removing an EcoRV-NdeI fragment from the 5'-end of the full-length PDE3A insert, causing a 608-aa deletion of the ORF. All three constructs were tagged with hemagglutinin (HA) epitope tags at the carboxy terminus by PCR and subcloning into pcDNA3.
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The sequence was confirmed by sequencing on both strands using the dideoxy chain termination method (Cyclist ExpoPfu DNA Sequencing kit; Strategene). All plasmids were purified using a Maxi plasmid preparation kit (Qiagen, Inc., Valencia, CA). DNA concentrations were estimated by reading optical density at 260 nm and confirmed using ethidium bromide staining with agarose gel electrophoresis.
Northern Blotting
Mouse PDE3A cDNA was digested by EcoRI, and an 875-bp fragment was recovered (nt 2678–3552). This fragment was labeled to a specific activity of 1 x 109 cpm/µg of DNA using [-32P]dCTP and Random Primers DNA labeling system (Gibco-BRL). The blot was hybridized with this probe using ExpressHyb Hybridization Solution (Clontech Laboratories, Inc., Palo Alto, CA) following the manufacturer's procedure. The membrane was washed several times for 10 min in 2x SCC + 0.005% SDS and three times for 10 min at 68°C in 0.1x SCC + 0.1% SDS. Autoradiographs were obtained after 72 h of exposure at -80°C.
Transient Transfection of MA-10 Cells
The Leydig cell tumor cell line MA-10 [22] was generously provided by Dr. Mario Ascoli, University of Iowa. MA-10 cells were seeded onto 90-mm dishes (Corning, Inc., Corning, NY) in Waymouth medium supplemented with 20 mM Hepes and 15% horse serum. Twenty-four hours after seeding, cells were transfected with 10 µg of each pcDNA3-PDE3A construct using the CaPO4 method [23].
Twenty-four hours after transfection, cells were harvested and disrupted by Dounce homogenization in lysis buffer consisting of 10 mM Na-phosphate buffer, pH 7.2, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 5 mM 2-mercaptoethanol, 1 µM microcystin, 3 mM benzamidine, 5 µg/ml leupeptin, 20 µg/ml pepstatin, 30 mM pyrophosphate, and 2 mM PMSF. For immunoprecipitation experiments, 0.5% Triton X-100 was added to the lysis buffer. The homogenate obtained was centrifuged at 12 000 x g for 30 min. The supernatant was collected as the soluble fraction. The pellet was washed twice with the same buffer, resuspended, and collected as the particulate fraction.
Isolation of Cumulus-Oocyte Complexes
Cumulus-oocyte complexes (COCs) were isolated from immature Sprague-Dawley female rats injected s.c. with 10 IU of eCG [14]. The COCs were washed three times, then transferred by aspiration with a minimum volume of medium to a 1.5-ml centrifuge tube. They were diluted to 1 COC/µl of the same lysis buffer as above with 0.5% Triton X-100. The cells were disrupted by pipetting, then centrifuged at 12 000 x g for 30 min at 4°C. The supernatant was collected and frozen in liquid nitrogen immediately. The oocytes were stored at -80°C until use in the PDE assay.
In the experiments where the effect of inhibitors on GVBD was determined, cumulus-enclosed oocytes (CEOs) were collected in fresh minimal essential medium containing 4 mM hypoxanthine and washed twice before transfer to the test medium. The CEOs were cultured with or without different PDE inhibitors in 4-well multidish at 37°C and 100% humidity with 5% CO2 in air. At the end of the culture, the oocytes were examined for the meiotic status by an inverted microscope equipped with Hoffman modulation contrast (Nikon, Tokyo, Japan). The presence of intact GV or GVBD was scored as previously reported [24].
Phosphodiesterase Assay
Phosphodiesterase activity was assayed in triplicate using 1 µM cAMP as substrate according to the method of Thompson and Appleman [25] with minor modifications. Phosphodiesterase inhibitors were dissolved in DMSO and were used at concentrations ranging between 10-10 and 10-2 M. In no instance did the DMSO concentration in the assay exceed 1%. Inhibition curves thus generated were analyzed using nonlinear regression analysis (GraphPad Prism, GraphPad Software).
Antibody Generation
The PDE3A antibody was prepared using a fragment of rat cardiac PDE3A cDNA isolated by RT-PCR with specific primers (5'-CGT-GGA-TTC-CTC-TTT-GCC-ACT-CCT-ACG-AC-3' and 5'-CGT-GAA-TTC-ACA-GGT-CTG-GTT-GTG-GAG-C-3'). The fragment corresponding to nt 3030–3426 (GenBank accession number RNU38179) was subcloned into the bacterial expression vector pGEX-3X (Amersham Pharmacia Biotech) between BamHI and EcoRI, in frame with the glutathione-S-transferase (GST) coding sequence. Expression of this construct in Escherichia coli produces a protein that is the result of fusion of the coding region of the GST and the carboxy-terminal portion of PDE3A. This fusion protein was isolated on single-step affinity chromatography on 4B glutathione-Sepharose according to the instructions (Amersham Pharmacia Biotech). The fusion protein was injected into three rabbits, and serum was collected from each animal.
Immunoprecipitation and Western Blot Analysis
The monoclonal IgG or anti-HA (12CA5) antibodies (Boehringer Mannheim, Indianapolis, IN) were used at a 1:250 dilution. Adsorption of the antibody to Protein G-Sepharose followed the procedure described by Meacci et al. [16] with minor modifications. The supernatants of cell homogenates were precleared by incubation for 2 h with purified monoclonal IgG adsorbed to Protein G-Sepharose. Supernatants were then immunoprecipitated overnight at 4°C by gentle mixing using anti-HA antibody immobilized on Protein G-Sepharose. At the end of the incubation, the samples were centrifuged at 1000 x g for 2 min. The pellets were washed three times with PBS and 0.1% BSA. The samples were dissolved in 1x sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% [w/v] SDS, 0.7 M 2-mercaptoethanol, and 0.0025% [w/v] bromphenol blue) and boiled for 5 min. After separation on an 8% SDS-polyacrylamide gel, proteins were transferred to an Immobilon membrane, and nonspecific binding sites were blocked by incubating the membrane overnight in 10% BSA (w/v) dissolved in TBS-T solution (0.2% Tween 20, 20 mM Tris-HCl, 14 mM NaCl, pH 7.6). The following day, the membrane was incubated in 1:200 PDE3A antisera in TBS-T for 1 h. After incubation with the primary antibody, the membrane was washed and incubated for 1 h with peroxidase-conjugated secondary antibody (Amersham Corp.) diluted in 1:5000 in TBS-T. Blots were visualized by the ECL-procedure (Amersham Corp.) followed by autoradiography.
RESULTS
Cloning of PDE3A cDNA from a Mouse Oocyte Library
Primers R3A1941 and R3A2615R (Fig. 1 and Table 1) were designed on the basis of the most conserved sequences of rat and human PDE3s. Using these primers and DNA derived from a mouse oocyte cDNA library as a template for PCR, we obtained an initial 675-bp fragment (fragment 1941–2615). From the sequence of this PCR product, two mouse-specific PDE3A primers, M3A2144R and M3A2078R (Table 1), were designed. Additional PCR amplification on the library was done using primer M3A2144R together with vector T7 primer and a nested PCR using M3A2078R and primers downstream to the T7. These amplifications produced fragments of 1.3 and 1.2 kb (fragments 847–2144 and 851–2078). Using the 675-bp and 1300-bp fragments as probes in a screening of 7 x 105 colonies of the mouse oocyte cDNA library, we obtained two clones (clones A and B). Their sequences showed high homology with the 5' (320–954) and 3' (2510–4270) regions of rat and human PDE3A, respectively. Several additional overlapping PCR products containing the fragments between the two clones were obtained and sequenced to confirm the overlap between the different fragments obtained by the two methods (Fig. 1). A meld of the overlapping clones produced an ORF of 3423 bp, corresponding to a protein of 1141 aa (GenBank accession number AF099187). Conceptual translation of PDE3A cDNA derived from the oocyte revealed that its deduced protein sequence has a homology of 96.1% to rat PDE3A and 81.1% to human PDE3A (Fig. 2), with the greatest residue conservation in the catalytic domain. The NH2-terminal region of PDE3A contains at least six potential transmembrane helices and several potential sites for PKA and PKB phosphorylation in the region downstream from the putative transmembrane domain (Fig. 2). Considerable divergence in sequence from the three species was present at the amino and carboxyl termini of the protein.
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Northern blot analysis of mouse heart with the full-length PDE3A detected a transcript of approximately 8 kb (Fig. 3). However, only very faint bands of 8.0, 6.0, and 3.3 kb could be detected by this method in the ovary mRNA. To clarify further whether the entire ORF is expressed in mouse oocytes, PCR analysis was performed with three sets of primers and oocyte mRNA (Fig. 4). Heart and granulosa cells were used as positive and negative controls, respectively. All three sets of primers yielded products of the expected size using heart mRNA. Although identical products were amplified from oocyte RNA, primers at the 5'-end of the mRNA consistently produced less amplification. This finding opens the possibility that an additional transcript with a different 5'-end is expressed in the oocyte. However, additional screening or PCR 5' extension did not yield new cDNAs. Confirming our previous observations, no amplification was observed with granulosa cell mRNA (Fig. 4).
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Transient Transfection of Recombinant PDE3A
To investigate the properties of the protein encoded by the mouse oocyte ORF, three different constructs were generated and subcloned in pcDNA3: the full-length (1141-aa) PDE3A, and two different NH2-terminal truncations, Delta 1 (346aa) of 795 aa and Delta 2 (608aa) of 533 aa. Expression of the cDNAs corresponding to full-length PDE3A and the two truncated PDE3A induced a 5- to 15-fold increase in PDE activity compared to mock transfected cells (see below, Fig. 6), confirming that the clones encode an active protein. The full-length PDE3A had kinetic properties similar to those of the human and rat PDE3A with a Km for cAMP of 0.2 µM, whereas the Km of the Delta 1 PDE3A was 0.5 µM (data not shown).
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Recombinant PDEs were immunoprecipitated with an anti-HA antibody and analyzed by Western blotting using anti-PDE3A antibody. Full-length PDE3A and Delta 1 and Delta 2 constructs were translated as 135 kDa, 100 kDa, and 70 kDa proteins, respectively (Fig. 5). These data indicate that the mouse PDE3A size is similar to those determined for human and rat heart PDE3A.
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Subcellular Localization of the Recombinant PDE3A Protein
When the properties of PDE recovered from oocytes are compared to those of the recombinant PDE3A, a major difference was observed in the compartmentalization of the two proteins (Fig. 6). Although the full-length PDE3A was consistently recovered in the particulate fractions of transfected cells (Fig. 6), the rat and mouse oocyte PDE behaved as an entirely soluble molecule (Fig. 6A). Because the PCR analysis suggested that the 5'-end of the PDE3A is underrepresented, we investigated the solubility of the truncated PDE3A products. In both cases, the truncated PDEs behaved as entirely soluble proteins, suggesting that the PDE3A expressed in the oocyte may represent a truncated PDE3A (Fig. 6). Numerous attempts to determine the molecular weight of the PDE3A recovered from oocytes using available antibodies have been, however, unsuccessful.
Comparison of the Pharmacological Properties of the Recombinant PDE3A and the Oocyte PDE
It is widely accepted that PDE inhibition blocks spontaneous and LH-induced oocyte maturation. Furthermore, previous data from our laboratories have indicated that PDE3 inhibitors are effective in blocking oocyte maturation in vitro and in vivo. To determine conclusively whether PDE3A is the PDE responsible for cAMP degradation and resumption of meiosis in oocytes, we have determined the rank of potency of different PDE inhibitors in blocking the activity of recombinant PDE3A. The rank of potency was then compared with the potency to inhibit the PDE activity derived from COCs and their inhibitory effect on oocyte maturation. Because rat and mouse oocyte PDE activity were identical, rat COCs were used for this comparison. In addition, cilostamide inhibited the PDE activity of both full-length and Delta 1 constructs with an IC50 of 0.05 µM (Fig. 7). Therefore, the full-length protein was used for further comparison. As shown in Figure 8, there was a highly significant correlation (P 
0.001) between the inhibition (IC50) of the recombinant PDE3A and the PDE derived from oocytes, as well as the inhibition of oocyte maturation. Among the compounds tested, hypoxanthine, a putative natural suppressor of oocyte maturation, inhibited the recombinant PDE3A, the oocyte-derived PDE activity, and oocyte maturation with an IC50 of 0.5–1 mM.
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DISCUSSION
The data described above demonstrate that PDE3A is the predominant PDE active in the mouse oocyte. Moreover, biochemical and pharmacological evidence indicate that this PDE isoform is involved in the control of oocyte maturation. Although a message encoding a full-length PDE3A was retrieved from the oocyte, the data collected suggest the presence of a truncated and soluble PDE3A in rodent oocytes.
Two PDE3 genes are present in the mammalian genome [13, 19]. The PDE3 proteins encoded by these genes are characterized by a high affinity for cAMP and cGMP and are sensitive to cilostamide and other inhibitors that affect cardiac function, platelet aggregation, and smooth muscle contractility. Complementary DNAs encoding two different proteins have been retrieved from human and rat tissues [15–17]. Here we have identified a mouse PDE3 that is hortologous to rat and human PDE3A. An alignment of human, rat, and mouse PDE3A protein sequences indicated a high degree of structural homology, including the presence of putative membrane-association domains and several downstream consensus sequences (RRxS or RxRxxS) for phosphorylation by PKA and PBK. Six predicted helical transmembrane segments were identified using the pSPORT II Prediction algorithm in mouse PDE3A. That these sequences are required for binding to particulate structures is indicated by the finding that removal of these sequences produces a protein recovered mostly in the soluble fraction of the cell. This finding is consistent with the observation made with human PDE3 where deletion mutagenesis has identified these domains as critical for membrane association [26].
The recovery of the oocyte PDE exclusively in the soluble fraction of the homogenate is inconsistent with the behavior of the full-length PDE3A that was recovered mostly in the particulate fraction of the transiently transfected cells. This discrepancy suggests that the PDE3A expressed in oocytes may not contain the 5'-end of the ORF that encodes the membrane anchoring function. At present it is unclear whether a novel splicing variant is expressed in these cells, or whether the soluble form is generated posttranslationally. Although the expression of the full-length PDE3A mRNA in mouse and rat oocytes was confirmed by RT-PCR analysis and is consistent with previous in situ hybridization data, the amplification of the 5'-end of the sequence was not as efficient as that for the core or the 3'-end of the ORF. From this finding it can be inferred that not all PDE3A messages contain the entire mouse PDE3A ORF that we have identified. Furthermore, Northern blot analysis of ovary mRNA indicated the presence of additional transcripts different from that found in the heart. However, a more detailed analysis of the properties of these lower molecular weight species has failed to reveal the significance of these shorter mRNAs. A message distinct from the full-length mRNA has been observed in placenta where an intronic promoter controls the expression from an AUG internal to the ORF [27, 28]. Whether the same mechanism is responsible for the generation of the oocyte form remains to be determined.
The presence of a PDE3 in oocyte is consistent with other published data on the properties of the oocyte hydrolytic activity. Early studies on resumption of meiosis have shown that injection of cGMP blocks oocyte maturation in mouse oocytes [29]. Considering that cGMP, although hydrolyzed by PDE3A, acts as a competitive inhibitor of cAMP hydrolysis, it is likely that the mechanism of cGMP action is to inhibit this enzyme and to increase cAMP which, in turn, blocks oocyte maturation. This cGMP inhibitory effect on PDE3A and the consequent increase in cAMP has been, for instance, demonstrated in platelets [30]. Along the same line, in Xenopus oocytes, PDE3 inhibitors block oocyte maturation suggesting that a PDE with the pharmacological properties of PDE3 is expressed in amphibian eggs [31]. Bornslaeger et al. [29] also suggested the presence of a calmodulin-regulated PDE in mouse oocytes; however, the evidence thus far accumulated indicates that this form is expressed at low levels in rodent oocytes and is not the form hydrolyzing the pool of cAMP that controls oocyte maturation.
A pharmacological profiling of different compounds demonstrates a highly significant correlation between their potency in inhibiting recombinant PDE3A and PDE3 retrieved from COCs in a cell-free assay and their efficacy in inhibiting resumption of meiosis. These data again are consistent with the conclusion that PDE3A is the form involved in the control of the cAMP pool that inhibits oocyte resumption of meiosis. In view of the possible use of PDE3 inhibitors as contraceptives [24], it remains to be determined whether the pharmacological properties of the oocyte PDE3A allow discrimination from the PDE3 expressed in somatic cells.
It should be noted that the correlation for pimobendan was not as clear as that for the other PDE3 inhibitors. In the heart, pimobendan has calcium-sensitizing effects in addition to PDE3 inhibition [32]. It is then possible that the higher potency in inhibiting GVBD may be due to additional effects on Ca2+ signaling in the oocyte. Finally, the IC50 of hypoxanthine on COCs should be regarded with some caution because this compound is a nonselective PDE inhibitor and therefore also inhibits the PDEs derived from cumulus cells.
It has been proposed that a naturally occurring PDE inhibitor is present in the follicular fluid and that this is the substance responsible for maintaining the oocyte maturation arrest [1, 33]. Downs et al. [34] have identified this substance as hypoxanthine, and among its proposed mechanisms of action is the inhibition of the oocyte PDE. Under our assay conditions hypoxanthine inhibits the PDE3A derived from the oocyte. At the concentration of hypoxanthine most frequently used (4 mM), and assuming complete equilibration with the intraoocyte environment, this concentration of hypoxanthine inhibits PDE3A by more than 80%. This inhibition should be sufficient to increase cAMP levels substantially and maintain the meiotic block in most of the oocytes.
In conclusion, we have provided evidence that PDE3A is the major PDE expressed in both mouse and rat oocytes. The pharmacological and biochemical properties of the native oocyte PDE are identical to those of a recombinant PDE3A encoded in an oocyte PDE3 clone. Moreover, pharmacological studies demonstrate that this PDE3A is the form involved in the control of oocyte maturation. The mechanisms yielding soluble PDE3A from the mRNA coding full-length PDE3A with the transmembrane region remain to be determined.
ACKNOWLEDGMENTS
The authors thank Drs. J. Eppig and H. Hidaka for the mouse oocyte cDNA library and cilostamide, respectively, and Dr. Alex Tsafriri for the stimulating discussions. We are also indebted to Caren Spencer for editorial assistance.
FOOTNOTES
First decision: 5 January 2001.
1 The work described was, in part, supported by National Institutes of Health P50 HD31398 grant and by a grant from N.V. Organon (both to M. Conti). 
2 Correspondence: Marco Conti, Division of Reproductive Biology, Stanford University School of Medicine, 300 Pasteur Dr., Room A344, Stanford, CA 94305-5317. FAX: 650-725-7102; marco.conti{at}stanford.edu
3 Current address: Department of Obstetrics and Gynecology, Tokushima Prefectural Central Hospital, 1-10-3, Kuramoto, Tokushima 770-8539, Japan.
Accepted: March 5, 2001.
Received: December 8, 2000.
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D. Nogueira, R. Ron-El, S. Friedler, M. Schachter, A. Raziel, R. Cortvrindt, and J. Smitz Meiotic Arrest In Vitro by Phosphodiesterase 3-Inhibitor Enhances Maturation Capacity of Human Oocytes and Allows Subsequent Embryonic Development Biol Reprod, January 1, 2006; 74(1): 177 - 184. [Abstract] [Full Text] [PDF]
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 L. M Mehlmann Stops and starts in mammalian oocytes: recent advances in understanding the regulation of meiotic arrest and oocyte maturation Reproduction, December 1, 2005; 130(6): 791 - 799. [Abstract] [Full Text] [PDF]
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D Nogueira, R Cortvrindt, B Everaerdt, and J Smitz Effects of long-term in vitro exposure to phosphodiesterase type-3 inhibitors on follicle and oocyte development Reproduction, August 1, 2005; 130(2): 177 - 186. [Abstract] [Full Text] [PDF]
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 J. Cheng, M. A. Thompson, H. J. Walker, C. E. Gray, M. M. Diaz Encarnacion, G. M. Warner, and J. P. Grande Differential regulation of mesangial cell mitogenesis by cAMP phosphodiesterase isozymes 3 and 4 Am J Physiol Renal Physiol, November 1, 2004; 287(5): F940 - F953. [Abstract] [Full Text] [PDF]
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D. Nogueira, R. Cortvrindt, D.G. De Matos, L. Vanhoutte, and J. Smitz Effect of Phosphodiesterase Type 3 Inhibitor on Developmental Competence of Immature Mouse Oocytes In Vitro Biol Reprod, December 1, 2003; 69(6): 2045 - 2052. [Abstract] [Full Text] [PDF]
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D. Nogueira, C. Albano, T. Adriaenssens, R. Cortvrindt, C. Bourgain, P. Devroey, and J. Smitz Human Oocytes Reversibly Arrested in Prophase I by Phosphodiesterase Type 3 Inhibitor In Vitro Biol Reprod, September 1, 2003; 69(3): 1042 - 1052. [Abstract] [Full Text] [PDF]
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J.T. Jensen, K.M. Schwinof, M.B. Zelinski-Wooten, M. Conti, L.V. DePaolo, and R.L. Stouffer Phosphodiesterase 3 inhibitors selectively block the spontaneous resumption of meiosis by macaque oocytes in vitro Hum. Reprod., August 1, 2002; 17(8): 2079 - 2084. [Abstract] [Full Text] [PDF]
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F. J. Richard, A. Tsafriri, and M. Conti Role of Phosphodiesterase Type 3A in Rat Oocyte Maturation Biol Reprod, November 1, 2001; 65(5): 1444 - 1451. [Abstract] [Full Text] [PDF]
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