New insight into the role of phosphodiesterase 3A in porcine oocyte maturation
© Sasseville et al; licensee BioMed Central Ltd. 2006
Received: 15 June 2006
Accepted: 12 October 2006
Published: 12 October 2006
The ovulatory surge of gonadotropins triggers oocyte maturation and rupture of the ovarian follicle. The resumption of nuclear maturation in the oocyte from the prophase stage is characterized by germinal vesicle breakdown (GVBD). It has previously been shown that specific inhibition of cAMP degradation by PDE3 prevents the resumption of oocyte meiosis. However, no report has characterized the activity of PDE3 in the porcine oocyte, or the implication of the cAMP-PDE3 pathway in the entire nuclear maturation process. In this study, PDE3 activity in the oocyte was assessed during in vitro maturation (IVM) and the possible roles of the cAMP-PDE3 pathway in the resumption and progression of meiosis were investigated in terms of different models of oocyte maturation.
Cyclic AMP-degrading PDE activity was detected in the cumulus-oocyte complex (COC) and was partially inhibited by a specific PDE3 inhibitor, cilostamide. When measured only in the denuded oocyte, PDE activity was almost completely inhibited by cilostamide, suggesting that cAMP-PDE3 activity is the major cAMP-PDE in porcine oocytes. PDE3A mRNA was detected by RT-PCR. PDE3 activity did not vary significantly during the early hours of IVM, but a maximum was observed at 13 hours. In cumulus-oocyte complexes, meiosis resumed after 20.81 hours of culture. PDE3 inhibition no longer maintained meiotic arrest if sustained beyond 17.65 hours of IVM, 3 hours prior to resumption of meiosis. Thereafter, PDE3 inhibition progressively lost its efficacy in GVBD. When the protein phosphatase 1 and 2A inhibitor okadaic acid was continuously or transiently (3 hours) present during IVM, meiosis resumed prematurely; PDE3 inhibition was unable to prevent GVBD. However, PDE3 inhibition in COC treated with OA for 3 hours significantly delayed meiosis at the intermediate stage.
The present investigation has demonstrated that PDE3A is the major cAMP-degrading PDE in the oocyte. It regulates the resumption of meiosis until 3 hours prior to GVBD and transiently affects meiotic progression.
Oogonia entering meiosis progress to the diplotene stage of the first meiotic prophase, at which they display a characteristic nucleus commonly known as the germinal vesicle (GV). Oocytes arrested at the GV stage, prophase I of meiosis, enter a growth phase during which the follicle differentiates from the primary to the preovulatory stage. Oocytes acquire the capacity to resume meiosis during this period and rely on 3'5'-cyclic adenosine monophosphate (cAMP) to prevent premature resumption. Cyclic AMP is produced in the somatic granulosa cells and transferred through gap-junction channels . Recent findings have shown that cAMP-producing capacity is also important in maintaining the prophase I-arrested state in oocytes . Cyclic AMP activates the cAMP-dependent protein kinase (PKA) by binding to the two regulatory subunits, liberating the two active catalytic subunits. High concentrations of cAMP and correspondingly high PKA activity within the oocyte prevent the resumption of meiosis, as shown using cAMP analogues [3, 4], adenylyl cyclase activators [5, 6] and invasive adenylyl cyclase treatments . Microinjection of Xenopus oocytes with the catalytic subunit of PKA also maintains them in the GV stage . However, the PKA-mediated mechanism that maintains meiotic arrest is not fully understood, although interesting advances have recently been made on possible PKA targets in mouse oocytes, such as CDC25B phosphatase and Wee1B kinase, two cell cycle regulators .
Cyclic AMP is degraded by members of the phosphodiesterase (PDE) family . PDEs are important regulators of ovarian physiology. During the past few years, PDE3 family-specific inhibitors (e.g. cilostamide, milrinone, Org 9935) have been shown to block the resumption of meiosis efficiently in murine , rat , bovine [13, 14], porcine [15, 16], macaque  and human  oocytes. PDE3A- and PDE4D-null female mice respectively show infertility and impaired fertility [19, 20]. Interestingly, PDE3A-deficient mice ovulate fully-grown oocytes, but they fail to resume meiosis . In rat oocytes, a cAMP-degrading activity sensitive to cilostamide (PDE3-specific) is increased 2-fold prior to resumption of meiosis, strongly suggesting that cAMP degradation is actively regulated in the oocyte and that regulation of PDE3A is part of the mechanism controlling resumption of meiosis .
Nuclear maturation in the oocyte and rupture of the follicle are triggered by an ovulatory surge of gonadotropins in vivo. The latter is characterized by breakdown of the germinal vesicle (GVBD). GVBD is correlated with an increased activity of M phase-promoting factor (MPF). MPF is essential for GVBD, chromosome condensation, formation of microtubules around the condensed chromosomes and their organization into a bipolar structure . The complete molecular pathways linking the endocrine cues to the first activation of MPF in the oocyte during nuclear maturation are unclear. However, it is known that gonadotropins trigger multiple phosphorylation cascades and second messenger signalling pathways in the somatic compartment of the ovarian follicle, and these are critical for the correct timing of oocyte maturation. Simultaneously with activation of the MPF, mitogen-activated protein kinase (MAPK) activates phosphorylation in the oocyte and is believed to be implicated in chromosome condensation and microtubule organisation . After its initial increase, MPF activity stays high until metaphase I before decreasing prior to homologous chromosome segregation and extrusion of the first polar body . A second increase in MPF activity maintains metaphase II prior to sperm penetration. Although this second activation is known to depend on factors such as cyclin B synthesis, presence of the nucleus and MAPK activity, the precise implication of the PDE3A/cAMP/PKA pathway remains unknown [22, 24]. Our laboratory has recently described physiological evidence showing the pivotal role of cAMP and its modulation by PDE3 in regulating the resumption of meiosis in porcine oocytes . Since no study has reported the progression of PDE3 activity during porcine oocyte nuclear maturation, the goal of the present study was to measure PDE3 activity in the oocyte during in vitro maturation (IVM) and to characterize the effect of PDE3 inhibition on different steps of nuclear maturation.
Contributions of different cAMP-degrading PDEs in the oocyte
Time-dependent ability of specific PDE3 inhibition to maintain COC in meiotic arrest
Effects of short OA exposure in the presence of PDE3 inhibition on oocyte nuclear maturation
This study demonstrates that in porcine oocytes, PDE3A is the major active cAMP-PDE; it efficiently regulates the resumption of meiosis until 3 hours before GVBD and transiently affects meiotic progression. Four lines of evidence support this conclusion: (1) cilostamide-sensitive cAMP-PDE activity in the oocyte represents the main cAMP-PDE activity; (2) PDE3A mRNA was detected in oocyte cDNA by RT-PCR; (3) PDE3 inhibition after 15 hours of IVM still prevents the resumption of meiosis; (4) PDE3 inhibition delays nuclear maturation in OA-treated oocytes.
Total and cilostamide-sensitive (PDE3) PDE activities in porcine COC and denuded oocytes freshly recovered from the ovary (figure 1A and 1B) were about the same as reported for mouse and rat oocytes [11, 21]. Furthermore, PDE3 is the predominant PDE activity in the oocyte (figure 1A and 1B), as reported for rodents [11, 21]. This result is also consistent with the presence of PDE3A protein detected by Western blotting in porcine oocytes . PDE3A messenger RNA was detected in the oocyte by in situ hybridization in rat and by RT-PCR in mouse and these findings are consistent with the presence of PDE3A mRNA in porcine oocyte (figure 1D) [11, 27]. The reported levels of cAMP during IVM range from 2 to 10 fmol , whereas the level of cAMP-degrading activity in denuded porcine oocytes reported in this study is around 2.5 fmol of cAMP degraded per minute per oocyte throughout IVM (figure 1B and 2). These results suggest that porcine oocytes replenish their cAMP content approximately every minute. Under our culture conditions, despite the maximum at 13 hours, total and cilostamide-sensitive (PDE3) phosphodiesterase activities were not statistically different during IVM (figure 2). One explanation could be that the timing of GVBD is regulated by multiple cAMP-generating mechanisms. The modulation of cAMP level in the oocyte involves both the permeability of the gap-junctions and adenylyl cyclase activity [1, 29]. In addition, the present results do not exclude a possible modulation of PDE3A activity by phosphorylation, which would be difficult to measure because highly synchronized GVBD oocytes would be needed. In the rat, a clear increase of PDE3A activity occurs 30 minutes prior to GVBD . In the pig, the present study shows a peak in cAMP-PDE3 activity 8 hours before GVBD and a loss of the capacity of a PDE3-specific inhibitor to prevent the resumption of meiosis 3 hours before GVBD. Control of oocyte PDE activity has been reported in Xenopus, where microinjection of constitutively active PKB has been shown to increase activity and the GVBD rate . Implication of PKB in the modulation of PDE activity remains a possible mechanism for the resumption of meiosis. From the 6 putative PKA and PKB serine phosphorylation sites in the N-terminus of the murine PDE3A protein sequence (NM_018779.1) described by Shitzukawa and coworkers , 5 are present in rat (NM_017337.1), pig (NM_213736.1) and human (NM_000921.3). In the other gene of the same family, PDE3B, at least two of these sites are conserved between rodents and human, and serine-302 is reportedly targeted by PKA and PKB in vitro [31, 32]. PDE3 has also been reported to be activated by partially purified MAPK and PKB in rat hepatocytes . PKB activation prior to GVBD has never been shown in pig oocytes.
Because cAMP is probably involved in controlling re-entry into the cell cycle in oocytes, the next step was to investigate the role of cAMP in meiotic progression. It was confirmed that OA treatment overcame PDE3-specific inhibition, suggesting that PP1/2A inhibition activates signalling molecules downstream of cAMP-PDE3 in the pig oocyte (figure 5B). This hypothesis is supported by previous reports showing that IBMX-arrested or milrinone (PDE3 inhibitor)-arrested mouse oocytes resume meiosis when PP1/2A is inhibited with OA [25, 34]. In the latter study, PDE3 inhibition of OA-treated oocytes delayed GVBD and reduced MPF activation . Interestingly, when using a spontaneous denuded oocyte maturation model, PDE3 inhibition of 3 hours OA-treated oocytes result in higher rates of intermediate stage oocyte compared to OA-treated oocytes alone after 48 hours of IVM (data not shown). These results support our findings, showing that PDE3 inhibition affects meiotic progression in OA-treated oocytes probably by delaying the decrease of MPF activity that allows nuclear maturation to be completed (figures 8 and 9).
In vertebrates, the direct MPF regulators CDC25B and Myt1 are candidate targets for PP1/2A . In fission and budding yeasts, a specific form of PP2A (PP2A-π) directly protects centromeric cohesins from phosphorylation and cleavage, preventing premature sister chromatin separation . The cohesion of centromeres in sister chromatids during metaphase I is of critical importance. In the present study, fifty percent of oocytes remained at the intermediate stage after 36 and 48 hours of IVM (figure 9F), suggesting that inhibition of PP1/2A with OA affects the segregation machinery in a way that impairs homologous chromosome segregation without causing complete meiotic spindle dissolution (figure 6). Similarly, transient PP1/2A inhibition with a relatively high dose of OA (1.24 μM) compromised anaphase initiation and stopped meiosis in metaphase I in mouse oocytes, while low doses (0.124 μM and 0.0124 μM) allowed normal meiosis to continue until metaphase II arrest but with high rates of chromosomal abnormalities . In the present study, the transient OA exposure approach allowed meiosis to proceed to the mature stage. This approach was used successfully in a previous study to reduce the incidence of chromosome segregation defects and to improve the percentage of oocytes reaching metaphase II compared to the continuously OA-treated group .
Wang and coworkers have shown that PKA inhibition is one of the earliest events in progesterone-induced resumption of meiosis in Xenopus oocytes . Our results suggest that effective PKA inhibition occurs late after the start of IVM, since stimulation of the cAMP/PKA pathway by PDE3 inhibition after 15 hours of IVM prevents GVBD completely (figure 3A). PDE3 inhibition after 19 hours of IVM has no effect on the completion of meiosis (figure 4). In Xenopus, a similar experiment also demonstrated that reactivating the cAMP/PKA pathway after GVBD has no effect on the completion of meiosis . OA-stimulated oocytes react differently by being delayed at an intermediate stage in the presence of the PDE3 inhibitor (figures 8 and 9). The reason for this is unclear; however, the capacity of the cumulus cells to transfer cAMP to the oocyte could explain the effect. PDE3 inhibition, combined with insufficient cAMP transfer, could prevent intracellular cAMP levels reaching a certain threshold and compromise PKA activation. The level of connexin 43 and its phosphorylation status have been shown to vary in porcine cumulus cells during IVM . Cyclic AMP has also been shown to inhibit proteasomal proteolysis in rat sertoli cells, COS-1 African monkey kidney cells and Y1 mouse adrenocortical tumour cells [41, 42]. Interestingly, porcine oocytes stop their nuclear maturation in metaphase I when proteasomal degradation is inhibited . It is possible that PDE3 inhibition in OA-treated oocytes leads to a rise in cAMP level sufficient to prevent proteasomal degradation and consequently delays the transition from intermediate to mature stage. The mechanism by which PDE3 inhibition maintains oocytes in the intermediate stage under OA-treated conditions will require further investigation.
In summary, the present investigation has revealed new insights into cAMP regulation during the resumption of meiosis in porcine oocytes. It has demonstrated that PDE3A is the major cAMP-degrading PDE activity and regulates meiotic resumption until 3 hours prior to GVBD. It has also demonstrated that PDE3 inhibition affects meiotic progression in OA-treated oocytes. We suggest that cAMP-PDE3A modulates meiotic resumption via a factor located upstream of PP1/2A substrates. This study highlights a distinctive characteristic of porcine oocyte nuclear maturation that makes it a valuable model for studying cAMP modulation during maturation: the very long latency period before the resumption of oocyte meiosis and the late release of the control maintained by the cAMP-PDE3 pathway over prophase arrest. Further experiments will provide a better understanding of the mechanisms involved in the control of PDE3A activity during IVM and the fundamental role of the cAMP/PKA pathway in the molecular mechanisms leading to meiotic maturation.
Unless otherwise notified, all chemicals were purchased from Sigma Chemical Co (St. Louis, MO, USA).
Ovaries were collected as previously described . Briefly, ovaries were recovered from a local slaughterhouse, placed in saline (0.9% NaCl) containing antibiotics and antimycotics (100,000 IU/l penicillin G, 100 mg/l streptomycin, 250 μg/l amphotericin B) and maintained at 34°C. Upon arrival in the laboratory, they were rinsed in saline containing antibiotics and antimycotics at 34°C.
COC were suspended in hypotonic buffer (Tris-HCl 20 mM, pH 7.4, ethylenedioxy-diethylene-dinitrilo-tetraacetic acid (EDTA) 1 mM, ethylene glycol-bis-(2-aminoethyl)-N, N, N', N'-tetraacetic acid (EGTA) 0.2 mM, NaF 50 mM, benzamidine 50 mM, sodium pyrophosphate 10 mM, aprotinin 4 μg/ml, pepstatin 0.7 μg/ml, soybean trypsin inhibitor 10 μg/ml, leupeptin 0.5 μg/ml and phenylmethylsulfonyl fluoride 2 mM) and homogenized by 9 freezing-thawing cycles along with vortex agitation. In all experiments, the hypotonic buffer contained 0.5% Triton X-100. Tris-HCl was purchased from Fisher Scientific Limited (Nepean, ON, Canada). The homogenate was centrifuged for 20 min at 14000 × g to obtain the supernatant. PDE activity was assessed at 34°C in 200 μl final volume with 1 μM cAMP as substrate following the method of Thompson et al.  with minor modifications ; the solution comprised 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, QC, Canada) (1 × 105 cpm/tube; 30 Ci/mmol). Measurements were performed in the presence of PDE inhibitors: 3-isobutyl-methylxanthine (IBMX) (1mM, non-specific), cilostamide (Cil) (10 μM, PDE3-specific) and rolipram (Rol) (10 μM, PDE4-specific). Cilostamide and rolipram were purchased from Biomol (Plymouth Meeting, PA, USA). To determine the contributions of different PDEs to the overall PDE activity, the activity measured in the presence of the respective inhibitors was subtracted from the total. Each enzyme assay was performed in triplicate for each experiment and each experiment was repeated at least three times.
RNA extraction and RT-PCR
Primers used for PCR reactions. The sequences of the primers used to amplify PDE3A and β-actin mRNA in the oocyte cDNA are shown, along with the expected sizes of the PCR products. Sasseville et al. 2006.
PCR product expected size (bp)
Culture of cumulus-oocyte complexes (COCs) and assessment of meiotic status
Media, COC collection, selection and culture conditions were described previously . Briefly, a group of 30 to 50 COCs were selected and washed three times with HEPES-buffered Tyrode medium containing 0.01% (w/v) polyvinyl alcohol (PVA-HEPES) . They were then cultured in Nunclon™Δ 4-well dishes in 500 μl standard porcine IVM culture medium, namely; BSA-free NCSU 23 medium  containing 25 μM beta-mercaptoethanol (Bio Rad), 0.1 mg/ml cysteine, 10% (v/v) filtered porcine follicular fluid and gonadotropin supplements at final concentrations of 2.5 IU/ml hCG (Ayerst Laboratories, Inc., Philadelphia, PA, USA) and 2.5 IU/ml PMSG (Intervet, Whitby, ON, Canada). Wells were covered with a mineral oil overlay. Oocyte nuclear status was assessed using aceto-orcein as previously described . Oocyte nuclear maturation status was classified as (1) GV stage oocyte when displaying a germinal vesicle, (2) intermediate stage oocyte when displaying germinal vesicle breakdown and condensed chromatin or metaphase I organised chromatin, or (3) mature stage oocyte when displaying anaphase I, telophase I or metaphase II DNA configuration.
Contributions of different cAMP-degrading PDEs in the oocyte
In experiment 1, cAMP-degrading PDE activity was measured in the COC after recovery from the ovary, and the individual contributions of the PDE3 and PDE4 isoenzymes were determined by the method described above. To evaluate the distribution of PDE activity further, overall PDE activity and specific PDE3 activity were measured in both COC and denuded oocytes. The results are presented as total and cilostamide-sensitive (PDE3) cAMP-PDE activities. The presence of PDE3A mRNA was confirmed in the oocyte by RT-PCR amplification. Total RNA was extracted and reverse transcribed from 100 denuded oocytes. The resulting cDNA was used as template for PDE3A PCR amplification. To verify the absence of contaminating genomic DNA from the RNA extract, PCR was also performed directly on the RNA sample (data not shown). In experiment 2, PDE activity was measured in the oocyte during IVM. After the IVM culture, COC were stripped of their cumulus cells and the denuded oocytes were rinsed in PVA-HEPES and snap-frozen in liquid nitrogen. Cyclic AMP-degrading PDE activity was measured and total, IBMX-sensitive and cilostamide-sensitive PDE activities are presented.
Time-dependent ability of specific PDE3 inhibition to maintain COC in meiotic arrest
In experiment 3, oocytes were cultured and meiotic maturation was monitored every 6 hours to evaluate the moment of resumption of meiosis. On the basis of previous studies, it is known that inhibiting PDE3 with cilostamide at the beginning of IVM prevents GVBD . To determine the cAMP-sensitive period during which oocytes were not irrevocably committed to resuming meiosis and could still be maintained at the GV stage, addition of cilostamide to the IVM medium was postponed and nuclear status was assessed after 24 hours of IVM. In experiment 4, to determine whether PDE3 inhibition affected meiotic progression, COC were kept in normal IVM conditions for the first 19 hours. Cilostamide was added to half of them and the other half were maintained under normal IVM conditions. The oocytes were denuded and were fixed for assessment of nuclear maturation after 19, 24, 30, 36 and 48 hours of IVM. Oocyte nuclear maturation status was classified as described above.
Effects of short OA exposure in the presence of PDE3 inhibition on oocyte nuclear maturation
In experiment 5, oocytes were continuously exposed to OA during IVM in order to study the effect of PDE3 inhibition on the resumption of meiosis in premature-GVBD oocytes treated with OA. COC were cultured in OA-supplemented media for 6 and 12 hours and the nuclear status of the oocytes was evaluated. The effect of PDE3-specific inhibition on the resumption of meiosis was assessed by exposing OA-treated oocytes to cilostamide for 24 hours of IVM. The results are reported as mean percentages of arrested, intermediate or mature stage oocytes. In experiment 6, to study the effect of PDE3 inhibition on the progression of meiosis past GVBD, three conditions of transient OA exposure were tested and compared to control conditions: (1) 1 hour OA-exposure followed by 11 or 23 hours of normal IVM; (2) 3 hours OA exposure followed by 9 or 21 hours of normal IVM; and (3) continuous OA exposure for 12 or 24 hours. The percentages of arrested, intermediate and mature stage oocytes, as described for experiment 4, are reported here after 12 hours and 24 hours of IVM. The percentages of oocytes displaying a scattered condensed chromatin configuration were also evaluated and are included in the intermediate stage percentage. Representative pictures of normal metaphase I and OA-treated condensed scattered chromatin oocytes are shown. In experiment 7, to assess the effect of PDE3 inhibition on 3 hours OA-treated COC, the COC were treated with OA for three hours followed either by normal culture conditions or treatment with the PDE3 inhibitor cilostamide. Oocytes were collected after 24 hours of IVM for assessment of nuclear maturation. The percentages of oocytes in arrested, intermediate or mature stages after IVM are reported here. In experiment 8, the effects of PDE3 inhibition on OA-treated COC were studied throughout the IVM period. Oocytes were exposed for 3 hours to OA followed by PDE3 inhibition up to 48 hours. Samples were fixed at 12, 24, 36 or 48 hours for assessment of nuclear status.
Statistical analyses were performed using Prism 4.00 GraphPad for Windows (GraphPad Software, San Diego, CA, USA). The onsets of 50% GVBD and of 50% cilostamide efficacy were calculated using non-linear regression with a sigmoidal dose-response equation. Statistical significances of PDE activity measurements and nuclear maturation status were assessed using one-way ANOVA analysis and the Bonferroni multiple comparison post-hoc test to identify individual differences between means. Probabilities of less then 0.05 were considered statistically significant.
3'5'-cyclic adenosine monophosphate
cAMP-dependent protein kinase
M-phase promoting factor
mitogen-activated protein kinase
in vitro maturation
protein phosphatase 1/2A
Thanks to Dr Mario Mayes for a critical review of the manuscript. Special thanks to Isabelle Laflamme for professional technical assistance. We also thank Richard Prince and O'Neil Fecteau for collecting ovaries at the slaughterhouse. MS is supported by a NSERC and FQRNT PhD fellowship. This project was supported by the Canadian Institutes for Health Research in the Program for Oocyte Health.
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