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BOR - Papers in Press, published online ahead of print December 13, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.057828
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BIOLOGY OF REPRODUCTION 76, 589–597 (2007)
DOI: 10.1095/biolreprod.106.057828
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Adenosine 5'-Monophosphate Kinase-Activated Protein Kinase (PRKA) Activators Delay Meiotic Resumption in Porcine Oocytes1

Mario A. Mayes 3 4, Martin F. Laforest 3 4, Christine Guillemette 4, Robert B. Gilchrist 5, and François J. Richard 2 4

Centre de Recherche en Biologie de la Reproduction,4 Département des Sciences Animales, Faculté des Sciences de l'Agriculture et d'Alimentation, Université Laval, Québec, Canada G1K 7P4 Research Centre for Reproductive Health,5 Discipline of Obstetrics and Gynaecology, Medical School, University of Adelaide, Adelaide, South Australia 5005, Australia

ABSTRACT

Adenosine monophosphate-activated kinase (PRKA) is a serine/threonine kinase that functions as a metabolic switch in a number of physiological functions. The present study was undertaken to assess the role of this kinase in nuclear maturation of porcine oocytes. RT-PCR and immunoblotting revealed the expression of the PRKAA1 subunit in granulosa cells, cumulus-oocyte complexes (COC), and denuded oocytes (DO). Porcine COC and DO contained transcripts that corresponded to the expected sizes of the designed primers for PRKAB1 and PRKAG1. The PRKAA2 subunit was detected in granulosa cells and COC, whereas the PRKAG3 subunit was not detected in granulosa cells, COC or DO, whereas it was detected in the heart. The PRKAA1 protein was detected in granulosa cells, COC, DO, and zona pellucida (ZP). In the presence of the pharmacological activator of PRKA 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP), COC were transiently maintained in meiotic arrest in a fully reversible manner. This inhibitory effect was not observed in DO. Other known PRKA activators, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and metformin, also blocked meiotic resumption in COC. In contrast to mouse oocytes, in which PRKA activators reverse the inhibitory effect of PDE3 inhibitors, this combination still blocked meiotic resumption in porcine COC. These results demonstrate that the meiotic resumption of porcine COC is transiently blocked by PRKA activators in a dose-dependent manner, and that this effect is dependent on PRKA activity in cumulus cells. The present study describes a new role for PRKA in regulating meiotic resumption in COC and strongly suggests that cumulus cells play an essential role in the control of porcine oocyte maturation through the PRKA metabolic switch.

follicle, gamete biology, meiosis, ovum

INTRODUCTION

Meiosis in mammalian oocytes begins during fetal life [1]. It progresses to prophase I and stops at the dictyate stage or germinal vesicle stage (GV) for up to several years. The oocyte resumes meiosis in vivo following the ovulatory surge in lutenizing hormone [2, 3], whereas in vitro, oocytes spontaneously resume meiosis once they are removed from the follicular environment [4]. The first morphological sign of meiotic resumption is the breakdown of the nuclear membrane or germinal vesicle breakdown (GVBD). The second messenger cAMP plays a key role in the regulation of oocyte maturation [5]. However, the exact mechanism whereby cAMP maintains the oocyte in meiotic arrest is not fully understood. In most species, a high intracellular level of cAMP in the oocyte maintains meiotic arrest [6–9], whereas a low level of cAMP allows meiotic resumption [10–12]. Phosphodiesterases (PDEs) are enzymes that hydrolyze cyclic nucleotides (cGMP and cAMP) to their inactive forms (5'-GMP and 5'-AMP, respectively). Inhibitors of cyclic nucleotide PDEs [13–20] and membrane-permeable analogs of cAMP, such as dibutyril-cAMP and 8-bromo-3',5'-cAMP [5, 7, 11, 21–23], prevent spontaneous meiotic resumption.

Until recently, adenosine monophosphate (AMP) was considered to be an inactive product of cAMP degradation by PDEs. However, it has become clear that AMP is a potent allosteric activator of adenosine monophosphate kinase (PRKA; formerly known as AMPK) [24]. This member of the PRKA/SNF1 protein kinase family is a well-conserved heterotrimeric protein with a 63-kDa catalytic {alpha} subunit (PRKAA) and regulatory ß (PRKAB) and {gamma} subunits (38 kDa and 35 kDa, respectively) [25, 26]. The {alpha} (PRKAA1 and PRKAA2) and ß (PRKAB1 and PRKAB2) subunits have two known isoforms, whereas the {gamma} subunit has three isoforms (PRKAG1, PRKAG2 and PRKAG3). There are at least 12 possible heterotrimeric combinations, since all of the isoforms appear to be capable of forming complexes [25, 27, 28]. This ser/thr kinase phosphorylates a range of metabolic enzymes [29] and has been implicated in an increasing number of physiological functions, including exercise [30], glucose uptake [31, 32], glycolysis [33], transcriptional regulation [34], lipolysis, fatty acid oxidation, and sterol synthesis [35].

Typically, PRKA is activated by an increase in the ratio of AMP to ATP [29, 36]. However, activation of PRKA can occur by allosteric activation, stimulation of phosphorylation of the {alpha} subunit on Thr-172 by upstream kinase(s) or inhibition of dephosphorylation by phosphatases [37–39]. There is good evidence that some, if not all, of these effects are antagonized by high concentrations of ATP [36]. In intact cells, PRKA can be activated with the adenosine analog 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) [40]. This adenosine analog, which is cell-permeable, is phosphorylated by adenosine kinase and accumulates in the cytoplasm as ZMP (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranosyl 5'-monophosphate), which in turn activates PRKA without disturbing the cellular AMP:ATP ratio [40]. Recently, it has been shown that metformin also stimulates PRKA without altering the AMP:ATP ratio [41–43]. Metformin is the most widely used drug for reducing blood sugar in Type 2 diabetic patients [44] and for the treatment of anovulation in women with polycystic ovary syndrome (PCOS) [45].

Recent reports implicate PRKA in the regulation of mouse oocyte maturation [21, 46, 47]. The activation of PRKA results in meiotic resumption of mouse oocytes maintained in meiotic arrest by a cAMP analog [46, 47]. Since the molecular mechanisms that govern oocyte maturation are notably different between rodents and non-rodent animals, the present study was undertaken to evaluate the role of PRKA activators in porcine oocyte maturation.

MATERIALS AND METHODS

Collection of Ovaries

Ovaries were collected from pre-pubertal gilts at a local slaughterhouse and transported in a thermos that contained saline solution (0.9% NaCl) supplemented with 100 000 IU/L penicillin G, 100 mg/L streptomycin, and 250 mg/L amphotericin B. Ovaries were maintained at 34°C and rinsed with saline upon arrival.

Chemicals

The specific PDE3 inhibitor cilostamide (CIL) was purchased from Biomol (Plymouth Meeting, PA) and the non-specific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) was obtained from Sigma Chemical Co. (St. Louis, MO). Millimolar stock concentrations of PDE inhibitor were dissolved in dimethylsulfoxide (DMSO) and stored at –20°C. The chemicals were added to the maturation medium approximately 4 h before the initiation of oocyte culture. The final concentrations of DMSO in culture medium never exceeded 0.1%. The control treatment consisted of oocytes incubated in medium without inhibitors or DMSO. Millimolar stocks of ZMP and AICAR were dissolved in the maturation medium and stored at –20°C. AICAR was purchased from Toronto Research Chemicals (North York, ON). Metformin and all the other chemicals used in the present study were purchased from Sigma.

Media

Oocytes were matured in BSA-free NCSU 23 (North Carolina State University) medium [48] that was supplemented with 25 µM beta-mercaptoethanol (Bio-Rad, Hercules, CA), 0.1 mg/ml cysteine, 10% (v/v) porcine follicular fluid, and gonadotropins (final concentrations of 2.5 IU/ml for hCG [APL; Ayerst Laboratories Inc., Philadelphia, PA] and 2.5 IU/ml for eCG [Folligon; Intervet, Whitby, ON, Canada]), as previously described [15, 49]. Porcine follicular fluid (pFF) was collected from follicles (2–6 mm in diameter) of pre-pubertal gilt ovaries using an 18G needle and a 10-ml syringe. After centrifugation at 1500 x g for 30 min at room temperature, the supernatant was filtered though 0.8-µm and 0.45-µm syringe filters and stored at –20°C until used [15].

Collection of Cumulus-Oocyte Complexes

Porcine cumulus-oocyte complexes (COC) were collected as described by Bureau and collaborators [49]. Follicles measuring 2–6 mm in diameter were aspirated with a 10-ml syringe and an 18G needle. The follicular contents were pooled in 50-ml conical tubes (Falcon, Franklin Lakes, NJ). After sedimentation, the pellet was washed twice in pFF. COC were recovered with the use of a stereomicroscope and transferred to a Petri dish that contained the supernatant of the follicular fluid. The COC were washed three times with Hepes-buffered Tyrode medium that contained 0.01% (w/v) polyvinyl alcohol (PVA-TLH) [50], and then subjected to their respective treatments. Groups of 25 to 30 COC were transferred into the wells of four-well multi-dishes (Nunc, Roskilde, Denmark) that contained 500 µl of maturation medium and 500 µl of mineral oil. The COC were cultured at 38.5°C in an atmosphere of 5% CO2 in air and 100% humidity. The culture medium was supplemented with gonadotropins once the oocytes were in culture.

Selection of COC and Preparation of Denuded Oocytes

Cultured COC were rigorously selected as previously described [15]. Briefly, the selected COC had at least three layers of clear and compact cumulus cells surrounding the oocyte. Oocytes with dark, pycnotic or expanded cumulus cells at the time of selection were discarded. In addition, oocytes with a very clear cytoplasm or small diameter were discarded. Denuded oocytes (DO) were obtained by removing the cumulus cells from selected COC before culture. Briefly, COC were transferred into a 2-ml centrifuge tube that contained 60 µl of PVA-TLH and repeatedly pipetted up and down until the cumulus cells were completely removed. The oocytes were rinsed twice in maturation medium. The DO were recovered under a stereomicroscope. Completely denuded oocytes with homogeneous cytoplasm were then allocated to their respective treatments. Porcine zona pellucidas (ZPs) were obtained by forcibly passing DO through a 27G needle and a 1 ml syringe in PVA-TLH until the oocytes were ruptured. The ZPs were recovered under a stereomicroscope, rinsed three times in PVA-TLH, and stored at –80°C until needed.

Evaluation of Nuclear Maturation

The stages of nuclear maturation were evaluated as previously described [15]. After 24 h in a fixative solution (ethanol:acetic acid, 3:1), the stages of nuclear maturation of the oocytes were evaluated under a contrast microscope at 100x and 400x magnification immediately after staining with 1% aceto-orcein [51]. Oocytes that contained a nuclear membrane were classified as being at the germinal vesicle (GV) stage, while those without a nuclear membrane were classified as having undergone meiotic resumption or germinal vesicle breakdown (GVBD).

RNA Extraction and RT-PCR

Total RNA was extracted according to the protocol provided with the absolutely RNA Microprep Kit (Stratagene, La Jolla, CA). Total RNA isolated was reversed-transcribed into cDNA using poly(dT) primer (Ambion, Austin, TX) with the Omniscript RT kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. PCR reactions were carried out under the following conditions: denaturation at 95°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 2 min, for a total of 30 (ACTB) or 35 (other genes) cycles, with a final extension step of 10 min at 72°C. The Taq polymerase was purchased from New England BioLabs (Beverly, MA). The PCR primers (Table 1) were designed according to the database sequences: Bos taurus ACTB (accession number AY_141970.1; 242 bp); Homo sapiens PRKAA1 (NM_006251; 523 bp); Homo sapiens PRKAA2 (NM_006252, 255 bp); Homo sapiens PRKAB1 (NM_006253, 230 bp); Homo sapiens PRKAG1 (NM_002733, 244 bp); and Homo sapiens PRKAG3 (AJ_249977, 268 bp). Additional amplifications were performed on an equivalent amount of RNA to exclude genomic DNA contamination. Amplified fragments were visualized by 1% agarose gel electrophoresis and staining with ethidium bromide. In order to validate the purity of the preparations, PCR reactions were carried out with 1 µl of the extracted total RNA serving as a template. The potential introduction of foreign DNA was monitored by performing the PCR reactions without adding any template to the master mix. The PCR products were sequenced and aligned with the known sequences from which the primers were designed.


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  		TABLE 1. PCR Primers used in the present study.

Immunoblotting

Protein samples were homogenized in hypotonic buffer (20 mM Tris-HCl, 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, 5 mg/ml Triton X-100, 2 mM PMSF). Protein samples (COC, DO, granulosa cells, heart tissues, and ZP fragments) were electrophoresed on an 8% SDS-polyacrylamide gel. The proteins were transferred to a Hybond-P membrane (GE Healthcare) using the Mini Protean 3 Cell apparatus (Bio-Rad). Membranes were blocked for 1 h with PBS that contained 0.05% (v/v) Tween-20 and 2% (v/v) ECL blocking agent (GE Healthcare). The first hybridization was performed overnight at 4°C in blocking buffer that contained the primary antibody anti-PRKAA1 (ab3759; Abcam, Cambridge, UK) diluted 1:50 000. The membranes were then washed three times in PBS-Tween and hybridized for 1 h at room temperature with the secondary antibody, peroxidase-conjugated anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA) diluted 1:100 000 in the blocking buffer. Detection was performed with ECL Advance (GE Healthcare) and the membranes were exposed on autoradiographic films.

Experimental Design

Effect of increasing concentrations of ZMP on the percentages of oocytes at the GV stage. COC were incubated in maturation medium in the presence of increasing concentrations (0, 0.10, 0.25, 0.50, 1.00 mM) of ZMP for 24 h.

Effects of culturing COC in the presence of ZMP for 22 and 44 h on the percentages of oocytes at the GV stage. COC were cultured in the presence or absence of ZMP (1 mM) for 22 h and 44 h.

Effects of AICAR and metformin on the percentages of oocytes at the GV stage. Other known activators of PRKA (AICAR and metformin) were also tested for their effects on oocyte nuclear maturation. COC were incubated in maturation medium for 24 h in presence of AICAR (1 mM) or metformin (0.10, 0.25, 0.50, 1.0 mM). Reversibility of exposure to the compound was also evaluated by an additional culture period of 24 h without the PRKA activators.

Effect of ZMP in combination with PDE inhibitors on the percentage of oocytes at the GV stage. COC were incubated for 24 h in maturation medium that was supplemented with the specific PDE3 inhibitor cilostamide (CIL 20 µM) or the non-specific PDE inhibitor IBMX (500 µM), both in the presence and absence of ZMP (2 mM).

Effects of different doses of ZMP in combination with a specific PDE3 inhibitor on the percentages of oocytes at the GV stage. COC and DO were incubated in maturation medium for 24 h in the presence of different doses of ZMP (0.10, 0.25, 0.50, 1.00 mM) and cilostamide (20 µM). Treatment reversibility was assessed in COC by a second culture period of 24 h without the inhibitors. At the end of the culture period, the oocytes were fixed and stained to evaluate the status of nuclear maturation, as described previously [15].

Statistical Analyses

The percentage of oocytes at the GV stage is expressed as the mean ± SEM of a minimum of three replicates. The data were analyzed by one-way ANOVA using GraphPad Prism ver. 4.0 for Windows (GraphPad Software, San Diego, CA). When ANOVA indicated a significant effect of treatment (P < 0.05), individual treatment differences were compared by the Bonferroni multiple comparison post-hoc test.

RESULTS

Localization of PRKA Transcripts and Isoforms in the Ovarian Follicle

RT-PCR of the control genes indicated the presence of ß-actin in all the tissues tested (Fig. 1). The presence of CYP2C19 (CYP-19 P450 aromatase) was detected only in granulosa cells and COC, and GDF9 was detected in granulosa cells, COC, and DO (data not shown). The presence of GDF9 in porcine granulosa cells has been shown previously [52]. The COC and DO contained transcripts that corresponded to the expected sizes of the designed primers for PRKAA1, PRKAB1, and PRKAG1. However, the transcript for PRKAA2 was only detected in the heart, granulosa cells, and COC. The PRKAG3 transcript was detected in the heart, as reported previously for porcine myoblasts [53]. The PCR products were sequenced using the 3130 XL Genetic Analyzer (Applied Biosystems, Foster City, CA). The following percentages sequence identity were obtained: for PRKAA1 (NM_006251, 96%); PRKAA2 (NM_006252, 100%); PRKAB1 (NM_006253, 91%); and PRKAG1 (NM_002733, 100%) (Table 1). Western blot analysis of PRKAA1 showed a positive signal at the expected molecular weight of 63 kDa in control porcine heart [54] and granulosa cells (Fig. 2A). The presence of PRKAA1 in the porcine heart [55] and skeletal muscle [56] has been reported previously. The PRKAA1 protein was also detected in porcine COC, DO, and cumulus cells (Fig. 2B). The PRKA immunoblots were carried out with COC and DO at the GV stage. The signal for PRKAA1 in porcine COC could be detected with as few as 12 COC (Fig. 2C). There was a linear increase in signal strength relative to the amount of sample loaded (Fig. 2, B, C, D and F). The signal for PRKAA1 was also detected in the ZP (Fig. 2E), porcine follicular fluid (Fig. 2F), and serum (data not shown). Together, these results indicate the presence of transcript and protein for PRKAA1 in the porcine ovarian follicle.


Figure 01
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  		FIG. 1. Localization of PRKA transcripts in the ovarian follicle. RT-PCR of ACTB and PRKA isoforms was carried out for the heart (H), granulosa cells (GC), COC, and DO. The negative control (-) involved PCR amplification without cDNA. The expected fragment length (bp) is indicated on the right. Representative results of three replicates are shown.


Figure 02
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  		FIG. 2. Expression pattern of PRKAA1 protein in ovarian tissues. Immunoblots of PRKAA1 in porcine tissues. A) The 63-kDa PRKAA1 subunit is detected in the heart (lane 1) and granulosa cells (lane 2) (25 µg each). B) Increasing numbers of COC (6, 12, 25, and 50) were loaded in lanes 1–4, respectively. C) Cumulus cells obtained from 6, 12, 25, 50, and 100 COC were loaded in lanes 1–5, respectively. D) Denuded oocytes (50 and 125) were loaded in lanes 1 and 2, respectively. E) Denuded oocytes (100 DO, lane 1) and ZPs (100 ZP, lane 2). F) Increasing doses (0.4, 0.8, 1.6, 3.2, 6.4, 12.8, and 25 µg) of pFF (lanes 1–7, respectively). The apparent shift in molecular weight of the PRKAA1 in some samples of pFF is due to the high protein concentration. The PRKA immunoblots were carried out using porcine COC and DO at the GV stage.

Meiosis Inhibition by PRKA Activators

Incubation of porcine COC in the presence of increasing concentrations of the PRKA activator ZMP (0, 0.10, 0.25, 0.50, 1.00 mM) increased the percentage of oocytes at the GV stage. The effect of this PRKA activator was significant at 0.50 and 1.00 mM ZMP (Fig. 3). The effective concentration (EC50) of ZMP was estimated to be 0.41 mM. The inhibitory effect of 1 mM ZMP in porcine oocyte maturation was observed after 22 h of culture (Fig. 4). However, after 44 h of continuous exposure to ZMP, almost all the oocytes had resumed meiosis and no significant difference was observed with the control. These data suggest that ZMP transiently blocks meiotic resumption in porcine oocytes. Other known activators of PRKA, such as AICAR and metformin, were also effective in blocking oocyte meiotic resumption. COC treated with 1 mM of either AICAR (Fig. 5A) or metformin (Fig. 5B) showed a significant increase (P < 0.05) in the percentage of COC arrested at the GV stage compared to the control treatment. In these same experiments, meiotic resumption occurred after a second culture period of 24 h in PRKA activator-free medium (Fig. 5, A and B). These data support the efficacy of three PRKA activators to block reversibly nuclear maturation in porcine COC. The efficacies with which ZMP and metformin activated PRKA were confirmed using the SignalScout kinase profiling system for PRKA (Stratagene). No PRKA activity was detected in COC at 0 h. However, a twofold increase in PRKA activity was measured in COC treated with ZMP or metformin after 3 h of culture, as shown previously in several cell types [40, 42, 43, 46].


Figure 03
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  		FIG. 3. Effect of increasing concentrations of ZMP on the percentages of oocytes at the GV stage. COC were incubated in the presence of increasing concentrations of ZMP (0, 0.10, 0.25, 0.50, 1.00 mM) for 24 h. Different letters indicate significant differences between treatments according to Bonferroni multiple comparison post-hoc tests. Each data-point represents the mean ± SEM of four replicates. The numbers of oocytes per treatment are indicated in parentheses.


Figure 04
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  		FIG. 4. The effect of ZMP on meiotic arrest is transient. The effect of ZMP on oocyte meiotic resumption was assessed by culturing COC in the presence of ZMP for 22 h and 44 h. ANOVA showed a significant effect of treatment (P < 0.05). Different letters indicate significant differences according to Bonferroni multiple comparison post-hoc tests. Each data-point represents the mean ± SEM of three replicates. The numbers of oocytes per treatment are indicated in parentheses.


Figure 05
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  		FIG. 5. Effects of AICAR and metformin on the percentages of oocytes at the GV stage. COC were incubated in maturation medium in the presence of either AICAR (A; 1 mM) or increasing concentrations of metformin (B; 0.10, 0.25, 0.50, and 1.00 mM) for 22 h. The reversibility of the 1 mM treatment groups on nuclear maturation was assessed by an additional culture period of 22 h without the PRKA activator. ANOVA showed a significant effect of treatment (P < 0.05). Different letters indicate significant differences according to Bonferroni multiple comparison post-hoc tests. Each data-point represents the mean ± SEM of three replicates. The numbers of oocytes per treatment are indicated in parentheses.

Effects of ZMP with PDE Inhibitors on Meiotic Resumption

To date, the only reports of PRKA regulation of oocyte maturation are in the mouse, in which PRKA activators induce resumption of meiosis, including oocytes arrested with phosphodiesterase (PDE) inhibitors [47]. The following experiments were designed to test this mechanism in a non-rodent oocyte model. COC treated with cilostamide (specific PDE inhibitor) or IBMX (broad-spectrum inhibitor) remained in meiotic arrest compared to control- and DMSO-treated COC (Fig. 6A). COC incubated in presence of 2 mM of ZMP also remained in meiotic arrest (Figs. 3 and 6A). The combination of this high dose of ZMP with PDE inhibitors (cilostamide or IBMX) did not show any statistical difference from their respective controls (P > 0.05). Together, these results suggest that the 2 mM ZMP does not reverse the inhibitory effects of broad-spectrum and specific PDE inhibitors on porcine oocytes.


Figure 06
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  		FIG. 6. Effect of ZMP in combination with PDE inhibitors on the percentage of oocytes at the GV stage. A) COC were incubated for 24 h in maturation medium alone or in the presence of cilostamide (20 µM) or IBMX (500 µM), or each of these drugs with ZMP (2 mM). B) COC were incubated in the presence of cilostamide (20 µM) and increasing concentrations (0, 0.10, 0.25, 0.50, 1.00 mM) of ZMP for 24 h, to evaluate the effects of increasing doses of ZMP on the percentages of oocytes at the GV stage. C) Reversibility of meiotic inhibition was evaluated after a second culture period of 24 h without the pharmacological compounds. ANOVA showed a significant effect of treatment (P < 0.05). Different letters indicate significant differences according to Bonferroni multiple comparison post-hoc tests. Each data-point represents the mean ± SEM of three replicates. The numbers of oocytes per treatment are indicated in parentheses.

Porcine COC were incubated in the presence of 20 µM cilostamide and different doses of ZMP to evaluate whether a specific dose of ZMP could reverse the inhibitory effect of the specific PDE3 inhibitor on oocyte maturation. In COC, the specific PDE3 inhibitor cilostamide maintained oocytes at the GV stage (68.1 ± 7.7%; Fig. 6B). A range of ZMP doses did not have any significant effect when treated in combination with cilostamide (Fig. 6B). All treatments were fully reversible, since treated oocytes resumed meiosis when incubated for an additional 24 h in compound-free medium (Fig. 6C).

ZMP alone maintained oocytes in meiotic arrest when administered to COC (Figs. 3 and 4; Fig. 6, A and B). However, in DO, ZMP did not have a significant effect (P > 0.05) on the percentage of oocytes at the GV stage compared to the control and DMSO (Fig. 7). The inefficiency of PRKA activator at maintaining oocytes in meiotic arrest is not due to the absence of PRKA protein in the porcine oocyte (Fig. 2D), ZP (Fig. 2E) or pFF (Fig. 2F). These data provide evidence that ZMP activates PRKA and transiently delays meiotic resumption in porcine oocytes, but requires the presence of cumulus cells to exert its effect on oocyte maturation.


Figure 07
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  		FIG. 7. Effects of increasing concentrations of ZMP and a specific PDE3 inhibitor on the percentages of denuded oocytes at the GV stage. DO were incubated in the presence of cilostamide (20 µM) and different doses (0, 0.10, 0.25, 0.50, 1.00 mM) of ZMP for 24 h. ANOVA showed a significant effect of treatment (P < 0.05). Different letters indicate significant differences according to Bonferroni multiple comparison post-hoc tests. Each data-point represents the mean ± SEM of three replicates. The numbers of oocytes per treatment are indicated in parentheses.

DISCUSSION

The present study demonstrates that the meiotic resumption of porcine COC is transiently blocked by PRKA activators in a dose-dependent manner. The lack of effect of PRKA activators on denuded oocytes strongly suggests that cumulus cells play an essential role in the control of porcine oocyte maturation via the PRKA signaling cascade.

Recently, the role of PRKA in the regulation of oocyte maturation in mice has been reported [47]. Mouse oocytes remain in meiotic arrest when treated with cAMP analogs or PDE inhibitors, such as hypoxanthine [57–60]. Activators of PRKA reverse the inhibitory effects of cAMP analogs and PDE inhibitors, thereby inducing meiotic resumption [47]. Our results using porcine oocytes dramatically contrast with these findings in the mouse. Several lines of evidence presented in the present study demonstrate that well-known activators of PRKA transiently inhibit meiotic resumption (Figs. 3–5). Furthermore, these agents do not overcome the inhibitory effect of PDE inhibitors on oocyte maturation (Fig. 6, A and B), as they do in mouse oocytes [47]. In addition, PRKA activators did not have an effect on the maturation of porcine DO (Fig. 7). Collectively, these findings demonstrate a novel role for PRKA in the regulation of oocyte maturation in a non-rodent model.

The activation of PRKA is generally achieved in vitro with either AICAR or ZMP. Most studies use AICAR to activate PRKA. However, the activation of PRKA by AICAR is dependent upon the activity of adenosine kinase, which transforms AICAR into ZMP. The present study uses ZMP, which is an analog of 5'-AMP, to activate directly PRKA. The role of PRKA in porcine meiotic resumption was validated using both PRKA activators (Figs. 3 and 5A). The concentration of AICAR commonly used for other cell types, such as skeletal muscle cells [61, 62], endothelial cells [63], and dermal fibroblasts [64], is 1 mM. Furthermore, the findings that the treatments were reversible and within the range of the effective concentration (EC50) strongly suggest that the observed results are not due to product toxicity. Metformin is reported to activate PRKA in rat hepatocytes and rat skeletal muscles [41, 43]. Our results show that metformin also maintains porcine COC in a state of meiotic arrest (Fig. 5B). The fact that all of the PRKA activators used in the present study reversibly maintained porcine COC at the GV stage strongly supports the role of PRKA in the regulation of porcine oocyte maturation.

Important differences exist in the regulation of oocyte meiotic resumption between rodent and non-rodent animals. The first difference is the time it takes for oocytes to undergo meiotic resumption. In mice, meiotic resumption occurs within 2 to 3 h of IVM, depending on the culture conditions, whereas in pigs, meiotic resumption is observed only after 20 h. The second important difference is that mouse oocytes resume meiosis in the presence of protein synthesis inhibitors [65, 66], whereas porcine and bovine oocytes remain in meiotic arrest [66–69], which demonstrates a requirement for protein synthesis to resume meiosis. The third difference is the role that cumulus cells play in the control of meiotic resumption. In mice, cumulus cells secrete a meiosis-activating substance [70, 71]. Porcine cumulus cells secrete progesterone to modulate meiotic resumption [72, 73]. The fourth difference is that RNA transcription is also required in porcine cumulus cells [74]. Furthermore, PRKA activators are effective in mouse COC and DO [47], whereas in pigs they are effective in COC but not in DO, as shown in the present study. The inability of PRKA activators to prevent meiotic resumption in porcine DO is not due to the lack of a functional PRKA, since immunoblotting revealed the presence of PRKAA1 in porcine DO, ZP, and follicular fluid. Thus, it appears that mammalian oocyte maturation is efficiently modulated by PRKA, whereby the oocyte is the effective site of action in the mouse [47], whereas the cumulus cells are the target in porcine COC.

PRKA has several physiological roles. PRKA is involved in the purine biosynthesis pathway. Mutations of the AICAR transformylase, which transforms AICAR into FAICAR, induce errors in purine biosynthesis [75]. FAICAR can enter the purine biosynthesis pathway through its conversion by inosine monophosphate cyclohydrolase into inosine monophosphate [76]. The PRKA activators AICAR and ZMP can enter the purine biosynthesis pathway. A convergent point of these compounds may be inosine monophosphate [77, 78]. Newly synthesized purines from these pharmacological compounds could then produce inosine and block meiotic resumption. In vitro, inosine does not block meiotic resumption of porcine oocytes (data not shown), which suggests that this pathway is not involved in blocking porcine oocyte maturation.

A second activity of PRKA is its ability to regulate transcription [34]. Interestingly, activation of PRKA reduces the activity of RNA polymerase I (POL I), which carries out the transcription of rRNA [79]. The activation of PRKA by AICAR inhibits key enzymes in protein synthesis, e.g., mTOR [80], p70S6K [81], and EF2 [82]. In rat hepatocytes, PRKA activation controls protein synthesis [82]. An inhibitor of protein synthesis, cycloheximide, maintains porcine oocytes in meiotic arrest even in the absence of cumulus cells [83]. Given that PRKA activators transiently maintain porcine COC in meiotic arrest and that cumulus cells are required, this suggests that PRKA activators do not directly inhibit protein synthesis in the oocyte.

The maturation of mammalian COC requires the availability of energy sources [84]. It is known that glucose uptake in bovine oocytes is very low [85, 86], whereas glucose is highly metabolized in the cumulus cells [87]. Cumulus cells increase glucose utilization by up to 25% during in vitro maturation [87]. In the present study, porcine COC were matured in NCSU-23 [48], which contains glucose (5.5 mM) and glutamine (1 mM) as energy sources. Glucose availability has a significant effect on the progression of oocyte maturation [88, 89] and it appears to be required for the completion of meiosis [88, 90]. The culture of porcine oocytes in low levels of glucose delays oocyte maturation but has little consequence in terms of developmental competence [88, 91]. These findings suggest that there is a correlation between the levels of energy substrates and meiotic progression. Since PRKA is the key metabolic sensor of cellular energy status [30, 37, 39], treatment of cells with ZMP triggers the physiological response of cells under energy stress and redirects the use of glucose. Thus, cumulus cells are essential to mediate the effect(s) of ZMP. It is possible that the transient delay in meiotic resumption is the result of a physiological response of porcine cumulus cells to signals that indicate low energy levels. These results suggest that modulation of the energy status of the cumulus cells in porcine COC is an effective way to delay meiotic resumption. Thus, it will be important to investigate the energetic needs of cumulus cells to improve in vitro maturation media.

In conclusion, the results presented in the present study demonstrate the presence of PRKAA1 in porcine granulosa cells, COC, and DO. PRKA activators transiently block meiotic resumption in COC. The differential actions of PRKA activators on COC versus DO demonstrate the requirement for cumulus cells to mediate the inhibitory effect. Additional experiments are necessary to identify the inhibitory factor(s) produced by the cumulus cells that control nuclear maturation in porcine oocytes. The present study further illustrates fundamental differences between species in the regulation of oocyte maturation. The role of PRKA in the maturation of other non-rodent oocytes, including human oocytes, remains to be elucidated.

ACKNOWLEDGMENTS

The authors would like to express their gratitude to Dr. Sylvie Bilodeau-Goeseels for her critical review of the manuscript and to Maxime Sasseville for helpful discussions. Special thanks to Gabrielle Darou for technical assistance. Special thanks to the slaughterhouse Olymel for providing gilt ovaries and to Michel Pépin, Richard Prince, and O'Neil Fecteau for collecting the ovaries at the slaughterhouse.

FOOTNOTES

3These authors contributed equally to this work. Back

1Supported by the Natural Sciences and Engineering Research Council of Canada and by the National Health and Medical Research Council of Australia. M.A.M. is the recipient of the Serono Foundation for the Advancement of Medical Science postdoctoral fellowship in Biomedicine. Back

Correspondence: 2FAX: 418 656 3766; e-mail: Francois.Richard{at}crbr.ulaval.ca

Received: 28 September 2006.

First decision: 23 October 2006.

Accepted: 8 December 2006.

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