Introduction
In mammalian oocytes, meiosis is initiated during fetal life and is subsequently arrested at the diplotene stage of the first meiotic division. Completion of the first meiotic division in pre-ovulatory oocytes is triggered by the gonadotropin surge and meiosis progresses to the metaphase II stage where it is arrested again until fertilization. Competent oocytes can resume meiotic maturation spontaneously without hormonal stimulation when they are removed from follicles and cultured in suitable media (Pincus & Enzmann, Reference Pincus and Enzmann1935), a finding that suggests that the follicle provides an inhibitory factor(s) that prevents meiosis.
The mechanisms involved in the control of meiotic arrest and resumption have not been completely elucidated. Cyclic AMP levels within the oocyte have an important role in the control of nuclear maturation, with high levels maintaining meiotic arrest and low levels being permissive for meiotic maturation (Horner et al., Reference Horner, Livera, Hinckley, Trinh, Storm and Conti2003; Kalinowsli et al., 2004; Thomas et al., Reference Thomas, Armstrong and Gilchrist2004). It has been suggested recently that AMP, the product of degradation of cAMP by phosphodiesterase enzymes, could have an active role in the control of oocyte meiotic maturation. The nucleotide AMP is a potent regulator of adenosine monophosphate-activated protein kinase (AMPK), a key regulatory enzyme which controls the activity of enzymes of the carbohydrate and fat metabolism in response to changes in cellular energy levels (Hardie & Carling, Reference Hardie and Carling1997) or environmental stresses (Corton et al., Reference Corton, Gillespie and Hardie1994). Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) resulted in the induction of nuclear maturation in arrested cumulus-enclosed oocytes (CEOs) and denuded mouse oocytes (Downs et al., Reference Downs, Hudson and Hardie2002; Chen et al., Reference Chen, Hudson, Chi, Chang, Moley, Hardie and Downs2006). Moreover, active AMPK was demonstrated in both FSH-treated and amphiregulin (AR)-treated oocytes within 3 h, a finding that supported a role for AMPK in FSH-treated and AR-induced maturation in mouse oocytes (Chen & Downs, 2008). In contrast, the AMPK activators AICAR and metformin (MET) inhibited meiosis in bovine denuded and CEO and in porcine cumulus-enclosed oocytes (Bilodeau-Goeseels et al., Reference Bilodeau-Goeseels, Sasseville, Guillemette and Richard2007; Mayes et al., Reference Mayes, Laforest, Guillemette, Gilchrist and Richard2007). Metformin is one of the most commonly used drugs for the treatment of type II diabetes and exerts its therapeutic effects, at least in part, through AMPK activation (Zhou et al., Reference Zhou, Myers, Li, Chen, Shen, Fenyk-Melody, Wu, Ventre, Doebber, Fujii, Musi, Hirshman, Goodyear and Moller2001). However, it is not clear whether or not the inhibitory effect of AICAR and MET on bovine oocyte meiosis was mediated through AMPK activation. This is because the AMPK inhibitor compound C (CC) has either stimulatory (Tosca et al., Reference Tosca, Uzbekova, Chabrolle and Dupont2007) or inhibitory effects (Bilodeau-Goeseels et al., Reference Bilodeau-Goeseels, Panich and Kastelic2011) on bovine oocyte meiosis, however AICAR and MET did not increase AMPK activation (assessed by Thr172 phosphorylation) in bovine cumulus cells and oocytes (Bilodeau-Goeseels et al., Reference Bilodeau-Goeseels, Panich and Kastelic2011).
The objective of this study was to characterize the inhibitory effect of MET on porcine oocyte meiosis by: (1) determining the effects of an AMPK inhibitor and of inhibitors of signalling pathways involved in MET-induced AMPK activation in other cell types on MET-mediated meiotic arrest in porcine CEO; (2) determining whether MET and AICAR treatments lead to increased activation of porcine oocyte and/or cumulus cell AMPK as measured by phosphorylation of its substrate acetyl-CoA carboxylase; and (3) determining the effects of inhibition of the AMPK kinase, Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), and Ca2+ chelation on oocyte meiotic maturation and AMPK activation in porcine oocytes and cumulus cells.
Materials and methods
Chemicals and antibodies
The AMPK inhibitor compound C (6-[4-(2-piperidine-1-yl-ethoxy)-phenyl)]-3-pyridine-4-yl-pyyrazolo[1,5-a] pyrimidine) was purchased from Calbiochem®. A 12.5 mM-stock solution was prepared in dimethyl sulphoxide (DMSO), which was further diluted 1/10 and 1/100 in culture medium before being added to the culture wells. The AMPK activator 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) was purchased from Toronto Research Chemicals, Inc. A stock solution was prepared in water at 100 mM. Metformin (MET; 1,1-dimethylbiguanide hydrochloride, Sigma-Aldrich Canada) was prepared in water as 200 mM or 1 M solutions. The eNOS inhibitor L-NAME, the c-Src inhibitor PP2, the PI-3 kinase inhibitors LY294002 and wortmannin, and the complex I inhibitor rotenone were purchased from Enzo Life Sciences, Inc. The CaMKK inhibitor STO-609 and the Ca2+ chelator BAPTA-AM were obtained from Tocris and okadaic acid was bought from Cedarlane. All chemicals were diluted in appropriate solvents and final concentration of ethanol, DMSO or additional water never exceeded 1% in the culture medium. This concentration of solvent was tested and was without effects on oocyte nuclear maturation (results not shown). Rabbit polyclonal antibodies against phospho-acetyl-CoA carboxylase (Ser79) and ACC were obtained from Cell Signaling Technology, Inc. All other reagents were from Sigma-Aldrich, Canada unless otherwise indicated.
Oocyte recovery and culture
Gilt ovaries were collected at an abattoir about 30 min after slaughter. Cumulus-enclosed oocytes (CEO) were recovered from the ovaries by aspiration of follicles larger than 2 mm. Oocytes with an unexpanded mass of cumulus cells and homogeneous cytoplasm were recovered under a stereomicroscope and were washed twice in 199-HEPES-BSA medium [medium 199 with Earle's salts and l-glutamine, supplemented with 20 mM HEPES, 0.4% bovine serum albumin (BSA), and 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco)] and once in culture medium. For all experiments, CEO were cultured in 500 μl of culture medium, which consisted of medium 199 supplemented with 10% porcine follicular fluid, 35 μg/ml of porcine follicle-stimulating hormone (FSH; Follitropin-V, Vetrepharm Inc.), 0.5 μg/ml leuteinizing hormone (LH; Lutropin, Vetrepharm Inc.), 10 ng/ml human epidermal growth factor, 0.022 mg/ml pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin. Oocytes were cultured at 39°C in a moisture-saturated atmosphere of 5% CO2 in air.
Granulosa cell recovery and culture
Porcine granulosa cells were recovered by cutting open follicles larger than 2 mm in diameter in phosphate-buffered saline (PBS) medium. The cells were filtered through a 50-micron pore size filter (CellMicroSieves™, BioDesign Inc.) to remove clumps, they were then washed by centrifugation and cells were seeded in 1 ml of medium 199 supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, Inc.), pyruvate (0.022 mg/ml), testosterone (100 ng/ml) and 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were cultured at 39°C in a moisture-saturated atmosphere of 5% CO2 in air. After 48 h of culture, the medium was changed (same medium as above but with 1 mg/ml BSA instead of FBS). Cells were then left to recover for at least 2 h before being exposed to treatments.
Fixation of oocytes and evaluation of nuclear maturation
At the end of the culture period, CEO were transferred to 1.5 ml tubes that contained approximately 400 μl of trypsin solution (2.5 mg/ml PBS) and vortex-agitated for 1 min to remove cumulus cells. The oocytes were then recovered under a stereomicroscope and transferred to glass slides. A mixture of Vaseline and paraffin was used to maintain a coverslip in contact with the oocytes. Coverslips were secured with epoxy glue and the slides immersed in a fixative solution (ethanol:acetic acid, 3:1, v:v) for at least 72 h, stained with 1% aceto-orcein and examined at ×320 magnification. Oocytes were classified as being at one of the following stages: germinal vesicle stage (GV), germinal vesicle breakdown (GVBD), condensed chromosomes, metaphase I (MI), anaphase I, telophase I or metaphase II (MII). Oocytes with an abnormal chromatin configuration were classified as degenerated.
Western blot analysis
Oocytes and cumulus cells were frozen directly in lysis buffer [10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Igepal (ICN Biochemicals Inc.)] supplemented with protease inhibitors (2 mM phenylmethyl sulfonylfluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin) and phosphatase inhibitors (100 mM NaF, 10 mM sodium pyrophosphate, 2 mM Na orthovanadate). Cell extracts were electrophoresed in 10% (w/v) polyacrylamide gels (Bio-Rad) under reducing conditions. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P, Millipore) over a period of 3–4 h at 400 mA. Membranes were incubated at room temperature for 2.5 h in Tris-buffered saline that contained 0.1% Tween-20 (TTBS) and supplemented with 5% non-fat dried milk powder to saturate non-specific sites. Membranes were then incubated overnight at 4°C with the appropriate antibody at a final dilution of 1:2000 in TTBS supplemented with 5% BSA. Membranes were washed in TTBS and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) (final dilution, 1:10,000), protected from light. After washing with TTBS, the signal was detected by enhanced chemoluminescence (ECL Plus, GE Healthcare). The films were scanned and the signals were quantified with the Quantity One software (Bio-Rad). The membranes were stripped before being re-hybridized with the other primary antibody.
Experimental design
Experiment 1: Effect of the AMPK inhibitor Compound C on porcine oocyte nuclear maturation
Porcine CEO were incubated for 24 h in control culture medium alone or supplemented with the AMPK inhibitor compound C (CC, 0.5, 1 or 5 μM), AICAR (1 mM), AICAR (1 mM) + CC (1 μM), metformin (MET, 2 mM), and MET (1 mM) + CC (1 μM) and were then fixed for evaluation of nuclear maturation. To determine if the inhibitory effect of CC was reversible, CEO were cultured with CC at 1 μM for 8 h, washed twice and cultured in control medium for 16 h, and then fixed for evaluation of nuclear maturation.
Experiment 2: Effect of signalling pathways inhibitors on metformin-mediated inhibition of meiosis
Porcine CEO were incubated for 24 h in control culture medium alone or supplemented with the nitric oxide synthase (NOS) inhibitor L-NAME (1 mM), the Src family tyrosine kinase inhibitor PP2 (10 μM), the PI 3-kinase inhibitors LY294002 (50 μM) and wortmannin (100 nM) and the mitochondrial complex I inhibitor rotenone (100 nM) by themselves or in combination with MET (2 mM). After culture, the oocytes were fixed for evaluation of nuclear maturation.
Experiment 3: Effect of okadaic acid on the inhibitory effect of AICAR, MET and CC
Okadaic acid (OA) is an inhibitor of protein phosphatase-1 (PP1) and PP2A. It accelerates GVBD in cattle and pig oocytes (Kalous et al., Reference Kalous, Kubelka, Rimkevicova, Guerrier and Motlik1993). As activators (AICAR and MET) and an inhibitor (CC) of AMPK all had inhibitory effects on oocyte nuclear maturation and these agents probably acted through phosphorylation/dephosphorylation of target proteins, we tested whether the effect of AICAR, MET and CC would be reversed by OA to determine if they acted by different mechanisms. Porcine CEO were incubated for 24 h in control medium alone or supplemented with the phosphatase inhibitor OA (0.1 μM), AICAR (1 mM), AICAR (1 mM) + OA (0.1 μM), MET (2 mM), MET (2 mM) + OA (0.1 μM), CC (5 μM) and CC (5 μM) + OA (0.1 μM). After culture, the oocytes were fixed for evaluation of nuclear maturation.
Experiment 4: Effects of AICAR, MET and CC on phosphorylation of Ser79 of Acetyl-CoA carboxylase
Acetyl-CoA carboxylase (ACC) is a substrate of AMPK and AMPK activation can be monitored by phosphorylation of Ser79 of ACC (Davies et al., Reference Davies, Carling and Hardie1989). Cumulus-enclosed oocytes were cultured for 3 h in control medium alone, or supplemented with AICAR (1 mM), MET (2 mM) or CC (1 or 10 μM). Following culture, CEO were mechanically separated into oocytes and cumulus cells (some CEO were also frozen intact). Cells were lysed and protein extracts were subjected to western blotting as described above with an antibody against phospho-ACC (Ser79), followed by hybridization with a secondary antibody and detection. The membranes were stripped and re-hybridized with an antibody against ACC and a secondary antibody followed by detection. The signals were quantified and for each sample, the phosphorylated ACC/total ACC ratio was calculated.
Experiment 5: Effects of the AMPK kinase inhibitor STO-609 and the Ca2+ chelator BAPTA-AM on meiotic maturation and phosphorylation of Ser79 of acetyl-CoA carboxylase
Cumulus-enclosed oocytes were cultured for 3 or 24 h in control medium alone, or supplemented with STO-609 acetate (20 μg/ml) or BAPTA-AM (25 μM). Oocytes cultured for 24 h were fixed for evaluation of nuclear maturation while complexes cultured for 3 h were separated mechanically into oocytes and cumulus cells and proteins were subjected to western blotting as described above.
Experiment 6: Effects of the AMPK activators MET and AICAR, the AMPK kinase inhibitor STO-609 and the Ca2+ chelator BAPTA-AM on phosphorylation of Ser79 of acetyl-CoA carboxylase in porcine granulosa cells
Porcine granulosa cells monolayers were cultured in the presence of the AMPK activators AICAR (2 mM) and MET (2 mM), the AMPK kinase inhibitor STO-609 (20 μg/ml) and the Ca2+ chelator BAPTA-AM (25 μM) for 2 h. The culture medium was then removed and 100 μl of lysis buffer was added to each well. Cells were lysed on ice for 20 min then the extracts were collected, centrifuged and stored at –80°C until used for western blot analysis.
Statistical analysis
At least three replicates were performed for each treatment for each maturation experiment. Data for oocyte maturation are expressed as mean ± standard error of the mean (SEM). They were arcsin-transformed and were analysed with a General Linear Model (GLM) of SAS (SAS Institute, Inc.). When GLM indicated a significant effect of treatment, differences were located with Duncan's multiple range test. Western blot analysis ratios are presented as mean ± SEM of three or four determinations and the effects of treatments were analysed by GLM followed by Duncan's multiple range test if significant differences were detected. For all analyses, a P-value <0.05 was considered to be significant.
Results
Experiment 1: Effect of the AMPK inhibitor compound C on porcine oocyte nuclear maturation
The majority of CEO cultured in control medium had resumed meiosis after 24 h of culture but 27.2 ± 9.6% were still at the GV stage (Fig. 1). The AMPK activators AICAR (1 mM) and MET (2 mM) increased (P < 0.05) the proportion of CEO remaining at the GV stage after 24 h of culture to 85.8 ± 6.7 and 94.8 ± 2.1%, respectively (Fig. 1). The AMPK inhibitor CC at 1 and 5 μM also increased (P < 0.05) the proportion of CEO remaining at the GV stage compared with control, although the lower concentration, 0.05 μM, had no significant effect (Fig. 1). Additionally, when CC (1 μM) was included in combination with AICAR or MET, the inhibitory effect of these AMPK activators was not reversed (Fig. 1). All oocytes underwent GVBD when they were transferred to control medium following 8 h of culture with CC at 1 μM (results not shown).
Effects of the adenosine monophosphate-activated protein kinase (AMPK) inhibitor compound C (CC; 0, 0.5, 1 or 5 μM), alone or in combination with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) (1 mM) or metformin (MET, 2 mM), on the percentage of porcine cumulus-enclosed oocytes (CEO) remaining at the germinal vesicle (GV) stage after 24 h of culture. Values represent means ± standard error of the mean (SEM) of at least three replicates with totals of 46 to 68 CEO per treatment. A,BTreatments without a common superscript differed (P < 0.05).
Experiment 2: Effect of signalling pathway inhibitors on metformin-mediated inhibition of meiosis
Of all the signalling pathway inhibitors tested, only rotenone, an inhibitor of complex I of the respiratory chain, significantly increased the percentage of CEO remaining at the GV stage compared to control after 24 h of culture (Fig. 2). The percentage of CEO remaining at the GV stage was significantly different between the two PI-3 kinase inhibitors, with LY2940002 resulting in a higher proportion of CEO remaining at the GV stage compared with wortmannin (Fig. 2). None of the pathway inhibitors tested reversed the inhibitory effect of metformin on porcine oocyte meiosis (Fig. 2).
Effects of the nitric oxide synthase (NOS) inhibitor L-NAME (1 mM), the Src family tyrosine kinase inhibitor PP2 (10 μM), the PI-3 kinase inhibitors LY94002 (50 μM) and wortmannin (100 nM) and the complex I inhibitor rotenone (100 nM) by themselves (grey bars) or in combination with metformin (MET; 2 mM, white bars) on the percentage of cumulus-enclosed oocytes (CEO) remaining at the germinal vesicle (GV) stage after 24 h of culture. Values represent means ± standard error of the mean (SEM) of at least four replicates with totals of 50 to 79 CEO per treatment. A,B,CTreatments without a common superscript differed (P < 0.05).
Experiment 3: Effect of okadaic acid on the inhibitory effect of AICAR, MET and CC
As previously observed, AICAR, MET and CC inhibited GVBD in porcine CEO (Fig. 3). Okadaic acid increased the percentage of CEO reaching the MI stage after 24 h of culture (36.5 ± 7.2 and 77.1 ± 16.6% for control and OA, respectively, P < 0.05) and reversed the inhibitory effect of AICAR 1 mM and CC 5 μM (P < 0.05) but not of MET 2 mM in CEO (Fig. 3).
Effects of okadaic acid (OA; 0.1 μM) alone or in combination with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR; 1 mM), metformin (MET; 2 mM) or compound C (CC, 5 μM) on the percentage of porcine cumulus-enclosed oocytes (CEO) at the germinal vesicle (GV) stage after 24 h of culture. Values represent means ± standard error of the mean (SEM) of at least three replicates with totals of 49 to 89 CEO per treatment. A,B,C,DTreatments without a common superscript differed (P < 0.05).
Experiment 4: Effects of CC, AICAR and MET on phosphorylation of Ser79 of Acetyl-CoA carboxylase
The culture of CEO in the presence of the AMPK inhibitor CC at 1 or 10 μM reduced the ratio of phospho-ACC over total ACC protein in oocytes but the difference was not significant (Fig. 4A). There were no significant changes in ACC phosphorylation in cumulus cells in response to CC (Fig. 4B). To determine whether the separation of cumulus cells from oocytes would affect the phosphorylation status of ACC, we performed western blot analysis of proteins extracted from whole cumulus–oocyte complexes cultured with CC for 3 h. As was the case with isolated oocytes and cumulus cells, the reduction in ACC phosphorylation in response to CC 10 μM was not significant (results not shown). The level of ACC phosphorylation in oocytes and cumulus cells did not change in response to culture in the presence of MET or AICAR for 3 h (Fig. 5A, B).
Effects of culture in control conditions or with compound C (CC 1 and 10 μM) for 3 h on the phosphorylation level of Ser79 of acetyl-CoA carboxylase (ACC) in oocytes (A) and cumulus cells (B). The results are presented as means ± standard error of the mean (SEM) of three (oocytes) or four (cumulus cells) determinations.
Effects of culture in control conditions or with metformin (MET; 2 mM) or 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR; 1 mM) for 3 h on the phosphorylation level of Ser79 of acetyl-CoA carboxylase (ACC) in oocytes (A) and cumulus cells (B) from cumulus–oocyte complexes (COC). The results are presented as means ± standard error of the mean (SEM) of four determinations.
Experiment 5: Effects of the AMPK kinase inhibitor STO-609 and the Ca2+ chelator BAPTA-AM on meiotic maturation and phosphorylation of Ser79 of acetyl-CoA carboxylase
The AMPK kinase inhibitor STO-609 increased (P < 0.05) the percentage of porcine CEO remaining at the GV stage after 24 h of culture while the Ca2+ chelator BAPTA-AM was without effect (Fig. 6). These two agents did not have any significant effect on ACC phosphorylation in oocytes and cumulus cells after 3 h of culture (Fig. 7A, B). BAPTA-AM was also without effect on ACC phosphorylation in whole cumulus-oocyte complexes (results not shown).
Effects of the adenosine monophosphate-activated protein kinase (AMPK) inhibitor STO-609 (20 μg/ml) and the Ca2+ chelator BAPTA-AM (25 μM) on the percentage of porcine cumulus-enclosed oocytes (CEO) at the GV stage after 24 h of culture. Values represent means ± standard error of the mean (SEM) of at least three replicates with totals of 52–82 CEO per treatment. A,BTreatments without a common superscript differ (P < 0.05).
Effects of culture in control conditions or with STO-609 (20 μg/ml) or BAPTA-AM (25 μM) for 3 h on the phosphorylation level of Ser79 of acetyl-CoA carboxylase (ACC) in oocytes (A) and cumulus cells (B) from cumulus–oocyte complexes (COC). The results are presented as means ± standard error of the mean (SEM) of three (oocytes) or four (cumulus cells) determinations.
Experiment 6: Effects of the AMPK activators MET and AICAR, the AMPK kinase inhibitor STO-609 and the Ca2+ chelator BAPTA-AM on phosphorylation of Ser79 of acetyl-Co carboxylase in porcine granulosa cells
Previous experiments showed that these treatments had limited effects on ACC phosphorylation in porcine oocytes and cumulus cells. To verify our procedure, we performed experiments in granulosa cells. The culture of porcine granulosa cells in the presence of the AMPK activators AICAR (2 mM) and MET (2 mM) for 2 h increased ACC phosphorylation (Fig. 8A). The AMPK kinase inhibitor STO-609 (5, 10 and 20 μg/ml) was without effect on ACC phosphorylation in granulosa cells (Fig. 8B), while the Ca2+ chelator BAPTA-AM (25 μM) completely abolished ACC phosphorylation in granulosa cells (Fig. 8C).
Effects of culture in control conditions or treatment with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) (2 mM) or MET (2 mM) (A), STO-609 (B) or BAPTA-AM (C) for 2 h on the phosphorylation level of Ser79 of ACC in porcine granulosa cells.
Discussion
Studies with mouse oocytes suggested that AMPK could be involved in the control of meiosis. Pharmacological activation of AMPK with AICAR resulted in induction of nuclear maturation in arrested cumulus-enclosed and denuded mouse oocytes (Downs et al., Reference Downs, Hudson and Hardie2002; Chen et al., Reference Chen, Hudson, Chi, Chang, Moley, Hardie and Downs2006). Mouse oocyte AMPK was also activated by hormones and stress; this activation preceded GVBD, and inhibiting AMPK activity blocked the effects of these stimuli (Chen et al., Reference Chen, Hudson, Chi, Chang, Moley, Hardie and Downs2006; Chen & Downs, 2008; LaRosa & Downs, Reference LaRosa and Downs2006, Reference LaRosa and Downs2007). A recent study provided evidence that AMPK is also involved after GVBD to promote the completion of mouse oocyte meiosis and to prevent premature activation (Downs et al., Reference Downs, Ya and Davis2010). Different results were obtained in livestock species in which pharmacological activation of AMPK inhibited GVBD in bovine cumulus-enclosed and denuded oocytes (Bilodeau-Goeseels et al., Reference Bilodeau-Goeseels, Sasseville, Guillemette and Richard2007) and in porcine CEO (Mayes et al., Reference Mayes, Laforest, Guillemette, Gilchrist and Richard2007). In the present study, the inhibitory effect of AICAR and MET on porcine cumulus-enclosed oocyte meiosis was confirmed. However, the AMPK inhibitor compound C (CC) did not reverse the inhibitory effect of AICAR and MET on porcine oocyte nuclear maturation. Additionally, CC itself had an inhibitory effect on meiosis suggesting that, as in bovine oocytes (Bilodeau-Goeseels et al., Reference Bilodeau-Goeseels, Panich and Kastelic2011), the inhibitory effect of AICAR and MET on porcine oocytes may not be through AMPK activation. The decrease in ACC phosphorylation in oocytes in response to CC at concentrations that inhibited meiosis was not significant suggesting that the inhibitory effect of CC on meiosis may also not be due to an effect on AMPK. Additionally, in the present study, no changes in ACC phosphorylation were detected in porcine cumulus cells and oocytes in response to AICAR or MET at concentrations that inhibited meiosis suggesting again that the inhibitory effect of AICAR and MET on porcine oocyte meiosis may not be due to AMPK activation. Similar results were obtained with bovine oocytes and cumulus cells (Bilodeau-Goeseels et al., Reference Bilodeau-Goeseels, Panich and Kastelic2011) and indicated that the effects of AMPK activators and inhibitors as well as the extent of involvement of AMPK in the control of meiosis is more similar between bovine and porcine oocytes compared to mouse oocytes. It should be mentioned that different results were obtained in another laboratory where MET inhibited GVBD in cumulus-enclosed but not in denuded oocytes (MET inhibited GVBD in bovine denuded oocytes (Bilodeau-Goeseels et al., Reference Bilodeau-Goeseels, Sasseville, Guillemette and Richard2007) but was not tested on porcine denuded oocytes), and increased phosphorylation of Thr172 of AMPK in cumulus cells and oocytes from complexes, plus CC accelerated GVBD in CEO (Tosca et al., Reference Tosca, Uzbekova, Chabrolle and Dupont2007). These different results could be due to different culture medium used and/or the fact that a higher MET concentration (10 mM) was used by Tosca et al. (Reference Tosca, Uzbekova, Chabrolle and Dupont2007).
What are the putative mechanisms of action of AICAR, MET and CC to inhibit meiosis if not through modulation of AMPK activity? It is known that AICAR and MET can have AMPK-independent effects. For example, AICAR induced apoptosis in Jurkat cells, but did not activate AMPK (López et al., Reference López, Santidrián, Campàs and Gil2003) and metformin and AICAR inhibited the glucose-induced translocation of glucokinase from the nucleus to the cytoplasm in hepatocytes independently of AMPK activation (Guigas et al., Reference Guigas, Bertrand, Taleux, Foretz, Wienernsperger, Vertommen, Andreelli, Viollet and Hue2006). The pharmacological agents probably acted via different mechanisms to inhibit meiosis, as okadaic acid reversed the inhibitory effect of AICAR and CC but not of MET in cumulus-enclosed porcine oocytes and indicated that OA-sensitive phosphatases are involved in AICAR- and CC-mediated inhibition of meiosis in porcine oocytes. Okadaic acid reversed the inhibitory effect of MET and CC in bovine CEO (Bilodeau-Goeseels et al., Reference Bilodeau-Goeseels, Panich and Kastelic2011), a finding that indicated that although these agents had the same effect on meiosis in both species, the molecular mechanisms are potentially different.
AMPK has been shown to be up-regulated by nitric oxide (NO), the c-Src or the PI-3 kinase pathway in a variety of cell types (Fryer et al., Reference Fryer, Hajduch, Rencurel, Salt, Hundal, Hardie and Carling2000; Lira et al., Reference Lira, Soltow, Long, Betters, Sellman and Criswell2007; Hwang et al., Reference Hwang, Kim, Jeong, Jeong and Jeong2011; Mizrachy-Schwartz et al., Reference Mizrachy-Schwartz, Cohen, Klein, Kravchenko-Balasha and Levitzki2011). In bovine aortic endothelial cells (BAEC), reactive nitrogen species generated in response to MET were required for AMPK activation and inhibition of NO synthase, c-SRC or PI-3 kinase abolished the MET-enhanced AMPK activity. This suggests that, in these cells, activation of AMPK by MET might be mediated by mitochondria-derived reactive nitrogen species and by activation of the c-SRC/PI-3 kinase pathway (Zou et al., Reference Zou, Hou, Shi, Kirkpatrick, Liu, Goldman and Cohen2003, Reference Zou, Kirkpatrick, Davis, Nelson, Wiles IV, Schlattner, Neumann, Brownlee, Freeman and Goldman2004). In the present study, addition of an eNOS inhibitor, a c-Src inhibitor or two PI-3 kinase inhibitors did not reverse the inhibitory effect of MET on porcine oocyte meiosis and suggested that these pathways are not involved in the inhibitory effect of MET on oocyte meiosis irrespective of AMPK involvement.
Activation of AMPK requires the phosphorylation of Thr172 of the α-subunit by an upstream AMPK kinase (AMPKK) (Hawley et al., Reference Hawley, Selbert, Goldstein, Edelman, Carling and Hardie1995). Two AMPKK have been identified; these are LKB1 and Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). LKB1, which was originally recognized as a tumor suppressor, is expressed ubiquitously in mammalian cells and phosphorylates and activates AMPK in response to energy depletion and MET in various cell types (Hawley et al., Reference Hawley, Boudeau, Reid, Mustard, Udd, Makela, Alessi and Hardie2003; Shaw et al., Reference Shaw, Kosmatka, Bardeesy, Hurley, Witters, DePinho and Cantley2004, Reference Shaw, Lamia, Vasquez, Koo, Bardeesy, DePinho, Montminy and Cantley2005; Gormand et al., Reference Gormand, Henriksson, Ström, Jensen, Sakamoto and Göransson2011). However, AMPK phosphorylation and activation by CaMKK is AMP-independent and is instead triggered by a rise in intracellular Ca2+ concentrations (Hurley et al., Reference Hurley, Anderson, Franzone, Kemp, Means and Witters2005; Gormand et al., Reference Gormand, Henriksson, Ström, Jensen, Sakamoto and Göransson2011). Several studies have shown that CaMKK-mediated AMPK activation may play an important role in how cells respond to nutrients, hormones and certain drugs in skeletal muscles (Jensen et al., Reference Jensen, Rose, Jorgensen, Brandt, Schjerlingt, Wojtaszewski and Richter2007; Shen et al., Reference Shen, Zhu, Tong, Ren and Du2007). CaMKK is expressed in pig oocytes at different maturation stages (Xu et al., Reference Xu, Xiong, Lin, Zhu, Hou, Chen and Sun2009) and, in the present study, we showed that selective inhibition of CaMKK inhibited meiosis but had no effect on ACC phosphorylation in porcine oocytes and cumulus cells. Additionally, chelation of Ca2+ was without effect on meiosis and ACC phosphorylation, a finding that indicated that CaMKK is probably not the upstream kinase responsible for AMPK activation in porcine oocytes and cumulus cells. A specific LKB1 inhibitor was not available and studies on the role of LKB1 are usually performed with LKB1−/− mutant cells.
As in bovine oocytes and cumulus cells (Bilodeau-Goeseels et al., Reference Bilodeau-Goeseels, Panich and Kastelic2011), most of the treatments applied to CEO resulted in very little change in ACC phosphorylation, even though they were supposed to modulate AMPK activation and most had an effect on oocyte maturation. This finding could be due to the fact that the level of ACC phosphorylation in oocytes and cumulus cells cultured in control conditions was already high and a slight increase or decrease could have been undetected. However, to determine whether the absence of effects (or detection of effects) of AMPK modulators was a characteristic of oocytes and cumulus cells or was due to experimental conditions, we performed basic experiments with porcine granulosa cell monolayers that showed that, in these cells, the pharmacological activators AICAR and MET can increase AMPK activation (as measured by ACC phosphorylation). Inhibition of CaMKK did not have any significant effect on ACC phosphorylation, however Ca2+ chelation completely abolished ACC phosphorylation. These results indicate that Ca2+ plays a crucial role in AMPK activation in porcine granulosa cells.
In conclusion, the inhibitory effect of AICAR and MET on porcine CEO was probably not mediated via activation of AMPK, as an AMPK inhibitor also prevented meiosis and AMPK activation (assessed by ACC phosphorylation) was not demonstrated after culture in the presence of AICAR and MET at inhibitory concentrations. The inhibitory effect of metformin on meiosis was not modulated by eNOS, the Src or the PI-3 kinase pathway and, as in bovine oocytes, the AMPK activators AICAR and MET probably inhibited meiosis through different pathways.
Acknowledgements
The authors thank Paul Panich for technical assistance and Sandra Hindson for graphics.
