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BOR - Papers in Press, published online ahead of print June 13, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.060848
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BIOLOGY OF REPRODUCTION 77, 452–465 (2007)
DOI: 10.1095/biolreprod.107.060848
© 2007 by the Society for the Study of Reproduction, Inc.

Possible Role of 5'AMP-Activated Protein Kinase in the Metformin-Mediated Arrest of Bovine Oocytes at the Germinal Vesicle Stage During In Vitro Maturation1

Lucie Tosca 3, Svetlana Uzbekova 3, Christine Chabrolle , and Joëlle Dupont 2

Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Nouzilly, France

ABSTRACT

The 5'AMP-activated protein kinase (AMPK) activation is involved in the meiotic maturation of oocytes in the ovaries of mice and pigs. However, its effects on the oocyte appear to be species-specific. We investigated the patterns of AMPK and mitogen-activated protein kinases (MAPK3/1) phosphorylation during bovine in vitro maturation (IVM) and the effects of metformin, an AMPK activator, on oocyte maturation in cumulus-oocyte complexes (COCs) and denuded bovine oocytes (DOs). In bovine COCs, PRKAA Thr172 phosphorylation decreased, whereas MAPK3/1 phosphorylation increased in both oocytes and cumulus cells during IVM. Metformin (5 and 10 mM) arrested oocytes at the GV stage in COCs but not in DOs. In COCs, this arrest was associated with the inhibition of cumulus cell expansion, an increase in PRKAA Thr172 phosphorylation, and a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. However, the addition of compound C (10 µM), an inhibitor of AMPK, accelerated the initiation of the GV breakdown (GVBD) process without any alteration of MAPK3/1 phosphorylation in oocytes from bovine COCs. Metformin decreased AURKA and CCNB1 protein levels in oocytes. Moreover, after 1 h of IVM, metformin decreased RPS6 phosphorylation and increased EEF2 phosphorylation, suggesting that protein synthesis rates were lower in oocytes from metformin-treated COCs. Most oocytes were arrested after the GVBD stage following the treatment of COCs with the MEK inhibitor, U0126 (100 micromoles). Thus, in bovine COCs, metformin blocks meiotic progression at the GV stage, activates PRKAA, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during IVM. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK3/1 phosphorylation was not.

5'AMP-activated protein kinase, cumulus cells, granulosa cells, mechanisms of hormone action, metformin, mitogen-activated protein kinases, oocyte, ovary, signal transduction

INTRODUCTION

In mammals, such as cattle, oocyte maturation is characterized by periods of meiotic arrest and resumption. In vivo, the oocyte remains at the immature stage or germinal vesicle stage (GV, i.e., prophase of meiosis I) until the ovulatory LH surge [1]. However, oocytes may also spontaneously resume meiosis when cumulus-oocyte complexes (COCs) are removed from the follicles and cultured in vitro [1, 2]. During nuclear maturation, immature oocytes undergo GV breakdown (GVBD) and proceed through metaphase II (Meta II) of meiosis. Meiotic resumption and maturation are regulated by various factors, including protein kinases, which modulate cellular processes by phosphorylation. These kinases include MAPK3/MAPK1 or MAPK3/1, formerly known as MAPK ERK1/2 (mitogen-activated protein kinase extracellular regulated kinase 1/2), also known as p44MAPK for ERK1 and p42MAPK for ERK2. For example, the MAPK1 activity increases throughout oocyte maturation, with a peak at the Meta II stage [3]. In several species, cAMP is an important regulator of meiotic maturation [4–6]. For example, it inhibits meiotic maturation in mammals by activation of protein kinase A [7]. However, cAMP is also converted by phosphodiesterases into 5'-AMP, which in turn activates the 5'AMP-activated protein kinase (AMPK) [8].

AMPK is a key regulatory enzyme of cellular energy homeostasis [9] and is involved, for example, in the regulation of fatty acid and cholesterol synthesis [10]. It is a serine/threonine protein kinase consisting of a catalytic subunit {alpha} (PRKAA1 or PRKAA2; formerly known as the AMPK {alpha}1 and {alpha}2 isoforms) and two regulatory subunits: ß (PRKAB1 or PRKAB2; formerly known as the AMPK ß1 and ß2 isoforms) and {gamma} (PRKAG1, PRKAG2, or PRKAG3; formerly known as the AMPK {gamma}1, {gamma}2, and {gamma}3 isoforms) [11]. AMPK activity results partly from phosphorylation of the Thr172 residue of the {alpha} subunit by two known upstream kinases: LKB1 and calcium-calmodulin-dependent kinase kinase (CaMKK) [11, 12]. AMPK is activated by a change in AMP:ATP ratio in response to several stimuli, including exercise [13], hypoxia [14], hormones [15, 16], and pharmacological drugs such as metformin [17] and 5-aminoimidazole-4-carboxamide-riboside-5-phosphate (AICAR) [18]. AMPK is a multisubstrate enzyme well characterized in many tissues, including the liver, muscle, lung, heart, kidney, brain [19], and ovary [20–22].

In mice, AMPK activation stimulates the resumption of meiosis in oocytes maintained in meiotic arrest by cAMP analogs, suggesting that this kinase plays a key role in oocyte maturation [23–26]. However, the data for mice conflict with those obtained with porcine oocytes [27], as some activators of AMPK, such as AICAR and metformin, which significantly increase the percentage of porcine COCs arrested at the GV stage [27]. These findings were obtained with COCs but not with DOs (denuded oocytes), suggesting that porcine cumulus cells are responsible for inhibiting the meiotic progression induced by AMPK activation or that cumulus cells block AICAR from coming into contact with the oocyte. We recently characterized AMPK in bovine ovary and demonstrated that metformin-induced AMPK activation decreased the levels of MAPK3/1 phosphorylation and basal and FSH- or insulin-like growth factor-1-induced progesterone and estradiol secretion by granulosa cells [28]. Metformin is an insulin-sensitizing agent, which has been used over a number of years for the clinical treatment of type 2 diabetes mellitus [29] and polycystic ovary syndrome (PCOS). PCOS is the most common cause of anovulation and infertility [30]. It is characterized by hyperandrogenism and chronic anovulation, often associated with insulin resistance [30, 31]. Patients with PCOS also display impaired folliculogenesis and poor oocyte quality [32]. Metformin improves oocyte maturation rate [33], ovulation, fertilization, and pregnancy rates in patients with PCOS [34, 35]. However, its mechanism of action remains unclear. Ovarian follicular cysts are the most important ovarian disorders in high-lactating dairy cows [36, 37]. They reduce reproductive efficiency by increasing the calving interval by about 22–64 days [38]. Oocyte quality may also be affected in the ovary [39], as in PCOS.

AMPK activation and the effects and mechanism of action of metformin have not, to our knowledge, ever been investigated in bovine oocytes. We therefore analyzed the patterns of AMPK and MAPK3/1 phosphorylation in bovine oocyte and cumulus cells during the in vitro maturation (IVM) of COCs. We then investigated the effects and possible molecular mechanisms of metformin on the meiotic progression of bovine oocytes in COCs and in DOs, to evaluate the role of cumulus cells in the effects of metformin on the oocyte.

MATERIALS AND METHODS

Reagents

TCM199 medium, hyaluronidase, trypan blue, and metformin were obtained from Sigma. The MEK-specific inhibitor U0126 and the AMPK inhibitor compound C (Comp C) were obtained from Calbiochem. Metformin was dissolved in sterile water, and U0126 and Comp C were dissolved in dimethyl sulfoxide (DMSO). The concentration of DMSO in the medium was kept below 0.1%.

Antibodies

Rabbit polyclonal antibodies against phospho-PRKAA (Thr172), phospho-MAPK3/1 (Thr202/Tyr204), phospho-EEF2 for phospho-Eukaryotic Elongation Factor 2 (Thr56), phospho-RPS6 for phospho-Ribosomal Protein S6 (Ser 235/236), and EEF2 were purchased from New England Biolabs. Rabbit polyclonal antibodies against MAPK1 (C14) and RPS6 were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against PRKAA1/2 and PRKAA1 were obtained from Upstate Biotechnology. Mouse monoclonal antibody against vinculin was obtained from Sigma. Mouse monoclonal antibody against recombinant full-length human AURKA kinase (formerly known as Aurora A kinase) was produced and characterized in bovine oocytes, as previously described [40]. Mouse CCNB1 formerly known as cyclin B1 (Ab-3) monoclonal antibody, was purchased from Lab Vision.

Oocyte Collection and IVM

All procedures were approved by the Agricultural Agency and the Scientific Research Agency and conducted in accordance with the guidelines for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Ovaries collected from slaughtered cows were transported, in physiological saline, at 37°C, to the laboratory. COCs were obtained by aspiration from 2- to 6-mm antral follicles. Only oocytes surrounded by more than three layers of compact cumulus cells were selected. COCs were washed four times in modified PBS (supplemented with 36 mg/L pyruvate, 50 mg/L gentamycin, and 0.5 g/L BSA), and groups of 35–40 COCs were then transferred to four-well plates containing 500 µl of maturation medium and incubated for 1, 3, 6, 10, or 22 h, at 39°C, in a humidified atmosphere consisting of 5% CO2 in air. The maturation medium routinely used was TCM199 supplemented with 10 ng/ml epidermal growth factor (EGF, for permissive maturation), as previously described [40]. COCs for experiments were sampled as follows: 1) just after collection at T0 (immature oocytes at the GV stage) or 2) after 1, 3, 6, 10, or 22 h of culture in the presence or absence of metformin (0.1–10 mM) or in the presence or absence of Comp C (10 µM) or U0126 (100 µM). Immature and matured oocytes were then denuded by mechanical and enzymatic (hyaluronidase 0.5%) treatments. The samples (cumulus cells and oocytes) were frozen at –80°C until use.

Evaluation of Oocyte Meiotic Stages

Oocyte meiotic stages were determined by Hoechst staining of chromatin and by immunofluorescence with a lamin A/C antibody, as previously described [41].

For chromatin staining, oocytes were fixed on slides in 100% ethanol and mounted in Moviol supplemented with 1 µg/µl Hoechst 33258 (Sigma). Immunofluorescence was observed with an Axioplan Zeiss fluorescence microscope equipped with the appropriate filter. For lamin A/C staining, oocytes were fixed by incubation for 10 min in 50% saturated picric acid, 3.7% formaldehyde, and 5% acetic acid and then washed four times in PBS supplemented with 0.2% BSA. They were incubated in PBS supplemented with 0.2% BSA/10% goat serum/0.1% Triton X-100 for 1 h for blocking. Oocytes were incubated overnight at 4°C with anti-lamin A/C antibodies (1/100, Ozyme), with constant shaking. They were washed four times, for 30 min each, in PBS-0.2% BSA and then with secondary Alexa Fluor 594 goat anti-mouse antibody (1/200, Molecular Probes) for 1 h at room temperature. They were then washed four times, for 30 min each. Immunofluorescence was observed with a confocal microscope equipped with the appropriate filters.

Western Blotting

Oocytes or cumulus cells were frozen directly in lysis buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 0.5% Igepal) supplemented with various protease inhibitors (2 mM phenylmethyl sulfonyl fluoride, 10 mg/ml leupeptin, and 10 mg/ml aprotinin) and phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium orthovanadate; Sigma). Cell extracts were subjected to electrophoresis in a 12% (w:v) polyacrylamide gel under reducing conditions. Proteins were electrotransferred onto nitrocellulose membranes (Schleicher and Schuell) over a period of 1.5 h at 80 V. Membranes were incubated for 1 h at room temperature with Tris-buffered saline (TBS; 2 mM Tris-HCl, pH 8.0, and 15 mM NaCl, pH 7.6) supplemented with 5% nonfat dry milk powder (NFDMP) and 0.1% Tween-20 to saturate nonspecific sites. Membranes were then incubated overnight at 4°C with the appropriate antibodies (final dilution, 1:1000) in TBS supplemented with 0.1% Tween-20 and 5% NFDMP. Membranes were washed in TBS-0.1% Tween-20 and incubated for 2 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG) (final dilution, 1:10 000; Diagnostic Pasteur) in TBS-0.1% Tween-20. They were washed in TBS-0.1% Tween-20, and the signal was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). The films were analyzed, and the signals were quantified with Scion Image software Beta 4.0.2 (Fuji PhotoFilm).

Immunohistochemistry

Ovary biopsy samples were fixed by incubation for 12 h in 50% saturated picric acid, 3.7% formaldehyde, and 5% acetic acid. Samples were dehydrated by passage through a graded series of alcohols and embedded in paraffin. Serial sections (7 µm) were cut, and the paraffin was removed; they were then rehydrated and incubated for 5 min in antigen unmasking solution (Vector Laboratories) in the microwave. They were then left to cool to room temperature. Sections were washed in PBS for 5 min and then immersed in peroxidase blocking reagent for 10 min at room temperature to quench endogenous peroxidase activity (DAKO Cytomation; DAKO). They were washed three times, for 5 min each, in PBS and blocked by incubation with 5% goat serum in PBS for 20 min. They were then incubated overnight at 4°C with PBS-0.1% BSA supplemented with the anti-PRKAA1 antibody. The sections were washed three times, for 5 min each, in PBS and were incubated for 30 min at room temperature with a biotinylated goat anti-rabbit antibody. After several washes in PBS, the sections were incubated in streptavidin peroxidase solution (Lab Vision) for 10 min and then with 3,3'-diaminobenzidine (Lab Vision) at room temperature. Slides were counterstained with Meyer hematoxylin (Sigma) and mounted in Depex (Sigma). For negative controls, the primary antibodies were replaced with rabbit IgG. Slides were observed under an Axioplan Zeiss transmission microscope. The specificity of the antibody has been demonstrated in rat tissues, in experiments involving the incubation of primary antibody with blocking peptide (20 µg/ml SC-19128P from Santa Cruz for PRKAA1 [20]) before incubation with sections.

Progesterone RIA

The concentration of progesterone in the culture medium of cumulus cells from COCs or detached cumulus cells without oocytes was measured after 3, 6, or 22 h of maturation in vitro, with or without 10 mM metformin, by RIA, as previously described [20]. The detection limit for progesterone was 12 pg/tube (60 pg/well), and the intra- and interassay coefficients of variation were less than 10% and 11%, respectively. Results are expressed as the amount of steroids (nanograms per milliliter) secreted over 3, 6, or 22 h per 35 COC-equivalent cumulus cells.

Statistical Analysis

All experimental data are presented as mean ± SEM. One-way ANOVA was used to test differences. If ANOVA detected significant effects, the means were compared by the Newman test, with P < 0.05 considered significant.

RESULTS

Localization of PRKAA1 in Bovine COCs During Folliculogenesis

Consistent with previous reports [28], immunohistochemical methods localized the PRKAA1 subunit to bovine follicles (Fig. 1). PRKAA1 was detected in the oocyte (Oo) and its surrounding cumulus cells in primary (Fig. 1a) and preantral (Fig. 1b) follicles and in theca cells, granulosa cells, cumulus cells, and oocytes in early antral (Fig. 1, c and d) and antral (Fig. 1, f and g) follicles. Thus, PRKAA1 is present in COCs throughout folliculogenesis.


Figure 01
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  		FIG. 1 Localization by immunohistochemistry of PRKAA1 in cumulus-oocyte complexes (COCs) from bovine ovarian follicles during folliculogenesis. DAB-immunoperoxidase staining was performed on paraffin-embedded bovine ovary sections with antibodies against PRKAA1 (a, b, c, d, f, and g) or no primary antibody (e and h). Specific immunostaining is shown in brown. The sections were counterstained with Meyer hematoxylin. PRKAA1 is detected in oocyte (Oo) and cumulus cells (CC) from primary (a) and preantral (b) follicles and in theca cells (Tc), granulosa cells (Gc), oocyte (Oo), and cumulus cells (CC) from early antral (c and d) and antral (f and g) follicles. Black rectangles in (c) and (f) are magnified in (d) and (g), respectively. Bars = 50 µm (a, b, d, g) and 100 µm (c, f, e, h).

Effects of IVM on PRKAA and MAPK3/1 Phosphorylation Levels in Bovine COCs

As shown in Figure 2 (left panel), PRKAA was phosphorylated on Thr172 in immature oocytes at the GV stage (Oo T0). Interestingly, PRKAA phosphorylation in oocytes was very weak or undetectable after 22 h of COC IVM. As expected [3], IVM induced MAPK3/1 phosphorylation in oocytes (Oo IVM 22 h, Fig. 2, left panel). Similar results were obtained with cumulus cells, except that the MAPK3/1 kinases were already phosphorylated in immature cells, and the increase in MAPK3/1 phosphorylation, although significant, was smaller in cumulus cells than in oocytes after 22 h of IVM (Fig. 2, right panel). Similarly, the decrease in PRKAA phosphorylation, although significant, was also smaller in cumulus cells than in oocytes after 22 h of IVM (Fig. 2, right panel). Thus, after 22 h of IVM, phosphorylated PRKAA levels were found to have decreased in COCs, whereas phospho-MAPK3/1 levels had increased in both oocytes and cumulus cells.


Figure 02
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  		FIG. 2 PRKAA and MAPK3/1 phosphorylation levels in oocyte (Oo) and cumulus cells (CC) from in vitro bovine immature and matured COCs. Immunodetection was made of phospho-PRKAA, PRKAA1/2, phospho-MAPK3/1, and MAPK3/1 in GV oocytes just after collection at T0 (Oo T0) or after 22 h of IVM with cumulus cells (Oo IVM 22 h) in TCM199 supplemented with EGF. After 22 h of IVM, each COC was mechanically separated into oocyte (Oo IVM 22 h, left panel) and cumulus cells (CC IVM 22 h, right panel) (35 oocytes/cumulus per lane). Oocyte and cumulus cells were then lysed and subjected to Western blotting with antibodies against phospho-PRKAA (Thr172), PRKAA1/2, phospho-MAPK3/1, and MAPK1. Representative blots from three independent experiments are shown. Blots were quantified, and the phosphorylated protein:total protein ratio is shown. The phosphorylation states at 22 h of IVM were normalized to the phosphorylation levels at T0. The results are presented as mean ± SEM. Different letters indicate significant differences with P < 0.05. Different capital letters indicate significant differences in the phosphorylation of MAPK3/1, whereas different lower-case letters indicate significant differences in the phosphorylation of PRKAA. A rat ovary sample was used as a positive control (C).

Effects of Metformin Treatment on the Nuclear Maturation of Bovine Oocytes in COCs

We studied the effects of metformin (10 mM), a well-known AMPK activator, on the meiotic progression during IVM of bovine oocytes in COCs. After oocyte collection (T0), all oocytes had an intact nucleus (GV stage, Fig. 3, A.a and B). As shown in Figure 3A.a, GV chromatin was either diffuse or condensed into a perinucleolar ring, as previously described [42]. Moreover, as previously shown [41], the bovine GV stage oocyte was stained with antibodies against lamin A/C (Fig. 3A.a). After 6 h of IVM, about 25% of oocytes underwent GVBD (Fig. 3A.b). After 22 h of COC culture in IVM medium, all oocytes underwent GVBD, about 80% of oocytes went up to meiotic arrest at Meta II (Fig. 3A.c), and only about 5% remained at the GV stage (Fig. 3B). However, if COCs were matured for 22 h in IVM medium supplemented with metformin (5 or 10 mM), more than 80% of the oocytes remained at the GV stage, with chromatin condensation into a perinucleolar ring (Fig. 3, A.d and B). Thus, metformin treatment of COCs during IVM resulted in the arrest of most of the bovine oocytes at the GV stage.


Figure 03
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  		FIG. 3 Effects of metformin treatment on bovine oocyte nuclear maturation. A) Hoechst (blue) and anti-lamin A/C (red) staining before (T0, GV stage just after oocyte collection; a), during (IVM 6 h, GVBD stage; b), and after maturation in the absence (IVM 22 h, MII stage; c) or in the presence of metformin at a concentration of 10 or 5 mM (IVM 22 h, GV stage; d). Bars = 50 µM (a–d). B) Bovine oocytes were allowed to mature for 22 h in the presence or absence of various concentrations of metformin (0.1, 5, and 10 mM). The percentage of oocytes at the GV stage in the various conditions is shown. Different letters indicate significant differences with P < 0.05. The results are presented as mean ± SEM of three independent experiments. We used 40 bovine oocytes for each set of conditions in each experiment.

Effects of Metformin Treatment on PRKAA and MAPK3/1 Phosphorylation Levels in Bovine Oocytes in COCs after IVM for Various Periods of Time

We investigated the molecular mechanisms involved in the effects of metformin on the nuclear maturation of bovine oocytes in COCs by determining the levels of PRKAA and MAPK3/1 phosphorylation in the presence or absence of metformin (10 mM) in oocytes allowed to mature in vitro for various periods of time (6, 10, and 22 h). As shown in Figure 4A, the level of PRKAA phosphorylation decreased, whereas that of MAPK3/1 increased over time in the oocyte during IVM. The addition of metformin (10 mM) to the maturation medium for 6 h doubled PRKAA phosphorylation but decreased MAPK3/1 phosphorylation by a factor of three in oocytes from COCs. This increase in PRKAA phosphorylation in oocytes in response to metformin was abolished after 22 h of IVM, whereas the level of MAPK3/1 phosphorylation remained about one third of that in untreated cells. Thus, metformin treatment during IVM increased PRKAA phosphorylation and decreased MAPK3/1 phosphorylation in COCs.


Figure 04
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  		FIG. 4 Effects of metformin treatment on the level of phosphorylation of PRKAA and MAPK3/1 in bovine oocytes from COCs during IVM. Bovine COCs were cultured for various lengths of time (6, 10, or 22 h) in maturation medium in the presence or absence of metformin (10 mM). COCs were then mechanically separated into oocyte and cumulus cells. Denuded oocytes (40 oocytes per lane) were lysed and subjected to Western blotting with antibodies against phospho-PRKAA (Thr172) and PRKAA1/2 (A) and phospho-MAPK3/1 and MAPK1 (B). Representative blots from three independent experiments are shown. Blots were quantified, and the phosphorylated protein:total protein ratio is shown. Different letters indicate significant differences with P < 0.05. The results are presented as mean ± SEM.

Effects of Comp C Treatment on Nuclear Maturation and PRKAA and MAPK3/1 Phosphorylation Levels in Bovine Oocytes in COCs after IVM

To determine whether PRKAA phosphorylation is involved in the metformin-induced meiotic arrest, we investigated the effects of Comp C, a well-known inhibitor of AMPK, on nuclear maturation. After 6 h of IVM in COCs, the Comp C treatment (10 µM) significantly decreased the percentage of oocytes at the GV stage from 57.9% ± 2.5% to 32.5% ± 1.8% compared with the control group with DMSO (Fig. 5A). The viability of oocytes as determined by Hoechst staining was not affected by the treatment (data not shown). By lamin A/C staining, we observed that the oocytes that were not at the GV stage were at the GVBD stage. After 3 h of IVM, immunoblot analysis confirmed that Comp C treatment significantly decreased PRKAA phosphorylation compared with the control group with DMSO (Fig. 5B). However, the Comp C treatment had no effect on the MAPK3/1 phosphorylation (Fig. 5C). Thus, Comp C accelerates the initiation of the GVBD during the first 6 h of IVM and inhibits PRKAA phosphorylation without any variation of MAPK3/1 phosphorylation after 3 h of IVM in bovine COCs.


Figure 05
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  		FIG. 5 Effects of compound C (Comp C) treatment on oocyte nuclear maturation (A) and on the phosphorylation levels of PRKAA and MAPK3/1 in oocytes from bovine COCs during IVM. Three independent groups of bovine COCs were cultured for 3 or 6 h in maturation medium in the presence or absence of Comp C (10 µM). After the culture, oocytes and cumulus cells were separated. A) At T0 and after 6 h of IVM, the percentage of oocytes at the GV was determined by Hoechst and lamin A/C stainings. Different letters indicate significant differences with P < 0.05. B, C) At T0 and after 3 h of IVM in the presence or absence of Comp C (10 µM), 30 oocytes from COCs were lysed and subjected to Western blotting with antibodies against phospho-PRKAA (Thr 172) and PRKAA1/2 (B) and phospho-MAPK3/1 and MAPK1 (C). Representative blots for the three experiments are shown. Blots were quantified, and the phosphorylated protein:total protein ratio is shown. The results are mean ± SEM. Different letters indicate significant differences with P < 0.05.

Effects of Metformin Treatment on Levels of CCNB1 and AURKA Protein and on the Level of Phosphorylation of RPS6 and EEF2 in Bovine Oocytes in COCs During IVM

We investigated whether metformin supplementation during oocyte maturation affected the levels of two proteins involved in oocyte maturation in several species: CCNB1 and AURKA. The levels of CCNB1 and AURKA in bovine oocytes were found to have increased significantly after the culture of COCs for 22 h in IVM medium (Fig. 6A). This increase was totally abolished in the presence of metformin (10 mM). Protein synthesis is crucial for the initiation of oocyte maturation in large domestic species, including cattle [43]. We therefore investigated the effects of metformin on the phosphorylation state of ribosomal protein S6 (RPS6) and the EEF2 elongation factor, two factors involved in protein synthesis [44, 45] (Fig. 6B). EEF2 activity is essential for the elongation step of translation, and this activity is negatively regulated by phosphorylation [46]. IVM for 1 h significantly increased the phosphorylation of RPS6 and decreased that of EEF2 with respect to the T0 state (at the collection time). Metformin (10 mM) totally abolished the increase in RPS6 phosphorylation and the decrease in EEF2 phosphorylation induced after 1 h of IVM, which suggests that metformin inhibits protein synthesis in bovine oocytes (Fig. 6B).


Figure 06
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  		FIG. 6 Effects of metformin treatment on CCNB1 and AURKA protein levels and on EEF2 and RPS6 phosphorylation levels in bovine oocytes from COCs. A) Bovine COCs were cultured for 22 h in maturation medium in the presence or absence of 10 mM metformin. COCs were then mechanically separated into oocyte and cumulus cells. Denuded oocytes were lysed and subjected to Western blotting (40 oocytes per lane) with antibodies against AURKA, CCNB1, and VLC. As a control of meiotic maturation, we also probed the membranes with antibodies against phospho-MAPK3/1 and MAPK1. Representative blots from three independent experiments are shown. Blots were quantified, and the CCNB1:VLC and AURKA:VLC ratios are shown. Different letters indicate significant differences with P < 0.05. The results are presented as mean ± SEM. B) Bovine COCs were cultured for 1 h in maturation medium in the presence or absence of 10 mM metformin. COCs were then mechanically separated into oocyte and cumulus cells. Denuded oocytes were lysed and subjected to Western blotting (40 oocytes per lane) with antibodies against phospho-EEF2, phospho-RPS6, EEF2, and RPS6. Representative blots from three independent experiments are shown. Blots were quantified, and the phospho-EEF2:EEF2 and phospho-RPS6:RPS6 ratios are shown. Different letters indicate significant differences with P < 0.05. The results are presented as mean ± SEM.

Effects of the MEK Inhibitor U0126 on Bovine Oocytes During IVM in COCs

We investigated whether the arrest of bovine oocyte maturation in response to metformin was due to inhibition of the MAPK3/1 signaling pathway. We used the MEK-specific inhibitor U0126 (100 µM) to block MAPK3/1 phosphorylation and observed the nuclear status of bovine oocytes in COCs after 22 h of IVM. We first checked that U0126 (100 µM) totally inhibited MAPK3/1 phosphorylation in bovine oocytes during IVM without affecting the viability of cumulus cells, as determined by trypan blue staining and the viability of oocytes evaluated by Hoechst staining as previously described ([47], data not shown). Hoechst and anti-lamin A/C staining showed that most of the oocytes (about 65%) underwent GVBD and were arrested in pro-Meta I after incubation with U0126 for 22 h of IVM (Fig. 7, A and B). Whereas about 80% of the oocytes were arrested at the GV stage after metformin treatment, only about 20% were arrested at the GV stage after U0126 treatment (Fig. 7, A and B). This suggests that the inhibition of MAPK3/1 by metformin is not the main molecular mechanism involved in the effects of metformin on bovine oocyte maturation.


Figure 07
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  		FIG. 7 Effects of the MEK inhibitor U0126 on oocyte nuclear maturation. A) Bovine oocytes were either studied immature (T0) or allowed to mature for 22 h in the presence or absence of U0126 (100 µM) or metformin (10 mM). The percentage of oocytes at the GV stage in the various conditions is shown. Different letters indicate significant differences with P < 0.05. The results are presented as mean ± SEM for three independent experiments. We used 40 bovine oocytes in each set of conditions in each experiment. B) Hoechst (blue) and anti-lamin A/C (red, small rectangles) staining after maturation in the absence (IVM 22 h, Meta II stage; a) or presence of 100 µM U0126 (IVM 22 h + U0126 (GVBD/Pro-Meta I; b) or 10 mM metformin (IVM 22 h + MetF, GV; c). Bars = 50 µm (a–c).

Effects of Metformin Treatment on Bovine DOs During IVM

We assessed the importance of cumulus cells in the effects of metformin by allowing oocytes to undergo IVM in the presence (COCs) or absence (DOs) of cumulus cells. The percentage of bovine oocytes at the GV stage after 22 h of IVM was similar (about 5%) in COCs and DOs (Fig. 8A). The addition of metformin during bovine oocyte maturation clearly inhibited meiotic progression in COCs but not in DOs. Indeed, about 80% of the bovine oocytes in COCs treated with metformin during 22 h of IVM were arrested at the GV stage, whereas only about 7% of the bovine DOs matured for 22 h were at the GV stage (Fig. 8A). IVM for 22 h led to the induction of MAPK3/1 phosphorylation but completely abolished PRKAA phosphorylation in oocytes from COCs and DOs (Fig. 8B). Unlike oocytes from COCs (Fig. 8, A and B), DOs displayed no effect of metformin treatment on PRKAA and MAPK3/1 phosphorylation levels after 6 or 22 h of IVM (Fig. 8C, right and left panels). Furthermore, metformin treatment had no effect on PRKAA phosphorylation levels in oocytes from DOs after 1 h of IVM (Fig. 8D). These data suggest that cumulus cells play an essential role in the effects of metformin on the meiotic progression of bovine oocytes.


Figure 08
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  		FIG. 8 Effects of metformin on oocyte nuclear maturation and on the levels of PRKAA and MAPK3/1 phosphorylation in denuded oocytes (DOs) during IVM. Bovine oocytes were allowed to mature for 22 h in the presence (COCs) or absence (DOs) of cumulus cells and in the presence or absence of 10 mM metformin. COCs were then mechanically separated into oocyte and cumulus cells. A) Some of the oocytes (COCs and DOs) were fixed and mounted on slides with Hoechst stain to visualize the chromatin. The percentage of oocytes at the GV stage is shown. Different letters indicate significant differences with P < 0.05. The results are presented as mean ± SEM for three independent experiments. B) Other oocytes were frozen and analyzed by Western blotting with antibodies against phospho-PRKAA (Thr172), PRKAA1/2 phospho-MAPK3/1, and MAPK1, as previously described. Representative blots from three independent experiments are shown. C) Bovine DOs were cultured for various periods of time (6 or 22 h) in maturation medium in the presence or absence of metformin (10 mM). DOs (40 oocytes per lane) were lysed and subjected to Western blotting with antibodies against phospho-PRKAA (Thr172), PRKAA1/2, phospho-MAPK3/1, and MAPK1. Representative blots from three independent experiments are shown. Blots were quantified, and the phosphorylated protein:total protein ratio is shown. Different letters indicate significant differences with P < 0.05. The results are presented as mean ± SEM. D) Bovine DOs were cultured for 1 h or not (T0) in maturation medium in the presence or absence of metformin (10 mM). DOs (40 oocytes per lane) were lysed and subjected to Western blotting with antibodies against phospho-PRKAA (Thr172) and PRKAA1/2. One representative blot from three independent experiments is shown. Blots were quantified, and the phosphorylated protein:total protein ratio is shown. Different letters indicate significant differences with P < 0.05. The results are presented as mean ± SEM.

Effects of Metformin Treatment on the Cumulus Cells of Bovine COCs During IVM (Cell Expansion, Cell Viability, and Levels of Phosphorylation of PRKAA and MAPK3/1)

We then studied the effect of metformin on cumulus cells, as these cells seem to be essential for the effects of metformin on the maturation of bovine oocytes in vitro. The addition of metformin (10 mM) to the maturation medium markedly reduced the expansion of cumulus cells observed after 22 h of IVM (Fig. 9). However, metformin (0.1, 5, or 10 mM) had no effect on the viability of cumulus cells, as shown by trypan blue staining (Fig. 9B). We also determined the levels of phosphorylation of PRKAA and MAPK3/1 in cumulus cells treated with various doses of metformin (0.1, 5, and 10 mM) during IVM for 22 h (Fig. 9C) or with 10 mM metformin for various periods of IVM (Fig. 10, A and B). Metformin treatment increased the level of phosphorylation of PRKAA and decreased those of MAPK3/1 in a dose-dependent manner after 22 h of IVM (Fig. 9C). However, these effects of metformin occurred very early, as PRKAA phosphorylation tripled within 6 h of the onset of IVM, whereas the level of MAPK3/1 phosphorylation was halved (Fig. 10, A and B).


Figure 09
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  		FIG. 9 Effect of metformin treatment on bovine cumulus cells after 22 h of IVM in COCs. Bovine COCs were cultured in maturation medium for 22 h in the presence or absence of various doses of metformin (0.1, 5, and 10 mM). Oocytes and cumulus cells were then separated. A) The expansion of bovine cumulus cells is shown before (T0) and after IVM (22 h) in the absence (IVM) or presence of 10 mM metformin (IVM+MetF). Original magnification x10. B) The percentage of the cumulus cells viable after IVM (22 h) in the presence of various doses of metformin (0.1, 5, and 10 mM) was determined by trypan blue staining. The results are represented as mean ± SEM for three independent experiments. C) The levels of phosphorylation of PRKAA (Thr172) and MAPK3/1 in cumulus cells in response to various doses of metformin (0.1, 5, and 10 mM) were also measured as described in the legend to Figure 2. Representative blots from three independent experiments are shown. The results are presented as mean ± SEM. Different letters indicate significant differences with P < 0.05. Different capital letters indicate significant differences in MAPK3/1 phosphorylation, whereas different lower-case letters indicate significant differences in PRKAA phosphorylation.


Figure 10
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  		FIG. 10 Effects of metformin treatment on PRKAA (Thr172) (A) and MAPK3/1 (B) phosphorylation and progesterone secretion (C) by bovine cumulus cells after IVM for various periods of time. Bovine COCs were cultured for various periods of time (6, 10, or 22 h) in maturation medium in the presence or absence of 10 mM metformin. Oocytes and cumulus cells were then separated. Cumulus cells (20 COC-equivalent cumulus cells per lane) were lysed and subjected to Western blotting with antibodies against phospho-PRKAA (Thr172) and PRKAA1/2 (A), phospho-MAPK3/1, and MAPK1 (B). Representative blots from three independent experiments are shown. Blots were quantified, and the phosphorylated protein:total protein ratio is shown. Different letters indicate significant differences with P < 0.05. The results are presented as mean ± SEM. C) Cumulus cells from bovine COCs were cultured for 3, 6, or 22 h in maturation medium in the presence or absence of 10 mM metformin and in the absence (CC) or presence of oocytes (COCs). The culture medium was then collected, and its progesterone content was analyzed by RIA, as described in Materials and Methods. Results are expressed as nanograms per milliliter of 35 COC-equivalent cumulus cells. Results are mean ± SEM for three independent experiments. Different letters indicate significant differences with P < 0.05. Different capital letters indicate significant differences for the COCs, whereas different lower-case letters indicate significant differences for cumulus cells.

Effects of Metformin on the Progesterone Secretion of Cumulus Cells During the IVM of Bovine COCs

We have shown that AMPK activation inhibits the phosphorylation of MAPK3/1 and decreases progesterone secretion in bovine granulosa cells [28]. Moreover, progesterone secretion by cumulus cells is known to play a key role in bovine oocyte maturation [48, 49]. We therefore investigated the effects of metformin on progesterone secretion by COCs and dissociated cumulus cells. As expected, progesterone levels gradually increased over the course of IVM in the presence (COCs) or absence (cumulus cells, Fig. 10C) of oocytes. The addition of metformin to the maturation medium for 22 h significantly decreased progesterone secretion in cumulus cells matured without oocytes but not in those matured within COCs (Fig. 10C).

DISCUSSION

In the present study, we show that PRKAA1 is present in bovine COCs throughout folliculogenesis. The level of PRKAA phosphorylation on Thr172 is high in immature oocytes and cumulus cells (GV stage) and decreases strongly during IVM. These findings contrast with those for MAPK3/1 phosphorylation (Fig. 2). The addition of metformin (10 mM) to the maturation medium arrested most of the bovine oocytes at the GV stage in COCs, but not in DOs, which suggests that cumulus cells are essential for the effects of metformin on bovine oocyte maturation. Moreover, in the presence of metformin, PRKAA phosphorylation increased, whereas MAPK3/1 phosphorylation decreased in oocyte and cumulus cells from COCs during IVM (Fig. 2). Metformin increased PRKAA phosphorylation, inhibited RPS6 phosphorylation, and increased EEF2 phosphorylation in oocytes after 1 h of IVM in COCs, suggesting that metformin blocks protein synthesis in bovine oocytes. Unlike metformin, the MEK inhibitor U0126 (100 µM) arrested oocytes after GVBD. Furthermore, the Comp C treatment (10 µM) decreased PRKAA phosphorylation and accelerated the initiation of GVBD but did not affect MAPK3/1 phosphorylation. These results suggest that PRKA activation can regulate nuclear maturation in bovine oocyte during IVM. Furthermore, MAPK3/1 is not essential for the inhibitory effects of metformin on the meiotic progression from GV to GVBD stages of bovine oocytes (Fig. 6).

PRKAA phosphorylation levels in both oocytes and cumulus cells decreased during the IVM of bovine COCs. This decrease in phosphorylation was inversely correlated with MAPK3/1 phosphorylation levels. MAPK3/1 activation occurs during the first hours of maturation in many species, including cattle [3]. In bovine oocytes, it is associated with the GVBD stage [3]. We recently showed that PRKAA activation decreases MAPK3/1 phosphorylation in rat and bovine granulosa cells [20, 28]. Our hypothesis is that the decrease in PRKAA phosphorylation during the first hours of IVM is one of the events leading to the increase in MAPK3/1 phosphorylation in bovine oocytes during IVM. To determine the involvement of the MAPK3/1 pathway in the nuclear maturation, we used the MEK inhibitor U0126. We show that MAPK3/1 activation does not appear necessary for the initiation of GVBD, but it is crucial for the Meta I stage in bovine oocytes. The increase in the MAPK3/1 activation at the Meta I stage could be a result of the GVBD initiation induced by PRKAA dephosphorylation. We investigated the role of PRKAA activation in bovine oocyte maturation by studying the effects of metformin, which is known to activate AMPK in various systems, including porcine oocytes [27]. We found that the addition of metformin (10 mM) to the maturation medium of bovine COCs arrested oocytes at the GV stage. Our results are consistent with previous results obtained for porcine oocytes [27] but not with those obtained for mouse oocytes [24]. Mayes et al. [27] recently demonstrated that AMPK activators, such as AICAR (1 mM) and metformin (1 mM), transiently block the resumption of meiosis in pigs (more than 70% of porcine oocytes were arrested at the GV stage after 22 h of IVM in the presence of these molecules). In contrast, in mouse oocytes, PRKAA activation in response to AICAR promotes the resumption of meiosis in oocytes following arrest in response to cAMP analogs, phosphodiesterase inhibitors, or hypoxanthine [24]. The timing of meiotic resumption may account for theses differences in results for rodents and other animals. Meiotic resumption (GVBD) occurs very early in IVM (within 2 h [50]) in mice, whereas at least 6 h are required in bovine oocytes [51].

In the present study, we show that metformin-induced arrest at the GV stage of the oocyte after 22 h of IVM is associated with concomitant increases in PRKAA phosphorylation and decreases in MAPK3/1 phosphorylation in both oocyte and cumulus cells. We also show that levels of CCNB1 and AURKA are significantly lower in the presence of metformin than in its absence. These two factors are induced during IVM in bovine oocytes [40, 52]. With the MEK inhibitor, U0126 (100 µM), we demonstrated that most of the bovine oocytes were blocked after the GVBD stage, suggesting that the decrease in MAPK3/1 phosphorylation levels was not the main event accounting for the inhibitory effect of metformin on bovine oocyte maturation and is not a direct result of PRKAA dephosphorylation. This is confirmed by the results obtained with the Comp C experiments in the present study. Indeed, the Comp C (10 µM) inhibited PRKAA phosphorylation and accelerated the initiation of GVBD stage without increasing MAPK3/1 phosphorylation before GVBD. Thus, AMPK might act before the beginning of the GVBD process independently on MAPK3/1 and might be involved later with active MAPK3/1 for chromatin condensation and/or spindle formation during the Meta I stage. Metformin treatment during 1 h of IVM in COCs increased PRKAA phosphorylation, inhibited RPS6 phosphorylation, and increased EEF2 phosphorylation in oocytes. This suggests that metformin inhibits protein synthesis in bovine oocytes. RPS6 and EEF2 are key molecules in protein synthesis [44, 45]. Moreover, metformin-induced PRKAA activation has been shown to inhibit several factors involved in protein synthesis, such as P70S6K [53] and EEF2 [45]. Thus, metformin treatment, by activating PRKAA, may inhibit oocyte protein synthesis, leading to the arrest of bovine oocytes at the GV stage. Indeed, new protein synthesis during the first 8 h of culture is required for spontaneous meiotic resumption in bovine oocytes [52, 54], whereas there is no such requirement in mouse oocytes [55]. This may also account for the differences between mice and cattle in the effect on oocyte maturation of AMPK activators.

In the present study, we found that metformin increased PRKAA phosphorylation in bovine oocytes and arrested oocytes at the GV stage only if oocytes were matured in COCs, with no such effect observed in oocytes separated from their cumulus cells (DOs). These results are similar to those obtained for porcine oocytes [27]. Indeed, the percentage of oocytes that reach the Meta II stage was similar in DOs treated with metformin and in the control group without metformin treatment. However, we could expect that even if this percentage is the same after 22 h of IVM, the DOs treated with metformin could undergo IVM at a faster rate. However, opposite results are observed. Indeed, Bilodeau-Goeseels et al. [56] showed that after 7 h of IVM with 2 mM metformin, the percentage of bovine DOs at the GV stage was higher than the control group. Thus, because metformin does not increase PRKAA phosphorylation in DOs (even after 1 h) and does not regulate the maturation process, we can suggest that cumulus cells are required for the meiotic maturation arrest effect of metformin. The oocyte is known to form a syncytium with the surrounding somatic cells, indicating that the cells communicate with each other. Moreover, communication between the oocyte and the cumulus cells is supported by gap junctions, which are known to be important in controlling oocyte maturation [57, 58]. Thus, metformin could act first on cumulus cells to regulate key factors involved in the oocyte meiotic maturation, such as AMPK. The "signal" induced by AMPK activation might then go through gap junctions to reach the oocyte compartment and allow nuclear maturation. PRKAA1 has been immunolocalized inside these gap junctions in bovine COCs [56]. Thus, oocyte nuclear progression may be directly related to the AMPK activity in the oocyte during IVM. Calcium might be a link between these two processes (AMPK activation and nuclear maturation). Indeed, the CaMKK regulates PRKA activity [11], and intracellular calcium oscillations are known to play an important role in bovine oocyte maturation [59]. Several studies have shown that progesterone stimulates oocyte maturation [48, 49]. We have shown that metformin-induced PRKAA activation reduces progesterone secretion in bovine mural granulosa cells [28]. We therefore hypothesized that metformin-induced PRKAA phosphorylation in cumulus cells decreased progesterone secretion, resulting in the arrest of bovine oocytes at the GV stage. However, the addition of metformin to the bovine COCs maturation medium increased PRKAA phosphorylation during IVM in cumulus cells without decreasing progesterone secretion. Conversely, metformin treatment inhibited the secretion of progesterone by dissociated cumulus cells without oocytes. Thus, a decrease in progesterone production by cumulus cells is not the main mechanism underlying the inhibitory effects of metformin on bovine oocyte maturation. We demonstrated in the present study that metformin treatment for 22 h during IVM inhibited the expansion of the cumulus cells within bovine COCs without affecting their viability. One previous study showed that the administration of metformin together with dehydroepiandrosterone prevented the increase in ovarian COX2 expression in prepuberal BALB/c mice and increased the level of phosphorylation of PRKAA [60]. Moreover, in HT-29 colon cancer cells, PRKAA activation decreases cox2 expression in response to a combination of 5-fluorouracil and genistein [61]. In cattle, cox2 is expressed by cumulus cells during in vivo maturation and IVM [62]. Higher levels of in vitro cox2 expression are associated with higher rates of cumulus expansion and proportions of oocytes at Meta II after 24 h of culture [62]. Thus, metformin-induced PRKAA activation may decrease cox2 expression in bovine cumulus cells, inhibiting cumulus cell expansion and the progression of meiosis.

In conclusion, the results presented show that PRKAA phosphorylation decreases, whereas MAPK3/1 phosphorylation increases in both oocyte and cumulus cells during the IVM of bovine COCs. The addition of metformin to bovine COCs maturation medium induced PRKAA phosphorylation and decreased MAPK3/1 phosphorylation in oocytes and cumulus cells, inhibited cumulus cell expansion, inhibited activation of two key factors (RPS6 and EEF2) involved in protein synthesis in oocytes, and arrested most of the oocytes at the GV stage. However, these effects of metformin were observed only in bovine oocytes matured in COCs, not in oocytes separated from their cumulus cells. Further investigations are required to identify the factors and molecular mechanisms in cumulus cells associated with the inhibitory effects of metformin on oocyte maturation.

FOOTNOTES

3These authors contributed equally to this work. Back

1Supported by the GIS-AGENA, ANR, and Apis-GENE. L.T. is supported by the Région Centre. Back

3These authors contributed equally to this work. Back

Correspondence: 2FAX: 33 2 47 42 77 43; e-mail: jdupont{at}tours.inra.fr

Received: 12 February 2007.

First decision: 15 March 2007.

Accepted: 12 June 2007.

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