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Am J Physiol Endocrinol Metab 293: E1198-E1206, 2007. First published August 7, 2007; doi:10.1152/ajpendo.00097.2007
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Maternal diabetes adversely affects AMP-activated protein kinase activity and cellular metabolism in murine oocytes

Ann M. Ratchford,* Aimee S. Chang,* Maggie M.-Y. Chi, Rachael Sheridan, and Kelle H. Moley

Department of Obstetrics and Gynecology, Washington University in St. Louis, St. Louis, Missouri

Submitted 12 February 2007 ; accepted in final form 3 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Maternal diabetes is associated with an increased risk of miscarriages and congenital anomalies. Preovulatory oocytes in murine models also experience maturational delay and greater granulosa cell apoptosis. The objective of this study was to examine whether maternal diabetes influences preovulatory oocyte metabolism and impacts meiotic maturation. Streptozotocin-induced diabetic B6SJLF1 mice were superovulated, and oocytes were collected at 0, 2, and 6 h after human chorionic gonadotropin (hCG) injection. Individual oocyte concentrations of ATP, 5'-AMP, glycogen, and fructose-1,6-phosphate (FBP) and enzyme activities of glucose-6-phosphate dehydrogenase (G6PDH), adenylate kinase, hydroxyacyl-CoA dehydrogenase (Hadh2), and glutamic pyruvate transaminase (Gpt2) were measured. Protein levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were also measured. ATP levels were significantly lower in oocytes from diabetic mice, and the percent change in the AMP-to-ATP ratio was significantly higher in these oocytes. In contrast, activities of Hadh2 and Gpt2, two enzymes activated by AMPK, were significantly less in these oocytes. Additionally, glycogen and FBP levels, both endogenous inhibitors of AMPK, were elevated. Phosphorylated ACC, a downstream target of AMPK, and phosphorylated AMPK were both decreased in diabetic oocytes, thus confirming decreased AMPK activity. Finally, addition of the activator AICAR to the in vitro maturation assay restored AMPK activity and corrected the maturation defect experienced by the oocytes from diabetic mice. In conclusion, maternal diabetes adversely alters cellular metabolism leading to abnormal AMPK activity in murine oocytes. Increasing AMPK activity in these oocytes during the preovulatory phase reverses the metabolic changes and corrects delays in meiotic maturation.

oocyte; meiotic maturation

MATERNAL DIABETES has been found to adversely affect preimplantation embryo development and pregnancy outcomes (32, 33). Previous work from our laboratory and others has suggested that an insult may occur before the one-cell zygote stage, and that this insult negatively impacts the outcome of the resulting embryo (17, 34). In prior studies, we reported that oocyte meiotic maturation rate or progression to germinal vesicle breakdown (GVBD) is delayed in oocytes obtained from streptozotocin-induced diabetic mice (34). A delay in oocyte resumption of meiosis in mice with diabetes has also been associated with abnormal metabolism in other studies as well as with increased apoptosis in the surrounding granulosa cells (9). Poor fetal outcomes experienced by women with diabetes, including an increased risk of congenital anomalies and miscarriage rate, may be associated with these early detrimental alterations that occur in the oocyte. We hypothesize that diabetic conditions result in abnormalities in oocyte metabolism, potentially preprogramming the oocyte for poor outcomes after fertilization.

The oocyte depends on the metabolism of lactate and pyruvate via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation for most of its energy stores (4, 29, 38). The cumulus oocyte complex (COC) metabolizes glucose via several important pathways, including the pentose phosphate pathway and glycolysis, and this metabolism is necessary for successful oocyte maturation and the resumption of meiosis at the appropriate time (19, 21, 41, 42). Cyclic adenosine monophosphate (cAMP), generated by adenylate cyclase downstream of the pentose phosphate pathway, is an important negative regulator of meiotic maturation via its activation of cAMP-dependent protein kinase (PKA) (20). Depletion of cAMP by phosphodiesterases triggers GVBD. Alternatively, AMP has been shown to be involved in meiotic maturation via its activation of AMP-activated protein kinase (AMPK) (18). Both {alpha}1- and {alpha}2-subunits of AMPK are expressed in COCs and denuded oocytes. The AMP-to-ATP (AMP/ATP) ratio acts as a measure of cellular energy stores. As AMP levels rise and ATP levels fall, this ratio increases, and AMPK is activated as if in a stress situation (22). With its activation, AMPK phosphorylates and inhibits key enzymes in several energy-consuming biosynthetic pathways and stimulates energy-producing pathways, thereby conserving ATP. AMPK and cAMP appear to be important opposing regulators of meiotic regulation (18). Maternal embryonic leucine zipper kinase (MELK) is a recently identified member of the SNF1/AMPK serine-threonine kinase family (23) that is highly expressed in the murine oocyte and may be the isoform of AMPK responsible for this meiotic regulation.

Because meiotic maturation is closely linked to oocyte metabolism via AMP and cAMP, and because glucose metabolism is altered in many diabetic tissues, we examined the activities of key enzymes and the major metabolites in oocytes from diabetic and control mice. Although previous investigations have shown that there is no change in flux through the TCA cycle or the glycolytic pathway in diabetic oocytes, and only de novo purine synthesis and flux through the pentose phosphate pathway are decreased (12), these studies were done in vitro and with radiolabeled substrates on pooled oocytes. We hypothesize in this study that metabolic abnormalities may be evident in diabetic oocytes because of alterations in AMPK activity induced by high glucose concentrations or other metabolic disturbances. This phenomenon has been described previously by others in beta-cells, where increasing concentrations of glucose lead to decreased AMPK activity in beta-cell-derived MIN-6 cells in culture (14, 27, 28). These AMPK activity differences in the oocytes may explain the meiotic maturation delay in diabetic mice and may carry over to embryo, manifesting as poor development competency, for example, preimplantation embryos from diabetic mice that, when removed from the diabetic milieu, still experience significant developmental delay and apoptosis by the blastocyst stage. Our rationale is that, if this hypothesis is valid, correction of these abnormalities with AMPK activators may reverse the defect in meiotic maturation and perhaps improve pregnancy outcomes in these conditions. Using the specialized microanalytical enzymatic cycling assays employed previously in our laboratory, we chose to measure metabolites and enzyme activities in individual oocytes, including ATP, 5'-AMP, phosphocreatinine (PCr), fructose-1,6-bisphosphate (FBP), glycogen, adenylate kinase, glucose-6-phosphate dehydrogenase (G6PDH), hydroxyacyl-CoA dehydrogenase (Hadh2), and glutamic pyruvate transaminase (Gpt2). We also examined the protein expression of phosphoacetyl-CoA carboxylase (pACACA) (also known as pACC) and phospho-AMPK (pAMPK) in diabetic vs. control oocytes. ACC acts as a substrate for AMPK, and pACC directly correlates with AMPK activity. Moreover, we investigated the effect of adding AMPK activators, both 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and metformin, on AMPK activity and meiotic maturation in diabetic vs. control oocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Oocyte retrieval. All mouse studies were approved by the Animal Studies Committee at Washington University School of Medicine and conform to the National Research Council's Guide for the Care and Use of Laboratory Animals. Female immature B6SJLF1 mice (age 20–24 days) were utilized for most experiments. Diabetic and age-matched controls received 10 IU of equine chorionic gonadotropin (eCG) by intraperitoneal injection, and 48 h later, they were either killed or given an injection of 5 IU of human chorionic gonadotropin (hCG). At the appropriate time points (t = 0, 2, and 6 h), the ovaries were removed and placed in a dish containing 1.5 ml of culture medium. Preovulatory oocytes were isolated by puncture of the antral follicles with sterile needles and then washed through several changes of medium. Before the puncturing, the ovaries were stored briefly in an incubator with the following settings: 5% CO2-5% O2-90% N2 atmosphere at 37°C. Cumulus-enclosed oocytes (CEOs) were denuded using a small bore pipette and repetitive pipetting.

Induction of hyperglycemia. To generate a diabetic model, female mice (age 20–24 days) received a single injection of streptozotocin at a dose of 190 mg/kg (dissolved in sodium citrate buffer, pH 4.4; Sigma, St. Louis, MO). Four days postinjection, a tail blood sample was measured for glucose concentrations via a Hemocue B glucose analyzer (Stockholm, Sweden). If glucose levels were >240 mg/dl, these mice were selected and received a priming injection of eCG. A few control mice were also randomly selected; their blood sugar was checked to ensure that it was <240 mg/dl.

Metabolic analyses. The ova were freeze-dried as described by Chi et al. (11). Briefly, they were transferred with 0.5–1 µl of the incubation medium onto a glass slide with a braking pipette, frozen by dipping the slide into isopentane, brought to –150°C with liquid nitrogen, and then freeze-dried on the slide in a glass vacuum tube in a cryostat at –35°C. The dry medium and crystals surrounding the ova were removed with a stiff hair point, and specimens were stored at –70°C in individual wells, measuring 0.5-mm deep and 3-mm wide, drilled into the surface of a microscopic glass slide.

Each oocyte was added to 1 µl of a special extraction medium (11) that permits storage at –70°C and repeated freezing and thawing without appreciable loss of any of the enzymes reported here. From each single oocyte, extract aliquots were obtained in nanoliter volumes under oil using constriction glass pipettes, as previously described for the assay of enzymes and metabolites from oocytes and embryos (10, 11). The assays are based on oxidation of NAD+ or NADP+ or reduction of NADH, either directly, as in the case of G6PDH (glucose-6-phosphate + NADP+ -> 6-phosphogluconate + NADPH), or more often indirectly with the aid of one or more auxiliary enzymes, as in the case of hexokinase (glucose + ATP -> 6-phosphogluconate + NADPH). The specific methods are minor modifications of published procedures: hexokinase (HK) and G6PDH (H6pd) from Lowry et al. (2), glutamic pyruvate transaminase (Gpt2) (previously referred to as SGOT) from Chan et al. (8), hydroxyacyl-CoA dehydrogenase (Hadh2) (previously referred to as BOAC) from Lowry et al. (31), and all others [PCr, FBP, glycogen, and adenylate kinase (Ak2)] from Passoneau and Lowry (35). Most of the modifications consisted of adjustments to meet the sensitivity needs, for example, changing the number of enzymatic cycling amplifications to obtain a more exact amount. ATP has previously been measured in embryos and oocytes in our studies (Fig. 1A) (10). The only assay not previously published for low concentrations as found in the oocyte is that of 5'-AMP, which is detailed below. The change in AMP/ATP ratio was calculated by comparing the ratio between nondiabetic and diabetic oocytes and then expressing the value as a percentage of the diabetic value.


Figure 1
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  		Fig. 1. ATP and AMP levels in diabetic denuded oocytes. A: the assay reaction for measuring ATP in individual oocyte extracts. B: the assay reaction for measuring 5'-AMP in individual oocyte extracts. C: ATP levels were compared between the denuded diabetic oocytes and nondiabetic oocytes at 0, 2, and 6 h after human chorionic gonadotropin (hCG) injection. *P < 0.03; **P < 0.01. D: AMP levels were compared between the denuded diabetic oocytes and nondiabetic oocytes at 0, 2, and 6 h after hCG injection. *P < 0.0001.

 
5'-AMP measurement (low concentration of 0.02–0.5 µM) by stimulation of phosphorylase a in individual oocytes. The method for the measurement of 5'-AMP is based on the observation that, when glycogen and Pi are present at low levels, the activity of muscle phosphorylase a becomes a sensitive and specific indicator of 5'-AMP concentration (Fig. 1B). This assay has previously been reported on a macrolevel (1) but not on a microanalytical level in oil wells, as is needed for these oocytes. Enzymes were from Sigma Chemical, Roche, and Calbiochem. Rabbit liver glycogen type III was from Sigma; other chemicals were reagent grade. The glycogen used in the assay was made, by weight, 8 g/100 ml (500 mM) and dialyzed overnight at 4°C against 50 mM (pH 4.6) acetate buffer to remove bound AMP (36) and stored at –20°C. Rabbit muscle phospho-glucomutase was centrifuged to remove (NH4)2SO4 and replaced with 20 mM imidazole buffer, pH 7, containing 0.02% BSA. Lyophilized Leuconostoc mesenteroides G6PDH was dissolved in the same imidazole buffer and BSA mixture. Phosphorylase a is activated before use by diluting to 1 mg/ml in 20 mM imidazole buffer, pH 7, containing 0.02% BSA and 5 mM beta-mercaptoethanol and heating at 37°C for 20 min (1).

Oil well procedures for tissue measuring 20–100 fmol are as follows. Each freeze-dried oocyte was extracted at ambient temperature in a 25-nl droplet of 0.3 N HClO4 under oil for 20 min to inactivate tissue enzymes; 0.2 µl of 0.1 M imidazole base was added for 10 min to buffer the HClO4 to pH 7.2. Double-strength specific reagent 1 kept at 0°C was added quickly in 0.2 µl for 60 min. Reagent 1 consisted of 50 mM imidazole acetic acid (HAc), pH 7.2 (30 mM free base-20 mM HAc), 0.02% BSA, 4 mM MgAc2, 5 mM beta-mercaptoethanol, 2 mM glycogen (as glucosyl residues), 1 µM glucose-1,6-bisphosphate, 10 µM P1,P5-di(adenosine-5')-pentaphosphate pentasodium salt (adenylate kinase inhibitor), 4 mg/ml G6PDH, 10 µg/ml P-glucomutase, and 4 µg/ml phosphorylase a, with 0.6 mM K2HPO4 added last to the cold mixture. Double-strength reagent 2 at 0.4 µl was added quickly for 30 min. The pH was raised to 9 to stop the phosphorylase a reaction and proceed with the next step with G6PDH. Reagent 2 consisted of 100 mM 2-amino-2-methyl-1,3-propanediol HCl buffer, pH 9.7 (95 mM free base-5 mM HCl), and 150 µM NADP+ (added just before use). One microliter of 0.1 N NaOH was added to stop the reaction with heating at 80°C for 20 min. Samples, standards, and blanks were carefully matched in terms of salt concentration. A 0.5-µl aliquot was added to 50 µl of NADP+ cycling reagent in a tube at 37°C for 60 min. It contained 100 mM imidazole HCl, pH 7, 7.5 mM {alpha}-ketoglutarate, 5 mM glucose-6-phosphate, 25 mM NH4Ac, 0.02% BSA, 100 µM ADP, 20 µg/ml beef liver glutamate dehydrogenase, and 4 µg/ml L. mesenteroides G6PDH. This enzyme ratio gives ~2,000-fold amplification. The reaction was heat stopped by boiling for 3 min. Finally, 1 ml of the indicator reagent consisting of 50 mM imidazole HAc, pH 7, 1 mM EDTA, 30 mM NH4Ac, 5 mM MgCl2, 100 µM NADP+, and 0.0125 U/ml 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus was added (1).

Detection of pACC in the CEOs by immunohistochemistry. CEOs were fixed on glass slides in 3% paraformaldehyde for 20 min and permeabilized with 0.1% Tween 20 for 10 min. The CEOs were first incubated in 20% normal donkey serum (Pierce, Rockford, IL) in PBS containing 2% BSA (PBS-BSA) for 1 h to block any nonspecific binding. They were then washed in PBS-BSA and incubated in the primary antibody to phosphorylated acetyl-CoA carboxylase (pACC) (10 µg/ml; Upstate Biotechnology, Lake Placid, NY) for 1 h at room temperature. Next, the CEOs were washed with PBS-BSA and incubated in the appropriate secondary antibody, goat anti-rabbit Alexa Fluor 488 antibody or goat anti-mouse Alexa Fluor 488 antibody for ~30 min. The slides were washed, and nuclear staining was performed in 4 µM To-Pro-3-iodide. Following the extensive washing in PBS-BSA, confocal immunofluorescent microscopy (Nikon C1 laser scanning microscope, x63 magnification) was used to detect fluorescence as described above. As a negative control, nondiabetic control CEOs were immunostained with rabbit preimmune sera. As a positive control, CEOs were incubated with another AMPK activator, metformin (50 µM), for 2 h and then fixed and labeled as above.

Expression of pACC and pAMPK by Western analysis. CEOs in equivalent groups of 80–100 were collected at 0, 2, and 6 h post-hCG as described above. The pooled samples were washed in several different washes of human tubal fluid (HTF) and then frozen. The CEOs were placed in Laemmli buffer with beta-mercaptoethanol. All samples were boiled for at least 5 min at 100°C. The samples were subjected to 7.5% SDS-PAGE and transferred to nitrocellulose. pACC or pAMPK protein was then detected with a rabbit polyclonal anti-pACC antibody (Upstate Cell Signaling Solutions, 1.5 µg/ml) or a rabbit polyclonal anti-pAMPK antibody (Upstate Cell Signaling Solutions, 4 µg/ml) as the primary antibody, and a horseradish peroxidase (HRP)-labeled donkey anti-rabbit antibody (Pierce, 1:20,000) was utilized as the secondary antibody. SuperSignal West Dura Luminol/Enhancer solution (Pierce) was used for detection. HRP-labeled bands were quantified utilizing NIH Image (version 1.6). All experiments were performed in triplicate. Mouse anti-actin (Chemicon International, 1:2,000) was utilized as the loading control for all blots. Blots were also stripped and probed with rabbit anti-ACC antibody (Upstate Cell Signaling Solutions, 4 µg/ml) to detect unphosphorylated ACC, and the blots were normalized to ACC. For the pAMPK assays, blots were stripped and probed with rabbit anti-AMPK-{alpha}1 (Upstate Cell Signaling) to detect unphosphorylated AMPK. All experiments were conducted at least three times on separate pools of oocytes to generate the data shown.

In vitro oocyte maturation experiments. Denuded oocytes were incubated in 300 µM dibutyryl-cAMP (dbcAMP)-supplemented medium with or without 250 µM AICAR. Incubation was carried out in 1 ml of medium in plastic culture dishes (Falcon no. 353037) and placed in a tissue culture incubator with the following settings: 5% CO2-5% O2-90% N2 atmosphere at 37°C. At least 35 oocytes were used per group per experiment, and oocyte maturation was analyzed at 0, 2, 4, and 6 h of culture time. To assess maturation, oocytes were examined on an inverted Nikon microscope and scored for GVBD or dissolution of the nuclear envelope surrounding the chromatin before reinitiation of meiosis. Maturation was expressed as a percentage of oocytes with GVBD per total oocytes recovered per mouse.

Statistical analysis. Statistical analysis was performed with Student's t-test for comparing protein expression between the diabetic and nondiabetic groups. When multiple groups were being compared, as with the ATP or enzyme activities between diabetics and nondiabetics at different time points, ANOVA was utilized with Fishers post hoc test. All metabolite and enzymes assays were performed on at least 25 oocytes for each individual group in each experiment. Results are expressed as means ± SE of at least three separate experiments. Significance was defined at P value <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ATP levels are lower and the AMP/ATP ratio is elevated in diabetic denuded oocytes. The first question asked in this study was whether ATP levels differed between diabetic and nondiabetic oocytes, perhaps because of metabolic differences induced by the hyperglycemic milieu. ATP levels were expressed as a concentration, millimoles per kilogram per wet weight of the oocyte, taking into account the volume of the oocytes (11). ATP levels were compared between the diabetic denuded oocytes and the nondiabetic oocytes at 0, 2, and 6 h after hCG injection. ATP levels were significantly lower in the diabetic oocytes at the 2- and 6-h time points compared with their time-matched controls, as seen in Fig. 1C (2 h, P < 0.03; 6 h, P < 0.01). Although these decreases are not dramatic, we have shown previously that ATP levels among different blastocyst stage treatment groups rarely vary more than 2–3% from control (10). Therefore, the ~18% decrease in ATP between diabetic and control oocytes at the 6-h time point seen here represents a relatively large difference and suggests that the metabolism of oocytes is different from that of embryos, and that this decrease reflects a dramatic metabolic aberration.

5'-AMP levels were also measured in both the diabetic and nondiabetic denuded oocytes (Fig. 1D). The AMP/ATP ratio acts as a measure of cellular energy stores. In both groups, AMP levels dropped significantly between 0 and 2 h post-hCG, suggesting increased conversion of AMP to ATP as the oocyte resumes meiosis. AMP levels, however, were significantly lower in the diabetic oocytes compared with nondiabetic oocytes at the 0 time point (P < 0.0001), suggesting a preexisting deficit in the oocytes before activation. Although the 5'-AMP levels did not vary significantly at the 6-h time point, the ATP levels were significantly lower in the diabetic oocytes at this time point. Thus the percent change in the AMP/ATP ratio between diabetic and nondiabetic groups was significantly higher at 6 h post-hCG (Fig. 2) (P < 0.01). This ratio serves as a general measure of energy stores. An elevated ratio suggests that the cell or group of cells may be in a stressful condition in which ATP is being used rapidly. These findings suggest that the diabetic oocyte is in a stress state with lower AMP stores to start with and a higher AMP/ATP ratio at 6 h, as seen in Fig. 2. Given this significantly higher AMP/ATP ratio, the AMPK activity would be expected to be greater in the diabetic oocyte.


Figure 2
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  		Fig. 2. Percent change in AMP/ATP ratio in diabetic vs. control denuded oocytes. The percent change in the AMP/ATP ratio in denuded oocytes from diabetic vs. control mice was recorded at 0, 2, and 6 h post-hCG. *P < 0.01.

 
Oocyte enzyme activity differs in diabetic oocytes vs. nondiabetic oocytes. An increase in AMPK activity results in the conservation of cellular ATP and upregulation of pathways that generate energy, including fatty acid oxidation and amino acid catabolism. To test whether AMPK activity was increased in the diabetic oocytes compared with the control oocytes, the activities of two enzymes thought to be involved in processes downstream of AMPK activation were measured. These include hydroxyacyl-CoA dehydrogenase (Hadh2), an enzyme involved in fatty acid metabolism, and glutamic pyruvate transaminase (Gpt2), involved in amino acid metabolism. To test whether AMPK activation led to increased activity of these enzymes in oocytes, a series of experiments were done. Control nondiabetic oocytes at 0-h hCG were incubated in medium containing AICAR for increasing periods of time, ranging from 2.5 to 5 h. AICAR at a concentration of 250 µM induced a peak increase in the activity of both Hadh2 and Gpt2 after 4.5 h of incubation (Fig. 3, A and B). This stimulation was reversed by adding 10 µM Compound C, an AMPK inhibitor (data not shown), confirming that this stimulation in activity was due to AMPK activation. Other enzymes were also measured and selected as representatives of key steps in glucose metabolism via the pentose phosphate pathway [G6PDH (H6pd), adenylate kinase (Ak2)]. It should be emphasized that the enzyme levels are given as in vitro activities under optimal conditions. They indicate enzyme maximum capacities. They do not indicate activities in vivo, which are subject to control by a variety of other tissue components. H6pd and Ak2 did not vary significantly between the diabetic and the nondiabetic group (data not shown). In contrast, two enzymes that are major representatives of fatty acid metabolism (Hadh2) and amino acid metabolism (Gpt2) and induced, as we have shown, by AMPK activity were significantly lower in the diabetic group (Fig. 4, A and B). This significant difference was seen at 6 h post-hCG (Hadh2, P < 0.05; Gpt2, P < 0.01), when the percent change in the AMP/ATP ratio was greatest in the diabetic oocytes. These two enzymes are normally activated with the increase in AMPK activity. This finding of decreased activity in the presence of an elevated ratio, however, suggests a block of AMPK activity. To explore this hypothesis, a metabolic inhibitor of AMPK activity, glycogen, was assayed in denuded oocytes from control vs. diabetic mice.


Figure 3
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  		Fig. 3. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) stimulated enzyme activity in nondiabetic oocytes. Nondiabetic oocytes at 0-h hCG were denuded and cultured in medium containing 300 µM dibutyryl-cAMP (dbcAMP) with or without 250 µM AICAR for 2.5–5 h. Enzyme activities were analyzed according to procedures listed in MATERIALS AND METHODS. A: hydroxyacyl-CoA dehydrogenase (Hadh2) activity in control vs. AICAR-treated oocytes. *P < 0.0001. B: glutamic pyruvate transaminase (Gpt2) activity in control vs. AICAR-treated oocytes. *P = 0.002. N = 120 oocytes for the 4.5-h time point and 20 oocytes for all other time points.

 

Figure 4
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  		Fig. 4. Oocyte enzyme activity in diabetic oocytes vs. nondiabetic oocytes. A: Hadh2 activity was measured and compared between the denuded diabetic oocytes and nondiabetic oocytes at 0, 2, and 6 h after hCG injection. *P < 0.05. B: Gpt2 activity was measured and compared between the denuded diabetic oocytes and nondiabetic oocytes at 0, 2, and 6 h after hCG injection. *P < 0.01.

 
Glycolytic metabolites are elevated in diabetic oocytes. Glycogen levels were significantly higher in the diabetic oocyte at the 0- and 2-h time points (Fig. 5A) (0 h, P < 0.01; 2 h, P < 0.001), whereas FBP levels were significantly higher at the late 6-h time point (Fig. 5B) (P < 0.01). This finding suggests that the hyperglycemic environment leads to accumulation of glucose stored as glycogen before maturation in the oocyte. This elevated glycogen level in the diabetic oocytes could inhibit and lead to blockade of AMPK activity in the face of an elevated AMP/ATP ratio. Recent reports in the literature have shown that glycogen suppresses AMPK activation in cardiac and skeletal muscle (7, 15, 44). The exact mechanism is not clear; however, a glycogen-binding domain has been identified in the beta-subunit of AMPK, where branch points are metabolized (24, 37), suggesting that glycogen or glycogen-like metabolites could regulate AMPK activity. We hypothesize that, once maturation begins, glycogen is metabolized to glucose via glycogenolysis within the oocyte. Glucose is further metabolized to FBP and accumulates as seen in these experiments, possibly because of a blockade in glycolysis. This blockade would appear to be at the level of glyceraldehyde dehydrogenase (GAPDH), preventing triose metabolism and leading to a buildup of upstream glycolytic metabolites such as FBP. GAPDH inhibition does occur in hyperglycemic conditions because of generation of oxygen free radicals, which directly block GAPDH activity (6).


Figure 5
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  		Fig. 5. Glycolytic metabolites in diabetic vs. control oocytes. A: glycogen levels were compared between the denuded diabetic oocytes and nondiabetic oocytes at 0, 2, and 6 h after hCG injection. *P < 0.01; **P < 0.001. B: fructose bisphosphate (FBP) levels were compared between the denuded diabetic oocytes and nondiabetic oocytes at 0, 2, and 6 h after hCG injection. *P < 0.01.

 
Phosphorylation of ACC and AMPK is decreased in diabetic CEOs by Western immunoblot and immunofluorescent microscopy. To test the hypothesis that AMPK activity was decreased in the oocytes from diabetic mice, AMPK activity was examined first by measuring downstream activation or phosphorylation of ACC. Phosphorylation and inactivation of ACC at Ser79 is thought to be accomplished exclusively by AMPK; the amount of pACC is often used as a downstream marker of AMPK activity (16, 26). The amount of pACC detected increased with time in CEOs recovered from control mice, comparing 0, 2, and 6 h post-hCG by immunofluorescent microscopy (Fig. 6A). pACC did not increase over the same period in CEOs from diabetic mice as assessed by immunofluorescent microscopy and appeared significantly different from control as time increased. Control Western immunoblots using preimmune sera revealed no protein staining. Control CEOs also were incubated in metformin (50 µM) or AICAR (250 µM) for 2 h and then fixed and labeled. There was a significant increase in detection of pACC by both immunohistochemistry and Western immunoblot (Fig. 6B, metformin only shown), suggesting that the phospho-antibody was detecting the phosphorylated protein. By Western immunoblot analysis, the difference in pACC levels normalized to total ACC between CEOs from control vs. diabetic mice was not significantly different at 0 h; however, it reached significance at 6 h post-hCG (P < 0.03, n = 3 experiments) (Fig. 6, C and D). Expression levels of ACC, the unphosphorylated protein, were not increased between diabetic and nondiabetic CEOs (Fig. 6D), suggesting that only activation of ACC by AMPK differed between the groups.


Figure 6
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  		Fig. 6. Protein levels of phosphorylated acetyl-CoA carboxylase (pACC) are lower in diabetic cumulus-enclosed complexes (CEOs) by immunofluorescent microscopy and Western immunoblot. A: protein expression of pACC was detected using immunofluorescent microscopy in CEOs from diabetic (A: D–F) vs. nondiabetic (A: A–C) mice at 0 (A: A and D), 2 (A: B and E), or 6 h (A: C and F) post-hCG. Green channel, pACC protein; blue, nuclear staining. Magnification x63. B: pACC detection in CEOs cultured in the absence (B: A) or presence (B: B) of 50 µM metformin for 2 h. C: Western immunoblotting was used to quantitate expression of pACC between these different groups of CEOs. *P < 0.03. D: representative Western immunoblot of control and diabetic CEOs at 6 h post-hCG from 2 of 3 experiments. pACC is seen in the top bands in each group with total ACC below. Blots were also stripped and GAPDH detected to assure equal loading of protein.

 
The same experiments were done using antibody to pAMPK to confirm AMPK activation. For these experiments CEOs were recovered and cultured for 2 h in vitro with or without AICAR (250 µM). The CEOS were evaluated by Western immunoblot for the presence of pAMPK. Less phosphorylated protein normalized to total AMPK was detected in diabetic vs. control CEOs, and this phosphorylation was increased in the presence of AICAR (P < 0.01, n = 3, control vs. control plus AICAR; P < 0.05, n = 3, control vs. diabetic; not significant, control plus AICAR vs. diabetic plus AICAR) (Fig. 7, A and B). Expression levels of AMPK, the unphosphorylated protein, were not increased between diabetic and nondiabetic CEOs (Fig. 7A), suggesting that only activation of AMPK differed between the groups and not total AMPK.


Figure 7
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  		Fig. 7. Phosphorylation of AMP-activated protein kinase (AMPK) in control and diabetic CEOs by Western immunoblot. A: representative Western immunoblot of control and diabetic CEOs cultured in the absence or presence of 250 µM AICAR for 2 h. Phospho-AMPK (pAMPK) is represented in the top set of immunoblots, with the band directly under it as a nonspecific band. Total AMPK is the single band in the bottom set of immunoblots and did not vary between groups. N = 3. B: Western immunoblotting was used to quantitate the differences in expression of pAMPK normalized to total AMPK. C, control; D, diabetic. *P < 0.01, control CEO vs. control plus AICAR; **P < 0.05, diabetic CEO vs. control CEO; N = 3 experiments. Not significant, control plus AICAR vs. diabetic plus AICAR.

 
Diabetic oocytes demonstrate retarded meiotic maturation in vitro that is partially rescued by AICAR. To test the hypothesis that decreased AMPK activity was responsible for the difference in resumption of meiosis in diabetic vs. control oocytes, we examined the ability of an AMPK activator to rescue the delayed meiotic maturation seen in the diabetic oocytes. Denuded oocytes were maintained in meiotic arrest with the addition of 300 µM dbcAMP in the culture medium. In diabetic oocytes, the basal level of GVBD (in dbcAMP medium) was found to be significantly lower than in control nondiabetic oocytes at 4 and 6 h of culture (Fig. 8). This finding agrees with the in vivo data previously reported in oocytes from diabetic mice recovered following hCG administration (5). In these studies, in vivo maturation was delayed, as seen in vitro in this study. The addition of AICAR, an activator of AMPK, to the culture medium stimulated an increase in GVBD compared with oocytes treated only with dbcAMP in both diabetic and nondiabetic groups (Fig. 8). A significant increase in the percentage of cells undergoing GVBD was seen in diabetic oocytes at 4 and 6 h after AICAR stimulation and in nondiabetic oocytes after 6 h of AICAR stimulation. Additionally, 6 h after AICAR stimulation, the percentage of diabetic oocytes that underwent GVBD is similar to the percentage of nondiabetic oocytes that underwent GVBD. This implies that AICAR is able to recover the innately impaired maturation ability in diabetic oocytes.


Figure 8
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  		Fig. 8. Diabetic oocytes demonstrate retarded meiotic maturation in vitro that is partially rescued by AICAR. Effect of AICAR on diabetic and nondiabetic mouse oocyte maturation. Oocytes from control, nondiabetic, and diabetic mice were cultured in medium containing 300 µM dbcAMP plus or minus 250 µM AICAR. Germinal vesicle breakdown (GVBD) was analyzed at 0, 2, 4, and 6 h of culture time. Data are expressed as the mean percent GVBD + SE from at least 4 experiments for each group. Groups with common letters are significantly different (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we propose that, before ovarian priming with eCG and triggering of in vivo maturation with hCG or in vitro maturation, exposure to the diabetic environment adversely affects oocyte metabolism. These changes may be responsible for the delays in meiotic maturation seen in diabetic mice and diabetic women. These studies demonstrate that ATP levels are significantly lower and AMP levels significantly higher in the denuded oocytes from diabetic mice. As a result, the AMP/ATP ratio is skewed, suggesting metabolic stress and activation of AMPK. AMPK activation should accelerate GVBD; however, a delay is demonstrated in these oocytes. We hypothesize that AMPK activity is decreased in the oocyte complex in response to the diabetic condition, as has been described in other tissues exposed to a diabetic milieu (14, 27, 28). This proposed dysregulation of AMPK is further supported by a decrease, rather than increase, in Gpt2 and Hadh2 activity, since both of these enzymes should have greater activity at times of AMPK activation. With AMPK activation, the oocyte seeks alternative energy sources as it conserves ATP stores, and thus amino acid catabolism and fatty acid oxidation should be increased (39, 40). Moreover, levels of activated or phosphorylated ACC should be elevated by AMPK activation, and instead lower levels are seen in these experiments. Given these findings, we conclude that AMPK activity is blocked or dysregulated in the diabetic oocyte.

Interestingly, a decrease in both Hadh2 and Gpt2 was seen previously using these microanalytical enzyme techniques to evaluate human oocytes (45). In a group of oocytes from older patients undergoing in vitro fertilization, studies revealed that Hadh2 and Gpt2 activity (previously referred to as BOAC and SGOT) negatively correlated with age and poorer oocyte quality. It is possible that these decreased activities represented decreased activation of AMPK because of skewed AMP/ATP ratio; however, these measurements were not made. For these reasons, intraoocyte ATP levels may prove to be accurate predictors of oocyte maturational competence. Noninvasive measures of ATP have been made in other tissue types, and therefore it may be possible to screen oocytes for ATP content to produce the best-quality embryos. In addition, other data suggest that a lack of Hadh2 activity may impart early developmental problems, leading to embryo lethality. In the long-chain acyl-CoA dehydrogenase null mouse, embryo lethality occurs at the morula-to-blastocyst transition (3). Fatty acid supplementation did not reverse this effect, suggesting that the deficiency may cause earlier developmental problems, possibly during oogenesis.

The reason for the decreased ATP levels is not clear. Previously, it was demonstrated that TCA cycle metabolism and glycolysis were not different in the diabetic oocyte matured in vitro compared with the nondiabetic oocyte; however, pentose phosphate pathway flux and de novo synthesis of nucleotides were significantly decreased in these oocytes (12, 13). It is possible that, following hCG administration, glycogen stores, which are significantly higher in the diabetic oocytes in this study, undergo glycogenolysis because of energy demands of the activated oocyte. This phenomenon would lead to an initial increase in glycolysis and an increase in the generation and leakage of oxygen free radicals from the mitochondria and result in inhibition of GAPDH, as previously reported in other diabetic tissues exposed to hyperglycemia (6). In addition, this would also result in lower ATP levels, as demonstrated in these oocytes at 6 h.

There are several possible factors responsible for the dysregulation of AMPK in these hyperglycemia-exposed oocytes. Elevated glycogen levels inhibit AMPK activation (25, 37, 44), and thus the elevated glycogen levels reported in this study may be responsible for this AMPK dysfunction. Direct evidence for this inhibition of AMPK is thought to occur via a glycogen-binding domain in the beta-subunit, although this hypothesis has been disputed (37, 44). Oocytes store glycogen as an energy source to prepare for fertilization and during the free-floating preimplantation period. Glycogen synthase activity is highest at the oocyte stage in human oocytes (45). It is possible that hyperglycemic conditions induce increased uptake of glucose or fructose into the oocyte, either of which is then stored as excess glycogen in the oocyte, and this accounts for the AMPK dysfunction. AICAR supplementation to the in vitro culture conditions reversed oocyte maturation kinetics, suggesting that AMPK may be involved in the decreased GVBD seen in the diabetic oocytes. The remaining question is, how does AMPK activation enhance GVBD? Recent studies suggest that vasodilator-stimulated phosphoprotein (VASP), an actin regulatory protein, is an AMPK substrate(5). Phosphorylation of VASP by AMPK leads to actin cytoskeleton rearrangements, and this signaling system is downregulated in diabetic blood vessels. Mammalian oocytes contain a nuclear actin network that participates in nuclear envelope breakdown and chromosome delivery to the microtubule spindle (30). In addition, polymerization of actin has been shown to be critical for oocyte maturation in a porcine model in other studies(43). Disruption of actin filaments during resumption of meiosis leads not only to delayed GVBD but also to abnormal development of the fertilized oocyte to blastocyst stage, suggesting poor developmental competence. It is possible that downregulation by decreased AMPK activity of this actin system plays a role in the delayed resumption of meiosis. Future studies can investigate this hypothesis.

In summary, this study demonstrates metabolic differences between oocytes from control vs. diabetic mice. These differences suggest that the oocytes exposed to a diabetic milieu begin their maturation process with increased glycogen and decreased ATP stores. As meiotic maturation is induced with hCG, excess glycogen levels may lead to AMPK dysregulation in the oocytes from the diabetic mice. This inhibition is reflected by decreased Gpt2 and Hadh2 activity, and this drop in AMPK activity may be the cause of delayed GVBD in these oocytes. The ability of AMPK activation with AICAR to correct the maturation defect in vitro in the diabetic oocytes further supports the important role of AMPK activation in this step. Continued elucidation of the mechanisms behind these metabolic changes in diabetic oocytes and determination of the manifestation, if any exists, of these changes later in development may lead to novel therapeutic treatments started before conception in women with hyperglycemia and diabetes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by the American Society for Reproductive Medicine/Ortho-McNeil Research Grant in Reproduction (A. S. Chang), National Institute of Child Health and Human Development Cooperative Program on Female Health and Egg Quality Grants U01-HD-044691 (K. H. Moley) and T32-HD-049305 (A. M. Ratchford), and The Berlex Foundation (A. S. Chang).


   ACKNOWLEDGMENTS
 
We thank the other members of the Cooperative Program for suggestions as well as Dr. Stephen Downs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Moley, Dept. of Obstetrics and Gynecology, Washington Univ. in St. Louis, 660 South Euclid Ave., St. Louis, MO 63110 (e-mail: moleyk{at}wustl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. M. Ratchford and A. S. Chang contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
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 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Barbehenn EK, Law MM, Brown JG, Lowry OH. Measurement of 5'-adenylic acid by stimulation of phosphorylase a. Anal Biochem 70: 554–562, 1976.[CrossRef][Web of Science][Medline]
  2. Barbehenn EK, Wales RG, Lowry OH. Measurement of metabolites in single preimplantation embryos: a new means to study metabolic control in early embryos. J Embryol Exp Morphol 43: 29–46, 1978.[Web of Science][Medline]
  3. Berger PS, Wood PA. Disrupted blastocoele formation reveals a critical developmental role for long-chain acyl-CoA dehydrogenase. Mol Genet Metab 82: 266–272, 2004.[CrossRef][Web of Science][Medline]
  4. Biggers JD, Whittingham DG, Donahue RP. The pattern of energy metabolism in the mouse oocyte and zygote. Proc Natl Acad Sci USA 58: 560–567, 1967.[Free Full Text]
  5. Blume C, Benz PM, Walter U, Ha J, Kemp BE, Renne T. AMP-activated protein kinase impairs endothelial actin cytoskeleton assembly by phosphorylating vasodilator-stimulated phosphoprotein. J Biol Chem 282: 4601–4612, 2007.[Abstract/Free Full Text]
  6. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813–820, 2001.[CrossRef][Medline]
  7. Carling D. The AMP-activated protein kinase cascade–a unifying system for energy control. Trends Biochem Sci 29: 18–24, 2004.[CrossRef][Web of Science][Medline]
  8. Chan AW, Perry SG, Burch HB, Fagioli S, Alvey TR, Lowry OH. Distribution of two aminotransferases and D-amino acid oxidase within the nephron of young and adult rats. J Histochem Cytochem 27: 751–755, 1979.[Abstract]
  9. Chang AS, Dale AN, Moley KH. Maternal diabetes adversely affects preovulatory oocyte maturation, development, and granulosa cell apoptosis. Endocrinology 146: 2445–2453, 2005.[Abstract/Free Full Text]
  10. Chi MM, Hoehn A, Moley KH. Metabolic changes in the glucose-induced apoptotic blastocyst suggest alterations in mitochondrial physiology. Am J Physiol Endocrinol Metab 283: E226–E232, 2002.[Abstract/Free Full Text]
  11. Chi MM, Manchester JK, Yang VC, Curato AD, Strickler RC, Lowry OH. Contrast in levels of metabolic enzymes in human and mouse ova. Biol Reprod 39: 295–307, 1988.[Abstract]
  12. Colton SA, Humpherson PG, Leese HJ, Downs SM. Physiological changes in oocyte-cumulus cell complexes from diabetic mice that potentially influence meiotic regulation. Biol Reprod 69: 761–770, 2003.[Abstract/Free Full Text]
  13. Colton SA, Pieper GM, Downs SM. Altered meiotic regulation in oocytes from diabetic mice. Biol Reprod 67: 220–231, 2002.[Abstract/Free Full Text]
  14. da Silva Xavier G, Leclerc I, Salt IP, Doiron B, Hardie DG, Kahn A, Rutter GA. Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene expression. Proc Natl Acad Sci USA 97: 4023–4028, 2000.[Abstract/Free Full Text]
  15. Davies JK, Wells DJ, Liu K, Whitrow HR, Daniel TD, Grignani R, Lygate CA, Schneider JE, Noel G, Watkins H, Carling D. Characterization of the role of gamma2 R531G mutation in AMP-activated protein kinase in cardiac hypertrophy and Wolff-Parkinson-White syndrome. Am J Physiol Heart Circ Physiol 290: H1942–H1951, 2006.[Abstract/Free Full Text]
  16. Davies SP, Carling D, Hardie DG. Tissue distribution of the AMP-activated protein kinase, and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay. Eur J Biochem 186: 123–128, 1989.[Web of Science][Medline]
  17. Diamond MP, Moley KH, Pellicer A, Vaughn WK, DeCherney AH. Effects of streptozotocin- and alloxan-induced diabetes mellitus on mouse follicular and early embryo development. J Reprod Fertil 86: 1–10, 1989.[Abstract/Free Full Text]
  18. Downs SM, Hudson ER, Hardie DG. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev Biol 245: 200–212, 2002.[CrossRef][Web of Science][Medline]
  19. Downs SM, Humpherson PG, Martin KL, Leese HJ. Glucose utilization during gonadotropin-induced meiotic maturation in cumulus cell-enclosed mouse oocytes. Mol Reprod Dev 44: 121–131, 1996.[CrossRef][Web of Science][Medline]
  20. Downs SM, Hunzicker-Dunn M. Differential regulation of oocyte maturation and cumulus expansion in the mouse oocyte-cumulus cell complex by site-selective analogs of cyclic adenosine monophosphate. Dev Biol 172: 72–85, 1995.[CrossRef][Web of Science][Medline]
  21. Downs SM, Mastropolo AM. The participation of energy substrates in the control of meiotic maturation in murine oocytes. Dev Biol 162: 154–168, 1994.[CrossRef][Web of Science][Medline]
  22. Fryer LG, Carling D. AMP-activated protein kinase and the metabolic syndrome. Biochem Soc Trans 33: 362–366, 2005.[CrossRef][Web of Science][Medline]
  23. Heyer BS, Kochanowski H, Solter D. Expression of Melk, a new protein kinase, during early mouse development. Dev Dyn 215: 344–351, 1999.[CrossRef][Web of Science][Medline]
  24. Hudson ER, Pan DA, James J, Lucocq JM, Hawley SA, Green KA, Baba O, Terashima T, Hardie DG. A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrhythmias. Curr Biol 13: 861–866, 2003.[CrossRef][Web of Science][Medline]
  25. Kawanaka K, Nolte LA, Han DH, Hansen PA, Holloszy JO. Mechanisms underlying impaired GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats. Am J Physiol Endocrinol Metab 279: E1311–E1318, 2000.[Abstract/Free Full Text]
  26. LaRosa C, Downs SM. MEK inhibitors block AICAR-induced maturation in mouse oocytes by a MAPK-independent mechanism. Mol Reprod Dev 70: 235–245, 2005.[CrossRef][Web of Science][Medline]
  27. Leclerc I, Rutter GA. AMP-activated protein kinase: a new beta-cell glucose sensor? Regulation by amino acids and calcium ions. Diabetes 53, Suppl 3: S67–S74, 2004.[Abstract/Free Full Text]
  28. Leclerc I, Woltersdorf WW, da Silva Xavier G, Rowe RL, Cross SE, Korbutt GS, Rajotte RV, Smith R, Rutter GA. Metformin, but not leptin, regulates AMP-activated protein kinase in pancreatic islets: impact on glucose-stimulated insulin secretion. Am J Physiol Endocrinol Metab 286: E1023–E1031, 2004.[Abstract/Free Full Text]
  29. Leese HJ, Barton AM. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil 72: 9–13, 1984.[Abstract/Free Full Text]
  30. Lenart P, Bacher CP, Daigle N, Hand AR, Eils R, Terasaki M, Ellenberg J. A contractile nuclear actin network drives chromosome congression in oocytes. Nature 436: 812–818, 2005.[CrossRef][Medline]
  31. Lowry OH, Berger SJ, Carter JG, Chi MM, Manchester JK, Knor J, Pusateri ME. Diversity of metabolic patterns in human brain tumors: enzymes of energy metabolism and related metabolites and cofactors. J Neurochem 41: 994–1010, 1983.[CrossRef][Web of Science][Medline]
  32. Moley K. Diabetes and preimplantation events of embryogenesis. Semin Reprod Endocrinol 17: 137–151, 1999.[Web of Science][Medline]
  33. Moley KH, Chi MMY, Mueckler M. Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos. Am J Physiol Endocrinol Metab 275: E38–E47, 1998.[Abstract/Free Full Text]
  34. Moley KH, Vaughn WK, DeCherney AH, Diamond MP. Effect of diabetes mellitus on mouse pre-implantation embryo development. J Reprod Fertil 93: 325–332, 1991.[Abstract/Free Full Text]
  35. Passonneau JL, Lowry OH. Enzymatic Analysis. Totowa, NJ: Humana, 1993.
  36. Passonneau JV, Lowry OH. The role of phosphofructokinase in metabolic regulation. Adv Enzyme Regul 2: 265–274, 1964.[CrossRef][Web of Science][Medline]
  37. Polekhina G, Gupta A, Michell BJ, van Denderen B, Murthy S, Feil SC, Jennings IG, Campbell DJ, Witters LA, Parker MW, Kemp BE, Stapleton D. AMPK beta subunit targets metabolic stress sensing to glycogen. Curr Biol 13: 867–871, 2003.[CrossRef][Web of Science][Medline]
  38. Roberts R, Franks S, Hardy K. Culture environment modulates maturation and metabolism of human oocytes. Hum Reprod 17: 2950–2956, 2002.[Abstract/Free Full Text]
  39. Rossmeisl M, Flachs P, Brauner P, Sponarova J, Matejkova O, Prazak T, Ruzickova J, Bardova K, Kuda O, Kopecky J. Role of energy charge and AMP-activated protein kinase in adipocytes in the control of body fat stores. Int J Obes Relat Metab Disord 28, Suppl 4: S38–S44, 2004.
  40. Sakoda H, Fujishiro M, Shojima N, Ogihara T, Kushiyama A, Fukushima Y, Anai M, Ono H, Kikuchi M, Horike N, Viana AY, Uchijima Y, Kurihara H, Asano T. Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity. Am J Physiol Endocrinol Metab 289: E474–E481, 2005.[Abstract/Free Full Text]
  41. Sutton ML, Cetica PD, Beconi MT, Kind KL, Gilchrist RB, Thompson JG. Influence of oocyte-secreted factors and culture duration on the metabolic activity of bovine cumulus cell complexes. Reproduction 126: 27–34, 2003.[Abstract]
  42. Sutton ML, Gilchrist RB, Thompson JG. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influence on oocyte developmental capacity. Hum Reprod Update 9: 35–48, 2003.[Abstract/Free Full Text]
  43. Wang WH, Abeydeera LR, Prather RS, Day BN. Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development. Biol Reprod 62: 1177–1183, 2000.[Abstract/Free Full Text]
  44. Wojtaszewski JF, Jorgensen SB, Hellsten Y, Hardie DG, Richter EA. Glycogen-dependent effects of 5-aminoimidazole-4-carboxamide (AICA)-riboside on AMP-activated protein kinase and glycogen synthase activities in rat skeletal muscle. Diabetes 51: 284–292, 2002.[Abstract/Free Full Text]
  45. Yazigi RA, Chi MM, Mastrogiannis DS, Strickler RC, Yang VC, Lowry OH. Enzyme activities and maturation in unstimulated and exogenous gonadotropin-stimulated human oocytes. Am J Physiol Cell Physiol 264: C951–C955, 1993.[Abstract/Free Full Text]



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