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research-article |
Biology Department, Marquette University, Milwaukee, Wisconsin 53233
ABSTRACT
In this study, we examined the effect of heat pulsing on oocyte maturation and assessed the possible role of stress-activated enzymes during heat stress-induced meiotic maturation. Denuded oocytes from immature eCG-primed mice were pulsed for 30 min at increasing temperatures from 40°C to 43°C in dibutyryl cAMP-containing medium and were subsequently cultured at 37°C for a total incubation time of 17–18 h. Oocytes exposed to 42°C showed the greatest stimulation of maturation, with no effect at 43°C. A heat pulse did not compromise progression to metaphase II as observed by polar body (PB) formation. The AMP-activated protein kinase (PRKA) inhibitors compound C and Ara-A each blocked the meiosis-stimulating effects of heat. Western blots showed that acetyl-CoA carboxylase, an important substrate of PRKA, was phosphorylated in heat-treated germinal vesicle-stage oocytes, indicating activation of PRKA before maturation. The mitogen-activated protein 2 kinase (MAP2K1) inhibitor PD98059 also prevented heat-induced maturation, but this effect was unrelated to MAPK1/3 activation, which was not observed until after germinal vesicle breakdown (GVB). Phosphorylated MAPK14 was not detected in the oocyte under any experimental condition, and only high concentrations of the MAPK14 inhibitor SB203580 blocked heat-stimulated maturation, suggesting that MAPK14 is not involved in meiotic induction. MAPK8/9 was activated by heat, and the MAPK8/9 inhibitor SP600125, but not JUN N-terminal kinase I, blocked heat-induced maturation. Heat treatment transiently suppressed GVB and PB formation in spontaneously maturing oocytes by a mechanism that is apparently different from its meiosis-inducing action. Collectively, these data show that an acute heat pulse stimulates GVB in meiotically arrested oocytes and suggest that this effect is mediated through the activation of PRKA.
gamete biology, meiosis, oocyte development, stress
In mammals, it is firmly established that cAMP maintains the oocyte in meiotic arrest and that the reinitiation of meiosis occurs after a decrease in oocyte cAMP levels [1]. The cAMP that is synthesized in the somatic cells possibly contributes to intraoocyte levels following transfer to the oocyte through gap junctions that interconnect these two cell types [2–5]. However, the oocyte is also capable of generating cAMP on its own, due to the presence of adenylate cyclase [6], the heterotrimeric G protein Gs [7, 8], and an orphan member of the G protein-coupled receptor family, GPR3 [9, 10]; in fact, all of these components are essential for maintaining the prophase I arrest. Therefore, the oocyte is likely the principal site for generating the cAMP that is involved in blocking meiotic resumption, with the granulosa cells serving a regulatory function.
Cyclic AMP binds to the regulatory subunits of cAMP-dependent protein kinase A (PRKACA), causing release of the active catalytic subunits. PRKACA is important in the regulation of oocyte meiotic arrest through substrate phosphorylation and sequestration of critical regulatory effectors [11]. Phosphodiesterase (PDE) is an enzyme that hydrolyzes cAMP and inactivates PRKACA and has been shown to be indispensable for meiotic resumption [11–13]. However, the loss of PRKACA activity may not be the only means by which germinal vesicle breakdown (GVB) can be triggered. The end product of PDE action, 5'-AMP, is a potent allosteric activator of an important stress response kinase, AMP-activated protein kinase (PRKA), which has been implicated in meiotic resumption in mouse oocytes [14, 15].
PRKA acts as a cellular fuel gauge and can be turned on when a cell is exposed to stressful conditions, particularly those that deplete ATP and consequently elevate the AMP:ATP ratio [16]. AMP is an allosteric activator of PRKA, triggering phosphorylation on threonine 172. Following AMP binding, PRKA becomes a better substrate for the upstream kinase and a weaker substrate for phosphatases that inactivate it [17]. In general, activation of PRKA switches off anabolic pathways of lipid and carbohydrate metabolism and switches on catabolic pathways to conserve intracellular ATP and to generate more ATP [18, 19].
5-Aminoimidazole-4-carboxamide 1-ß-ribofuranoside (AICAR) is an adenosine analog that is taken up by cells and is converted by adenosine kinase to the AMP analog ZMP which is a potent stimulator of PRKA. AICAR [14] and other adenosine analogs [20] are effective inducers of meiotic maturation in mouse oocytes maintained in meiotic arrest by a variety of inhibitors. An important substrate of PRKA is acetyl-CoA carboxylase (ACACA), a rate-determining enzyme in the fatty acid synthetic pathway [21], and Western blot analysis of the phosphorylation state of this enzyme is a useful assay for assessing the activation state of PRKA. In mouse oocytes, ACACA is phosphorylated before GVB in AICAR-treated oocytes, and this is accompanied by activation of PRKA as measured by Western blot analysis of threonine-172 phosphorylation and by an enzyme assay [15].
Physiological and pathophysiological stimuli that increase the AMP:ATP ratio within cells have been shown to activate PRKA and include heat shock, muscle contraction, metabolic stresses and poisons, ischemia, and osmotic and oxidative stress [17, 22–30]. In an earlier article, we reported that meiotic maturation was induced when mouse oocytes were exposed to metabolic, oxidative, or osmotic stress; moreover, GVB was preceded by activation of PRKA as determined by ACACA phosphorylation, and the stress-induced maturation was prevented by PRKA inhibitors [31]. Therefore, these results demonstrated the meiosis-inducing potential of stress mediated by PRKA.
In the present study, we continued our evaluation of stress effects on mouse oocytes by examining their response to heat stress. We also included an analysis of members of the mitogen-activated protein kinase (MAPK) family, including the stress-activated protein kinases (SAPKs) MAPK14 and MAPK8/9. The results show that heat treatment stimulates PRKA and subsequent meiotic resumption, but they fail to consistently implicate any member of the MAPK family in this heat response.
Oocyte Isolation and Culture Conditions
Animals were raised in the research colony of the principal investigator (S.M.D.). All experiments were carried out with prior approval of the Marquette University Institutional Animal Care and Use Committee.
Immature C57BL6/J x SJL/J F1 female mice, 19–23 days old, were used for all experiments. Mice were primed using 5 IU of eCG (National Institutes of Health) and were killed by cervical dislocation 2 days later. Ovaries were removed and placed in culture medium, and large antral follicles were pierced using sterile needles. Oocyte-cumulus cell complexes (OCCs) were collected, washed through several changes of fresh isolation medium, and transferred in a small volume to plastic culture tubes (Falcon 2058) or stoppered borosilicate glass tubes containing 1 ml of the appropriate test medium. Denuded oocytes (DOs) were prepared by repeated pipetting using a Pasteur pipette and were cultured alone without cumulus cells present. Cumulus cell-enclosed oocytes (CEOs) are defined as cumulus-free oocytes obtained by removal of cumulus cells after culture as OCCs. Culture tubes were gassed using a humidified mixture of 5% O2, 5% CO2, and 90% N2 before placement in a water bath. The medium used was Eagle minimum essential medium (MEM) supplemented with 0.23 mM pyruvate, penicillin, streptomycin sulfate, and 3 mg/ml of crystalized lyophilized bovine serum albumin (ICN ImmunoBiologicals, Lisle, IL).
For detection of MAPK proteins, DOs were washed and collected in a small volume and were added to an equal volume of 2x Laemmli buffer containing 20% beta-mercaptoethanol. Samples were placed in a 500-µl microfuge tube, heated at 100°C for 5 min, and stored at –80°C until used for Western blot. Before electrophoresis, tubes were vortexed and centrifuged, and the sample was loaded onto a NuPage 4%–12% Bis-Tris minigel (Invitrogen) [25]. DOs were loaded into each lane for MAPK1/3 detection, while the number of DOs or OCCs for MAPK14 and MAPK8/9 detection varied between experiments (see the figure legends). Following electrophoresis, proteins were transferred to a nitrocellulose membrane (Bioscience), and the membrane was washed for 10–15 min in Tris-buffered saline (TBS) and TBS-Tween 20. Membranes were blocked for 1 h using 5% nonfat dry milk in TBS-Tween 20 and were again washed for 10–15 min using TBS and TBS-Tween 20. To detect phospho-MAPK members (MAPK1/3, MAPK14, and MAPK8/9), blots were incubated overnight at 4°C in mouse monoclonal antiphospho-MAPK1/3 antiserum, antiphospho-MAPK14 (Sigma Chemical Co., St. Louis, MO), or antiphospho-JUN (Calbiochem) diluted 1:1000 for MAPK1/3 and 1:250 for MAPK14 and JUN in TBS/BSA. The membrane was then washed and probed for 1 h at room temperature using horseradish peroxidase (HRP)-conjugated anti-mouse antiserum (Bio-Rad Laboratories, Hercules, CA) diluted 1:3000 or goat anti-rabbit antiserum diluted 1:1000 (Pierce Biotechnology, Rockford, IL). MAPK1/3 proteins were detected using enhanced chemiluminescence reagents, and MAPK14 and MAPK8/9 were detected using West Pico/Dura/Femto SuperSignal (Pierce Biotechnology). Total MAPK was determined by stripping the blots at 30°C for 30 min in TBS containing 2% SDS and 0.7% Eagle basal medium. Blots were then washed, blocked in milk solution, washed again, and reprobed overnight using rabbit anti-MAPK1/3 antibody (Sigma Chemical Co.) diluted 1:5000 in TBS/BSA, MAPK14, or MAPK8/9 diluted 1:500 in TBS/BSA. The label was detected as already described following 1-h incubation using HRP-conjugated goat anti-rabbit antiserum (1:1000; Pierce Biotechnology).
Detection of phospho-ACACA was determined by running samples (the number of oocytes varies among groups; see the figure legends) on a 3%–8% Tris-acetate minigel (Invitrogen) and by immunoblotting overnight at 4°C using rabbit antiphospho-ACACA antibody at 1:250 dilution (Upstate). After blocking for 2 h in 5% nonfat dry milk (pH 7.3–7.4) at room temperature, the blots were washed and exposed to HRP-conjugated donkey anti-rabbit antiserum (1:2000; Pierce Biotechnology) for 1 h. Protein was detected using West Pico/Dura SuperSignal from Pierce Biotechnology. Blots were reprobed according to the procedure already described using sheep anti-ACACA antibody (1:2000) as the primary antibody.
Oocytes were removed from culture, washed in PBS/polyvinylpyrrolidone (3 mg/ml) and fixed for 1–2 h at 4°C in 4% paraformaldehyde. The oocytes were washed and stained using Hoechst 33342 (5 µg/ml in PBS; Sigma Chemical Co.).
All culture components, dibutyryl (db) cAMP, isobutylmethylxanthine (IBMX), hypoxanthine (HX), Ara-A, PD98059, SB203580, and SP600125 were obtained from Sigma Chemical Co. Compound C was obtained from Calbiochem (EMD Biosciences, San Diego, CA) and JUN N-terminal kinase 1 (JNK1) from Axxora (San Diego, CA). The eCG was obtained from A. F. Parlow at the National Hormone and Peptide Program.
All oocyte maturation experiments were repeated at least three times with at least 25 oocytes per group per experiment. Viability was maintained at a minimum of 93% for the duration of the culture [32]. Oocyte maturation frequencies were subjected to arcsin transformation and were statistically analyzed using ANOVA followed by Duncan multiple range test. For comparison between two groups, two-tailed paired t-test was used. For all statistical analyses, P < 0.05 was considered significant.
Characterization of Heat-Induced Meiotic Maturation
To determine if a heat pulse could induce maturation in meiotically arrested oocytes, DOs were cultured in medium containing 300 µM dbcAMP for 30 min at increasing temperatures up to 43°C and were then transferred to a 37°C water bath for 17–18 h before assessment of GVB. Pulsing was carried out because prolonged exposure to elevated temperatures is lethal. When oocytes were maintained at 37°C for the entire culture period, 11% of the oocytes resumed maturation (Fig. 1A). Increasing the pulsing temperature produced a progressive stimulation of maturation that peaked at 42°C with a 40% increase in GVB.
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To determine how pulse time affected meiotic induction, dbcAMP-arrested DOs were pulsed for increasing periods up to 60 min at 42°C. As shown in Figure 1B, there was a progressive increase in maturation induction as the pulse time was increased from 10 to 60 min, with a 60-min pulse producing a 65% increase in GVB over controls.
A kinetics experiment was performed to determine when meiotic resumption was initiated in heat-pulsed oocytes. The dbcAMP-arrested DOs, pulsed for 60 min at 42°C, were assessed at regular intervals for up to 16 h of culture. In control oocytes maintained at 37°C for the entire culture period, maturation rates were low, reaching 20% GVB at 16 h. Meiotic induction in heat-pulsed oocytes was initiated between 4 h and 7 h, with maturation peaking by 13 h at about 70% GVB (Fig. 1C).
To ensure that the heat-induced induction of maturation is not unique to dbcAMP-arrested oocytes and to explore the effects of cumulus cells, DOs and CEOs were cultured under several different inhibitory conditions (dbcAMP, HX, and IBMX) and were exposed to a 60-min heat pulse at 42°C before overnight culture at 37°C. HX is a weak meiotic inhibitor; therefore, it was supplemented with 25 µM dbcAMP to maintain low maturation percentages. In each treatment group, a significant stimulation of maturation resulted from the pretreatment at 42°C (Fig. 2). In addition, the presence of cumulus cells had no effect on the extent of meiotic induction brought about by heat treatment.
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Heat stress has been associated with many nuclear and cytoplasmic abnormalities [33, 34]; therefore, it was important to determine if heat pulsing allowed for normal metaphase II (MII) progression after the resumption of meiosis. DOs were heat pulsed for 60 min in 300 µM dbcAMP and were transferred to the control bath for 17–18 h before the assessment of GVB and polar body (PB) formation. Figure 3 shows that PB formation was not compromised in heat-treated oocytes because 80% of oocytes resuming maturation extruded a PB (70.5% GVB and 56.4% PBs).
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Role of PRKA in Heat-Induced Maturation
PRKA (formerly known as AMPK), a heterotrimeric protein that can be activated by numerous types of stress [35], has been shown to be involved in meiotic induction in mouse oocytes [14, 15, 20]. A commonly used indirect method to assess PRKA activity is Western blot analysis of the phosphorylation state of ACACA (formerly known as ACC), an enzyme that is important in fatty acid metabolism and is a key substrate of PRKA [15, 21, 36]. To determine if PRKA activation precedes GVB in heat-treated dbcAMP-arrested oocytes, Western blot analysis of extracts from germinal vesicle (GV)-stage oocytes was carried out using antiphospho-ACACA antibody. Extracts were prepared from the following four groups of DOs: 1) 1-h culture at 37°C, 2) 1-h culture at 42°C, 3) 2-h culture at 37°C, and 4) 1-h pulse at 42°C followed by 1-h pulse at 37°C. All media contained 300 µM dbcAMP. After a 1-h heat pulse, phospho-ACACA levels were increased compared with controls; the phospho-ACACA band was somewhat less intense after an additional hour at 37°C (Fig. 4A).
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The experiment was repeated using OCCs that had been heat pulsed for 1 h followed by 1 h or 3 h at 37°C (2 h or 4 h of total culture time). The heat-treated OCCs also exhibited increased PRKA activity, with the phospho-ACACA band at 4 h being considerably more intense (Fig. 4B). These data indicate that heat treatment activates PRKA before GVB is initiated.
To further examine a role for PRKA in heat-induced meiotic maturation, two inhibitors of PRKA, compound C [37] and Ara-A [38], were tested on heat-induced GVB. DOs were maintained in meiotic arrest with 300 µM dbcAMP at 37°C using a 30-min pretreatment of increasing concentrations of inhibitor. They were then pulsed for 60 min at 42°C, cultured overnight at 37°C, and assessed for GVB. Both inhibitors blocked heat-induced maturation in a dose-dependent manner (Fig. 5, A and C). At a concentration of 5 µM, compound C also blocked the heat-stimulated phosphorylation of ACACA (Fig. 5B). These results further implicate PRKA in the mediation of heat-induced maturation.
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Involvement of MAPK Family Members in Heat-Induced Meiotic Resumption
MAPK1/3. Initial experiments addressed the possible involvement of MAPK1/3 (formerly known as ERK1/2). To test if PD98059, a common MAP2K1 inhibitor [39], could block heat-induced GVB, DOs were maintained in meiotic arrest using 300 µM dbcAMP, and meiotic maturation was stimulated using a 60-min pulse at 42°C. Oocytes were pretreated for 30 min at 37°C using increasing concentrations of PD98059 before the addition of the heat stress, and GVB was assessed 17–18 h later. At 37°C, DOs resumed meiosis at a frequency of 15%, whereas a 60-min pulse at 42°C increased this to 82% (Fig. 6A). PD98059 blocked the maturation induction in a dose-dependent manner, with almost complete inhibition at 75 µM.
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To establish a relationship between the activation state of MAPK1/3 and the meiotic status of the heat-pulsed oocytes, extracts of control and 1-h heat-pulsed DOs were collected at different time points and were subjected to Western blot analysis using an antiphospho-MAPK1/3 antibody. As shown in Figure 6B, there was no detectable MAPK1/3 phosphorylation in GV-stage oocytes treated at 37°C or in GV-stage oocytes pulsed for 1 h at 42°C and returned to 37°C for 5, 7, or 9 h of culture. At 9 h, GVB-stage heat-pulsed oocytes exhibited a prominent phospho-MAPK1/3 band. This indicates that MAPK1/3 activation does not precede the resumption of maturation stimulated by heat but rather occurs only in association with it.
MAPK14. To determine if MAPK14 (formerly known as p38) is involved in heat-induced GVB, the MAPK14 inhibitor SB203580 was tested. DOs maintained in meiotic arrest using 300 µM dbcAMP were pretreated for 30 min at 37°C using increasing concentrations of SB203580 before a 60-min pulse at 42°C, and GVB was assessed 17–18 h later. These results are shown in Figure 7A. Heat treatment stimulated a 75% increase in GVB, and this was suppressed by SB203580, although the maximum inhibition achieved was only about 50% (at 50 µM).
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To determine how heat treatment affects the activation state of MAPK14, Western blot analysis was carried out using an antibody to phosphorylated MAPK14, the active form of the kinase. Extracts from DOs or OCCs following 1-h culture at 37°C or 42°C were prepared for analysis. Although DOs contained MAPK14, no phosphorylation was detected in any of the groups regardless of whether they were exposed to heat stress or to AICAR (Fig. 7B). Among OCCs, the control group exhibited a strong phospho-MAPK14 band, indicating a high level of active kinase, but this activity was unaffected by treatment with AICAR or by heat (Fig. 7C). These results suggest that MAPK14 activation is not induced or increased by a heat pulse or by AICAR treatment in DOs or in OCCs.
MAPK8/9. To determine if MAPK8/9 (formerly known as JNK) is activated in DOs and OCCs in response to stress, Western blot analysis was carried out using an antiphospho-JUN antibody. JUN (formerly known as c-Jun) is a protein component of the activating protein transcription factor and is a substrate of MAPK8/9; therefore, JUN phosphorylation is commonly used to assess MAPK8/9 activity [40]. Extracts were prepared from GV-stage DOs cultured for 1 h at 37°C or 42°C in 300 µM dbcAMP alone or at 37°C in dbcAMP plus 500 µM AICAR. Little phosphorylated JUN was detected in DOs cultured at 37°C in dbcAMP alone, but a prominent band was apparent in the other two oocyte groups (Fig. 8A). When extracts were analyzed from control or heat-pulsed OCCs after 1, 2, or 4 h of total culture, a strong band was apparent in heat-treated OCCs after 1 h that was greatly reduced by 2 h and was absent by 4 h (Fig. 8B). These results suggest a transient stimulation of MAPK8/9 activity in response to heat stress.
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Next, two inhibitors of MAPK8/9 were tested on the meiotic induction generated by heat treatment. DOs were maintained in meiotic arrest using 300 µM dbcAMP and were pretreated for 30 min at 37°C using increasing concentrations of SP600125 [41] or D-JNK1 [42] before pulsing for 60 min at 42°C, with GVB assessment 17–18 h later. SP600125 blocked the meiotic induction in a dose-dependent manner, resulting in a 38% decrease at 50 µM that was not significantly different from that of the untreated control group (Fig. 9A). On the other hand, D-JNK1 had no inhibitory effects on heat-induced maturation at concentrations up to 2.5 µM (Fig. 9B) within the range shown to significantly block MAPK8/9 in mouse embryos [43]. Both inhibitors blocked heat-stimulated MAPK8/9 activity as determined by Western blot analysis of phospho-JUN (Fig. 9C). These data suggest that heat-induced GVB is not dependent on activation of MAPK8/9 because only high concentrations of SP600125 and not D-JNK1 blocked this induction and because each inhibitor only partially suppressed JUN phosphorylation.
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To determine if these inhibitors could be acting on PRKA, Western blot analysis of ACACA phosphorylation was carried out in heat-pulsed DOs. As shown in Figure 10A, neither inhibitor appreciably affected the phosphorylation state of ACACA, indicating a lack of effect on PRKA. Similarly, compound C, an inhibitor of PRKA, did not block heat-stimulated MAPK8/9 activity (Fig. 10B), which fails to support PRKA as an upstream regulator of MAPK8/9.
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Effects of Heat Pulsing on Spontaneous Maturation
Our results demonstrate a positive meiosis-inducing effect of heat stress on oocyte maturation. However, much of the literature regarding heat stress in oocytes reveals a suppressive effect on spontaneous maturation. Because heat stress seems to have opposite effects on spontaneously maturing oocytes as opposed to those maintained in meiotic arrest, it was important to determine if meiotic inhibitors such as dbcAMP could protect against the negative heat shock effect on spontaneous maturation. To test this, DOs and CEOs were cultured for 60 min in 300 µM dbcAMP at 37°C or 42°C, washed free of dbcAMP, and placed into inhibitor-free medium for an additional 2 h or 3 h.
In the groups that were cultured in dbcAMP for the first hour, washed, and placed into inhibitor-free MEM, 53% of DOs and 49% of CEOs underwent spontaneous maturation when cultured at 37°C for 2 h, whereas only a negligible number of oocytes receiving a heat pulse underwent GVB (Fig. 11A). Increasing the culture time in inhibitor-free medium to 3 h resulted in further increases in GVB in control oocytes (91% of DOs and 74% of CEOs), but little change was seen in heat-treated oocytes. These findings demonstrate that culturing the oocytes using dbcAMP does not protect the oocyte from the inhibition of spontaneous maturation caused by the heat pulse.
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To examine the long-term effects of heat pulsing during spontaneous maturation, the previous experiment was repeated, but the postwashing culture time was increased to 18 h. With the extended culture time, all treatment groups exhibited identical maturation frequencies (92%–99% GVB; Fig. 11A), demonstrating that heat pulsing only slowed down the kinetics of spontaneous meiotic resumption.
To determine if progression to MII was affected by heat treatment, control and heat-treated oocytes were examined for PB formation 17–18 h or 22–23 h after washing out the dbcAMP and culturing in inhibitor-free medium. Oocytes from the group examined 22–23 h after washing were stained using Hoechst 33342 in case PB degeneration occurred during the longer culture period. Although PB formation was maintained at a high level in control oocytes at both time points (89%–94%), heat treatment reduced completion of maturation in the group examined 17–18 h after washing (32%), with no effect in the group examined 22–23 h after washing (86%) (Fig. 11B). Taken together, these data suggest that heat pulsing at 42°C retards spontaneous maturation but does not permanently block it.
Heat Treatment Attenuates AICAR-Induced Maturation
Because heat stress has inhibitory and stimulatory actions on oocyte maturation, it was important to determine if heat treatment could interfere with meiotic induction brought about by PRKA activation. To test this, DOs were cultured for 1 h at 37°C or 42°C in medium containing 300 µM dbcAMP; oocytes were then cultured at 37°C for 4 h in the presence or absence of 500 µM AICAR. In the absence of AICAR, the maturation percentage in both groups was low (10%–17% GVB) (Fig. 12). In the oocyte group not exposed to heat, AICAR effectively induced maturation (92% GVB) but failed to trigger maturation in the heat-pulsed oocytes (10% GVB). However, this suppressive effect of heat was only temporary as complete meiotic induction was achieved when cultures were extended to 17–18 h (Fig. 12).
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In this study, we showed that heat pulsing of meiotically arrested mouse oocytes triggers the resumption of meiosis with normal progression to MII but coincidentally attenuates spontaneous maturation. The activation of PRKA before GVB and the suppression of heat-induced maturation by the PRKA inhibitors compound C and Ara-A implicate PRKA in the meiotic induction observed. Although PD98059 produced a similar suppressive effect on meiotic induction, failure to detect phosphorylated MAPK1/3 until after GVB discounts MAPK1/3 as a causative agent in heat-induced maturation. MAPK14, one of the SAPK members of the MAPK family, showed no activation in DOs and no differential activation in OCCs with exposure to heat. However, MAPK8/9, another SAPK, was phosphorylated after a heat pulse and before GVB. The MAPK8/9 inhibitor SP600125 blocked heat-induced GVB, but JNK1, another MAPK8/9 inhibitor, had no effect on meiotic induction. Both inhibitors partially blocked heat-induced JUN phosphorylation, but neither inhibitor had any effect on ACACA phosphorylation, suggesting that the inhibitors are not acting on PRKA. These data indicate mediation of heat-induced maturation by PRKA but fail to convincingly support a role for MAPKs. The failure of AICAR to stimulate maturation in heat-stressed oocytes suggests that oocyte damage due to acute heat stress leads to cell cycle arrest that cannot be overcome by a meiosis-inducing stimulus until the damage has been repaired.
It was previously reported that a pharmacological activator of PRKA, AICAR, stimulates PRKA activation and GVB in meiotically arrested mouse oocytes [14]. In addition, recent experiments demonstrated a causal role for PRKA in eliciting GVB under a variety of meiosis-inducing conditions [15] and under a variety of stressful conditions in mouse oocytes [31]. PRKA is a stress response kinase that is activated by numerous stress signals that deplete energy reserves or increase the cellular AMP:ATP ratio [23]. Therefore, it was of interest to test if heat stress is able to stimulate 1) nuclear maturation in meiotically arrested mouse oocytes and 2) activation of PRKA or other stress response proteins.
It has been known for many years that exposure of females to elevated temperatures can lead to increased embryonic mortality [44–48]. In addition, oocytes exposed to heat stress during meiotic maturation exhibit abnormal spindles and progress to MII in reduced numbers [33, 49–51]. Curci et al. [52] reported that GVB and protein synthesis were suppressed in mouse oocytes incubated at 43°C for 20–40 min. Matsuzuka et al. [53] showed that heat stressing of mouse oocytes results in a decreased percentage of zygotes that develop to blastocysts for naturally fertilized and in vitro-fertilized oocytes but does not cause a reduction in early embryo developmental competence. Contrary to these findings, Kim et al. [54] failed to see a negative effect of acute heat shock (0.25–1 h) on GVB and on PB formation during spontaneous maturation in mouse oocytes, but a more prolonged shock (2–4 h) demonstrated prevention of almost all oocytes from resuming maturation as determined after 4 h, as well as severely retarded PB formation when checked after 18 h. This susceptibility to heat is thought to be due to the poor heat shock response capabilities of fully grown oocytes [55].
In the present study, short-term exposure of meiotically arrested oocytes to elevated temperatures led to GVB and to progression to MII, representing a unique response to heat shock. Meiotic induction stimulated by increasing temperatures peaked at 42°C, with complete elimination of the meiotic response at 43°C. Therefore, the specific temperature and duration of exposure determine whether meiotic maturation is promoted or is compromised. A 60-min pulse at 42°C triggered meiotic induction in DOs and in CEOs arrested using dbcAMP, IBMX, or hypoxanthine. The failure of cumulus cells to affect the meiotic response is consistent with our previous results [31] and demonstrates that the cumulus cells are not protecting the oocyte against the heat pulse. Furthermore, the meiosis-inducing action is not dependent on the inhibitor chosen to maintain meiotic arrest.
To determine if PRKA could be mediating the heat-induced maturation observed, Western blot analysis of ACACA phosphorylation, and therefore PRKA activation [36], was carried out. In DOs and in OCCs, more pronounced ACACA phosphorylation was observed in the heat-treated GV-stage oocytes than in the control oocytes maintained at 37°C. Stimulation of PRKA by heat before GVB supports a mediating role for this kinase in meiotic induction, an idea further bolstered by the suppression of heat-induced maturation using the PRKA inhibitors compound C and Ara-A. The degree of ACACA phosphorylation stimulated by heat stress was less than that achieved using AICAR, reflecting less effective activation of PRKA, and this is associated with a lower rate of meiotic induction (for comparison, see LaRosa and Downs [31]).
Stimulation of PRKA in some systems has been shown to increase MAPK1/3 activity [56, 57]. Therefore, it was important to determine if MAPK1/3 activation was involved in the heat-stimulated meiotic response in mouse oocytes. The MAP2K1 inhibitor PD98059 blocked oocyte maturation elicited by the heat pulse, consistent with a role for MAPK1/3 in the heat-induced maturation pathway. However, Western blot analysis revealed that MAPK1/3 was not phosphorylated until after GVB; therefore, it is not likely to be mediating the meiotic response. This is consistent with two recent studies in which we reported effective suppression of AICAR-induced maturation [58] or stress-induced maturation [31] by PD98059, although MAPK1/3 phosphorylation was not observed until after GVB. Because MAPK1/3 apparently does not participate in the stress-activated pathways leading to meiotic resumption, these results provide further evidence that the effects of PD98059 are not entirely specific for MAPK1/3 [59, 60].
It was also important to test the SAPK members of the MAPK family, MAPK14 and MAPK8/9. MAPK14 and MAPK8/9 are not only activated by a variety of different stress stimuli such as ischemia [61], hypoxia [62], DNP [63], proinflammatory cytokines, bacterial lipopolysaccharides [64], heat shock [65], high osmolarity [66], and H2O2 [67], but they also respond to physiological and pathological stimuli such as progesterone and sorbitol [68]. Moreover, the activation of MAPK14 and MAPK8/9 has been associated with the activation of PRKA [69–75].
To determine if the heat-induced increase in GVB is associated with MAPK14 activation, the MAPK14 inhibitor SB203580 was tested [76]. Higher concentrations of this drug incompletely blocked the maturation induction brought about by heat treatment. A commonly used concentration of SB203580 is 10 µM, which is sufficient to block AICAR-induced maturation in DOs and FSH-induced GVB in CEOs (data not shown); however, this concentration did not result in significant inhibition of heat-induced maturation. SB203580 is an ATP-competitive pyridinyl imidazole compound with a half-maximal inhibitory concentration (IC50) of 0.3–0.6 µM. There have been numerous reports questioning the specificity of the inhibitor when used in excess of 2 µM. It has been reported to block activity of not only MAPK14 but also MAPK8/9 [77, 78], MAPK1/3 [79], thromboxane synthase, cyclooxygenases 1 and 2 [60], phosphoinositide-dependent protein kinase 1, and protein kinase B [80]. In addition, it has been shown to block the effects of AICAR by interfering with the nucleoside transporter responsible for AICAR uptake [17, 81]. In our study, Western blot analysis determined that heat pulsing did not phosphorylate, and thus activate, MAPK14 in DOs or in OCCs above control levels at any of the chosen time points. We also failed to observe MAPK14 phosphorylation in oocytes treated with AICAR. These data indicate that MAPK14 is not activated by heat stress, nor is it linked to PRKA activation in the oocyte.
MAPK8/9 has been reported to induce maturation in Xenopus oocytes [82]. Also, MAPK8/9 is stimulated in preimplantation mouse embryos by shearing stress [83] or by suboptimal culture conditions [43, 84], but its activity is apparently not required for normal development. To determine if heat could activate MAPK8/9 in mouse oocytes, Western blot analysis was performed using an antibody to phosphorylated JUN, a common MAPK8/9 substrate. The results indicated a pronounced activation of MAPK8/9 in DOs and in OCCs after a 60-min pulse at 42°C. AICAR treatment also activated MAPK8/9 as measured by JUN phosphorylation, suggesting that MAPK8/9 is activated downstream of PRKA. However, the PRKA inhibitor compound C failed to block heat-induced JUN phosphorylation in oocytes, which suggests that heat-stimulated MAPK8/9 activation is not dependent on PRKA.
To further explore the role of MAPK8/9 in heat-induced maturation, two selective inhibitors were used. SP600125 is an ATP-competitive anthrapyrazolone compound with an IC50 of 5–10 µM [85] that has a 300-fold greater selectivity over other MAPK members [41]; however, other studies have shown SP600125 to have slight inhibitory potency on MAPK3, MAPK14, and a range of other kinases [86–89]. Bennett et al. [41] reported that 50 µM SP600125 significantly reduced MAPK8/9 and JUN phosphorylation; however, the phosphorylation of MAPK14 and its substrate ATF2 were also slightly blocked. Kefas et al. [90] reported that cells exposed to AICAR or cellular stress showed activation of PRKA followed by MAPK8/9 activation and apoptosis. Herein, 50 µM SP600125 effectively blocked the heat-induced maturation of mouse oocytes.
Another MAPK8/9 inhibitor, D-JNK1, has been used in a variety of cell types to suppress MAPK8/9 activity [43, 91, 92]. In mouse oocytes, concentrations as high as 2.5 µM did not block heat-stimulated GVB, although 1 µM was sufficient to block MAPK8/9 activity as assessed by JUN phosphorylation. These findings do not support a requirement for MAPK8/9 in heat-induced maturation, not do they discount a possible meiosis-inducing capability of the kinase. Yet, the inability of JNK1 to block heat-induced GVB suggests that SP600125 acts nonspecifically on components of meiotic induction that are unrelated to MAPK8/9. Neither inhibitor seems to interfere with PRKA signaling because ACACA phosphorylation in response to heat treatment was not affected. Therefore, PRKA is not acting downstream of MAPK8/9.
Our results demonstrate that the spontaneous maturation of mouse oocytes can be suppressed by a heat pulse but that this effect is only temporary if the pulsing occurs at 42°C or lower. Previous studies also reported suppression of spontaneous maturation in heat-stressed oocytes. Curci et al. [52] maintained mouse oocytes in dbcAMP and pulsed them for 60 min at 42°C. The oocytes were then washed and placed in control medium (without meiotic inhibitor) for 20 h, after which approximately 30% of the oocytes had undergone GVB. In the present study, almost 100% of the oocytes underwent GVB after a 60-min heat pulse at 42°C in dbcAMP followed by 17–18 h in control medium. The reason for this discrepancy is not clear, but a contributing factor might be the age of the mice: 3-week-old mice were used herein, and 40–60-day-old mice were used in the study by Curci et al. In a more recent study, Kim et al. [54] showed that chronic (2–4 h), but not acute (0.25–1 h), heat shock at 43°C suppressed GVB and PB formation in mouse oocytes. Oocytes exposed to acute heat shock (15 min or 30 min) showed accelerated maturation compared with controls 1 h later. Therefore, consistent with our results, stimulatory and inhibitory effects of heat shock were detected.
It is not clear why heat pulsing stimulates maturation in meiotically arrested oocytes while temporarily blocking GVB during spontaneous maturation. These may be two independent unrelated events in response to heat treatment that antagonize one another. The meiosis-suppressing action of heat may result from damage to the oocyte that requires repair or recovery before meiotic resumption is possible. Such a block to cell cycle progression is a common response to heat shock in many cell systems [93–95]. This idea is supported by the slow kinetics of meiotic resumption in heat-treated oocytes and by the failure of AICAR to trigger GVB in heat-treated oocytes during short-term culture. Protein synthesis may be involved because it is compromised in heat-stressed mouse oocytes [52] and because AICAR-induced maturation requires the synthesis of new proteins (LaRosa and Downs, unpublished results). Therefore, heat stress-induced maturation likely results from activation of kinases such as PRKA that can trigger GVB when there is no longer a heat-induced block to cell cycle progression. In the event of severe heat shock, cell damage is too extensive to repair, and the oocyte never recovers. In mouse oocytes, 43°C may be the threshold at which this occurs.
Correspondence: 1Stephen M. Downs, Biology Department, Marquette University, 530 North 15th Street, Milwaukee, WI 53233. FAX: 414 288 7357; e-mail: stephen.downs{at}marquette.edu
Received: 15 September 2006.
First decision: 10 October 2006.
Accepted: 2 November 2006.
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