Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print November 9, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.046524
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
74/3/585    most recent
biolreprod.105.046524v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by LaRosa, C.
Right arrow Articles by Downs, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by LaRosa, C.
Right arrow Articles by Downs, S. M.
Agricola
Right arrow Articles by LaRosa, C.
Right arrow Articles by Downs, S. M.
BIOLOGY OF REPRODUCTION 74, 585–592 (2006)
DOI: 10.1095/biolreprod.105.046524
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Stress Stimulates AMP-Activated Protein Kinase and Meiotic Resumption in Mouse Oocytes

Cean LaRosa , and Stephen M. Downs 1 

Biology Department, Marquette University, Milwaukee, Wisconsin 53233

ABSTRACT

This study examined the effects of three different cellular stresses on oocyte maturation in meiotically arrested mouse oocytes. Cumulus-cell enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured for 17–18 h in dbcAMP-containing medium plus increasing concentrations of the metabolic poison, sodium arsenite, or the free radical-generating agent, menadione. Alternatively, oocytes were exposed to osmotic stress by pulsing with sorbitol and returned to control inhibitory conditions for the duration of culture. Arsenite and menadione each dose-dependently induced germinal vesicle breakdown (GVB) in both DO and CEO. DO, but not CEO, pulsed for 60 min with 500 mM sorbitol were stimulated to resume maturation. The lack of effect in CEO suggests that the cumulus cells may be playing a protective role in osmotic stress-induced GVB. The AMP-activated protein kinase (PRKA; formerly known as AMPK) inhibitors, compound C and araA, completely blocked the meiosis-stimulating effects of all the tested stresses. Western blots showed that acetyl-CoA carboxylase, an important substrate of PRKA, was phosphorylated before GVB, supporting a role for PRKA in stress-induced maturation. Together, these data show that a variety of stresses stimulate GVB in meiotically arrested mouse oocytes in vitro and suggest that this effect is mediated through activation of PRKA.

gamete biology, meiosis, oocyte development

INTRODUCTION

In vivo, fully grown mammalian oocytes are maintained in the diplotene stage of the first meiotic prophase and are characterized by a large nucleus, termed the germinal vesicle (GV). These GV-stage oocytes are maintained in meiotic arrest until stimulated by the preovulatory gonadotropin surge to undergo GV breakdown (GVB). Meiosis progresses through anaphase I and polar-body extrusion to reach metaphase II, the stage at which the oocyte is ovulated and fertilized. Removal of the oocyte from the follicle and its culture in vitro allows maturation to occur spontaneously in the absence of hormonal stimulation.

The regulation of meiotic maturation is not well understood and remains an active area of research. It is firmly established that cAMP maintains the oocyte in meiotic arrest and that the reinitiation of meiosis is subsequent to a drop in oocyte cAMP levels [1]. High concentrations of cAMP activate cAMP-dependent protein kinase A (PRKACA; formerly known as PKA), important in the regulation of oocyte meiotic arrest through its substrate phosphorylation and sequestration of critical regulatory effectors [2]. The cAMP may be transferred from the granulosa cells to the oocyte through gap junctions that interconnect these two cell types. It has been proposed that, through phosphorylation of connexin proteins, the gap junctional communication is broken and the subsequent decrease in oocyte cAMP levels terminates meiotic arrest [1, 3]. Alternatively, it has been proposed that GVB is triggered by a positive signal, generated within the somatic compartment that reaches the oocyte through the gap-junction pathway or following secretion and diffusion [4, 5].

Phosphodiesterase (PDE) is an enzyme that hydrolyzes cAMP and inactivates PRKACA and has been shown to be indispensable for meiotic resumption [2, 6, 7]. Loss of PRKACA activity would satisfy the first model of meiotic resumption. However, it is possible that, in addition to removing an inhibitory influence, PDE generates a positive signal for oocyte maturation. The end product of PDE action, 5'-AMP, is a potent allosteric activator of an important stress-response kinase, AMP-activated protein kinase (PRKA; formerly known as AMPK). PRKA acts as a cellular fuel gauge of the cell and its activity is regulated by the AMP/ATP ratio [8]. When a cell is exposed to stressful conditions, stimulation of PRKA turns off energy-consuming processes and turns on those that generate ATP [9, 10]. PRKA is present in mouse oocytes, and AICAR, a pharmacological activator of PRKA, stimulates GVB in meiotically arrested oocytes [11]. In addition, other means of exposing the oocyte to higher levels of PRKA activation, including injection of active PRKA into the oocyte, also causes the resumption of meiotic maturation (unpublished data). It is possible that turning on PRKA, by a decrease in cAMP and coincident increase in AMP, could mediate mouse oocyte meiotic resumption in vitro [11].

Physiological and pathophysiological stimuli that increase the AMP/ATP ratio within cells have been demonstrated to activate PRKA and include muscle contraction, heat shock, metabolic stresses and poisons, ischemia, and osmotic and oxidative stress [12–22]. Considering the evidence that PRKA has a strong link to meiotic maturation in mouse oocytes, we tested the ability of three of these stresses to 1) elicit GVB in prophase I-arrested oocytes and 2) activate PRKA before GVB. We report that each of the three different stress treatments stimulates PRKA in GV-stage oocytes and induces meiotic resumption, lending further evidence that PRKA is a potent meiosis-inducing kinase in mouse oocytes.

MATERIALS AND METHODS

Oocyte Isolation and Culture Conditions

Animals were raised in the research colony of the principal investigator (S.M.D.), and all experiments were carried out with the prior approval of the Marquette University Institutional Animal Care and Use Committee.

Immature C57BL6/J x SJL/J F1 female mice, 19 to 23 days old, were used for all experiments. Mice were primed with 5 IU equine chorionic gonadotropin (NIH) and killed by cervical dislocation 2 days later. Ovaries were removed, placed in culture medium, and large antral follicles were pierced with sterile needles. Oocyte-cumulus cell complexes (OCC) 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 (DO) were prepared by repeated pipetting with a Pasteur pipette and were cultured alone without cumulus cells present. Cumulus cell-enclosed oocytes (CEO) are defined as cumulus-free oocytes obtained by removal of cumulus cells after culture as OCC. Culture tubes were gassed with a humidified mixture of 5% O2, 5% CO2, and 90% N2 before placement in a water bath at 37°C, unless stated differently. The medium used was Eagle minimum essential medium supplemented with 0.23 mM pyruvate, penicillin, streptomycin sulfate, and 3 mg/ml crystallized lyophilized bovine serum albumin (ICN ImmunoBiologicals, Lisle, IL).

Stress Treatments

To determine if stresses that have been reported to activate PRKA in other cells lines can also activate the kinase in oocytes, the following three stresses were chosen: 1) metabolic stress by arsenite suppression of the tricarboxylic acid cycle, 2) oxidative stress through menadione-stimulated free radical formation, and 3) hyperosmotic stress caused by pulsing with high concentrations of sorbitol. For experiments involving the inhibitors, compound C and araA, oocytes were pretreated with inhibitors for 30 min before administration of the stress. The poststress culture period for maturation experiments was 17–18 h, while that for Western blot analysis was 1 h. Control groups not receiving inhibitor were cultured for an equivalent period.

Western Blot Analysis

DO were washed and collected in a small volume and 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 blotting. Detection of phospho-acetyl-CoA carboxylase (ACACA; formerly known as ACC) was determined by running samples (350 GV-stage DO) on a 3–8% tris-acetate minigel (Invitrogen). Following electrophoresis, proteins were transferred to a nitrocellulose membrane (Bioscience). Membranes were blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline (TBS) and again washed for 10–15 min with TBS, then TBS-Tween 20. They were then immunoblotted overnight at 4°C with rabbit anti-phospho-ACACA antibody at a 1:250 dilution (Upstate). After blocking for 2 h in 5% nonfat dry milk in TBS (pH 7.3–7.4) at room temperature, the blots were washed and exposed to HRP-conjugated donkey anti-rabbit antiserum (Pierce Biotechnologies) (1:2000) for 1 h. Protein was detected using west pico super signal from Pierce Biotechnology (Rockford, IL). Blots were reprobed according to the procedure above using sheep anti-ACACA antibody (1:2000) as the primary antibody. Anti-ACACA antibody was generously provided by Dr. Graham Hardie from Dundee University, Dundee, Scotland.

Chemicals

All culture components, dbcAMP, araA, sorbitol, arsenite, and menadione were obtained from Sigma Chemical Co. (St. Louis, MO). Compound C was a gift from Merck & Co., Inc. (Rahway, NJ).

Statistical Analysis

All oocyte maturation experiments were repeated at least three times with at least 30 oocytes per group per experiment. Viability was maintained at a minimum of 93% for the duration of the culture [23]. Maturation data are presented as mean percent GVB ± SEM. Oocyte maturation frequencies were subjected to arcsine transformation and analyzed statistically by ANOVA followed by Duncan multiple range test. For all statistical analyses, a P-value less than 0.05 was considered significant.

RESULTS

Arsenite

Dose response and time course of meiotic maturation To test for a meiosis-inducing effect of arsenite, denuded and cumulus cell-enclosed mouse oocytes were cultured for 17–18 h in 300 µM dbcAMP plus increasing concentrations of sodium arsenite. While control oocytes in dbcAMP alone resumed maturation at a frequency of 13% and 15% in DO and CEO, respectively (Fig. 1A), arsenite dose dependently stimulated maturation, with greater potency in CEO. At the optimal dose of arsenite (10 µM), GVB was increased by 46% in DO and 73% in CEO. All of the control oocytes undergoing maturation progressed to metaphase II, but polar-body formation in arsenite-treated oocytes was reduced by over 75% (data not presented) despite significant stimulation of GVB.


Figure 1
View larger version (32K):
[in this window]
[in a new window]
  		FIG. 1. Effects of arsenite on meiotic maturation and the involvement of PRKA in arsenite-induced maturation. A) DO and CEO were cultured in 300 µM dbcAMP with increasing concentrations of sodium arsenite. GVB was assessed after 17–18 h. CEO and DO were analyzed separately for statistical differences. Within each category, groups with a different letter are significantly different. B) DO were cultured in 300 µM dbcAMP plus or minus 10 µM arsenite and maturation was assessed at the indicated time points. Groups with a different letter are significantly different. C) Inhibition of PRKA. The 250 µM araA and increasing concentrations of compound C completely blocked arsenite-induced maturation in dbcAMP-arrested DO, cultured for 17–18 h. Within each treatment set, groups with a different letter are significantly different. D) Western blot analysis of ACACA phosphorylation. DO were cultured for 1 h in medium containing 300 µM dbcAMP alone (control) or dbcAMP plus 250 µM AICAR or 10 µM arsenite

To determine the time course of arsenite-induced maturation, DO were cultured in medium containing 300 µM dbcAMP plus or minus 10 µM arsenite, and maturation was assessed at 6, 8, and 10 h of culture. In the absence of arsenite, maturation percentages remained low for the duration of the culture, increasing only 6% from 24% GVB at 6 h to 30% GVB after 10 h. In contrast, GVB induction was initiated after 6 h of arsenite treatment, and 73% of the oocytes had resumed maturation by 10 h. (Fig. 1B).

Effects of PRKA inhibitors on oocyte maturation and Western blot analysis of stress-induced ACACA phosphorylation An experiment was performed to determine the effect of the PRKA inhibitor, compound C, on arsenite-induced maturation in dbcAMP-arrested DO. As seen in Figure 1C, arsenite produced a 53% increase in GVB, and all concentrations of compound C completely blocked meiotic induction. Another PRKA inhibitor, araA, was examined. Again, the increase in GVB produced by arsenite was completely blocked by 250 µM araA.

Because the above experiments implicated PRKA in arsenite-induced GVB, Western blot analysis was carried out using an antibody specific for phosphorylated acetyl-CoA carboxylase (ACACA-P) to determine if PRKA was activated before GVB. The phosphorylation state of ACACA is often used to assess activation or PRKA because PRKA is the upstream kinase for this substrate [24]. Extracts were prepared from GV-stage DO cultured 1 h with or without 10 µM arsenite, and AICAR-treated oocytes served as a positive control. Immunoblots showed that ACACA became phosphorylated by arsenite treatment within 1 h of culture (Fig. 1D), but that the phosphorylation was less than that achieved with AICAR treatment.

Menadione

Dose response and time course of meiotic maturation To determine if menadione could stimulate oocyte maturation, DO and CEO were cultured for 17–18 h in 300 µM dbcAMP plus increasing concentrations of menadione. GVB was dose dependently stimulated in both DO and CEO, with maximal induction of 34% and 66%, respectively (Fig. 2A), but there was no effect of menadione on polar-body formation (data not presented). We have also achieved comparable meiotic induction with the oxidants hydrogen peroxide and diamide (data not presented).


Figure 2
View larger version (37K):
[in this window]
[in a new window]
  		FIG. 2. Effects of menadione on meiotic maturation and the involvement of PRKA in menadione-induced maturation. A) DO and CEO were cultured in 300 µM dbcAMP with increasing concentrations of menadione (Men). GVB was assessed after 17–18 h. CEO and DO were analyzed separately for statistical differences. Within each category, groups with a common letter are not significantly different. B) DO were cultured in 300 µM dbcAMP plus or minus 5 µM menadione and maturation was assessed at the indicated time points. Groups with a common letter are not significantly different. C) Inhibition of PRKA. The 500 µM araA and increasing concentrations of compound C completely blocked menadione-induced maturation in dbcAMP-arrested DO, cultured 17–18 h. Within each treatment set, groups with a different letter are significantly different. D) Western analysis of ACACA phosphorylation. DO were cultured for 1 h in medium containing 300 µM dbcAMP plus or minus 5µM menadione or 250 µM AICAR

A time-course experiment was performed to determine when meiotic resumption was initiated in menadione-treated oocytes. Meiotic induction increased slowly between 5 and 11 h of culture (11–30%), but showed a more dramatic increase (75%) by 19 h (Fig. 2B).

Effects of PRKA inhibitors on oocyte maturation and Western blot analysis of stress-induced ACACA phosphorylation To determine a possible role for PRKA in menadione-stimulated maturation, dbcAMP-arrested DO were exposed to menadione and treated with increasing concentrations of compound C. Oocytes treated with dbcAMP alone maintained a 25% rate of maturation, and the addition of menadione increased the percentage to 64% GVB (Fig. 2C). All concentrations of compound C completely blocked the meiosis-stimulating effects of menadione. The PRKA inhibitor, araA, was used to further examine the effect of PRKA inhibition on oocyte maturation. Figure 2C shows that the menadione-induced increase in maturation (40%) was completely blocked with 500 µM araA. AraA at a concentration of 250 µM was ineffective (data not presented).

To determine if PRKA activation precedes menadione-induced GVB, Western analysis using the anti-phospho-ACACA antibody was performed. As seen in Figure 2D, ACACA phosphorylation was increased by menadione in GV-stage oocytes after 1 h, but less than that seen in AICAR-treated oocytes.

Sorbitol

Dose response and time course of meiotic maturation To determine if osmotic stress could induce maturation in meiotically arrested oocytes, dbcAMP-arrested DO were pulsed with 250 or 500 mM sorbitol for increasing periods of time up to 60 min, then washed and returned to dbcAMP-containing medium for 17–18 h before assessment of GVB. Pulsing with sorbitol was carried out because continuous exposure at these concentrations is toxic to the oocytes. The control group was also washed after 60 min to eliminate any possible concern that a stimulating effect is due to washing. As shown in Figure 3A, a 45- and 60-min pulse of 500 mM sorbitol in DO significantly stimulated the resumption of meiosis by 23% and 27%, respectively. Interestingly, meiotic resumption was not triggered in CEO pulsed for 60 min in 500 mM sorbitol (data not shown). There was no effect of sorbitol on polar-body formation (data not shown) despite significant stimulation of GVB.


Figure 3
View larger version (35K):
[in this window]
[in a new window]
  		FIG. 3. Effects of sorbitol on meiotic maturation and the involvement of PRKA in sorbitol-induced maturation. A) DO were pulsed in 300 µM dbcAMP with 250 or 500 mM sorbitol for increasing time points, washed, and put into control medium. GVB was assessed after 17–18 h. CEO and DO were analyzed separately for statistical differences. Groups with an asterisk are significantly different from the control group not exposed to sorbitol. B) DO were pulsed for 60 min in 300 µM dbcAMP plus or minus 500 mM sorbitol, washed, and maturation was assessed at the indicated time points. Groups with a different letter are significantly different. C) Inhibition of PRKA. The 250 µM araA and increasing concentrations of compound C completely blocked menadione-induced maturation in dbcAMP-arrested DO. After a 60-min pulse with sorbitol, cultures were maintained for 17–18 h before assessment of GVB. Within each treatment set, groups with a different letter are significantly different. D) Western analysis of ACACA phosphorylation. DO were cultured for 1 h in medium containing 300 µM dbcAMP plus or minus 500 mM sorbitol or 250 µM AICAR

A time-course experiment determined that dbcAMP-arrested DO pulsed for 60 min with 500 mM sorbitol were induced to resume maturation after 5 h of culture (Fig. 3B). By 11 h, this stimulation had plateaued at about 25% above control.

Effects of PRKA inhibitors on oocyte maturation and Western blot analysis of stress-induced ACACA phosphorylation To determine the effect of compound C on sorbitol-induced maturation, dbcAMP-arrested DO treated with a 60-min pulse of sorbitol were exposed to increasing concentrations of compound C. As seen in Figure 3C, oocytes cultured in dbcAMP alone exhibited 18% GVB, and the pulse of sorbitol increased this maturation frequency to 51%. All concentrations of compound C completely blocked the meiosis-stimulating effects of sorbitol. AraA at 250 µM also completely blocked sorbitol-induced GVB, dropping the maturation percentage from 44% to 17% compared with 13% in the control.

Western blot analysis using anti-phospho-ACACA antibody revealed that a 60-min pulse of sorbitol stimulated the phosphorylation of ACACA in GV-stage DO, but to a lesser extent than in AICAR-treated oocytes (Fig. 3D). Upon washing out the sorbitol and culturing for an additional 60 min, the ACACA-P band was still evident in GV-stage DO (data not presented).

AICAR Pulsing

To determine if a transient exposure to AICAR could induce maturation, dbcAMP-arrested DO were pulsed for 20, 40, or 60 min with 250 µM AICAR, washed, and cultured 17–18 h in control medium. Assessment of GVB showed that a 60-min pulse with AICAR resulted in a maturation percentage comparable with the group continuously exposed to AICAR (74% and 85% GVB, respectively; Figure 4A).


Figure 4
View larger version (26K):
[in this window]
[in a new window]
  		FIG. 4. Effects of AICAR pulsing on oocyte maturation. A) DO were incubated 17–18 h with 300 µM dbcAMP plus 250 µM AICAR or were first pulsed for 20, 40, or 60 min with 250 µM AICAR, washed, and returned to AICAR-free control medium (17–18 h total) before assessment of GVB. B) DO were incubated 4 h with 300 µM dbcAMP plus or minus 250 µM AICAR or were first pulsed for 30, 60, 90, or 120 min with AICAR, washed, and returned to AICAR-free control medium (4 h total) before assessment of GVB. Within each treatment set, bars with a common letter are not significantly different

To test whether this transient increase in PRKA activation affects the rate of meiotic resumption, dbcAMP-arrested DO were pulsed for 0.5, 1, 1.5, or 2 h with 250 µM AICAR, washed, and placed into dbcAMP-containing control medium for a total of 4 h. As seen in Figure 4B, a continuous 4-h exposure to AICAR resulted in a 68% increase in maturation. Pulses of AICAR for 0.5 or 1 h had no effect on meiotic resumption, but increases in GVB were observed after 1.5- and 2-h pulses. Thus, a pulse of 1 h required more than 4 h to elicit a meiotic response. These results indicate that short-term activation of PRKA is able to induce maturation, but with slower kinetics than continuous activation.

DISCUSSION

This study has shown that exposing dbcAMP-arrested oocytes to metabolic, oxidative, or osmotic stress triggers the resumption of meiosis. In addition, activation of PRKA (AMPK) before GVB and the suppression of stress-induced maturation by compound C and araA implicate PRKA in the meiotic induction observed.

We have previously reported that a pharmacological activator of PRKA, AICAR, stimulates both PRKA activation and GVB in meiotically arrested mouse oocytes [11]. In addition, recent experiments demonstrated a causal role for PRKA in eliciting GVB under a variety of meiosis-inducing conditions (unpublished data). PRKA is a stress response kinase that is activated by numerous stress signals that deplete energy reserves and/or increase the cellular AMP/ATP ratio [14]. It was therefore of interest to test several well-known PRKA-activating stresses for their ability to stimulate PRKA activation and nuclear maturation in meiotically arrested mouse oocytes. Metabolic poisons that uncouple mitochondria and/or block respiration are well-established stimulators of PRKA and include arsenite [25], sodium azide [19], and dinitrophenol [26–29]. PRKA activity is also increased by oxidative stress brought about by oxidizing agents such as menadione [30] or hydrogen peroxide [13, 15, 31, 32]. A similar activation is triggered by osmotic stress when cells are exposed to high levels of sorbitol [19, 20, 33] or mannitol [26, 28].

Metabolic shock (arsenite), oxidative shock (menadione), and osmotic shock (sorbitol pulse) all triggered meiotic resumption in dbcAMP-arrested DO. These stresses, with the exception of sorbitol, also stimulated maturation in dbcAMP-arrested CEO. This suggests a protective role for the cumulus cells against osmotic shock, but not the other types of stresses, and may be due to the fact that sorbitol pulsing was the weakest stimulus for maturation. In DO, optimal treatment of arsenite and menadione stimulated maturation above control levels by 46% and 34%, respectively, compared with 26% with sorbitol.

To determine if PRKA activation was a common link among the three stress responses, two types of experiments were carried out—PRKA inhibition and Western blot analysis of ACACA (ACC) phosphorylation. Compound C and araA, PRKA inhibitors [34, 35], completely blocked the maturation induction brought about by each of the stresses. Moreover, Western blotting showed that, following each stress treatment, ACACA phosphorylation increased before GVB. These data provide strong evidence that PRKA mediates the meiosis-inducing action of these stresses.

Stimulation of PRKA in some systems has been shown to increase MAPK1/3 (formerly Erk 2/1) activity [36–38]. However, we have shown in a previous study that AICAR stimulation of PRKA and meiotic resumption in arrested mouse DO was not accompanied by activation of MAPK1/3 before GVB [39]. Moreover, PD98059 and UO126, inhibitors of MAPK1/3 activation, blocked AICAR-induced oocyte maturation, suggesting an effect of the inhibitors unrelated to MAPK1/3 activity [39]. When tested on stress-treated oocytes, PD98059 completely blocked meiotic induction by all three stresses, and, consistent with earlier results, MAPK1/3 was not stimulated before GVB (unpublished data). Because MAPK1/3 apparently does not participate in the stress-activated pathway(s) leading to meiotic resumption, these results provide further evidence that PD98059 effects are not entirely specific for MAPK1/3. In this light, it is important to point out that Dokladda et al. [40] have recently shown that PD98059 stimulates PRKA activity in HEK293 cells by a MAPK1/3-independent mechanism.

Mouse oocytes use pyruvate as a principle energy source for meiosis [41], a function apparently mediated by ATP generation via oxidative phosphorylation [42]. The process of meiotic maturation, from GVB to polar-body extrusion and arrest at metaphase II, is accompanied by an increased pyruvate consumption [43], presumably due to increased demands for ATP. The metabolic poison, arsenite, deprives cells of ATP and leads to serious physiological repercussions. For example, in mouse oocytes and embryos, arsenite treatment causes chromosome abnormalities, increased free radicals, altered DNA repair and methylation patterns, gene amplification, altered cell division, and inhibition of p53 and telomerase [44, 45]. Bovine embryos treated with arsenite responded with increased caspase activity, apoptosis, and compromised development to blastocyst [46]. Treatment of oocytes from a number of different species with metabolic poisons has been shown to prevent GVB [42, 47, 48]. However, in the present study, exposure of mouse oocytes to arsenite stimulated meiotic resumption. We propose that this is due to a drop in the AMP/ATP ratio and subsequent activation of PRKA, with sufficient ATP present to support meiotic resumption. Further support that arsenite was having the desired metabolic effect was indicated by a decrease in polar-body formation, consistent with the need for ATP in maintaining the meiotic spindle [45] and completion of meiosis [43].

Oxidative stress has been shown to have detrimental effects on germ cells, including loss of meiotic spindle integrity and errors in chromosome segregation [49–51], increased apoptosis [52], and low fertilization and developmental rates [53]. The presence of antioxidants or lowered oxygen levels during oocyte growth and maturation improves developmental capacity [54–60] and prevents the negative effects of gamete aging [50, 61]. Also, elevated levels of reactive oxygen species (ROS) are associated with developmental arrest and apoptosis in preimplantation embryos [62–64], and scavenging of oxygen radicals can reverse embryonic blocks observed during in vitro culture [65–67]. Oocyte maturation is also affected by ROS, although evidence indicates a potential positive, meiosis-promoting effect. Studies in rats have demonstrated an inhibitory action of antioxidants on spontaneous maturation [68, 69]. Moreover, intrafollicular levels of ascorbic acid, a natural antioxidant, are rapidly depleted following preovulatory gonadotropin stimulation [70], and these changes have been implicated in meiotic resumption. In the present study, subjecting oocytes to oxidative stress had a stimulatory effect on meiosis, and high levels of the oxidants were detrimental to oocyte viability and maturation. Thus, a window of stimulation apparently exists whereby lower levels of ROS generation turn on PRKA and induce meiosis reinitiation, while higher levels overwhelm the oocyte, leading to its demise.

Exposure of cells to high solute concentrations is usually harmful to viability, and cells stressed in this way often accumulate organic osmolytes to protect against these conditions [71]. Such molecules help to maintain cell volume by stabilizing ionic strength within the cell. Subjecting mouse embryos to elevated sodium perturbs normal transcription and translation [72, 73], and osmotic stress in human oocytes results in increased spindle abnormalities [74]. The importance of osmoregulation during oocyte maturation or preimplantation embryo development is well documented, with many studies showing the benefits of reducing inorganic ion concentrations or adding osmolytes, such as taurine, hypotaurine, or betaine to the culture medium [72, 75–80]. We have previously shown that oocytes from diabetic mice exhibit abnormal meiotic regulation [81, 82] that may be due to metabolic transit of glucose through the sorbitol pathway under hyperglycemic conditions [83]. Nevertheless, while long-term exposure to elevated sorbitol is detrimental to the oocyte, in the present study, short-term pulsing with sorbitol stimulated PRKA-mediated meiotic resumption in dbcAMP-arrested oocytes. Interestingly, Yamauchi et al. [84] showed that meiotic induction of porcine oocytes was negatively affected by hyperosmotic conditions but that hypoosmotic conditions accelerated GVB. Thus, osmotic stress dramatically influences oocyte maturation, with a brief stress capable of turning on the meiosis-inducing pathway in a manner that does not compromise the health of the oocyte.

The dbcAMP-arrested DO treated with AICAR exhibit a strong phospho-ACACA immunoblot band within 1 h of culture compared with controls and undergo rapid maturation kinetics, with complete meiotic induction achieved within 4 h [11]. In contrast, the kinetics of maturation in stress-treated oocytes are considerably attenuated, and immunoblots showed less intense ACACA-P bands when compared with the AICAR-treated positive control group. These data suggest that the extent and kinetics of maturation induction are directly related to the level of PRKA activation reached in the oocyte. Moreover, ACACA-P bands were not readily seen in stress-treated oocytes if sampling was carried out after longer culture periods, closer to the time of GVB (data not shown). These observations suggested that a transient pulse of PRKA activity may suffice to exceed the threshold required for meiotic induction.

To test this idea, dbcAMP-arrested oocytes were pulsed with AICAR for varying periods of time and returned to AICAR-free medium for either an overnight (17–18 h) or short-term (4 h) culture period. In the overnight culture, 20- and 40-min pulses resulted in levels of GVB that were not significantly different from controls. However, a 60-min pulse of 250 µM AICAR resulted in a maturation percentage similar to that seen in the continuous exposure group. Yet when oocytes were pulsed with AICAR for 60 min and assessed for maturation 4 h later, there was no significant increase in maturation compared with controls, whereas maturation had already increased by 68% in oocytes continuously exposed to AICAR. Thus, longer culture times were required to obtain high levels of meiotic induction after AICAR pulsing. These data indicate that short pulses of PRKA activity can stimulate GVB, but that such limited activation leads to attenuated maturation kinetics. Consequently, we propose that the three stresses tested in this study stimulate oocyte maturation by activation of PRKA, but that their relative effectiveness is directly related to the intensity and duration of PRKA activity. The lack of increased polar-body formation in menadione- and sorbitol-treated oocytes is likely due to the limited period of time available to complete maturation after the delayed meiotic resumption.

To summarize, these data implicate PRKA in stress-induced maturation and provide further evidence that PRKA within the mouse oocyte can serve as a potent inducer of meiotic maturation. It is therefore prudent to consider the potential meiotic repercussions when exposing oocytes to stressful conditions.

FOOTNOTES

1 Correspondence. FAX: 414 288 7357; Stephen.Downs{at}marquette.edu Back

Received: 10 August 2005.

First decision: 12 September 2005.

Accepted: 2 November 2005.

REFERENCES

  1. Dekel N, Cellular, biochemical and molecular mechanisms regulation oocyte maturation. Mol Cell Endo 2005 234:19-25[CrossRef][Medline]
  2. Masciarelli S, Horner K, Liu C, Park S, Hinckley M, Hockman S, Nedachi T, Jin C, Conti M, Manganiello V, Cyclic nucleotide phosphodiesterase 3A-deficient mice as a model of female infertility. J Clin Invest 2004 114:196-205[CrossRef][Medline]
  3. Thomas R, Armstrong D, Gilchrist R, Bovine cumulus cell-oocyte gap junctional communication during in vitro maturation in response to manipulation of cell-specific adenosine 3',5'-monophosphate levels. Biol Reprod 2004 70:548-556[Abstract/Free Full Text]
  4. Downs S, Daniel S, Eppig J, Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stimulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin. J Exp Zool 1988 245:86-96[CrossRef][Medline]
  5. Byskov A, Anderson C, Nordholm L, Thogersen H, Xia G, Wassmann O, Anderson J, Guddal E, Roed T, Chemical structure of sterols that activate oocyte meiosis. Nature 1995 374:559-562[CrossRef][Medline]
  6. Conti M, Anderson C, Richard F, Mehats C, Chun S, Horner K, Jin C, Tsafriri A, Role of cyclic nucleotide signaling in oocyte maturation. Mol Cell Endocrinol 2002 187:153-159[CrossRef][Medline]
  7. Bilodeau-Goeseels S, Effects of phosphodiesterase inhibitors on spontaneous nuclear maturation and cAMP concentrations in bovine oocytes. Theriogenology 2003 60:1679-1690[CrossRef][Medline]
  8. Hardie D, Hawley S, AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays 2001 23:1112-1119[CrossRef][Medline]
  9. Hardie D, Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 2003 144:5179-5183[Abstract/Free Full Text]
  10. Carling D, The AMP-activated protein kinase cascade—a unifying system for energy control. Trends Biochem Sci 2004 29:18-24[CrossRef][Medline]
  11. Downs SM, Hudson ER, Hardie DG, A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev Biol 2002 245:200-212[CrossRef][Medline]
  12. Fryer L, Parbu-Patel A, Carling D, The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem 2002 277:25226-25232[Abstract/Free Full Text]
  13. Toyoda T, Hayashi T, Miyamoto L, Yonemitsu S, Nkano M, Tanaka S, Ebihara K, Masuzaki H, Hosoda K, Inoue G, Otaka A, Sato K, et al Possible involvement of the alpha 1 isoform of 5'AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle. Am J Physiol Endocrinol Metab 2004 287:E166-173[Abstract/Free Full Text]
  14. Hardie G, Roles of the AMP-activated/SNF1 protein kinase family in the response to cellular stress. Biochem Soc Symp 1999 64:13-27[Medline]
  15. Choi S, Kim S, Lee K, Kim J, Mu J, Birnbaum M, Soo Kim S, Ha J, The regulation of AMP-activated protein kinase by H2O2. Biochem Biophys Res Commun 2001 287:92-97[CrossRef][Medline]
  16. Zou M, Hou X, Shi C, Kirkpatrick S, Liu F, Goldman M, Cohen R, Activation of 5'-AMP-activated kinase is mediated through c-src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. J Biol Chem 2003 278:34003-34010[Abstract/Free Full Text]
  17. Marsin A, Bouzin C, Bertrand L, Hue L, The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase. J Biol Chem 2002 277:30778-30783[Abstract/Free Full Text]
  18. Marsin A, Bertrand L, Rider M, Deprez J, Beauloye C, Vincent M, Van der Berghe G, Carling D, Hue L, Phosphorylation and activation of heat PFK-2 by AMPK has a role in the stimulation of glycolysis during ischemia. Curr Biol 2000 10:1247-1255[CrossRef][Medline]
  19. Barnes K, Ingram J, Porras O, Barros F, Hudson E, Fryer L, Foufelle F, Carling D, Hardie D, Baldwin S, Activation of GLUT1 by metabolic and osmotic stress: potential involvement of AMP-activated protein kinase (AMPK). J Cell Sci 2002 115:2433-2442[Abstract/Free Full Text]
  20. Mao X, Bravo I, Cheng H, Alonso A, Multiple independent kinase cascades are targeted by hyperosmotic stress but only one activates stress kinase p38. Exp Cell Res 2004 292:304-311[CrossRef][Medline]
  21. Voellmy R, Transduction of the stress signal and mechanisms of transcriptional regulation of heat shock/stress protein gene expression in higher eukaryotes. Crit Rev Eukaryot Gene Expr 1994 4:357-401[Medline]
  22. Fryer L, Foufelle F, Barnes K, Baldwin S, Woods A, Carling D, Characterization of the role of the AMP-activated protein kinase in the stimulation of glucose transport in skeletal muscle cells. Biochem J 2002 363: (pt 1) 167-174[CrossRef][Medline]
  23. Downs S, Mastropolo A, Culture conditions affect meiotic regulation in cumulus cell-enclosed mouse oocytes. Mol Reprod Dev 1997 46:551-566[CrossRef][Medline]
  24. Carling D, Fryer L, Woods A, Daniel T, Jarvie S, Whitrow H, Bypassing the glucose/fatty acid cycle: AMP-activated protein kinase. Biochem Soc Trans 2003 31:1157-1160[Medline]
  25. Corton J, Gillespie J, Hardie D, Role of the AMP-activated protein kinase in the cellular stress response. Curr Biol 1994 4:315-324[CrossRef][Medline]
  26. Hayashi T, Hirshman M, Fujii N, Habinowski S, Witters L, Goodyear L, Metabolic stress and altered glucose transport. Diabetes 2000 49:527-531[Abstract]
  27. Fryer L, Hajduch E, Rencurel F, Salt I, Hundal H, Hardie D, Carling D, Activation of glucose transport by AMP-activated protein kinase via stimulation of nitric oxide synthase. Diabetes 2000 49:1978-1985[Abstract/Free Full Text]
  28. Patel N, Khayat Z, Ruderman N, Klip A, Dissociation of 5'AMP-activated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2+ and protein kinase C inhibition. Biochem Biophys Res Commun 2001 285:1066-1070[CrossRef][Medline]
  29. Pelleteir A, Joly E, Prentki M, Coderre L, Adenosine 5'-monophosphate-activated protein kinase and p38 mitogen-activated protein kinase participate in the stimulation of glucose uptake by dinitrophenol in adult cardiomyocytes. Endocrinology 2005 146:2285-2294[Abstract/Free Full Text]
  30. Jin Q, Jhun B, Lee S, Pi Y, Cho Y, Baik H, Kang I, Differential regulation of phosphatidylinositol 3-kinase/Akt, mitogen-activated protein kinase, and AMP-activated protein kinase pathways during menadione-induced oxidative stress in the kidney of young and old rats. Biochem Biophys Res Commun 2004 315:555-561[CrossRef][Medline]
  31. Walker J, Jion H, Churchill T, Kulka M, Madsen K, Activation of AMP-activated protein kinase reduces cAMP-mediated epithelial chloride secretion. Am J Gastrointest Liver Physiol 2003 285:G850-G860[Abstract/Free Full Text]
  32. Jung J, Lee J, Ha J, Kim S, Cho Y, Baik H, Kang I, 5-Aminoimidazole-4-carboxamide-ribonucleoside enhances oxidative stress-induced apoptosis through activation of nuclear factor-kappaB in mouse neuro 2a neuroblastoma cells. Neurosci Lett 2004 354:197-200[CrossRef][Medline]
  33. Hardie D, Pan D, Regulation of fatty acid synthesis and oxidation by the AMP-activated protein kinase. Biochem Soc Trans 2002 30:1064-1070[CrossRef][Medline]
  34. Zhou G, Myers R, Li Y, Chen Y, Shen X, Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman M, et al Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 2001 108:1167-1174[CrossRef][Medline]
  35. Henin N, Vincent M, Van den Burghe G, Stimulation of rat liver AMP-activated protein kinase by AMP analogues. Biochim Biophys Acta 1996 1290:197-203[Medline]
  36. Kim J, Yoon M, Choi S, Kang I, Kim S, Kim Y, Choi Y, Ha J, Effects of stimulation of AMP-activated protein kinase on insulin-like growth factor 1- and epidermal growth factor-dependent extracellular signal-regulated kinase pathway. J Biol Chem 2001 276:19102-19110[Abstract/Free Full Text]
  37. Turcotte L, Raney M, Todd M, ERK1/2 inhibition prevents contraction-induced increase in plasma membrane FAT/CD36 content and FA uptake in rodent muscle. Acta Physiol Scand 2005 184:131-139[CrossRef][Medline]
  38. Chen H, Bandyopadhyay G, Sajan M, Kanoh Y, Standaert M, Farese R, Farse R, Jr, Activation of the ERK pathway and atypical protein kinase C isoforms in exercise- and aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR)-stimulated glucose transport. J Biol Chem 2002 277:23554-23562[Abstract/Free Full Text]
  39. LaRosa C, Downs S, MEK inhibitors block AICAR-induced maturation in mouse oocytes by a MAPK-independent mechanism. Mol Reprod Dev 2005 70:235-245[CrossRef][Medline]
  40. Dokladda K, Green K, Pan D, Hardie DG, PD98059 and UO126 activate AMP-activated protein kinase by increasing the cellular AMP:ATP ratio and not via inhibition of the MAP kinase pathway. FEBS Lett 2005 579:236-240[CrossRef][Medline]
  41. Biggers D, Whittingham D, Donahue R, The pattern of energy metabolism in the mouse oocyte and zygote. Proc Natl Acad Sci U S A 1967 58:560-567[Free Full Text]
  42. Zeilmaker G, Hulsmann W, Wensinck F, Verhamme C, Oxygen-triggered mouse oocyte maturation in vitro and lactate utilization by mouse oocytes and zygotes. J Reprod Fertil 1972 29:151-152[Medline]
  43. Downs S, Humpherson P, Leese H, Pyruvate utilization by mouse oocytes is influenced by meiotic status and the cumulus oorphus. Mol Reprod Dev 2002 62:113-123[CrossRef][Medline]
  44. Liu L, Trimarchi J, Navarro P, Blasco M, Keefe D, Oxidative stress contributes to arsenic-induced telomere attrition, chromosome instability, and apoptosis. J Biol Chem 2003 278:31998-32004[Abstract/Free Full Text]
  45. Navarro P, Liu L, Keefe D, In vivo effects of arsenite on meiosis, preimplantation development, and apoptosis in the mouse. Biol Reprod 2004 70:980-985[Abstract/Free Full Text]
  46. Krininger C, Stephens S, Hansen P, Developmental changes in inhibitory effects of arsenic and heat shock on growth of pre-implantation bovine embryos. Mol Reprod Dev 2002 63:335-340[CrossRef][Medline]
  47. Brachet J, Schutter P, Hubert E, Studies on maturation in Xenopus leavis oocytes. III. Energy production and requirements for protein synthesis. Differentiation 1975 3:3-14[CrossRef][Medline]
  48. Hull S, Schuetz A, Toole B, In vitro inhibition of oocyte and follicular maturation and spawning in starfish (Asterias forbesi) by 2,4-dinitrophenol. Differentiation 1979 15:127-132[CrossRef][Medline]
  49. Tarin J, Vendrell F, Ten J, Blanes R, van Blerkom J, Cano A, The oxidizing agent tertiary butyl hydroperoxide induces disturbances in spindle organization, c-meiosis, and aneuploidy in mouse oocytes. Mol Hum Reprod 1996 2:895-901[Abstract/Free Full Text]
  50. Tarin J, Vendrell F, Ten J, Cano A, Antioxidant therapy counteracts the disturbing effects of diamide and maternal aging on meiotic division and chromosomal segregation in mouse oocytes. Mol Hum Reprod 1998 4:281-288[Abstract/Free Full Text]
  51. Zuelke K, Jones D, Perreault S, Glutathione oxidation is associated with altered microtubule function and disrupted fertilization in mature hamster oocytes. Biol Reprod 1997 57:1413-1419[Abstract]
  52. Tatemoto H, Sakurai N, Muto N, Protection of porcine oocytes against apoptotic cell death caused by oxidative stress during in vitro maturation: role of the cumulus cells. Biol Reprod 2000 63:805-810[Abstract/Free Full Text]
  53. Takahashi T, Takahashi E, Igarashi H, Tezuka N, Kurachi H, Impact of oxidative stress in aged mouse oocytes on calcium oscillations at fertilization. Mol Reprod Dev 2003 66:143-152[CrossRef][Medline]
  54. Hashimoto S, Minami N, Takakura R, Yamada M, Imai H, Kashima N, Low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine cumulus-oocyte complexes. Mol Reprod Dev 2000 57:353-360[CrossRef][Medline]
  55. Eppig J, Hosoe M, O'Brian M, Pendola F, Requena A, Watanabe S, Conditions that affect acquisition of developmental competence by mouse oocytes in vitro: FSH, insulin, glucose and ascorbic acid. Mol Cell Endocrinol 2000 163:109-116[CrossRef][Medline]
  56. Tatemoto H, Ootaki K, Shigeta K, Muto N, Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatments with ascorbic acid 2-O-alpha-glucoside during in vitro maturation. Biol Reprod 2001 65:1800-1806[Abstract/Free Full Text]
  57. Luvoni G, Keskintepe L, Brackett B, Improvement in bovine embryo production in vitro by glutathione-containing culture media. Mol Reprod Dev 1996 43:437-443[CrossRef][Medline]
  58. Da Motos D, Gasparrini B, Pasqualini S, Thomson J, Effect of glutathione synthesis stimulation during in vitro maturation of bovine oocytes on embryos development and intracellular peroxide content. Theriogenology 2002 57:1443-1451[CrossRef][Medline]
  59. Sikka S, Rajasekaran L, Hellstrom W, Role of oxidative stress and antioxidants in male infertility. J Androl 1995 16:464-468[Abstract/Free Full Text]
  60. Miesel R, Drzejczak P, Kurpisz M, Oxidative stress during the interaction of gametes. Biol Reprod 1993 49:918-923[Abstract]
  61. Hamatani T, Flaco G, Carter M, Akutsu H, Stagg C, Sharvo A, Dudekula D, VanBuren V, Ko M, Age-associated alteration of gene expression patterns in mouse oocytes. Hum Mol Genetics 2004 13:2263-2278[Abstract/Free Full Text]
  62. Nasr-Esfahani M, Johnson M, The origin of reactive oxygen species in mouse embryos cultured in vitro. Development 1991 113:551-560[Abstract]
  63. Kwon H, Yang H, Hwang K, Effects of low oxygen condition on the generation of reactive oxygen species and the development in mouse embryos cultured in vitro. Obstet Gynecol Res 1999 25:359-366
  64. Yang H, Hwang K, Kwon H, Kim H, Choi K, Oh K, Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod 1998 13:998-1002[Abstract/Free Full Text]
  65. Noda Y, Embryo development in vitro. Assist Reprod Dev 1992 2:9-15
  66. Neuer A, Spandorfer S, Giraldo P, Dieterle S, Rosenwaks Z, Witkin S, The role of heat shock proteins in reproduction. Hum Reprod Update 2000 6:149-159[Abstract/Free Full Text]
  67. Johnson M, Nasr-Esfahani M, Radical solutions and cultural problems; could free oxygen radicals be responsible for the impaired development of preimplantation embryos in vitro?. BioEssays 1994 16:31-38[CrossRef][Medline]
  68. Takami M, Preston S, Toyloy V, Behrman H, Antioxidants reversibility inhibit the spontaneous resumption of meiosis. Am J Physiol Endocrinol Metab 1999 276:E684-688[Abstract/Free Full Text]
  69. Takami M, Preston S, Behrman H, Eicosatetraynoic and eicosatetriynoic acids, lipoxygenase inhibitors, block meiosis via antioxidant action. Am J Physiol Cell Physiol 2000 278:C646-650[Abstract/Free Full Text]
  70. Guarnaccia M, Takami M, Jones E, Preston S, Behrman H, Luteinizing hormone depletes ascorbic acid in preovulatory follicles. Fertil Steril 2000 74:959-963[CrossRef][Medline]
  71. Yancey P, Clark M, Hand S, Bowlus R, Somero G, Living with water stress: evolution of osmolyte systems. Science 1982 217:1214-1222[Abstract/Free Full Text]
  72. Anbari K, Schultz R, Effect of sodium and betaine in culture media on development and relative rates of protein synthesis in preimplantation mouse embryos in vitro. Mol Reprod Dev 1993 35:24-28[CrossRef][Medline]
  73. Ho Y, Doherty A, Schultz R, Mouse preimplantation embryo development in vitro: effect of sodium concentration and culture media on RNA synthesis and accumulation and gene expression. Mol Reprod Dev 1994 38:131-141[CrossRef][Medline]
  74. Mullen S, Agca Y, Broermann D, Jenkins C, Johnson C, Critser J, The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation. Hum Reprod 2004 19:1148-1154[Abstract/Free Full Text]
  75. Van Winkle L, Haghighat N, Campione A, Glycine protects preimplantation mouse conceptuses form detrimental effect on development on the inorganic ions in oviductal fluid. J Exp Zool 1990 253:215-219[CrossRef][Medline]
  76. Barnett D, Bavister B, Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts and production of term offspring from in vitro-fertilized ova. Biol Reprod 1992 47:297-304[Abstract]
  77. Takahashi Y, Kanagawa H, Effects of glutamine, glycine, and turine on the development of in vitro fertilized bovine zygotes in a chemically defined medium. J Vet Med Sci 1998 60:433-437[CrossRef][Medline]
  78. Dumoulin J, Evers J, Bras M, Piepers M, Geraedts J, Positive effect of taurine on preimplantation development of mouse embryos in vitro. J Reprod Fertil 1992 4:373-380
  79. Biggers J, Lawitts J, Lechene C, The protective action of betaine on the deleterious effects of NaCl on preimplantation mouse embryos in vitro. Mol Reprod Dev 1993 34:380-390[CrossRef][Medline]
  80. Funahashi H, Kim H, Stumpf T, Cantley T, Day B, Presence of organic osmolytes in maturation medium enhances cytoplasmic maturation of porcine oocytes. Biol Reprod 1996 54:1412-1419[Abstract]
  81. Colton S, Pieper G, Downs S, Altered meiotic regulation in oocytes from diabetic mice. Biol Reprod 2002 67:220-231[Abstract/Free Full Text]
  82. Colton S, Humpherson P, Leese H, Downs S, Physiological changes in oocyte-cumulus cell complexes from diabetic mice that potentially influence meiotic regulation. Biol Reprod 2003 69:761-770[Abstract/Free Full Text]
  83. Colton S, Downs S, Potential role for the sorbitol pathway in the meiotic dysfunction exhibited by oocytes from diabetic mice. J Exp Zool 2004 301: A 439-448[CrossRef]
  84. Yamauchi N, Sasada H, Soloy E, Dominko T, Kikuchi K, Nagai T, Effects of hormones and osmolarity in the culture medium on germinal vesicle breakdown of porcine oocytes. Theriogenology 1999 52:153-162[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Reproductive SciencesHome page
Wenjing Zhong, Yufen Xie, Yingchun Wang, J. Lewis, A. Trostinskaia, Fangfei Wang, E. E. Puscheck, and D. A. Rappolee
Use of Hyperosmolar Stress to Measure Stress-Activated Protein Kinase Activation and Function in Human HTR Cells and Mouse Trophoblast Stem Cells
Reproductive Sciences, September 1, 2007; 14(6): 534 - 547.
[Abstract] [PDF]

Home page
 Biol. Reprod.Home page
L. Tosca, S. Uzbekova, C. Chabrolle, and J. Dupont
Possible Role of 5'AMP-Activated Protein Kinase in the Metformin-Mediated Arrest of Bovine Oocytes at the Germinal Vesicle Stage During In Vitro Maturation
Biol Reprod, September 1, 2007; 77(3): 452 - 465.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
Y. Xie, W. Zhong, Y. Wang, A. Trostinskaia, F. Wang, E.E. Puscheck, and D.A. Rappolee
Using hyperosmolar stress to measure biologic and stress-activated protein kinase responses in preimplantation embryos
Mol. Hum. Reprod., July 1, 2007; 13(7): 473 - 481.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
C. LaRosa and S. M. Downs
Meiotic Induction by Heat Stress in Mouse Oocytes: Involvement of AMP-Activated Protein Kinase and MAPK Family Members
Biol Reprod, March 1, 2007; 76(3): 476 - 486.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
L. Tosca, C. Chabrolle, S. Uzbekova, and J. Dupont
Effects of Metformin on Bovine Granulosa Cells Steroidogenesis: Possible Involvement of Adenosine 5' Monophosphate-Activated Protein Kinase (AMPK)
Biol Reprod, March 1, 2007; 76(3): 368 - 378.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
L. Tosca, S. Crochet, P. Ferre, F. Foufelle, S. Tesseraud, and J. Dupont
AMP-activated protein kinase activation modulates progesterone secretion in granulosa cells from hen preovulatory follicles.
J. Endocrinol., July 1, 2006; 190(1): 85 - 97.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
74/3/585    most recent
biolreprod.105.046524v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by LaRosa, C.
Right arrow Articles by Downs, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by LaRosa, C.
Right arrow Articles by Downs, S. M.
Agricola
Right arrow Articles by LaRosa, C.
Right arrow Articles by Downs, S. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS