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Role of AMP-activated protein kinase (AMPK) during postovulatory aging of mouse oocytes1

Running title: AMPK facilitates oocyte aging

Summary sentence: AMPK facilitated oocyte aging through inhibiting MPF activities, and postovulatory oocyte aging activated AMPK with decreased cAMP by activating CaMKs via increasing ROS and cytoplasmic calcium.

Guang-Yi Sun2, Shuai Gong2, Qiao-Qiao Kong, Zhi-Bin Li, Jia Wang, Ming-Tao Xu, Ming-Jiu Luo3 and Jing-He Tan3

Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai’an City 271018, P. R. China Grant support: This study was supported by grants from the China National Natural Science Foundation (Nos. 31772599 and 31702114), the National Key R&D Program of China (Nos. 2017YFD0501904, 2017YFC1001602 and 2017YFC1001601), the Natural Science Foundation of Shandong Province (No. ZR2017BC025), and the Funds of Shandong Double Tops Program (No. SYL2017YSTD12).

2. Downloaded from https://academic.oup.com/biolreprod/advance-article-abstract/doi/10.1093/biolre/ioaa081/5843372 by Beurlingbiblioteket user on 27 May 2020
1. Guang-Yi Sun and Shuai Gong contributed equally to this work.
3. Correspondence: Jing-He Tan or Ming-Jiu Luo, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, Shandong Province, P R China, Post code: 271018, Phone: 0538-8249616, FAX: 0538-8241419, Email: [email protected] or [email protected]

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Abstract
Studies suggested that postovulatory oocyte aging might be prevented by maintaining a high maturation-promoting factor (MPF) activity. Whether AMP-activated protein kinase (AMPK) plays any role in postovulatory oocyte aging is unknown. Furthermore, while activation of AMPK stimulates meiotic resumption in mouse oocytes, it inhibits meiotic resumption in pig and bovine oocytes. Thus, the species difference in AMPK regulation of oocyte MPF activities is worth in-depth studies. This study showed that AMPK activation with metformin or AICAR and inactivation with compound C significantly increased and decreased, respectively, the activation susceptibility (AS) and other aging parameters in aging mouse oocytes. While AMPK activity increased, MPF activity and cAMP decreased significantly with time post ovulation. In vitro activation and inactivation of AMPK significantly decreased and increased the MPF activity, respectively. MPF upregulation with MG132 or downregulation with roscovitine completely abolished the effects of AMPK activation or inactivation on AS of aging oocytes, respectively. AMPK facilitated oocyte aging with increased reactive oxygen species (ROS) and cytoplasmic calcium. Furthermore, treatment with Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitors significantly decreased AS and AMPK activation. Taken together, the results suggested that AMPK facilitated oocyte aging through inhibiting MPF activities, and postovulatory oocyte aging activated AMPK with decreased cAMP by activating CaMKs via increasing ROS and cytoplasmic calcium.

Key words: aging; AMP-activated protein kinase; maturation-promoting factor; metformin; oocytes

Introduction

A time-dependent process of aging occurs when mammalian oocytes are not fertilized or activated in time following ovulation or in vitro maturation [1-3]. It has been found that this postovulatory oocyte aging process can impair embryo development [4-6] and cause abnormalities in offspring [7,8]. Furthermore, a statistically significant increase in the risk of early pregnancy loss was Downloaded from https://academic.oup.com/biolreprod/advance-article-abstract/doi/10.1093/biolre/ioaa081/5843372 by Beurlingbiblioteket user on 27 May 2020 observed with increased likelihood of postovulatory oocyte aging in human beings [9]. Thus, preventing postovulatory aging of the oocyte is of great importance for both natural and assisted reproduction. However, the mechanisms that underlie the postovulatory oocyte aging are currently largely unclear.

One of the earliest manifestations in the postovulatory aging oocytes is an increase in their activation susceptibility (AS) [10,11] due to a significant decline in the maturation-promoting factor (MPF) activity [3,12]. Zhu et al. [13] observed that advanced aging manifestations such as caspase-3 activation and cytoplasmic fragmentation were successfully prevented in postovulatory aging mouse oocytes when a high MPF activity was maintained by MG132 treatment. Thus, the postovulatory oocyte aging may be prevented by maintenance of a high MPF activity. Cyclic adenosine monophosphate (cAMP) is an important regulator of MPF activity in mammalian oocytes. For example, a decrease in cAMP, which is brought about by the action of cAMP phosphodiesterase (PDE), can activate MPF and initiate germinal vesicle (GV) breakdown through inactivation of PKA. Orthologues of adenosine monophosphate (AMP)-activated protein kinase (AMPK) are observed in essentially all eukaryotes. AMPK can regulate diverse metabolic and physiological processes and its dysregulation is associated with major chronic diseases [14]. Because the product of PDE activity, 5’-AMP, is a potent activator of AMPK [15], the role of AMPK activity in regulating the MPF activity of oocytes is worth exploring to understand the mechanisms for both oocyte maturation and aging. Furthermore, reports that both MPF and cAMP levels decreased in postovulatory aging oocytes [16] are contradictory with the notion that the cAMP-dependent activation of PKA inactivates MPF through activation of Wee1 and inactivation of Cdc25 [17]. Studies are required to solve this contradiction.

In mouse oocytes, activation of AMPK with 5-aminoimidazole- 4-carboxamide- 1-beta-d- ribofuranoside (AICAR) [18] or microinjection of constitutively active AMPK [19] induced resumption of meiosis in both cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) when meiotic arrest was maintained by cAMP analogs. In contrast, meiotic resumption in pig COCs was inhibited by the presence of AMPK activators, AICAR or metformin [20].
Bilodeau-Goeseels et al. [21] observed that metformin and AICAR prevented meiotic resumption in both bovine COCs and DOs. However, Mayes et al. [20] observed that AICAR or metformin could not block meiotic resumption in pig DOs, and Tosca et al. [22] found that metformin arrested bovine oocytes at the GV stage in COCs but not in DOs. In short, the above data suggest that while AMPK activation activates MPF and stimulates meiotic resumption in mouse oocytes, it inactivates MPF and inhibits meiotic resumption in pig and bovine oocytes. Whether cumulus cells are essential for AMPK to regulate MPF activity of oocytes remains in question. Thus, the species difference in AMPK regulation of oocyte MPF activities is worth in-depth studies, and particularly, the mechanisms by which AMPK regulates MPF activities in mouse oocytes must be studied using a system that involves neither cumulus cells nor any drug use to inhibit meiotic resumption. The results of such studies will definitely help to integrate the current conflicting results and contribute greatly to our understanding of the mechanisms for oocyte aging as well as maturation, as the dichotomy of this pathway between species for oocyte maturation is likely to indicate a similar difference in AMPK activity between species in oocyte aging. Furthermore, the results of such researches will be beneficial also to the improvement of the human IVF/ART technique, because in human, AMPK agonists may contribute to miscarriage or hinder IVF/ART when applied in vitro [23].

The aim of this study was to determine the role of the AMPK activity in postovulatory aging of mouse oocytes. To this end, effects of AMPK activation on early and advanced manifestations of oocyte aging were first observed. The relationship between AMPK and MPF activities were then studied using in vivo aging oocytes and the in vitro aging DOs involving neither cumulus cells nor the use of any drug to prevent MPF activation. Finally, the signaling events leading to AMPK activation were analyzed during aging of mouse oocytes. The results suggested that AMPK facilitated oocyte aging through inhibiting MPF activities, and postovulatory oocyte aging activated AMPK with decreased cAMP by activating CaMKs via increasing reactive oxygen species (ROS) and cytoplasmic calcium.

Materials and methods
Unless otherwise specified, all the chemicals and reagents used in this study were bought from the
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Sigma-Aldrich company.

Mice and oocyte recovery
Mice of the Kunming strain were kept in rooms with 14-h light and 10-h dark cycles (the dark began from 8 pm). All the procedures for animal care and handling were approved by the Animal Care and Use Committee of the Shandong Agricultural University P. R. China (Permit number: SDAUA-2001-001). Female mice were superovulated at the age of 8–10 weeks by intra-peritoneally injecting 10 IU equine chorionic gonadotropin (eCG) and 10 IU human chorionic gonadotropin (hCG) at 48 h intervals. Both eCG and hCG were purchased from Ningbo Hormone Product company limited, China. To recover oocytes for in vitro aging, the superovulated mice were sacrificed at 13 h after hCG administration, whereas they were sacrificed at 13, 18 or 24 h post hCG injection to recover in vivo aged oocytes. Oocytes were collected by breaking the oviductal ampullae. In this study, a total of 445 female mice were used in the in vitro experiments while a total of 123 female mice were used for in vivo aging experiments. After being washed three times in M2 medium, the oocytes were denuded of cumulus cells by pipetting in M2 containing 0.1% hyaluronidase and the DOs obtained were used for experiments.

Preparation of medium conditioned by cumulus cells at high oxygen tension (OCM)
Our previous studies demonstrated that cumulus cells secreted more FasL and TNF-alpha when cultured at high oxygen tension, and the OCM produced as such significantly facilitated oocyte aging [6,24]. Thus, this study adopted OCM to activate AMPK in experiments where AMPK was inhibited to demonstrate its role in oocyte aging. The OCM medium was prepared as follows. The cumulus cells that were released during preparation of DOs were harvested and cultured at 5–8×105 cells/ml for 24 h in regular Chatot–Ziomek–Bavister (CZB) medium (NaCl, 81.62 mM; KCl, 4.83 mM; KH2PO4, 1.18 mM; MgSO4, 1.18 mM; NaHCO3, 25.12 mM; CaCl2·2H2O, 1.7 mM; sodium lactate, 31.3 mM; sodium pyruvate, 0.27 mM; EDTA, 0.11 mM; glutamine, 1 mM; bovine serum albumin, 5 g/l; penicillin, 0.06 g/l; streptomycin, 0.05 g/l) containing 200 µM H2O2. The high oxygen treated cumulus cells were then incubated in the regular CZB medium for 24 h. At the end of the incubation, the conditioned medium was collected and centrifuged at 3000×g for 5 min to remove cells and debris. The OCM thus produced was frozen-stored at −80°C until use.

In vitro aging treatment of oocytes

The DOs were cultured for aging in CZB or OCM containing various concentrations of metformin, 5-aminoimidazole- 4-carboxamide- riboside- 5-phosphate (AICAR), compound C, MG132 or roscovitine. To prepare stock solutions, metformin (1.5 M) was dissolved in water; AICAR (10 mM) was dissolved in CZB; and compound C (10 mM), MG132 (1 mM), roscovitine (50 mM), STO-609 (5000 µg/ml) and KN-93 (1000 µM) were dissolved in DMSO. All the stock solutions were stored in aliquots at −20°C except for metformin, STO-609 and KN-93, which were stored at 4°C. The stock solutions were diluted to desired concentrations with corresponding aging media immediately prior to use. The aging culture was carried out in wells of a 96-well culture plate (about 30 oocytes per well containing 100 µl of medium) at 37°C under 5% CO2 in humidified air.

Activation treatment of oocytes

In this study, the ethanol plus 6-dimethylaminopurine (6-DMAP) activation protocol was adopted to assess oocyte activation susceptibility (AS) as described previously [11]. During the ethanol + 6-DMAP treatment, oocytes were first treated with 5% ethanol in M2 medium for 5 min at room temperature, and then cultured in 2 mM 6-DMAP in CZB medium for 6 h at 37.5°C in a humidified atmosphere containing 5% CO2 in air. To observe embryo development, oocytes were activated by SrCl2 treatment for 6 h, using a calcium-free CZB medium containing 10 mM SrCl2 and 5 mg/ml cytochalasin B. At the end of the 6-h culture, oocytes were observed under a inverted microscope for activation. Oocytes showing one or two pronuclei, or showing two cells each having a nucleus, were considered activated. About 30 Sr2+-activated oocytes were placed in a 100-µl drop of regular CZB medium and cultured for 4 days in at 37.5°C under 5% CO2 in humidified air. When embryos developed beyond 3- or 4-cell stages, 5.55-mM glucose was added to the medium.

Cytoplasmic fragmentation observation
Newly-ovulated DOs were treated for 6 h in CZB or OCM containing different drugs to regulate AMPK or MPF activities before post-treatment aging in CZB alone. At 24 or 36 h of the post treatment aging, oocytes were assessed for cytoplasmic fragmentation under a phase contrast microscope. Oocytes with a clear moderately granulate cytoplasm, and an intact first polar body, were considered as un-fragmented, and oocytes with more than two asymmetric cells were considered to be fragmented [6]. Oocytes with both degenerated and normal cells were also considered fragmented in this study.

Immunofluorescence microscopy
Procedures for immunofluorescence microscopy were carried out at room temperature unless mentioned otherwise. Oocytes (DOs) were always washed three times between treatments. Oocytes were (1) fixed for 30 min in PBS with 3.7% paraformaldehyde; (2) treated for 10 seconds in M2 with 0.5% protease to remove zona pellucida; (3) permeabilized at 37.5°C for 10 min with 0.1% Triton X-100 in PBS; and (4) blocked in PBS with 3% BSA at 37.5°C for 30 min. To detect active caspase-3, the blocked oocytes were incubated at 37.5°C for 1 h first in PBS containing rabbit anti-active caspase-3 (1:200, ab13847, Abcam) and 1% BSA, and then, in PBS containing Cy3-conjugated goat-anti-rabbit IgG (1:800, 111-165-144, Jackson ImmunoResearch) and 3% BSA. For tubulin staining, the blocked oocytes were incubated at 37°C for 1 h in PBS containing FITC-conjugated anti-α-tubulin monoclonal antibodies (1:50). To stain CGs, the blocked oocytes were incubated for 1 h in 100 µg/ml of FITC-labeled lens culinaris agglutinin (LCA) in M2.
Following the above FITC- or Cy3-conjugated antibody labeling, the oocytes were incubated for 5 min in M2 with 10 µg/ml Hoechst 33342 to stain chromatin. Samples in which the primary antibody was omitted were also processed to serve as negative controls. The stained oocytes were examined under a Leica confocal microscope, and blue diode (405 nm), argon (488 nm) and helium/neon (543 nm) lasers were used to excite Hoechst, FITC and Cy3, respectively.

Fluorescence was detected with 420-480 nm (Hoechst), 492–520 nm (FITC) and 560–605 nm (Cy3) filters, and the captured signals were recorded as blue (Hoechst), red (caspase-3) or green (tubulin and CGs). To quantify the active caspase-3 expression, the relative fluorescence intensities were measured on the raw images using Image-Pro Plus software (Media Cybernetics Inc., Silver Spring, MD) under fixed thresholds across all slides.

Western blotting for AMPK
Around 200 DOs were put in each 1.5 ml microfuge tube containing 20 µl sample buffer (20 mM Hepes, 100 mM KCl, 5 mM MgCl2, 2 mM DTT, 0.3 mM PMSF, 3 mg/ml leupetin, pH 7.5) and the tubes were frozen at −80°C until use. To run the gel, 5 µl of 5× SDS-PAGE loading buffer was added to each tube and the tubes were heated at 100°C for 5 min. The samples were separated on a 10% SDSPAGE and transferred onto PVDF membranes. The membranes were (a) washed twice in TBST (150 mM NaCl, 2 mM KCl, 25 mM Tris, 0.05% Tween-20, pH 7.4); (b) blocked for 1.5 h with TBST containing 3% BSA at room temperature; (c) incubated at 4°C overnight with primary antibodies at a dilution of 1:1000 in 3% BSA-TBST; (d) washed three times in TBST (5 min each); (e) incubated for 1 h at 37°C with second antibodies diluted to 1:2000 in 3% BSA-TBST; (f) washed 3 times in TBST; and (g) detected by a BCIP/NBT alkaline phosphatase color development kit (Beyotime Institute of Biotechnology, China). The relative quantities of proteins were determined by analyzing the sum density of each protein band image using Image J software. GAPDH or β-actin was used as internal controls. The primary antibodies used included rabbit Anti-AMPK alpha2 (phospho T172) monoclonal antibody (Abcam, ab133448), and mouse anti-GAPDH monoclonal antibody (ComWin Biotech, CW0100M) and anti-β-actin monoclonal antibody (ComWin Biotech, CW0096). The secondary antibodies included goat anti-rabbit IgG AP conjugated (ComWin Biotech, CW0111) and goat anti-mouse IgG AP conjugated (ComWin Biotech, CW0110).

Measurement for MPF (CDK1) activity

The CDK1 activity was assayed using a MESACUP cdc2 kinase assay kit (code 5234; MBL, Downloaded from https://academic.oup.com/biolreprod/advance-article-abstract/doi/10.1093/biolre/ioaa081/5843372 by Beurlingbiblioteket user on 27 May 2020 Nagoya, Japan). Briefly, about 60 DOs were placed in a plastic tube containing 10 µl cell lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 2 mM EDTA, 5 mM EGTA, 1% [v/v] Triton X-100,
2.5 mM sodium pyrophosphate, 1 mM β-Glycerophosphate, 1 mM Na3VO4, 1 µg/ml of leupeptin, and 1 mM PMSF). The tubes were then frozen at −80°C and thawed at room temperature three times. The cell extracts obtained were frozen stored at −80°C until use. Then, 10 µl of oocyte extracts were mixed with 35 µl kinase assay buffer B (25 mM Hepes buffer [pH 7.5, MBL], 10 mM MgCl2 [MBL], 10% [v/v] MV peptide solution [SLYSSPGGAYC; MBL], 0.1 mM ATP), and the mixture was incubated for 30 min at 30°C. Finally, 200 ml PBS containing 50 mM EGTA (MBL) were added to end the reaction. Phosphorylation of the MV peptides was detected at 492 nm using a plate reader (BioTek-ELx808, BioTek Instruments, Inc.). Data were expressed as the fold strength of CDK1 activity in control oocytes.

Intra-oocyte reactive oxygen species (ROS) measurement
The level of intra-oocyte ROS was quantified by measuring H2O2 levels using 2’,7’-dichloro- dihydro- fluorescein diacetate (DCHFDA) staining. A 1-mM stock solution of DCHFDA was prepared by dissolving DCHFDA in DMSO and was frozen-stored in the dark. The stock solution was diluted to 10 µM with M2 medium before use and DOs were stained with the resultant solution at 37°C for 10 min. After staining, the oocytes were washed in M2, placed on a slide, and observed under a Leica confocal microscope. Fluorescence was obtained by excitation 488 nm.
All the photographs were taken with fixed microscopic parameters to ensure data consistency. The fluorescence intensity value of each oocyte was analyzed using a Leica software.

Measurement of mitochondrial membrane potential (MMP)
The MMP was detected using an MMP detection (JC-1) kit (Beyotime Biotechnology Research Institute, China). Briefly, DOs were washed three times in M2 and placed in a drop of working solution consisting of 1 ml M2 and 1 ml JC-1 dye. The DOs were then incubated at 37°C for 25 min. After being washed three times with a JC-1 staining buffer, the oocytes were observed under a Leica laser scanning confocal microscope. The same oocytes were observed through both TRITC channel (red fluorescence) and FITC channel (green fluorescence). The aggregate JC-1 (red fluorescence) was detected at an emission wave length of 590 nm, whereas the monomeric JC-1 (green fluorescence) was monitored at 529 nm. The ratio of aggregated/ monomeric JC-1 was calculated to quantify MMP level, and a decreased red/green JC-1 ratio represented depolarization of the mitochondria.

Measurement of oocyte cytoplasmic calcium
Intracellular Ca2+ was measured using the Ca2+-sensitive dye fluo-2 as reported by Hagen et al. [25]. To load the Ca2+ probe, DOs were incubated at room temperature for 30 min in a Hepes-buffered CZB medium containing 1 µM Fluo-2 AM and 0.02% pluronic F-127. Drops of Hepes-buffered CZB medium were made in a Fluoro dish (FD35-100, World Precision Instruments) and covered with mineral oil. Oocytes were transferred into the Hepes-buffered CZB drops and observed with a Leica DMI 6000 inverted microscope at 37°C. A Fluo-2 fluorescence module was used for excitation, and a Leica LAS-AF calcium imaging module was used to calculate the F490/520 ratio. The oocytes were monitored for 10 min to record the F490/520 ratio, which represented the concentration of cytoplasmic calcium.

Measurement of intra-oocyte cAMP

We measured concentrations of cAMP using a mouse cAMP Elisa kit (BLUE GENE, Shanghai, China). We collected 120 oocytes and washed them three times in PBS. The oocytes were then re-suspended in PBS and subjected to ultrasonication for three times. After centrifugation at
1000×g for 15 minutes at 4°C to remove cellular debris, the samples were stored at −80°C. All kit components and samples were brought to room temperature (20-25°C) before use. Then, 50 µl of standards or samples were added to the appropriate well in the antibody pre-coated microtiter plate. Fifty microliters of PBS were added in the blank control well. We then dispensed 5 µl of balance solution into 50 µl specimens, and mixed them thoroughly. Then, we added 100 μl of conjugate to each well except for the blank control well, and mixed thoroughly. The plate was incubated for 1 h at 37°C. The microtiter plate was washed 5 times using wash solution. We then added 50 µl substrate A and 50 µl substrate B to each well including blank control well, and incubated the plate for 20 minutes at 37°C (Avoid sunlight). Finally, we added 50 µl of stop solution to each well, including the blank control well, and mixed well. The Optical Density (O.D) was determined immediately at 450 nm using a microplate reader.

Data analysis
Each treatment was repeated at least three times in all the experiments. Data were first arc sine transformed and then analyzed with ANOVA. A Duncan multiple test was carried out to locate the differences. All the data were analyzed using the software of SPSS 11.5 (SPSS Inc.). Data are expressed as mean ± SEM, and p <0.05 was considered significant.

Results

Activation of AMPK increased the activation susceptibility (AS) in aging mouse oocytes
To observe the effects of AMPK activity on AS of aging oocytes, newly ovulated DOs were aged for 12 h in CZB medium containing various concentrations of AMPK activators, metformin or AICAR, or in OCM supplemented with different concentrations of AMPK inactivator, compound C, before ethanol + 6-DMAP treatment for activation. The results showed that oocyte activation rates increased significantly (P<0.05) with increasing concentrations of metformin and AICAR, but decreased significantly (P<0.05) with increasing concentrations of compound C (Fig. 1A, B and C), suggesting that activation of AMPK facilitated aging of mouse oocytes. To ascertain that the AS-facilitating effect of metformin was indeed mediated through activation of AMPK,
newly-ovulated oocytes were aged for 12 h in CZB alone or with metformin or with both metformin and compound C before AS examination. The results showed that the presence of compound C completely eliminated the AS-facilitating effect of metformin (Fig. 1D), suggesting that the AS-facilitating effect of metformin was solely mediated through the activation of AMPK.

Effects of AMPK activity on cytoplasmic fragmentation, caspase-3 activation and developmental potential in aging mouse oocytes

To observe the effects of AMPK activity on cytoplasmic fragmentation of aging oocytes, newly ovulated DOs were treated for 6 h to regulate AMPK/MPF activity in CZB alone or with 15 mM metformin or with both metformin and MG132, or in OCM alone or with 10 µM compound C before post treatment aging in CZB alone. At 24 or 36 h of post-treatment aging, oocytes were examined for cytoplasmic fragmentation. The results showed that at both 24 h and 36 h of
post-treatment aging, AMPK activation with metformin and inactivation with compound C significantly (P<0.05) increased and decreased the percentages of fragmented oocytes, respectively (Fig. 2A). Furthermore, compared to treatment with metformin alone, supplementation with MG132 significantly (P<0.05) reduced fragmentation rates at 24 h post treatment (Fig. 2B). The results suggested that a significant decline in MPF activity at the early stage of oocyte aging was essential for the late events to occur in aged oocytes.

Expression of active caspase-3 was quantified by immunofluorescence microscopy. Newly ovulated DOs were cultured for 12 h in different media to regulate AMPK activity before examination for caspase-3 distribution and quantification under a confocal microscope. The results showed that the fine granules of active caspase-3 were evenly distributed throughout the ooplasm (Figs. 2E to H). Our quantification of fluorescence intensity (Fig. 2C) indicated that the level of active caspase-3 was significantly (P<0.05) higher when oocytes were cultured in CZB with than without metformin, but in contrast, the caspase level was significantly (P<0.05) lower when oocytes were cultured in OCM with than without compound C. Thus, activation of AMPK with metformin and its inactivation with compound C increased and decreased the level of active caspase-3 expression, respectively. As cytoplasmic fragmentation has been regarded as one of the signs for programmed cell death of aging oocytes [26], the results suggested that AMPK activation facilitated apoptosis of aging mouse oocytes.

To observe the effects of AMPK activity on developmental potential of aging oocytes, newly ovulated DOs were treated for 12 h in OCM alone or with compound C before SrCl2 activation for embryo development. Although % activated oocytes/ cultured oocytes (ranging from 97.1±0.5 to 98.3±0.8%) and % 2-cell embryos/ activated oocytes (ranging from 97.8±1.3 to 99.1±0.9%) did not differ significantly (P>0.05) between treatments, aging in OCM significantly (P<0.05) decreased percentages of 4-cell and blastocyst embryos (Fig. 2D). The presence of compound C in OCM, however, significantly (P<0.05) improved 4-cell and blastocyst development. The results suggested that AMPK activation impaired the developmental potential of aging mouse oocytes.

Effects of AMPK activity on morphology of spindle/chromosomes and distribution of cortical granules (CGs) in aging mouse oocytes
Newly ovulated DOs were treated for 6 h in different media before post-treatment culture for 24 h and confocal microscopic observation for spindle/chromosomes morphology and CGs distribution. Spindle/chromosomes morphology was classified into 4 types: focused-pole (F) or barrel-shaped
(B) spindles with chromosomes congressed (C) on the metaphase plate, or disintegrated (D) spindles with congressed (C) or misaligned (M) chromosomes. Percentages of oocytes with F/C (Fig. 3C) ranging from 50±2.1% to 56.6±1.9% did not differ (P>0.05) between treatments (Fig. 3A). Treatment with metformin in CZB decreased oocytes with B/C (Fig. 3D) while increasing the oocytes with D/C (Fig. 3E) or D/M (Fig. 3F) significantly (P<0.05). Treatment with compound C in OCM, however, increased B/C oocytes while decreasing the D/M oocytes significantly (P<0.05) (Fig. 3A). Three patterns of CGs distribution were observed. In the normal distribution (ND) pattern, all the CGs were tidily aligned beneath the cell membrane except for the CG-free domain above the spindle (Fig. 3G). In the early migration (EM) pattern, CGs migrated inwards and toward the vegetal pole to some extent (Fig. 3H). In the late migration (LM) pattern, CGs migrated further inwards and toward the vegetal pole (Fig. 3I). Treatment with metformin in CZB decreased percentages of oocytes with ND of CGs while increasing oocytes with EM and LM of CGs significantly (P<0.05), whereas treatment with compound C in OCM produced the opposite effect (Fig. 3B). Taken together, the results demonstrated that while activating AMPK with metformin disturbed, inactivating AMPK with compound C maintained spindle/chromosome morphology and CGs distribution in aged mouse oocytes, suggesting that AMPK facilitates the late events of oocyte aging.

Relationship between AMPK and MPF activities in aging mouse oocytes
To analyze the relationship between AMPK and MPF activities in regulating oocyte aging, the AMPK and MPF activities were first measured in oocytes aging for different times in vivo or after the newly-ovulated DOs were cultured for 12 h in different treatment media. The results showed that while the AMPK activity increased to maximum at 18 h post hCG injection (Fig. 4A), the MPF activity decreased gradually and significantly (P<0.05) during in vivo aging from 13 h to 24 h post hCG injection (Fig. 4B). Treatment with metformin in CZB and treatment with compound C in OCM significantly (P<0.05) up and down regulated AMPK activity, respectively, and the AMPK activity was significantly (P<0.05) higher following oocyte culture in OCM alone than in CZB alone (Fig. 4C). Furthermore, in vitro AMPK activation and inactivation significantly (P<0.05) decreased and increased the MPF activity, respectively (Fig. 4D).

Effects of modulating the AMPK and MPF activities on AS of aging oocytes were then observed. Newly-ovulated DOs were cultured for 12 h in different treatment media before examination for activation rates. When cultured in CZB medium, upregulation of MPF activity with MG132 decreased activation rates to the lowest level (4.5±2.0%) in spite of the activation of AMPK with metformin (Fig. 4E). Similarly, when cultured in OCM, downregulation of MPF with roscovitine significantly (P<0.05) increased activation rates in spite of the inactivation of AMPK with compound C (Fig. 4F). Taken together, the results suggested that (a) AMPK is activated with natural aging of oocytes; (b) metformin and compound C up and down regulated AMPK activity, respectively, in aging mouse oocytes; and (c) AMPK worked upstream of MPF and its activation facilitated aging of mouse oocytes by down regulating MPF activity.

Effects of AMPK activity on levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) of aging mouse oocytes

Since it is known that mitochondrial dysfunction may both result from and contribute to oxidative stress during cell apoptosis [27-29], we examined the effects of AMPK activity on levels of ROS and MMP in aging mouse oocytes. Newly-ovulated oocytes were treated for 12 h to regulate AMPK activity in CZB, CZB + metformin, OCM or OCM + compound C before examination for ROS and MMP. While activation of AMPK with metformin increased ROS while decreasing MMP, inactivation of AMPK with compound C decreased ROS while increasing MMP significantly (P<0.05) (Fig. 5), suggesting that AMPK activation facilitated oocyte aging with increased oxidative stress and impaired mitochondrial function.

Roles of cytoplasmic calcium and CaMKKβ/CaMKII in regulating AMPK activities during aging of mouse oocytes

Newly-ovulated oocytes were treated for 12 h to regulate AMPK activity in CZB, CZB + metformin, OCM or OCM + compound C before measurement for cytoplasmic calcium and activation rates. Oocytes showed a significantly higher calcium level along with a higher activation rate following aging in OCM than in CZB (Fig. 6A and B), confirming that increased AS was associated with elevated cytoplasmic calcium in our oocytes aging without metformin. However, although oocytes aging in CZB with metformin and those aging in OCM showed the same level of cytoplasmic calcium (Fig. 6A), activation rates were 35% higher (P<0.05) in the former than in the latter (Fig. 6B). Furthermore, while activation rates decreased from 50% to 6%, calcium level showed only a mild decrease from OCM to OCM +compound C. The results suggested that AMPK activation was only partially dependent on the calcium signaling to increase AS.

We then observed whether AMPK was activated through CaMKKβ and/or CaMKII in aging oocytes. Newly-ovulated oocytes were cultured for 12 h in CZB, CZB +metformin or OCM with or without CaMKKβ inhibitor STO-609 or CaMKII inhibitor KN-93 before examination for activation rates and level of AMPK activation (pT172). While the presence of STO-609 significantly (P<0.05) decreased activation rates in all treatments, KN-93 showed no effect in CZB containing metformin (Fig. 6C). Activation rates increased by 64% from 20% in CZB alone to 84% in CZB +metformin, but they decreased only by 20% from 84% to 64% in CZB +metformin + STO-609, suggesting again that the metformin-induced increase of AS was onlypartially dependent on calcium signaling. The presence of STO-609 significantly (P<0.05) decreased the level of active AMPK in both CZB + metformin and OCM (Fig. 6D), suggesting that AMPK activation in aging oocytes is dependent upon the calcium signaling and CaMKs. However, while STO-609 decreased the level of active AMPK by 30% in CZB +metformin, it decreased active AMPK by 66.7% in OCM, suggesting that metformin activation of AMPK was less dependent upon calcium signaling than the AMPK activation in OCM was. Taken together, our results suggested that metformin might activate AMPK both directly and indirectly by way of calcium signaling.

Metformin increased ROS production by activating AMPK and facilitating aging

Our above results suggested that metformin might activate AMPK (facilitate AS) through calcium signaling by increasing ROS, as our previous studies demonstrated that an increase in ROS might initiate cytoplasmic calcium rises in aging oocytes [30,31]. However, although some studies indicated that metformin induced apoptosis with increased oxidative stress [32], and oxidative stress can activate AMPK [33], others confirmed that metformin ameliorated oxidative damage in various cells [34,35]. To differentiate whether metformin activated AMPK by increasing ROS or it increased ROS by activating AMPK and facilitating aging, we measured the ROS level in oocytes aging for 6 h in CZB alone or with metformin. The results showed that the ROS level increased significantly (P<0.05) from ovulation to 6 h and 12 h of culture in CZB alone, but at 6 h of culture, it did not differ (P>0.05) between oocytes cultured with and without metformin (Fig.7A and D to G). Together with our above results that by 12 h of culture, the ROS level was significantly (P<0.05) higher in oocytes cultured with than without metformin (Fig. 5A), our results suggested that metformin increased ROS after it had activated AMPK and facilitated aging of oocytes.

Intracellular levels of cAMP decreased significantly with aging of mouse oocytes

In vivo aging oocytes were recovered at 13, 18 and 24 h post hCG injection, and for in vitro aging, oocytes recovered at 13 h post hCG injection were cultured for 5 or 11 h in regular CZB medium with their cumulus cells intact. Concentrations of cAMP were measured using a porcine cAMP Elisa kit. The results showed a steady and significant (P<0.05) decline in cAMP level in both in vivo (Fig. 7B) and in vitro aged oocytes (Fig. 7C) from ovulation to 18 and 24 h after hCG injection.

DiscussionAMPK activation facilitated both early and late aging manifestations during postovulatory aging of mouse oocytes

The present results showed that activation of AMPK facilitated both the early aging manifestations (such as an increase in AS and a decline in MPFactivities [3,10-12]), and the late aging phenomena (such as a loss of spindle/chromosomes integrity, disordered distribution of cortical granules, and cytoplasmic fragmentation [36,37]), in mouse oocytes. Furthermore, the current results suggested that a significant decline in MPF activities at the early stage of oocyte aging was essential for the late events to occur in aged oocytes. Both Choi [38] and Zhu et al. [13] found that maintaining a high MPF activity at the early stage of aging culture significantly reduced cytoplasmic fragmentation and caspase-3 activation later in aged mouse oocytes. Thus, it seems that a cell must be in interphase for apoptosis to occur. In fact, when cellular injuries such as DNA damage or oxidative stress accumulate in proliferating cells, the cell cycle is arrested in G1 or G2 phase before apoptosis begins [39]. Furthermore, treatment of cancer cells with roscovitine inhibited cyclin-dependent kinases and induced apoptosis with a progressive arrest at the G2 phase [40].

Relationship between AMPK and MPF during postovulatory aging of mouse oocytes

Previous studies observed that activation of AMPK prevented GVBD in pig and bovine COCs [20-22], suggesting that AMPK activation inactivated MPF in mammalian oocytes. However, those studies did not measure MPF activity directly, and nor they specified whether the AMPK signaling within the oocyte itself or that in cumulus cells modulates the intraoocyte MPF activity.By culturing DOs, this study has demonstrated for the first time and unequivocally that AMPK activation inhibits the intra-oocyte MPF activity. It is reported that in somatic cells, AMPK can regulate cell cycling [41,42]. In oocytes of the marine nemertean worm, activation of AMPK with AICAR inhibited GVBD by preventing MPF activation [43]. As for how AMPK activation inactivates MPF, Stricker [43] found that active AMPK might maintain the MPF inactive by inhibiting TOR in immature nemertean oocytes, because active TOR could induce mitosis in cancer cells [44]. Alternatively, AMPK activation might inactivate MPF by increasing TSC2 activity, which might not only inactivate TOR but also upregulate the MPF inhibitor Kip1 [43].

Furthermore, this study showed that upregulating MPF with MG132 and downregulating it with roscovitine significantly decreased and increased AS, respectively, in spite of activation and inactivation of AMPK with metformin and compound C, respectively, suggesting that AMPK worked upstream of MPF in aging oocytes. When nemertean oocytes were incubated in seawater solution containing roscovitine to inhibit MPF, AMPK deactivation continued, suggesting that AMPK inactivation occurred upstream to, and independently of, MPF activation [43].

Signaling pathways leading to AMPK activation in postovulatory aging oocytes
The current results showed that AMPK activation increased ROS and cytoplasmic calcium while decreasing MMP significantly in aging mouse oocytes. Previous studies suggested that oxidative stress might initiate the aberrations observed in aged oocytes. For example, oxidative stress in aging oocytes can decrease MPF and MAPK activities, impair calcium homoeostasis, cause mitochondrial dysfunction and damage intracellular components [36]. Previous studies also indicated that the increase in AS and ROS was always associated with cytoplasmic calcium rises in aging oocytes [30,31]. Furthermore, AS and AMPK activation were significantly decreased when we treated the aging oocytes with CaMK inhibitors, suggesting that the cytoplasmic calcium rises activated AMPK by activating CaMKs. Mungai et al. [45] demonstrated that
hypoxia triggered AMPK activation through increasing cytosolic calcium that activated CaMKKβ.
Mathew et al. [46] observed that both CaMKKβ and CaMKII contributed to the calcium-dependent activation of AMPK in muscle cells.

This study observed that the level of cAMP decreased significantly during both in vivo and in vitro aging of mouse oocytes from 13 h to 24 h after hCG injection. Drugs that elevate cAMP levels directly or indirectly slowed postovulatory aging of rat oocytes cultured in vitro [47]. Together with our results that AMPK activation increased significantly with postovulatory aging of mouse oocyte, we proposed that a reduction in the cAMP level had contributed to AMPK activation during postovulatory oocyte aging. There are several reports that protein kinase A (PKA) can inactivate AMPK. In primary mouse adipocytes, for example, PKA phosphorylated AMPKalpha1 at Ser-173 impeding Thr-172 phosphorylation and thus prevented AMPKalpha1 activation by LKB1 [48]. Hurley et al. [49] observed that cAMP-elevating agents downregulated AMPK activity by modulating its phosphorylation sites in clonal beta-cell line INS-1, mouse embryonic fibroblasts and COS cells.Both the present result and previous studies [16] observed a significant decline in the intra-oocyte cAMP level with increasing time of postovulatory oocyte aging. This is in conflict with the common findings that the MPF activity decreased significantly with postovulatory oocyte aging [3,12], as it is known that PKA inactivates MPF [17]. The present study has thus provided a perfect explanation for this conflict by showing that the decrease in cAMP contributed to activation of AMPK, which inactivates MPF as demonstrated in oocytes from both marine nemertean worm [46] and mammals [20-22].

Limitations related to use of metformin and compound C
In this study, metformin and compound C were used to activate and inhibit AMPK, respectively. It is known, however, both drugs are not specific enough. For example, metformin affects ETC complex 1 and other targets as well as AMPK [50], and compound C inhibits multiple kinases with similar or greater potency than AMPK [51]. However, although El-Mir et al. [50] suggested that high concentrations of metformin inhibited the respiratory chain complex 1, leading to a decrease in ATP and an increase in AMP, it is known that AMP causes activation of AMPK [15]. Although compound C might prevent metformin uptake as it did for AICAR [52], the end result was still its suppression on AMPK activity. We tested AICAR, another commonly used AMPKactivator, to confirm that metformin activated AMPK in aging oocytes. We found that during in vivo aging of ovulated oocytes without any drug treatment, while MPF activity and cAMP decreased, AMPK activity increased significantly with time post ovulation. Furthermore, we performed western blotting to confirm the specificity of metformin and compound C on AMPK activity in the aging oocytes, which showed that while metformin increased, compound C decreased the pT172 level of AMPK significantly. Thus, although our results using activators/inhibitors cannot exclude their non-specific effects, the whole study confirmed that the changes in cellular functions we observed were brought about mostly (though not exclusively) by their actions on AMPK activities.

In summary, our results suggested that AMPK facilitated oocyte aging through inhibiting MPF activities, and that postovulatory oocyte aging activated AMPK with decreased cAMP by activating CaMKs via increasing ROS and cytoplasmic calcium. Thus, on the one hand, postovulatory aging increases levels of ROS and cytoplasmic calcium, and on the other hand, it decreases the level of cAMP (Fig. 8). The elevation in cytoplasmic calcium activates AMPK by activating CaMKs, and the decline in cAMP facilitates AMPK activation as cAMP inactivates AMPK by activating PKA [48,49]. The decline in cAMP may also promote oocyte aging by generating AMP, a potent activator of AMPK [15]. The activated AMPK inactivates MPF leading to the early and late manifestations in aged oocytes. Metformin facilitates oocyte aging first by directly activating AMPK, and then, it activates AMPK further through increased ROS and the calcium signaling in aged oocytes, leading to severer aging manifestations. The data are important not only theoretically for elucidating the mechanisms but also practically for prevention of oocyte aging in both normal and assisted reproduction. However, our results suffered from limitations due to the use of nonspecific AMPK activators/inhibitors; future studies involving genetic (knockdown or depletion) approaches will definitely provide more convincing evidence for the role of AMPK in postovulatory oocyte aging.

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