Abstract
Unlike in mice, where the onset of oocyte maturation (germinal vesicle breakdown, GVBD) is blocked by cAMP and triggered by AMP-activated kinase (AMPK), oocytes of the marine nemertean worm Cerebratulus undergo GVBD in response to cAMP elevations and AMPK deactivation. Since the pathways underlying AMPK's effects on mammalian or nemertean GVBD have not been fully defined, follicle-free nemertean oocytes were treated with pharmacological modulators and subsequently analyzed via immunoblotting methods using phospho-specific antibodies to potential regulators and targets of AMPK. Based on such phosphorylation patterns, immature oocytes possessed an active LKB1-like kinase that phosphorylated AMPK's T172 site to activate AMPK, whereas during oocyte maturation, AMPK and LKB1-like activities declined. In addition, given that MAPK can deactivate AMPK in somatic cells, oocytes were treated with inhibitors of ERK1/2 MAPK activation. However, these assays indicated that T172 dephosphorylation during maturation-associated AMPK deactivation did not require MAPK and that an observed inhibition of GVBD elicited by the MAPK kinase blocker U0126 was actually due to ectopic AMPK activation rather than MAPK inactivation. Similarly, based on tests using an inhibitor of maturation-promoting factor (MPF), T172 dephosphorylation occurred upstream to, and independently of, MPF activation. Alternatively, active MPF and MAPK were necessary for fully phosphorylating a presumably inhibitory S485/491 site on AMPK. Furthermore, in assessing signals possibly linking AMPK deactivation to MPF activation, evidence was obtained for maturing oocytes upregulating target-of-rapamycin activity and downregulating the cyclin-dependent kinase inhibitor Kip1. Collectively, these findings are discussed relative to multiple pathways potentially mediating AMPK signaling during GVBD.
Introduction
In response to proper stimuli, oocytes start to mature by undergoing a process of nuclear disassembly referred to as germinal vesicle breakdown (GVBD). Based on comparisons across the animal kingdom, GVBD is differentially regulated by intraoocytic concentrations of the secondary messenger cAMP. In mammals, other vertebrates, and starfish, GVBD is characteristically blocked by elevated levels of cAMP ( Voronina & Wessel 2003, Mehlmann 2005, Richard 2007), whereas intraoocytic cAMP elevations can trigger, rather than inhibit, GVBD in several marine invertebrates, including worms of the phylum Nemertea ( Stricker & Smythe 2001).
In addition to cAMP, various protein kinases are also integrally involved in oocyte maturation via their eventual downstream effects on a proximate inducer of GVBD called maturation-promoting factor (MPF; Kishimoto 2003). In investigations of kinase-mediated processes during MPF-induced GVBD, the evolutionarily conserved AMP-activated kinase (AMPK) pathway has recently been analyzed, since in addition to integrating intracellular energy states, AMPK can also regulate cell cycling ( Carling 2005, Alessi et al. 2006, Motoshima et al. 2006, Hardie 2007). Moreover, given that cAMP hydrolysis not only reduces cAMP levels but also raises AMP levels that in turn help to activate AMPK, signaling pathways involving AMPK may play key roles both in normal oocytes ( Downs et al. 2002) and in those affected by diseases such as diabetes and polycystic ovary syndrome ( Ratchford et al. 2007, Dupont et al. 2008).
Accordingly, various analyses have shown that AMPK triggers GVBD in denuded or cumulus-enclosed oocytes of mice ( Chen et al. 2006, LaRosa & Downs 2006, 2007, Chen & Downs 2008). Alternatively, AMPK agonists can block GVBD in other cumulus-enclosed mammalian oocytes ( Bilodeau-Goeseels et al. 2007, Mayes et al. 2007, Tosca et al. 2007), although such blockage is apparently due to effects on follicle cells and/or to non-AMPK-mediated pathways ( Mayes et al. 2007, Tosca et al. 2007, Bilodeau-Goeseels et al. 2010). However, oocytes of nemertean worms lack follicle cells ( Stricker et al. 2001), and AMPK activations nevertheless inhibit nemertean GVBD ( Stricker et al. 2010a, 2010b), indicating that intraoocytic AMPK can stimulate or block MPF activation in different animals.
To determine how AMPK modulates GVBD, signaling pathways have been examined in follicle cells ( Tosca et al. 2005, 2006, 2010, Kayampilly & Menon 2009, Richardson et al. 2009) and within oocytes themselves. For example, recent studies of intraoocytic AMPK in mice have assessed the relationship between diabetes and AMPK-mediated targets in glucose metabolism ( Ratchford et al. 2007), the activation of AMPK by FSH and amphiregulin ( Chen & Downs 2008), and the interconnections between AMPK and fatty acid oxidative pathways ( Downs et al. 2009). Moreover, how AMPK interacts with MAPK and other signals has been evaluated in oocytes of cows and pigs ( Bilodeau-Goeseels et al. 2007, Mayes et al. 2007, Tosca et al. 2007).
However, in spite of these and other analyses, intraoocytic AMPK signaling is incompletely understood, particularly with regard to some of the key upstream regulators and downstream targets that are known to mediate AMPK's effects in somatic cells. Thus, this study aims to track activity-related phosphorylations of AMPK signaling proteins in nemertean oocytes that are stimulated to mature in the presence or absence of pharmacological modulators. Based on these methods, new findings are presented regarding such regulators of intraoocytic AMPK as the AMPK kinase (AMPKK) LKB1, MAPK, and MPF. Moreover, target-of-rapamycin (TOR) signaling and the cyclin-dependent kinase (CDK) inhibitor p27 Kip1 are evaluated for the first time as possible targets linking AMPK deactivation to MPF activation, and collectively such results are discussed in the context of multiple pathways potentially mediating AMPK's effects during GVBD.
Results
Phosphorylation patterns of an LKB1-like AMPKK during oocyte maturation
Given that LKB1 is the most common AMPKK to activate AMPK via T172 phosphorylation in AMPK's activation loop ( Shackelford & Shaw 2009, Ruderman et al. 2010), Cerebratulus oocytes were probed with a phospho-specific LKB1 antibody to track an S428 phosphorylation that can upregulate LKB1 ( Liangpunsakul et al. 2008, Xie et al. 2008). Based on these tests, immature oocytes had relatively high T172 phosphorylation and likewise exhibited a putatively active LKB1 at the appropriate MW of ∼52 kDa ( Fig. 1A and C). Moreover, consistent with GVBD-related reductions in the phosphorylations of both AMPK's T172 site and S79 on the AMPK target acetyl-CoA carboxylase (ACC; Stricker et al. 2010a), such S428 phosphorylation decreased significantly (44±22%; N=23; P<0.05) during maturation-associated AMPK deactivation ( Fig. 1B and C). Alternatively, as potential evidence for LKB1-independent AMPK activation such as described elsewhere ( Sakamoto et al. 2004, Sanders et al. 2007), seawater (SW) solutions of the AMPK agonists AICAR and resveratrol kept oocytes immature and AMPK activity high ( Stricker et al. 2010a), and yet such agonist treatments in either SW or SW+cAMP elevators yielded phospho-S428 signals that were significantly lower (P<0.05) than in pre-treatment controls ( Fig. 1B–E).
LKB1-like activity in nemertean oocytes. (A) Uncompressed living oocytes prior to maturation onset, showing intact germinal vesicles (GVs) in seawater (SW)+AICAR. (B) Mature oocytes after GVBD in SW+AICAR+the cAMP elevator IBMX; scale bar=50 μm. (C) Oocyte extracts dually probed with phospho-T172 and phosopho-S428 antibodies for tracking active forms of AMPK at 62 kDa and LKB1 at 52 kDa, showing a decrease in putative LKB1 activity during SW-induced GVBD (note: results were similar in separate blots using one antibody per blot). (D) Typical time-lapse sequence of decreasing phospho-S428 phosphorylations during SW-induced GVBD. (E) Even though T172 levels are elevated by SW solutions of the AMPK agonists AICAR and reseveratrol (res) (C), such treatments still allow as much LKB1 S428 dephosphorylation (P>0.05) as occurs in mature oocytes treated with either SW alone or with SW+AMPK agonists+cAMP elevators such as forskolin (for), suggesting that AMPK agonists may trigger an LKB1-independent activation of AMPK. BF=before treatment; other lanes on blots and graph in E=control and drug treatments at 1.5 h post-stimulation.
Citation: REPRODUCTION 142, 1; 10.1530/REP-10-0509
Effects of ERK1/2 MAPK on phosphorylations of AMPK's T172 and S485/491 sites
In somatic cells ( Zheng et al. 2009), AMPKK/AMPK can be downregulated by ERK1/2 MAPK (MAPK3/1 or simply MAPK hereafter), and such MAPK activity normally increases during nemertean GVBD ( Stricker and Smythe 2003, 2006a, Smythe & Stricker 2005). Thus, to determine if MAPK affects AMPK during GVBD, oocytes were treated with the MAPK kinase inhibitors CI1040, PD98059, and U0126. Based on such studies, CI1040- and PD98059-treated oocytes underwent GVBD in the absence of substantial MAPK activity and continued to deactivate AMPK, as evidenced by significant reductions (P<0.05) in both T172 and ACC phosphorylation, compared to immature controls ( Fig. 2A and B).
ERK1/2 mitogen-activated protein kinase (MAPK) and AMPK deactivation. (A and B) In spite of preventing MAPK activation, inhibitors of MAPK kinase (MAPKK; CI1040 (ci) or PD98059 (pd)) routinely allow GVBD as evidenced by reduced inhibitory Y15 phosphorylation on CDC2 or increased stimulatory phosphorylation at CDC2 T161. Similarly, based on control-like T172 dephosphorylation and decreased S79 phosphorylations on the AMPK target ACC, such oocytes lacking MAPK activity continue to undergo AMPK deactivation. (C) Alternatively, in some oocyte batches, SW solutions of the MAPKK blocker U0126 (uo) inhibit GVBD. However, T172 phosphorylations in these oocytes are significantly higher (P<0.05) than in SW controls, whereas co-treatments of uo plus cAMP elevators such as forskolin (for) further reduce T172 phosphorylation. Coupled with results for other MAPKK blockers and roscovitine ( Fig. 3B–D), such findings suggest uo may not block GVBD via MAPK inhibition but rather by ectopic AMPK activation. (D and E) Unlike the MAPK-independence of the phospho-T172 signal, phosphorylating AMPK's putatively inhibitory S485/491 site apparently requires MAPK, since all MAPKK blockers reduce such phosphorylations to significantly below (P<0.05) control levels in SW alone. BF=before treatment; other lanes on blots/graphs=control and drug treatments at 1.5 h post-stimulation.
Citation: REPRODUCTION 142, 1; 10.1530/REP-10-0509
Conversely, as noted previously ( Smythe & Stricker 2005, Stricker 2009b), some oocyte batches remained immature in SW solutions of U0126. Accordingly, U0126-treated oocytes had significantly higher (P<0.05) AMPK activity than did mature controls in SW alone ( Fig. 2A and B). Nevertheless, when stimulated by SW+cAMP-elevating drugs, U0126-treated specimens underwent GVBD ( Stricker 2009b) and further dephosphorylated (P<0.05) AMPK's T172 site ( Fig. 2C).
Oocytes treated with MAPKK inhibitors were also probed for S485/491 phosphorylation on AMPK, since such phosphorylation can inhibit AMPK in somatic cells ( Horman et al. 2006, Hurley et al. 2006, Funahashi et al. 2009). However, as opposed to the marked S485/491 phosphorylation increase that occurs during normal GVBD ( Stricker et al. 2010a), MAPKK blockers kept S485/491 phosphorylations in both immature and mature oocytes below (P<0.05) those exhibited by SW controls ( Fig. 2D and E).
AMPK–MPF interactions during oocyte maturation
Given that positive feedback is apparently established between MPF and MAPK during nemertean GVBD ( Stricker & Smythe 2006a), potential reciprocal interactions between AMPK and MPF were analyzed by treating immature oocytes with the MPF inhibitor roscovitine. In SW-stimulated oocytes kept immature by a roscovitine-mediated blockage of MPF activation, AMPK's T172 site continued to undergo control-like dephosphorylation, even in the absence of MAPK activity ( Fig. 3A–C). Alternatively, since MPF blockers can more effectively deactivate MAPK in nemertean oocytes stimulated by SW alone versus by SW+cAMP elevators ( Stricker & Smythe 2006a), roscovitine-mediated effects on S485/491 phosphorylations were also assayed. Accordingly, compared to roscovitine treatments in SW, elevated MAPK activations in SW solutions of roscovitine+cAMP elevators co-occurred with greater (P<0.05) S485/491 phosphorylation ( Fig. 3C and D), further indicating the MAPK-dependence of such phosphorylation. However, phospho-S485/491 signals were downshifted nearer to the phospho-T172 signals after treatment with either roscovitine ( Fig. 3C) or the less effective MPF inhibitor, olomoucine (data not shown). Moreover, in spite of relatively high MAPK activity ( Fig. 3D), phospho-S485/491 signals remained weaker (P<0.05) than in SW controls lacking roscovitine ( Fig. 3E), suggesting that along with MAPK-mediated phosphorylation, MPF may feed back on AMPK to facilitate S485/491 phosphorylation.
AMPK–MPF interactions. (A and B) T172 dephosphorylation continues in SW-stimulated oocytes that have low MPF activity owing to roscovitine (ros) treatments, as T172 phopsphorylations in such specimens are statistically the same (P>0.05) as in controls that matured in either SW or SW+forskolin. (C–E) As further evidence for the MAPK-independence of T172 dephosphorylations, T172 undergoes as much dephosphorylation (P>0.05) in roscovitine-treated oocytes as in SW controls (B) even though SW solutions of roscovitine cause significantly lower (P<0.05) MAPK phosphorylation than in SW alone (C and D). However, full phosphorylation of S485/491 apparently requires MPF in addition to MAPK, since in spite of control-like MAPK activities in forskolin (for)-stimulated oocytes (C and D), S485/491 phosphorylations in the presence of roscovitine are significantly lower (P<0.05; C and E) and downshifted (double arrowheads, C) compared to phospho-S485/491 bands of controls. BF=before treatment; other lanes on blots and graphs=control and drug treatments at 1.5 h post-stimulation; CaFSW=calcium-free seawater.
Citation: REPRODUCTION 142, 1; 10.1530/REP-10-0509
TOR signaling in maturing oocytes
Based on findings that AMPK can inhibit TOR signaling in somatic cells ( Bolster et al. 2002, Kimura et al. 2003, Wullschleger et al. 2006), oocytes were probed with a phospho-S2448 TOR antibody that can track TOR upregulation ( Rosner et al. 2010). In such blots, a putatively active TOR signal at the correct MW of ∼285 kDa was significantly reduced (P<0.05) in immature versus mature controls ( Fig. 4A–C). Moreover, SW solutions of the AMPK activators AICAR and resveratrol blocked GVBD while reducing (P<0.05) S2448 TOR phosphorylations below those of mature controls ( Fig. 4B and C). In addition, as further evidence for differing modes of AMPK signaling during SW- versus cAMP-induced GVBD ( Stricker et al. 2010a), adding cAMP elevators to SW solutions of AICAR or resveratrol restored GVBD and TOR phosphorylation to control-like levels in SW alone ( Fig. 4B and C).
Target-of-rapamycin (TOR) signaling. (A) Typical time-lapse sequence, showing an increase in putatively active TOR during seawater (SW)-induced GVBD. (B and C) AMPK agonists AICAR and resveratrol (res) block GVBD and significantly reduce (P<0.05) SW-induced elevations in S2448 TOR phosphorylations, whereas cAMP elevators (e.g. forskolin (for)) restore GVBD/S4228 phosphorylations and allow AMPK deactivation (see Fig. 1C), collectively suggesting that maturation-associated AMPK deactivation might induce TOR activation. (D and E) As further evidence for maturation-associated TOR activation during AMPK deactivation, a target of active TOR, p70/85 S6K, undergoes a significant (P<0.05) increase in its phosphorylation during maturation. Moreover, phospho-p85 S6K signals are reduced (P<0.05) by AICAR but restored by forskolin (for). BF=before treatment; other lanes on blots C, G and graphs B, H=control and drug treatments at 1.5 h post-stimulation.
Citation: REPRODUCTION 142, 1; 10.1530/REP-10-0509
To provide added support for maturation-associated TOR activation during AMPK deactivation, oocytes were probed with a phospho-specific antibody to Drosophila p70/p85 S6 kinase (S6K), since S6K represents both a target and, via positive feedback, an activator of TOR complex 1 ( Holz & Belnis 2006). For undetermined reasons, a weak and ephemeral phospho-p70 S6K signal was only occasionally observed during GVBD, whereas a strong phospho-S6K signal invariably occurred both at ∼85 kDa and at upshifted sites ( Fig. 4D), as noted for other phospho-S6Ks ( Petritisch et al. 1995, Kraiss et al. 2000). Moreover, as shown for phospho-S2448 TOR, phospho-S6K signals increased (P<0.05) during maturation, remained lower (P<0.05) in SW+AICAR than in SW controls, and re-elevated to control-like levels in SW solutions of AICAR+cAMP elevators ( Fig. 4E).
Maturation-associated changes in the CDK inhibitor p27 Kip1
In somatic cells, AMPK-mediated activation of a TOR inhibitor, called tuberous sclerosis complex 2 (TSC2; Inoki et al. 2003, Coradetti et al. 2004, Abraham 2005), can block cell cycling by TSC2’s stabilizing effects on p27 Kip1 ( Rosner et al. 2006), since Kip1 is a CDK downregulator that typically targets CDK2 ( Sherr & Roberts 1999) but can also inhibit the CDK1 (CDC2) of MPF ( Font de Mora et al. 1997, Nakayama et al. 2004). Thus, oocytes were probed with a phospho-Kip1 antibody against a T187 site that, when phosphorylated, allows more effective Kip1 degradation ( Vlach et al. 1997). In immature oocytes, such destabilizing Kip1 T187 phosphorylations at ∼27 kDa were relatively weak, but during maturation, the phospho-T187 signal rose significantly (P<0.05) along with increased AMPK deactivation ( Fig. 5A and B). Accordingly, signals from a pan-Kip1 antibody for tracking total Kip1 levels declined during maturation ( Fig. 5A). Moreover, the AMPK activator AICAR or resveratrol kept oocytes immature while preventing both T187-Kip1 phosphorylation and MPF activation ( Fig. 5C). Conversely, as further evidence for different AMPK signaling pathways mediating SW- versus cAMP-induced GVBD, adding cAMP elevators to SW solutions of AICAR or resveratrol restored AMPK deactivation ( Stricker et al. 2010a), T187 phosphorylation, and MPF activation to control levels ( Fig. 5C).
The cyclin-dependent kinase inhibitor p27 Kip1 during oocyte maturation. (A) Typical time-lapse sequence of a destabilizing (T187) phosphorylation on Kip1 (left) versus pan-Kip total protein levels (right), showing an increase and decrease, respectively, as oocytes mature and AMPK is deactivated by T172 dephosphorylation. (B) Quantification of the significant (P<0.05) increase in the destabilizing T187 phosphorylation on Kip1 during SW-induced maturation. (C) As with TOR signaling, AMPK agonists AICAR or resveratrol (res) prevent both SW-induced MPF activation (i.e. no decreases in inhibitory Y15-CDC2 phosphorylations) and phospho-187 Kip1 signals, whereas co-treatment with cAMP elevators (e.g. forskolin (for) or IBMX (ibm)) rescue such blockages. BF=before treatment; other lanes on blot C and graphs B, E=control and drug treatments at 1.5 h post-stimulation.
Citation: REPRODUCTION 142, 1; 10.1530/REP-10-0509
Discussion
Phosphorylation changes during AMPK deactivation in maturing nemertean oocytes
Based on data reported here and elsewhere ( Stricker et al. 2010a), AMPK activity declines during oocyte maturation in the nemertean worm Cerebratulus, and various AMPK agonists serve to block SW-induced GVBD, collectively indicating that AMPK deactivation is required for such GVBD. In this investigation, several proteins in the AMPK signaling pathway are shown to undergo phosphorylation changes during maturation. In general, such changes could involve either altered activities of upstream modulators acting upon already present proteins or changes in the overall amounts of proteins present in the oocyte. Currently, neither the levels of protein neosynthesis nor the proportions of phosphorylated pools relative to non-phosphorylated proteins are known for maturing oocytes, since total protein levels have yet to be analyzed, except for Kip1 where increased T187 phosphorylation is accompanied by decreased pan-Kip levels. However, given that high concentrations of protein synthesis inhibitors fail to keep nemertean oocytes from maturing ( Stricker & Smythe 2001, Smythe & Stricker 2005; SA Stricker, unpublished observation), any new proteins added following maturation stimulation are presumably not required for GVBD. Moreover, regardless of whether AMPK signaling utilizes extant or newly synthesized proteins during GVBD, the activities of such proteins are nevertheless modulated by phosphorylations that can be tracked by phospho-specific antibodies. Accordingly, this study reveals highly repeatable differences in the overall amounts of several phosphorylations, based on multiple comparisons of pre- and post-treatment levels to control values in similar amounts of total cellular protein and oocytes loaded per lane. Furthermore, coupled with concordant data obtained using pharmacological modulators as well as verifications involving appropriate changes in the phosphorylations of downstream targets, such findings can collectively provide insight into maturation-associated alterations in the activities of possible upstream modulators and downstream targets of AMPK, as discussed further below.
Potential upstream regulators of AMPK activity in nemertean oocytes
In somatic cells, LKB1 upregulates AMPK by phosphorylating AMPK's T172 site ( Alessi et al. 2006, Williams & Brenman 2008), and LKB1 homologues are also known to regulate polarity in oocytes ( Pellettieri & Seydoux 2002). However, as novel evidence for LKB1 potentially blocking GVBD, immature nemertean oocytes display elevated T172 phosphorylation on active AMPK along with an apparently active LKB1-like kinase that seems to decrease its activity as oocytes mature and T172 undergoes dephosphorylation. Precisely which components of the intraovarian milieu serve to activate LKB1 in immature nemertean oocytes remain unknown. Moreover, whether these oocytes possess other AMPKKs such as CaMKKβ ( Woods et al. 2005) or TAK1 ( Xie et al. 2006) awaits additional testing.
In any case, it seems unlikely that upregulating LKB1 activity is the only way that AMPK agonists activate AMPK, since the AMPK stimulator AICAR maintains T172 phosphorylation in SW-stimulated oocytes and yet fails to keep LKB1’s S428 site phosphorylated. It should be noted, however, that in some cells, S428 phosphorylation is either not required for LKB1 activity ( Fogarty & Hardie 2009) or associated with LKB1 deactivation ( Zheng et al. 2009), suggesting that reduced S428 phosphorylations in nemertean oocytes might not be indicative of decreased LKB1 activity. Alternatively, lowered S428 phosphorylation may indeed reflect LKB1 deactivation, and as reported for somatic cells, AICAR could stimulate AMPK in an AMPKK-independent manner by downregulating the phosphatase that normally dephosphorylates AMPK's T172 site ( Sakamoto et al. 2004, Sanders et al. 2007).
In the case of ERK1/2 MAPK which can deactivate AMPK in somatic cells ( Zheng et al. 2009), AMPK downregulation during nemertean GVBD occurs independently of such MAPK, given that MAPK inhibitions via CI1040 or PD98059 fail to prevent GVBD or AMPK deactivation. Alternatively, some oocyte batches treated with SW solutions of the MAPKK inhibitor U0126 do not undergo GVBD. Nevertheless, when co-stimulated with SW+cAMP elevators, nearly all U0126-treated oocytes mature in the absence of marked MAPK activity ( Stricker 2009b), and as is the case with somatic cell extracts where U0126 has been shown to activate AMPK ( Dokladda et al. 2005), U0126 reduces the amount of T172 dephosphorylation compared to controls in SW alone. Thus, U0126 does not seem to block GVBD by downregulating MAPK per se but rather by ectopically activating AMPK. Accordingly, although U0126-mediated AMPK activation is not universally reported ( Kayampilly & Menon 2009), results presented here and elsewhere ( LaRosa & Downs 2005) suggest that analyses relying exclusively on U0126 should be interpreted with caution.
Contrary to findings that T172 dephosphorylation proceeds independently of ERK1/2 MAPK, oocytes lacking such MAPK activity display reduced S485/491 phosphorylations on AMPK. These data suggest that MAPK is required for AMPK's S485/491 phosphorylations and also have implications for multiple pathways potentially regulating nemertean GVBD. For example, in previous tests of AMPK agonists ( Stricker et al. 2010a), immature nemertean oocytes always exhibited high phospho-T172 and low phospho-S485/491 signals, whereas mature specimens invariably displayed the opposite pattern of these phosphorylations. Moreover, GVBD and S485/491 phosphorylation were significantly reduced in SW solutions of the protein kinase B (PKB) inhibitor API-2, whereas maturation and S485/491 phosphorylations were restored, when API-2 was dissolved in SW+cAMP elevators ( Stricker et al. 2010a). Collectively, such findings indicated that S485/491 phosphorylation promoted AMPK deactivation and that such phosphorylation might be mediated by PKB in SW versus by cAMP-dependent protein kinase A (PKA) in the presence of cAMP elevators, based on the API-2 results and the fact that both PKB and PKA phosphorylate S485/491 in somatic cells ( Horman et al. 2006, Hurley et al. 2006).
However, given the results obtained here with MAPKK inhibitors, S485/491 phosphorylation is evidently non-essential for T172 dephosphorylation. Nevertheless, S485/491 phosphorylation might downregulate AMPK during normal GVBD where MAPK is activated, whereas in the absence of S485/491 phosphorylation, a different mode of dephosphorylating T172 such as phosphorylation at AMPK's S173 site ( Djouder et al. 2010) could compensate for such a loss. To test these hypotheses as well as to determine which signals downregulate AMPKK during AMPK deactivation requires additional analyses employing more precise means of altering and assessing AMPK phosphorylation status.
Potential downstream targets of AMPK deactivation during nemertean GVBD
Based on tests with SW solutions of the MPF inhibitor roscovitine, T172 dephosphorylation continues in SW-stimulated oocytes with low MPF levels, suggesting that T172 dephosphorylation occurs upstream to, and independently of, MPF activation. Conversely, roscovitine-mediated MPF deactivation causes a downward mobility shift and decrease in S485/491 phosphorylations that are not simply due to a lack of MAPK activity. Collectively, such findings suggest that, along with MAPK, MPF may facilitate full S485/491 phosphorylation via positive feedback between AMPK deactivation and MPF activation.
As for possible steps between AMPK deactivation and MPF activation, data presented here coincide with those reported for somatic cells ( Bolster et al. 2002, Kimura et al. 2003) by indicating that active AMPK inhibits TOR. Accordingly, TOR upregulation as AMPK levels decline in maturing oocytes may trigger meiotic resumption via MPF activation in a manner analogous to mitosis induction by constitutively active TOR in cancerous cells ( Wullschleger et al. 2006). However, exactly how TOR might activate MPF in nemertean oocytes remains unclear.
Alternatively, AMPK can inhibit TOR through TSC2 activation ( Inoki et al. 2003, Coradetti et al. 2004, Abraham 2005), which in turn also stabilizes the CDK inhibitor Kip1 ( Rosner et al. 2006). Accordingly, Kip1 levels appear to be elevated in immature oocytes and subsequently downregulated after AMPK deactivation in a manner resembling that described for rat follicle cells ( Kayampilly & Menon 2009). Such findings suggest that, as an alternative to possible TOR-mediated signaling, MPF activation could be due to decreased TSC2 activity in the wake of AMPK deactivation that might not only activate TOR but also downregulate the MPF inhibitor Kip1. However, currently no commercially available antibody for monitoring TSC2 activity has been found to successfully cross-react with nemertean oocytes. Thus, testing these hypotheses awaits genomic sequencing as well as more specific means to manipulate and track the activities of such potential mediators of AMPK signaling.
Nevertheless, regardless of how AMPK regulates MPF activity, the question remains as to why AMPK blocks GVBD in nemerteans and cumulus-enclosed oocytes of some mammals, whereas AMPK stimulates GVBD in mice. One possible explanation involves the fact that AMPKs are heterotrimers that comprise varying combinations of α, β, and γ subunits ( Hardie 2007). Accordingly, disparate effects of AMPK on GVBD could be due to alternative types of AMPKs, given that in somatic cells AMPKs can exhibit isotype-specific responses to modulating stimuli ( Salt et al. 1998, Higaki et al. 2001, Zierath 2002). In any case, data reported here and summarized in Fig. 6 not only provide added support for the view that multiple pathways can mediate AMPK activity during stimulation of nemertean oocytes by SW versus cAMP elevators ( Stricker et al. 2010a, 2010b) but also generate new findings as well as hypotheses to test regarding intraoocytic AMPK signaling in general.
Summary of multiple pathways potentially mediating intraoocytic AMPK signaling during GVBD in a marine nemertean worm. AMPK, AMP-activated kinase composed of α, β, γ subunits; GVBD, germinal vesicle breakdown; Kip1, p27 Kip1 cyclin-dependent kinase; LKB1, AMPK kinase; MAPK, ERK1/2 mitogen-activated kinase; MPF, maturation-promoting factor composed of Cyclin B and CDC2 kinase; P, phosphorylation; TOR, target of rapamycin; TSC2, tuberous sclerosis complex 2.
Citation: REPRODUCTION 142, 1; 10.1530/REP-10-0509
Materials and Methods
Fully-grown immature oocytes lacking follicle cells but containing an intact germinal vesicle ( Stricker & Schatten 1989) were obtained from gravid specimens of an undescribed species of the nemertean genus Cerebratulus that were collected during summers in an intertidal region on San Juan Island, WA, USA ( Stricker 1987, Stricker et al. 2010a, 2010b, 2010c). Isolated oocytes were pre-incubated ∼2 h in ice-cold calcium-free seawater (CaFSW) to reduce spontaneous GVBD and subsequently dejellied via a 150-μm Nitex filter before being gently pelleted. Using such methods, spontaneous GVBD levels were typically 0–10% before the start of experiments, based on inverted microscopy of uncompressed living oocytes. Moreover, at 12–14 °C, GVBD and metaphase-I arrest began by ∼20 and ∼90 min respectively, in dejellied controls that had been stimulated to mature by SW or cAMP elevators (10 μM forskolin (LC Labs, Woburn, MA, USA) or 150 μM IBMX (EMD Chemicals, Gibbstown, NJ, USA) dissolved in SW or CaFSW). For most runs, a drop of ∼200 dejellied oocytes suspended in CaFSW was either directly pelleted and frozen in liquid nitrogen (LN) after fluid removal (the ‘Before’ sample) or added to 1 ml of test solution kept at 12–14 °C for 90–120 min. The incubated samples were then assessed for GVBD, pelleted, and frozen in LN. Alternatively, for time-lapse sequences, 3–5 drops of oocytes were added to 4 ml test solution, and pelleted aliquots were frozen in LN several times during the run.
For manipulations of AMPK signaling, drugs were routinely dissolved in solutions of SW or SW+cAMP elevators, and to reduce the likelihood of off-target drug effects ( Davies et al. 2000), the following doses intermediate between half- and maximally effective concentrations were adopted: 300 μM AICAR, 20 μM resveratrol (Enzo Life Sciences, Plymouth Meeting, PA, USA); 30 μM roscovitine, 25 μM U0126 (LC Labs); 250 μM olomoucine, 50 μM PD98059 (Tocris Biosciences, Ellisville, MO, USA); and 25 μM CI1040 (a gift from Pfizer, Inc., Groton, CT, USA). In all experimental incubations, oocytes were continuously treated with a drug solution that had been pre-mixed ∼0.5–1 h before the onset of the run and equilibrated to 12–14 °C prior to addition of oocytes.
For immunoblotting analyses, LN-frozen samples were lysed in a protease- and phosphatase-containing buffer ( Carroll et al. 2000), and based on Bradford assays of total-cell lysates, 25 μg cellular protein/lane was loaded on 4%/10% SDS-PAGE gels run at 100 kV for 2–2.5 h. Following transfer to PVDF membranes for 75 min at 100 mV and subsequent blocking for 1 h in 5% dry milk in Tween 20-containing Tris-buffered saline (TTBS), blots were incubated overnight at 4 °C in TTBS containing 1–5% BSA plus primary antibody that was obtained from suppliers and typically diluted as follows: from Cell Signaling Technology (Danvers, MA, USA) – 1:1000 phospho-T172 AMPK #2535; 1:2500 phospho-S485/491 AMPK #4185; 1:2000 phospho-Y15 CDC2 #9111; 1:1500 phospho-T161 CDC2 #9114; 1:2000 phospho-S428 LKB1 #3482; 1:1500 phospho-S2448 mTOR #2971; 1:2000 phospho-T398 Drosophila p70/85 S6K #9209; 1:2500 phospho-T202/T204 ERK1/2 MAPK#9101; from Genscript (Piscataway, NJ, USA) – 1:1000 pan p27 Kip1 #A00436, 1:2000 phospho-T187 p27 Kip1#A00333; or from Millipore (Billerca, MA, USA) – 1:4000 phospho-S79 ACC #07-303. Thereafter, blots were treated 1.5 h with 1:5000 HRP-conjugated secondary antibody (#sc-2054, Santa Cruz Biotechnology, Santa Cruz, CA, USA) dissolved in milk-containing TTBS, and bands were visualized by ECL before being recorded on X-ray film using exposures typically 0.5–5 min long.
To assay for bands at expected MWs and ectopic sites, whole PVDF membranes were initially incubated with a single primary antibody. However, after confirming the absence of confounding signals for the following three antibody pairs, subsequent blots were often dually probed with phospho-T172+phospho-S485/491 AMPK, phospho-T172 AMPK+phospho-LKB1, or phospho-MAPK+a phospho-CDC2 antibody (Y15/T161). Alternatively, with MW markers on both sides of the membrane for guidance, each membrane was cut into 3 or 4 horizontal strips that were individually treated with either a single antibody or one of the three above-listed pairs, thereby allowing each composite figure presented here (i.e. stacked set of bands separated by a white line) to originate from a single gel transfer. Moreover, since each collection of strips was probed with antibodies whose signals have been shown to decrease (e.g. pT172 AMPK, pY15 MPF) versus increase (e.g. pS485/491 AMPK, pT161 MPF, pMAPK) during GVBD (Stricker et al. 2006a, 2010a), such parallel incubations with oppositely-trending antibodies helped to verify even gel loadings, since the occasional cases of markedly over- or under-loaded lanes tended to display inappropriately higher or lower signals across all strips and were thus excluded from analysis. In addition, to minimize female-specific variation and blot-to-blot differences in handling, densitometry of background-subtracted bands was quantified for at least three blots from two or more females, and band intensities were expressed relative to pre- and post-stimulation control levels on the same blot. For statistical significance, densitometric data were assessed by a Mann–Whitney U test ( Smythe & Stricker 2005).
Declaration of interest
The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
Parts of this study were funded by NSF Grant 0114319.
Acknowledgements
The use of research facilities at Friday Harbor Laboratories of the University of Washington and the technical assistance provided by L Fowles and P Soltani are gratefully acknowledged.
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