Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University, 300 Pasteur Drive, Stanford, CA 94305, USA.
The EMBO Journal (Impact Factor: 10.43). 01/2007; 25(24):5716-25. DOI: 10.1038/sj.emboj.7601431
Source: PubMed


cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

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Available from: Marco Conti, Aug 18, 2014
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    • "This observation is in accordance with similar findings in Xenopus, where PDE3 inhibition has been shown to attenuate insulin/IGFs action on oocyte GVBD (Sadler, 1991). In mammals, presence of PDE3 in oocytes (Shitsukawa et al., 2001), role of specific PDE3 inhibitor in oocyte maturation (Richard et al., 2001) and activation of PDE3A by Akt/PKB (Han et al., 2006 ) has been confirmed. However in fish, possible involvement of PDE3 in PI3K/Akt regulation of insulin-induced cdc2 activation is reported for the first time. "
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    ABSTRACT: Exposure of fully grown oocytes to growth factors (insulin/IGFs) initiates various signalling cascades that culminate to final stages of oocyte maturation. Regulation of signalling pathways during growth factor-induced meiosis resumption in fish is not well characterized. Here we studied the participation of PI3K/Akt signalling pathway during recombinant human insulin (rh-insulin)-induced meiotic maturation in zebrafish (Danio rerio) oocytes. Priming of defolliculated oocytes in vitro with rh-insulin promotes germinal vesicle breakdown (GVBD) in a dose- and time-dependent manner, an effect sensitive to translation but not transcription inhibition. More than 80% of the oocytes underwent GVBD due to 0.8 IU/ml rh-insulin within 10 h of incubation and the kinetics of p34cdc2 kinase activation corresponded well with GVBD data. PI3K inhibitors, wortmannin and LY294002 blocked insulin, but not 17α, 20β-DHP-induced GVBD. Immunoblot analyses of oocyte extract revealed that phospho-PI3K (p85α) was up regulated within 30-60 min of insulin stimulation followed by phospho-Akt (Ser473) at 60-120 min. Though PI3K/Akt phosphorylation was largely unaffected, pre-incubation with phosphodiesterase (PDE) inhibitors, IBMX and cilostamide, but not rolipram completely blocked rh-insulin-induced p34cdc2 activation and GVBD. These results suggest that PDE3 may be one potential downstream target to PI3K/Akt signalling necessary for rh-insulin-induced GVBD in zebrafish.
    Full-text · Article · Apr 2013 · Molecular and Cellular Endocrinology
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    • "The results of two recent studies (Norris et al, 2009; Vaccari et al, 2009) strongly support this idea and show that cGMP acts by blocking the activity of PDE3A, the primary oocyte cAMP phosphodiesterase (Richard et al, 2001; Shitsukawa et al, 2001). It follows that the decrease in ovarian cGMP at the time of the preovulatory gonadotropin surge accounts for the resulting increase in cAMP phosphodiesterase activity that drives meiotic resumption (Masciarelli et al, 2004, Han et al, 2006). "
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    ABSTRACT: Cumulus cell-enclosed oocytes (CEO), denuded oocytes (DO), or dissected follicles were obtained 44-48 hr after priming immature mice (20-23 days old) with 5 IU or immature rats (25-27 days old) with 12.5 IU of equine chorionic gonadotropin, and exposed to a variety of culture conditions. Mouse oocytes were more effectively maintained in meiotic arrest by hypoxanthine, dbcAMP, IBMX, milrinone, and 8-Br-cGMP. Atrial natriuretic peptide, a guanylate cyclase activator, suppressed maturation in CEO from both species, but mycophenolic acid reversed IBMX-maintained meiotic arrest in mouse CEO with little activity in rat CEO. IBMX-arrested mouse, but not rat, CEO were induced to undergo germinal vesicle breakdown (GVB) by follicle-stimulating hormone (FSH) and amphiregulin, while human chorionic gonadotropin (hCG) was ineffective in both species. Nevertheless, FSH and amphiregulin stimulated cumulus expansion in both species. FSH and hCG were both effective inducers of GVB in cultured mouse and rat follicles while amphiregulin was stimulatory only in mouse follicles. Changing the culture medium or altering macromolecular supplementation had no effect on FSH-induced maturation in rat CEO. The AMP-activated protein kinase (AMPK) activator, AICAR, was a potent stimulator of maturation in mouse CEO and DO, but only marginally stimulatory in rat CEO and ineffective in rat DO. The AMPK inhibitor, compound C, blocked meiotic induction more effectively in hCG-treated mouse follicles and heat-treated mouse CEO. Both agents produced contrasting results on polar body formation in cultured CEO in the two species. Active AMPK was detected in germinal vesicles of immature mouse, but not rat, oocytes prior to hCG-induced maturation in vivo; it colocalized with chromatin after GVB in rat and mouse oocytes, but did not appear at the spindle poles in rat oocytes as it did in mouse oocytes. Finally, cultured mouse and rat CEO displayed disparate maturation responses to energy substrate manipulation. These data highlight significant differences in meiotic regulation between the two species, and demonstrate a greater potential in mice for control at the level of the cumulus CEO.
    Full-text · Article · Oct 2011 · Molecular Reproduction and Development
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    • "Conversely, in response to maturation-inducing stimuli, increased intraoocytic PDE activity mediated by surrounding follicle cells would decrease intraoocytic cAMP levels (Norris et al., 2009) and presumably activate AMPK via both dephosphorylation of S485/491 and a concomitant increase in T172 phosphorylation once the phospho-S485/491 inhibition is lifted (Hurley et al., 2006). However, as opposed to its purported effects in nemerteans, PKB would presumably not be involved in phosphorylating S485/491 and thereby deactivating AMPK in mouse oocytes, since, as is the case with AMPK, PKB stimulates, rather than inhibits, GVBD in mice (Han et al., 2006; Kalous et al., 2006). As for the potential roles played by AMPK activity in the follicle cells of mice, it is known that such cells possess both a1 and a2 subunits of AMPK and that treatment with AMPK activators triggers GVBD in either cumulus-enclosed or denuded mouse oocytes (Downs et al., 2002). "
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    ABSTRACT: Previous studies have shown that elevations in intraoocytic cAMP prevent mammalian oocytes from maturing, whereas cAMP degradation allows these oocytes to begin maturation, as evidenced by the onset of oocyte nuclear disassembly (="germinal vesicle breakdown", GVBD). Moreover, such cAMP degradation not only reduces cAMP levels but also generates AMP, which in turn can stimulate AMP-activated kinase (AMPK), a well-documented inducer of GVBD in mice. Alternatively, in some marine invertebrates, intraoocytic cAMP triggers, rather than blocks, GVBD, and whether AMPK up- or downregulates maturation in these species has not been tested. Thus, AMPK was monitored in the nemertean worm Cerebratulus during GVBD stimulated by seawater (SW) or cAMP elevators. In oocytes lacking surrounding follicle cells, AMPK activity was initially elevated in immature oocytes but subsequently reduced during SW- or cAMP-induced GVBD, given that the catalytic alpha-subunit of AMPK in maturing oocytes displayed a decreased stimulatory phosphorylation at T172 and an increased inhibitory phosphorylation at S485/491. Accordingly, AMPK-mediated phosphorylation of acetyl-CoA carboxylase, a known target of active AMPK, also declined during maturation. Moreover, treatments with either ice-cold calcium-free seawater (CaFSW) or AMPK agonists dissolved in SW maintained AMPK activity and inhibited GVBD. Conversely, adding cAMP elevators to CaFSW- or SW-solutions of AMPK activators restored GVBD while promoting S485/491 phosphorylation and AMPK deactivation. Collectively, such findings not only demonstrate for the first time that intraoocytic AMPK can block GVBD in the absence of surrounding follicle cells, but these results also provide evidence for a novel GVBD-regulating mechanism involving AMPK deactivation by cAMP-mediated S485/491 phosphorylation.
    Preview · Article · Jun 2010 · Molecular Reproduction and Development
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