Rodent oocytes express an active adenylyl cyclase required for meiotic arrest

The intracellular levels of cAMP play a critical role in the meiotic arrest of mammalian oocytes. However, it is debated whether this second messenger is produced endogenously by the oocytes or is maintained at levels inhibitory to meiotic resumption via diffusion from somatic cells. Here, we demonstrate that adenylyl cyclase genes and corresponding proteins are expressed in rodent oocytes. The mRNA coding for the AC3 isoform of adenylyl cyclase was detected in rat and mouse oocytes by RT-PCR and by in situ hybridization. The expression of AC3 protein was confirmed by immunocytochemistry and immunofluorescence analysis in oocytes in situ. Cyclic AMP accumulation in denuded oocytes was increased by incubation with forskolin, and this stimulation was abolished by increasing intraoocyte Ca(2+) with the ionophore A23187. The Ca(2+) effects were reversed by an inhibitor of Ca(2+), calmodulin-dependent kinase II. These regulations of cAMP levels indicate that the major cyclase that produces cAMP in the rat oocyte has properties identical to those of recombinant or endogenous AC3 expressed in somatic cells. Furthermore, mouse oocytes deficient in AC3 show signs of a defect in meiotic arrest in vivo and accelerated spontaneous maturation in vitro. Collectively, these data provide evidence that an adenylyl cyclase is functional in rodent oocytes and that its activity is involved in the control of oocyte meiotic arrest.     

Lipoic acid stimulates cAMP production via G protein-coupled receptor-dependent and -independent mechanisms

Lipoic acid (LA) is a naturally occurring fatty acid that exhibits anti-oxidant and anti-inflammatory properties and is being pursued as a therapeutic for many diseases including multiple sclerosis, diabetic polyneuropathy and Alzheimer's disease. We previously reported on the novel finding that racemic LA (50:50 mixture of R-LA and S-LA) stimulates cAMP production, activates prostanoid EP2 and EP4 receptors and adenylyl cyclases (AC), and suppresses activation and cytotoxicity in NK cells. In this study, we present evidence that furthers our understanding of the mechanisms of action of LA. Using various LA derivatives, such as dihydrolipoic acid (DHLA), S,S-dimethyl lipoic acid (DMLA) and lipoamide (LPM), we discovered that only LA is capable of stimulating cAMP production in NK cells. Furthermore, there is no difference in cAMP production after stimulation with either R-LA, S-LA or racemic LA. Competition and synergistic studies indicate that LA may also activate AC independent of the EP2 and EP4 receptors. Pretreatment of PBMCs with KH7 (a specific peptide inhibitor of soluble AC) and the calcium inhibitor (Bapta) prior to LA treatment resulted in reduced cAMP levels, suggesting that soluble AC and calcium signaling mediate LA stimulation of cAMP production. In addition, pharmacological inhibitor studies demonstrate that LA also activates other G protein-coupled receptors, including histamine and adenosine but not the β-adrenergic receptors. These novel findings provide information to better understand the mechanisms of action of LA, which can help facilitate the use of LA as a therapeutic for various diseases.     

New insights into the regulation of cAMP synthesis beyond GPCR/G protein activation: implications in cardiovascular regulation

Regulation of intracellular concentrations of cyclic AMP is one of the most ubiquitous mechanisms for regulating cellular functions. Further, the manner in which cAMP production is regulated via G proteins at the level of adenylyl cyclase activation has been studied extensively. This review focuses instead on the recently identified mechanisms and roles for regulation of adenylyl cyclase functions beyond G protein activation. These mechanisms include: a) the coupling of particular isoforms of adenylyl cyclase to function within a single cell type b) regulation of membrane trafficking of higher order enzyme aggregates and c) raf kinase-dependent phosphorylation and sensitization of adenylyl cyclases--an important pathway for crosstalk between tyrosine kinase signaling cascades with regulation of cAMP-mediated responses.     

Generation of mouse oocytes defective in cAMP synthesis and degradation: endogenous cyclic AMP is essential for meiotic arrest

Although it is established that cAMP accumulation plays a pivotal role in preventing meiotic resumption in mammalian oocytes, the mechanisms controlling cAMP levels in the female gamete have remained elusive. Both production of cAMP via GPCRs/Gs/adenylyl cyclases endogenous to the oocyte as well as diffusion from the somatic compartment through gap junctions have been implicated in maintaining cAMP at levels that preclude maturation. Here we have used a genetic approach to investigate the different biochemical pathways contributing to cAMP accumulation and maturation in mouse oocytes. Because cAMP hydrolysis is greatly decreased and cAMP accumulates above a threshold, oocytes deficient in PDE3A do not resume meiosis in vitro or in vivo, resulting in complete female infertility. In vitro, inactivation of Gs or downregulation of the GPCR GPR3 causes meiotic resumption in the Pde3a null oocytes. Crossing of Pde3a^−/− mice with Gpr3^−/− mice causes partial recovery of female fertility. Unlike the complete meiotic block of the Pde3a null mice, oocyte maturation is restored in the double knockout, although it occurs prematurely as described for the Gpr3^−/− mouse. The increase in cAMP that follows PDE3A ablation is not detected in double mutant oocytes, confirming that GPR3 functions upstream of PDE3A in the regulation of oocyte cAMP. Metabolic coupling between oocytes and granulosa cells was not affected in follicles from the single or double mutant mice, suggesting that diffusion of cAMP is not prevented. Finally, simultaneous ablation of GPR12, an additional receptor expressed in the oocyte, does not modify the Gpr3^−/− phenotype. Taken together, these findings demonstrate that Gpr3 is epistatic to Pde3a and that fertility as well as meiotic arrest in the PDE3A-deficient oocyte is dependent on the activity of GPR3. These findings also suggest that cAMP diffusion through gap junctions or the activity of additional receptors is not sufficient by itself to maintain the meiotic arrest in the mouse oocyte.     

The G-protein-coupled receptors GPR3 and GPR12 are involved in cAMP signaling and maintenance of meiotic arrest in rodent oocytes

In mammalian and amphibian oocytes, the meiotic arrest at the G2/M transition is dependent on cAMP regulation. Because genetic inactivation of a phosphodiesterase expressed in oocytes prevents reentry into the cell cycle, suggesting autonomous cAMP synthesis, we investigated the presence and properties of the G-protein-coupled receptors (GPCRs) in rodent oocytes. The pattern of expression was defined using three independent strategies, including microarray analysis of GV oocyte mRNAs, EST database scanning, and RT-PCR amplification with degenerated primers against transmembrane regions conserved in the GPCR superfamily. Clustering of the GPCR mRNAs from rat and mouse oocytes indicated the expression of the closely related Gpr3, Gpr12, and Edg3, which recognize sphingosine and its metabolites as ligands. Expression of these mRNAs was confirmed by RT-PCR with specific primers as well as by in situ hybridization. That these receptors are involved in the control of cAMP levels in oocytes was indicated by the finding that expression of the mRNA for Gpr3 and Gpr12 is downregulated in Pde3a-deficient oocytes, which have a chronic elevation of cAMP levels. Expression of GPR3 or GPR12 in Xenopus laevis oocytes prevented progesterone-induced meiotic maturation, whereas expression of FSHR had no effect. A block in spontaneous oocyte maturation was also induced when Gpr3 or Gpr12 mRNA was injected into mouse oocytes. Downregulation of GPR3 and GPR12 caused meiotic resumption in mouse and rat oocytes, respectively. However, ablation of the Gpr12 gene in the mouse did not cause a leaky meiotic arrest, suggesting compensation by Gpr3. Incubation of mouse oocytes with the GPR3/12 ligands SPC and S1P delayed spontaneous oocyte maturation. We propose that the cAMP levels required for maintaining meiotic arrest in mouse and rat oocytes are dependent on the expression of Gpr3 and/or Gpr12.     

Altered cAMP signaling induced by lysophosphatidic acid in senescent human diploid fibroblasts

Lysophosphatidic acid (LPA) is a lipid mitogen that acts through G-protein-coupled receptors. LPA responsiveness has been reported to be dependent on the senescent state of the cells. To solve the mechanism underlying, we observed LPA-dependent cAMP status and found its age-dependent contrasting profile such as high level of cAMP in the senescent cells vs its low level in the young cells. In order to clarify the molecular mechanism of the ageing effect, we examined various molecular species involved in the cAMP signaling pathway by semi-quantitative RT-PCR. EDG-1 and EDG-4 were unchanged, but EDG-2 and EDG-7 were reduced with age. Senescent cells showed a partial reduction of Gi1, Gi2, and Gi3, but no change in the level of Gq. Decreased Gis and Gi-coupled LPA receptors may reduce the inhibitory effect of Gi alpha on adenylyl cyclases (ACs), resulting in cAMP accumulation via activation of adenylyl cyclase in senescent fibroblasts. We also observed an age-dependent increase in some of AC isoforms: II, IV, and VI. In conclusion, multiple changes in the cAMP signaling pathway of the senescent cells might explain the altered responsiveness to the mitogenic stimuli.     

A G(s)-linked receptor maintains meiotic arrest in mouse oocytes, but luteinizing hormone does not cause meiotic resumption by terminating receptor-G(s) signaling

The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a G(s) G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, G(s) is activated by a constitutively active orphan receptor, GPR3. To determine whether the action of luteinizing hormone (LH) on the mouse ovarian follicle causes meiotic resumption by inhibiting GPR3-G(s) signaling, we examined the effect of LH on the localization of Galpha(s). G(s) activation in response to stimulation of an exogenously expressed beta(2)-adrenergic receptor causes Galpha(s) to move from the oocyte plasma membrane into the cytoplasm, whereas G(s) inactivation in response to inhibition of the beta(2)-adrenergic receptor causes Galpha(s) to move back to the plasma membrane. However, LH does not cause a change in Galpha(s) localization, indicating that LH does not act by terminating receptor-G(s) signaling.     

Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblasts

Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3',5'-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs alpha/beta both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gialpha in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC alpha/beta and AC4/6.     

Role of an adenylyl cyclase isoform in ethanol's effect on cAMP regulated gene expression in NIH 3T3 cells

Previous research has indicated that the cyclic AMP (cAMP) signal transduction system plays an important role in the predisposition to and development of ethanol abuse in humans. Our laboratory has demonstrated that ethanol is capable of enhancing adenylyl cyclase (AC) activity. This effect is AC isoform-specific; type 7 AC (AC7) is most enhanced by ethanol. Therefore, we hypothesized that the expression of a specific AC isoform will play a role on the effect of ethanol on cAMP regulated gene expression. We employed NIH 3T3 cells transfected with AC7 or AC3 as a model system. To evaluate ethanol's effects on cAMP regulated gene expression, a luciferase reporter gene driven by a cAMP inducing artificial promoter was utilized. Stimulation of AC activity leads to an increase in the reporter gene activity. This increase was enhanced in the presence of ethanol in cells expressing AC7, while cells expressing AC3 did not respond to ethanol. cAMP reporter gene expression was increased in the presence of 8-bromo-cAMP; this expression was not enhanced by ethanol. These observations are consistent with our hypothesis. The basal level of CREB phosphorylation was high and did not change by cAMP stimulation or in the presence of ethanol. However, there were significant changes in the TORC3 amount in nuclei depending on stimulation conditions. The results suggest that nuclear translocation of TORC3 plays a more important role than CREB phosphorylation in the observed changes in the cAMP driven reporter gene activity.     

Effects of phosphodiesterase inhibitors on spontaneous nuclear maturation and cAMP concentrations in bovine oocytes

It was previously demonstrated that inhibition of cAMP degradation with phosphodiesterase type 3 (PDE3) inhibitors resulted in the maintenance of bovine cumulus–oocyte complexes (COC) and denuded oocytes (DO) in meiotic arrest, while a PDE4 inhibitor was without effect. In this study, different inhibitors of PDE3 and PDE4 were tested for their effects on bovine oocyte nuclear maturation. Bovine COC and DO were cultured in TCM-199+10% fetal bovine serum (FBS) with or without different concentrations of the PDE inhibitors. The PDE3 inhibitor trequinsin significantly increased the percentage of COC remaining at the germinal vesicle (GV) stage after 7 h of culture (19.3, 60.3, and 67.8% GV for control and trequinsin 10 and 50 nM, respectively) while Ro 20-1724 (a PDE4 inhibitor) was without effect. In DO, only trequinsin at 10 nM had a significant effect after 7 h of culture (51.3 and 86.1% GV for control and trequinsin 10 nM, respectively). Trequinsin reduced the percentage of COC reaching the mature phase after 22 h, but was without effect on DO. The protein kinase A (PKA) inhibitor H-89 reversed the inhibitory effect of trequinsin in COC and DO, indicating that inhibition of nuclear maturation by trequinsin involves activation of PKA. Trequinsin increased cAMP concentrations in COC but not in DO, suggesting that cumulus cells may also contain a PDE3 isoenzyme.     

Galphaq reduces cAMP production by decreasing Galphas protein abundance

The heterotrimeric guanine nucleotide-binding protein Gαq transduces signals from heptahelical transmembrane receptors (e.g., α₁-adrenergic, endothelin 1A, and angiotensin II) to stimulate generation of inositol-1,4,5-trisphosphate and diacylglycerol. In addition, Gαq decreases cAMP production, through unknown mechanisms, and thus affects physiological responsiveness of cardiac myocytes and other cells. Here, we provide evidence that Gαq expression increases Gαs ubiquitination, decreases Gαs protein content, and impairs basal and β₁-adrenergic receptor-stimulated cAMP production. These biochemical and functional changes are associated with Akt activation. Expression of constitutively active Akt also decreases Gαs protein content and inhibits basal and β₁-adrenergic receptor-stimulated cAMP production. Akt knockdown inhibits Gαq-induced reduction of Gαs protein. In addition, MDM2, an E3 ubiquitin ligase, binds Gαs and promotes its degradation. Therefore, increased expression of Gαq decreases cAMP production through Akt-mediated Gαs protein ubiquitination and proteasomal degradation.     

Adenylyl Cyclase Activity in Postmortem Human Brain: Evidence of Altered G Protein Mediation in Alzheimer''s Disease

The effects of agonal status, postmortem delay, and age on human brain adenylyl cyclase activity were determined in membrane preparations of frontal cortex from a series of 18 nondemented subjects who had died with no history of neurological or psychiatric disease. Basal and guanosine 5'-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were not significantly reduced over an interval from death to postmortem of between 3 and 37 h and were also not significantly different between individuals dying with a long terminal phase of an illness and those dying suddenly. Basal and aluminum fluoride-stimulated enzyme activities showed a negative correlation with increasing age of the individual. In subsequent experiments, basal and guanosine 5'-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were compared in five brain regions from a series of eight Alzheimer's disease and seven matched nondemented control subjects. No significant differences were observed between the groups for either basal activity or activities in response to forskolin stimulation of the catalytic subunit of the enzyme. In contrast, enzyme activities in response to stimulation with guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride were significantly reduced in preparations of neocortex and cerebellum from the Alzheimer's disease cases compared with the nondemented controls. Lower guanosine 5'-O-(3-thiotriphosphate)-, but not aluminum fluoride-, stimulated activity was also observed in preparations of frontal cortex from a group of four disease controls compared with nondemented control values. The disease control group, which contained Parkinson's disease and progressive supranuclear palsy patients, showed increased forskolin-stimulated activity compared with both the nondemented control and the Alzheimer's disease groups. These findings indicate a widespread impairment of G protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain, which occurs in the absence of altered enzyme catalytic activity and which is unlikely to be the result of non-disease-related factors associated with the nature of terminal illness of individuals.     

The involvement of cAMP in the growth inhibition of filamentous fungus Rhizopus nigricans by steroids

Several steroids, in particular progesterone, are toxic for the filamentous fungus Rhizopus nigricans and, at high concentrations, inhibit its growth. Previous studies on this microorganism revealed progesterone specific receptors coupled to G proteins at the plasma membrane. In this study, the next step of steroid signalling in R. nigricans following G protein activation is investigated, together with the possible impact of this pathway on fungal growth inhibition. The intracellular level of cAMP decreased in the presence of steroids, demonstrating the probable involvement of cAMP signalling in the response of R. nigricans to steroids. Results of the growth analysis in the presence of cAMP increasing agents suggest that the role of cAMP in fungal growth inhibition by steroids cannot be ruled out, but it would appear to be minor and not make a major contribution to growth inhibition.     

Targeting G protein-coupled receptor signaling in asthma

The complex disease asthma, an obstructive lung disease in which excessive airway smooth muscle (ASM) contraction as well as increased ASM mass reduces airway lumen size and limits airflow, can be viewed as a consequence of aberrant airway G protein-coupled receptor (GPCR) function. The central role of GPCRs in determining airway resistance is underscored by the fact that almost every drug used in the treatment of asthma directly or indirectly targets either GPCR–ligand interaction, GPCR signaling, or processes that produce GPCR agonists. Although many airway cells contribute to the regulation of airway resistance and architecture, ASM properties and functions have the greatest impact on airway homeostasis. The theme of this review is that GPCR-mediated regulation of ASM tone and ASM growth is a major determinant of the acute and chronic features of asthma, and multiple strategies targeting GPCR signaling may be employed to prevent or manage these features.     

GTPase-activating protein Elmod2 is essential for meiotic progression in mouse oocytes

Meiotic failure in oocytes is the major determinant of human zygote-originated reproductive diseases, the successful accomplishment of meiosis largely relay on the normal functions of many female fertility factors. Elmod2 is a member of the Elmod family with the strongest GAP (GTPase-activating protein) activity; although it was identified as a possible maternal protein, its actual physiologic role in mammalian oocytes has not been elucidated. Herein we reported that among Elmod family proteins, Elmod2 is the most abundant in mouse oocytes, and that inhibition of Elmod2 by specific siRNA caused severe meiotic delay and abnormal chromosomal segregation during anaphase. Elmod2 knockdown also significantly decreased the rate of oocyte maturation (to MII, with first polar body extrusion), and significantly greater numbers of Elmod2-knockdown MII oocytes were aneuploid. Correspondingly, Elmod2 knockdown dramatically decreased fertilization rate. To investigate the mechanism(s) involved, we found that Elmod2 knockdown caused significantly more abnormal mitochondrial aggregation and diminished cellular ATP levels; and we also found that Elmod2 co-localized and interacted with Arl2, a GTPase that is known to maintain mitochondrial dynamics and ATP levels in oocytes. In summary, we found that Elmod2 is the GAP essential to meiosis progression of mouse oocytes, most likely by regulating mitochondrial dynamics.     

Developmentally acquired PKA localisation in mouse oocytes and embryos

Localisation of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs) is known to coordinate localised signalling complexes that target cAMP-mediated signalling to specific cellular sub-domains. The cAMP PKA signalling pathway is implicated in both meiotic arrest and meiotic resumption, thus spatio-temporal changes in PKA localisation during development may determine the oocytes response to changes in cAMP. In this study we aim to establish whether changes in PKA localisation occur during oocyte and early embryo development.Using fluorescently-labelled PKA constructs we show that in meiotically incompetent oocytes PKA is distributed throughout the cytoplasm and shows no punctuate localisation. As meiotic competence is acquired, PKA associates with mitochondria. Immature germinal vesicle (GV) stage oocytes show an aggregation of PKA around the GV and PKA remains co-localised with mitochondria throughout oocyte maturation. After fertilisation, the punctuate, mitochondrial distribution was lost, such that by the 2-cell stage there was no evidence of PKA localisation. RT-PCR and Western blotting revealed two candidate AKAPs that are known to be targeted to mitochondria, AKAP1 and D-AKAP2. In summary these data show a dynamic regulation of PKA localisation during oocyte and early embryo development.     

Effect of dibutyryl cAMP on the cAMP content, meiotic progression, and developmental potential of in vitro matured pre-pubertal and adult pig oocytes

Pre-pubertal pig oocytes display reduced developmental competence compared with adult oocytes following in vitro maturation (IVM). Exposure to dibutyryl cyclic adenosine monophosphate (dbcAMP) for the first 20 hr IVM improves development of pre-pubertal oocytes, suggesting that their cAMP content may be inadequate. This study examined the effect of 1 mM dbcAMP treatment for the first 22 hr of IVM on the cAMP content, meiotic progression, and embryo development of pre-pubertal and adult oocytes. In control groups, a two-fold increase in cAMP was observed in adult oocytes after 22 hr IVM, with no change in pre-pubertal oocyte cAMP content. At 22 hr IVM, dbcAMP treatment resulted in two- and five-fold increases in pre-pubertal and adult oocyte cAMP, respectively. After 22 hr control IVM, a greater proportion of pre-pubertal oocytes occupied metaphase I (MI) compared with adult oocytes (69% vs. 49%). dbcAMP treatment reduced the proportion of pre-pubertal and adult oocytes in MI stage at 22 hr. Despite dbcAMP treatment, the proportion of pre-pubertal oocytes in the MI stage at 22 hr remained higher than that of adult oocytes. In control groups, adult oocytes displayed a greater ability to form blastocysts compared with pre-pubertal oocytes following either parthenogenetic activation (59% vs. 25%) or in vitro fertilization (IVF) (47% vs. 19%). dbcAMP treatment increased subsequent blastocyst formation rates of pre-pubertal oocytes, whereas blastocyst formation rates of adult oocytes remained unchanged. Our results suggest that the reduced developmental capacity of pre-pubertal oocytes may be a consequence of their reduced ability to accumulate cAMP during IVM.     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.