Cyclic nucleotide phosphodiesterase 3A–deficient mice as a model of female infertility
Silvia MasciarelliKathleen C. HornerChengyu LiuSun Hee ParkMary D HinckleySteven HockmanTaku NedachiCatherine JinMarco ContiVincent C. Manganiello
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Since cAMP blocks meiotic maturation of mammalian and amphibian oocytes in vitro and cyclic nucleotide phosphodiesterase 3A (PDE3A) is primarily responsible for oocyte cAMP hydrolysis, we generated PDE3A-deficient mice by homologous recombination. The Pde3a–/– females were viable and ovulated a normal number of oocytes but were completely infertile, because ovulated oocytes were arrested at the germinal vesicle stage and, therefore, could not be fertilized. Pde3a–/– oocytes lacked cAMP-specific PDE activity, contained increased cAMP levels, and failed to undergo spontaneous maturation in vitro (up to 48 hours). Meiotic maturation in Pde3a–/– oocytes was restored by inhibiting protein kinase A (PKA) with adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) or by injection of protein kinase inhibitor peptide (PKI) or mRNA coding for phosphatase CDC25, which confirms that increased cAMP-PKA signaling is responsible for the meiotic blockade. Pde3a–/– oocytes that underwent germinal vesicle breakdown showed activation of MPF and MAPK, completed the first meiotic division extruding a polar body, and became competent for fertilization by spermatozoa. We believe that these findings provide the first genetic evidence indicating that resumption of meiosis in vivo and in vitro requires PDE3A activity. Pde3a–/– mice represent an in vivo model where meiotic maturation and ovulation are dissociated, which underscores inhibition of oocyte maturation as a potential strategy for contraception.Keywords:
Germinal vesicle
Cyclic nucleotide phosphodiesterase
Hyper- and hypothyroid states occasionally induce skeletal muscle dysfunction i.e. periodic paralysis and thyroid myopathy. The etiology of these diseases remains unclear, but several findings suggest that the catecholamine-beta-receptor-cAMP system or other messenger systems are disturbed in these diseases. In this context, we evaluated changes in the cyclic 3',5'-nucleotide metabolic enzyme, cyclic 3',5'-nucleotide phosphodiesterase (PDE) and calmodulin concentrations in skeletal muscles of hyper- and hypothyroid rats. Activities of cyclic AMP-PDE were low in skeletal muscle both from hyper- and hypothyroid rats, and calmodulin concentration was high in hyperthyroid and low in hypothyroid rats, as compared with normal rats. DE-52 column chromatographic analysis showed that the cGMP hydrolytic activity in peak I and the cAMP hydrolytic activity in peak II were decreased in hypothyroid rats, whereas cAMP hydrolytic activity in peak III was unchanged. The cAMP hydrolytic activity in peak III was decreased in hyperthyroid rats, but the activities in peaks I and II were unchanged. These findings indicate that cAMP and calmodulin may have some role in skeletal muscle function in the hyperthyroid state, and that cAMP and calmodulin-dependent metabolism may be suppressed in the hypothyroid state.
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Trifluoperazine
Germinal vesicle
Oocyte activation
Cyclic nucleotide phosphodiesterase
EGTA
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Evidence has been presented (1, 2) that the effects of thyrotropin (TSH) and long-acting thyroid stimulator (LATS) on thyroid iodine uptake and discharge may be mediated by cyclic 3′,5′-adenosine monophosphate (C-AMP). Although dibutyryl C-AMP (DBC), concentration 5 mg/0.5 ml, consistently stimulated 131I release in the mouse bioassay in the studies reported herein, addition of the cyclic nucleotide to diluted LATS serum resulted in modest but significant reduction in the bioassay response to the abnormal stimulator. Since both TSH and LATS stimulate thyroidal glucose oxidation and phospholipogenesis, we studied the effects of C-AMP and DBC on 14CO2 production from glucose-1-14C and 32P incorporation into phospholipids in sheep and dog thyroid slices. C-AMP (100–500 μg/ml) failed to increase glucose-1-14C oxidation but DBC at 150–250 μg/ml was active (mean increase 20%). C-AMP at 250–500 μg/ml had a variable stimulatory effect on phospholipogenesis (5/8 experiments, mean increase 16%); BDC at 50–150 μg/ml consistently stimulated 32P incorporation into phospholipids (8/8 experiments, mean increase 55%); lower or higher DBC concentrations were inactive and the maximal response was obtained at 50 μg/ml. However, neither C-AMP nor DBC was shown to potentiate TSH or LATS effects on glucose oxidation and phospholipogenesis in sheep thyroid slices. Indeed, DBC at 50 μg/ml lowered TSH- and LATS-stimulated 32P incorporation into phospholipids (mean 15 and 19% reduction, respectively) although submaximal doses of TSH (10 mU/ml) and LATS (0.5 MRC mU/ml) were used. The failure of C-AMP and DBC to potentiate effective doses of TSH or LATS does not confirm the thesis that the action of these stimulators is, in fact, mediated via increased thyroidal C-AMP. The use of DBC as a tool for investigating mechanisms of hormone action is discussed.
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Basal (medicine)
Adenosine monophosphate
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Protein kinase C(PKC) is a family of Ser/Thr protein kinases commonly distributed in eukaryotic cells.It plays a pivotal role in the regulation of germinal vesicle breakdown(GVBD),chromatin condensation, spindle assembly and extrusion of the first polar body.Activity of PKC regulates GVBD,which characterizes the reinitiation of Meiosis Ⅰ.The activity of PKC rises gradually during the early maturation process and descends during the shift from metaphase to anaphase,which induces the extrusion of the first polar body and characterizes the finish of Meiosis Ⅰ and the transition to Meiosis Ⅱ.Here in this article,the recent advances of research in PKC in the process of Meiosis Ⅰ during mammalian oocyte maturation is reviewed.
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We report the results of experiments which support the hypothesis that, in mouse oocytes, a decrease in intraoocyte cyclic AMP (cAMP) initiates meiotic maturation; oocytes microinjected with cyclic nucleotide phosphodiesterase (PDE) underwent germinal vesicle breakdown (GVBD) in the presence of 3-isobutyl-1-methylxanthine (IBMX), which inhibited GVBD both in oocytes not injected with PDE and in oocytes injected with heat-inactivated PDE. Cyclic AMP-dependent protein kinase (PK) has been proposed to mediate maintenance of meiotic arrest by cAMP. In support of this hypothesis is the observation that 2′-deoxy cAMP, which does not activate PK, did not maintain meiotic arrest as did cAMP; this result was obtained both by microinjection of these compounds and by incubating oocytes in the presence of their membrane-permeable N6-monobutyryl derivatives. Furthermore, microinjection into oocytes of the heat-stable inhibitor of PK, PKI, induced GVBD in the presence of either dibutyryl cAMP (dbcAMP) or IBMX. Meiotic arrest was maintained in the absence of dbcAMP or IBMX, however, by microinjected catalytic subunit of PK, but not by catalytic subunit coinjected with PKI. In addition, specific changes in oocyte phosphoproteins that preceded resumption of meiosis were induced, in the presence of dbcAMP, by microinjected PKI; these changes were also tightly coupled with commitment of oocytes to resume meiosis. These results are discussed in terms of our model for regulation of meiotic arrest and maturation.
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Phosphodiesterase 3
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In the follicles of the mammalian and amphibian ovary, oocyte maturation is arrested at the prophase of the first meiotic division. Prior to ovulation, oocytes reenter the cell cycle, complete the meiotic division, and extrude the first polar body. Work from several laboratories including ours has provided evidence that the cAMP-mediated signal transduction pathway plays an important role in regulation of meiosis, the cyclic nucleotide acting as a negative regulator of maturation. Since cAMP can be regulated both at the level of synthesis and degradation, our laboratory is investigating the role of phosphodiesterases (PDE) in the control of cAMP levels of oocytes. Using pharmacological and molecular tools, we have determined that a PDE3 is the enzyme involved in the control of cAMP levels in the oocytes. In vitro and in vivo studies have established that inhibition of the oocyte PDE3 blocks resumption of a PDE is per se sufficient to cause resumption of meiosis in an amphibian oocyte model. The pathways regulating this PDE isoform expressed in the oocyte is under investigation, as they may uncover the physiological signals controlling meiosis.
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Establishment of cellular polarity is one of the key events during oocyte maturation. Inscuteable (Insc) has been identified as a key regulator of cell polarity during asymmetric division in Drosophila. However, the function of its evolutionarily conserved mammalian homologue, mInscuteable (mInsc), in mouse meiotic maturation is not clear. In this study, we investigated the roles of mInsc in mouse oocyte maturation. mInsc was detected at all stages of oocyte maturation. The protein level of mInsc was slightly higher at the germinal vesicle breakdown (GVBD) stage and remained constant during mouse oocyte maturation. The subcellular localization of mInsc overlapped with spindle microtubules. Disruption of microtubules and microfilaments caused changes in the localization of mInsc. Depletion or overexpression of mInsc significantly decreased the maturation rates of mouse oocytes. Depletion of mInsc significantly affected asymmetric division, spindle assembly, alignments of chromosomes and actin cap formation. Taken together, our results demonstrated that mInsc regulates meiotic spindle organization during mouse meiotic maturation.
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Cell polarity
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Since cAMP blocks meiotic maturation of mammalian and amphibian oocytes in vitro and cyclic nucleotide phosphodiesterase 3A (PDE3A) is primarily responsible for oocyte cAMP hydrolysis, we generated PDE3A-deficient mice by homologous recombination. The Pde3a–/– females were viable and ovulated a normal number of oocytes but were completely infertile, because ovulated oocytes were arrested at the germinal vesicle stage and, therefore, could not be fertilized. Pde3a–/– oocytes lacked cAMP-specific PDE activity, contained increased cAMP levels, and failed to undergo spontaneous maturation in vitro (up to 48 hours). Meiotic maturation in Pde3a–/– oocytes was restored by inhibiting protein kinase A (PKA) with adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) or by injection of protein kinase inhibitor peptide (PKI) or mRNA coding for phosphatase CDC25, which confirms that increased cAMP-PKA signaling is responsible for the meiotic blockade. Pde3a–/– oocytes that underwent germinal vesicle breakdown showed activation of MPF and MAPK, completed the first meiotic division extruding a polar body, and became competent for fertilization by spermatozoa. We believe that these findings provide the first genetic evidence indicating that resumption of meiosis in vivo and in vitro requires PDE3A activity. Pde3a–/– mice represent an in vivo model where meiotic maturation and ovulation are dissociated, which underscores inhibition of oocyte maturation as a potential strategy for contraception.
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Human chorionic gonadotropin
Cyclic nucleotide phosphodiesterase
Cyclic adenosine monophosphate
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Abstract To investigate the effect of thyroid hormone on cardiac muscle dysfunction in hyper- and hypothyroid states, we evaluated cyclic 3′, 5′-nucleotide metabolism by measuring cyclic 3′, 5′-nucleotide phosphodiesterase activity and calmodulin concentrations in the cardiac muscles of hyper- and hypothyroid rats. Cyclic AMP (cAMP) concentration was significantly high in the cardiac muscle of hyperthyroid rats and low in that from hypothyroid rats compared with control rats. Cyclic AMP and cyclic GMP phosphodiesterase activities were significantly decreased in the soluble fraction of cardiac muscle from hyperthyroid rats and markedly increased in this fraction in hypothyroid rats compared with normal animals. Calmodulin concentration was high in hyperthyroid and low in hypothyroid rats. It was concluded from these findings that low cAMP-phosphodiesterase activity might, in part, bring about the high concentration of cAMP. Calmodulin was sigificantly high in the cardiac muscle of hyperthyroid rats and the reverse was the case in hypothyroid rats compared with normal rats. The implication is that, in hyper- and hypothyroid states, these changes may play an important role in cardiac function via their effect on cyclic nucleotide and Ca 2+ metabolism. Journal of Endocrinology (1994) 143 , 515–520
Cyclic nucleotide phosphodiesterase
PDE10A
Cardiac muscle
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Regulation of cyclic AMP-dependent protein kinase, cyclic AMP-receptor activity and intracellular cyclic AMP concentrations by choriogonadotropin was studied in ovarian cells prepared from 26-day-old rats. A close correlation was observed between phospho-transferase activity and cyclic AMP-receptor activity in 12000g supernatant fractions from rat ovarian homogenate. The apparent activation constant (K(a)) and I(50) (concentration required to produce 50% inhibition) of different cyclic nucleotides for phosphotransferase and cyclic AMP receptor activities respectively were also determined. Cyclic AMP and 8-bromo cyclic AMP were most effective, giving K(a) values of 0.08 and 0.09mum and I(50) of 0.12 and 0.16mum respectively. Other nucleotides were also effective, but required higher concentrations to give a comparable effect. An increased concentration of cyclic AMP produced by choriogonadotropin (1mug/ml) treatment was accompanied by decreased cyclic AMP binding as early as 5min after hormone addition. Choriogonadotropin also stimulated the protein kinase activity ratio (-cyclic AMP/+cyclic AMP) under identical experimental conditions. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated the action of choriogonadotropin on the three parameters measured in a dose- and time-dependent manner. The maximal cyclic AMP-binding capacity, as determined by cyclic AMP-exchange assay, remained unchanged before and after hormone addition. The endogenously bound cyclic AMP was determined from the difference between the maximal binding capacity and the exogenously bound cyclic AMP. With different choriogonadotropin concentrations, a quantitative correlation was established between maximal binding capacity, exogenous binding and endogenous binding activities. Approx. 60% of total binding sites were endogenously occupied in untreated cells, and choriogonadotropin (1mug/ml) treatment fully saturated available binding sites with a parallel 10-fold increase in cellular cyclic AMP. The present results provide evidence for a probable intracellular compartmentalization of cyclic AMP in the ovarian cell, and suggest that in the unstimulated state all cyclic AMP present in the ovarian cell may not be available for protein kinase activation.
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Cyclic adenosine monophosphate
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