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Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa.

ABSTRACT: In mammals, adenosine 3', 5'-cyclic monophosphate (cAMP) is known to play highly important roles in sperm motility and acrosomal exocytosis. It is known to act through protein phosphorylation via PRKA and through the activation of guanine nucleotide exchange factors like EPAC. Sperm intracellular cAMP levels depend on the activity of adenylyl cyclases, mostly SACY, though transmembrane-containing adenylyl cyclases are also present, and on the activity of cyclic nucleotide phosphodiesterases (PDE) whose role is to degrade cAMP into 5'-AMP. The PDE superfamily is subdivided into 11 families (PDE1 to 11), which act on either cAMP or cGMP, or on both cAMP and cGMP although with different enzymatic properties. PDE10, which is more effective on cAMP than cGMP, has been known for almost 15 years and is mostly studied in the brain where it is associated with neurological disorders. Although a high level of PDE10A gene expression is observed in the testis, information on the identity of the isoforms or on the cell type that express the PDE10 protein is lacking. The objective of this study was to identify the PDE10A isoforms expressed in the testis and germ cells, and to determine the presence and localization of PDE10A in mature spermatozoa. As a sub-objective, since PDE10A transcript variants were reported strictly through analyses of bovine genomic sequence, we also wanted to determine the nucleotide and amino acid sequences by experimental evidence. Using RT-PCR, 5'- and 3'-RACE approaches we clearly show that PDE10A transcript variants X3 and X5 are expressed in bovine testis as well as in primary spermatocytes and spermatids. We also reveal using a combination of immunological techniques and proteomics analytical tools that the PDE10A isoform X4 is present in the area of the developing acrosome of spermatids and of the acrosome of mature spermatozoa.

SUBMITTER: Goupil S

PROVIDER: S-EPMC4993467 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa.

Goupil Serge S Maréchal Loïze L El Hajj Hassan H Tremblay Marie-Ève MÈ Richard François J FJ Leclerc Pierre P

PloS one 20160822 8

In mammals, adenosine 3', 5'-cyclic monophosphate (cAMP) is known to play highly important roles in sperm motility and acrosomal exocytosis. It is known to act through protein phosphorylation via PRKA and through the activation of guanine nucleotide exchange factors like EPAC. Sperm intracellular cAMP levels depend on the activity of adenylyl cyclases, mostly SACY, though transmembrane-containing adenylyl cyclases are also present, and on the activity of cyclic nucleotide phosphodiesterases (PDE ...[more]

PMID: 27548062

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Project description:BackgroundHeart failure is a leading cause of death worldwide. Cyclic nucleotide phosphodiesterases (PDEs), through degradation of cyclic nucleotides, play critical roles in cardiovascular biology and disease. Our preliminary screening studies have revealed PDE10A upregulation in the diseased heart. However, the roles of PDE10A in cardiovascular biology and disease are largely uncharacterized. The current study is aimed to investigate the regulation and function of PDE10A in cardiac cells and in the progression of cardiac remodeling and dysfunction.MethodsWe used isolated adult mouse cardiac myocytes and fibroblasts, as well as preclinical mouse models of hypertrophy and heart failure. The PDE10A selective inhibitor TP-10, and global PDE10A knock out mice were used.ResultsWe found that PDE10A expression remains relatively low in normal and exercised heart tissues. However, PDE10A is significantly upregulated in mouse and human failing hearts. In vitro, PDE10A deficiency or inhibiting PDE10A with selective inhibitor TP-10, attenuated cardiac myocyte pathological hypertrophy induced by Angiotensin II, phenylephrine, and isoproterenol, but did not affect cardiac myocyte physiological hypertrophy induced by IGF-1 (insulin-like growth factor 1). TP-10 also reduced TGF-? (transforming growth factor-?)-stimulated cardiac fibroblast activation, proliferation, migration and extracellular matrix synthesis. TP-10 treatment elevated both cAMP and cGMP levels in cardiac myocytes and cardiac fibroblasts, consistent with PDE10A as a cAMP/cGMP dual-specific PDE. In vivo, global PDE10A deficiency significantly attenuated myocardial hypertrophy, cardiac fibrosis, and dysfunction induced by chronic pressure overload via transverse aorta constriction or chronic neurohormonal stimulation via Angiotensin II infusion. Importantly, we demonstrated that the pharmacological effect of TP-10 is specifically through PDE10A inhibition. In addition, TP-10 is able to reverse pre-established cardiac hypertrophy and dysfunction. RNA-Sequencing and bioinformatics analysis further identified a PDE10A-regualted transcriptome involved in cardiac hypertrophy, fibrosis, and cardiomyopathy.ConclusionsTaken together, our study elucidates a novel role for PDE10A in the regulation of pathological cardiac remodeling and development of heart failure. Given that PDE10A has been proven to be a safe drug target, PDE10A inhibition may represent a novel therapeutic strategy for preventing and treating cardiac diseases associated with cardiac remodeling.

Project description:Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5±7.8% vs. 83.9±4.7%) as well as compensatory expansion of left ventricular diameter (4.19±0.19mm vs. 3.10±0.18mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (β-AR) response in TG mice. Importantly, the myocardial infarct size (4.0±1.8% vs. 24.6±3.8%) and apoptotic cell number (1.3±1.0% vs. 5.6±1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates β-AR signaling and protects against I/R injury by inhibiting cardiomyocyte apoptosis.

Dataset Information

Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa.

Publications

Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa.

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