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.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…B) SPERM ACTIVATION AND OOCYTE RECOGNITION. During fertilization, cAMP plays critical roles in regulating sperm motility, capacitation, and acrosome reaction (358). EPACs, both EPAC1 and EPAC2 along with their downstream effector Rap1, are identified to be expressed in mouse male germ cells (8,21,74), as well as in stallion and boar spermatozoa (705).…”

The protein phosphatase with EF‐hand domain 1 is a calmodulin‐binding protein that interacts with proteins involved in sperm capacitation, binding to the zona pellucida, and motility

“…In some experiments, sperm were resuspended in the presence of protease inhibitors (10 µg/ml each of leupeptin, aprotinin and pepstatin A, and 0.25 mM phenylmethylsulfonyl fluoride [PMSF]) before being subjected to subcellular fractioning protocol as previously described (Goupil et al, 2016). Plasma membranes were detached from sperm cells by nitrogen cavitation (750 psi, 10 min, 4°C).…”

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin‐binding proteins in bovine ejaculated spermatozoa

“…In some experiments, sperm were resuspended in D‐PBS in the presence of protease inhibitors (10 µg/ml each of leupeptin, aprotinin and pepstatin A, and 0.25 mM phenylmethylsulfonyl fluoride [PMSF]) before being subjected to subcellular fractionation protocol to obtain enriched fractions of plasma and acrosomal membranes, cytosol, heads, and flagella, as previously described (Goupil et al, ). Protein concentration from each subcellular fraction was evaluated using a micro bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, Il) after precipitation with trichloroacetic acid (TCA).…”

“…In some experiments, sperm were resuspended in D-PBS in the presence of protease inhibitors (10 µg/ml each of leupeptin, aprotinin and pepstatin A, and 0.25 mM phenylmethylsulfonyl fluoride [PMSF]) before being subjected to subcellular fractionation protocol to obtain enriched fractions of plasma and acrosomal membranes, cytosol, heads, and flagella, as previously described (Goupil et al, 2016).…”