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

<div><p>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 <i>PDE10A</i> 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 <i>PDE10A</i> 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 <i>PDE10A</i> 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.</p></div

Detection of bull sperm PDE10A at the electron microscopic level.

<p>Freshly ejaculated bull spermatozoa were washed, fixed with acrolein and pelleted in agarose. Sections of 50 μm were obtained and processed for immunodetection as described in Materials & Methods using monoclonal anti PDE10A antibody (A & B) or non-immune commercial mouse IgG (C & D). Sections were then post-fixed, embedded, and ultrathin sections were prepared and observed by transmission electron microscopy. Note that there is some non-specific signal in the midpiece region that is observed with both the PDE10A antibody and the non-immune IgG (arrowheads in A, B & C). Photographs were taken with magnification of 6800 X (A & C) and 9300 X (B & C). Acrosomal matrix (acr), plasma membrane (pm), and outer acrosomal membrane (oam; lining the acrosomal matrix) are indicated in the sperm head; mitochondria (mit), microtubules (mit) and outer dense fibers (odf) are indicated in the sperm midpiece. In all the panels, the bar scale represents 1 μm.</p

Immunoprecipitation and identification of bull sperm PDE10A.

<p>A) Ejaculated bull spermatozoa were processed for Immunoprecipitation as described in Materials & Methods using a PDE10A monoclonal antibody and protein G-coupled sepharose. The immune-complex on the beads was next solubilized with sample buffer, subjected to electrophoresis and transferred onto PVDF membrane. Proteins were next probed with a monoclonal anti-PDE10A antibody and revealed with a secondary antibody conjugated to horseradish peroxidase, enhanced chemiluminescence, and film exposure. Molecular weight standards (kDa) are indicated on the left. B) Coverage of the PDE10A isoform X4 amino acid sequence by the trypsin generated peptides identified by LC-MS/MS. Sequence sections highlighted in yellow matched the peptides identified. Amino acids highlighted in green show variable post-translational modifications (Oxidation of M, Deamination of Q or N).</p

5’-RACE of cDNA encoding PDE10A in bull testis.

<p>Total RNA isolated from bull testes was reverse transcribed and processed for 5’-RACE as described in Materials & Methods. The size of the amplified products was visualized after electrophoresis on an agarose gel. Three major amplicons of ≈1100, 700 and 450 bp were obtained. The position of the 1000 and 500 bp standards is indicated on the left. Similar results were obtained with RNA isolated from the testes of 3 different bulls.</p

Alignment with the genomic sequence.

<p>Alignment with the genomic sequence.</p

PDE10A in cryopreserved bull spermatozoa.

<p>Cryopreserved spermatozoa were thawed and washed by centrifugation through a two-layer (45 and 90%) Percoll density gradient and sperm from the 45/90% interface (45) and within the 90% Percoll fraction (90) were recovered. <b>A)</b> Spermatozoa from each fraction were processed for electrophoresis and immunoblotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161035#pone.0161035.g005" target="_blank">Fig 5</a>, the intensity of the positive signal was normalized to the intensity of tubulin and, for each bull, expressed as a percentage of the sum of the ratios calculated for the cells recovered at the 45/90% Percoll interface and within the 90% Percoll layer. <b>B)</b> Spermatozoa from each fraction were fixed/permeabilized in methanol and then assessed for the presence of the acrosome by PNA-FITC binding. Only sperm with complete absence of acrosomal staining or with staining restricted to the equatorial segment were considered as acrosome reacted (AR). * and ** are significantly different, p <0.05 and p<0.01, respectively from the value obtained in sperm from the 45/90% Percoll interface, (n = 12; paired t test).</p

Localization of PDE10A in bovine testis.

<p>Bull testicular sections were processed for immunohistochemistry as detailed in Materials & Methods using a monoclonal anti-PDE10A antibody (3G9; Panel A, C, D, E, and F) or non-immune commercial mouse IgG (Panel B). Positive signal (arrows in all Panels) appears as a brownish precipitate. Panels A and B show 400X magnification and Panels C—F show 1000X magnification. Sc stands for primary spermatocytes, Sd stands for round spermatids, and Es stands for elongated spermatids.</p

Oligonucleotide primers used for PCR amplification.

<p>Oligonucleotide primers used for PCR amplification.</p

Immunolocalisation of PDE10A in acrosome intact and acrosome reacted ejaculated bull spermatozoa.

<p>Washed spermatozoa were fixed with formaldehyde and permeabilized with Triton X-100. They were next incubated overnight with a monoclonal anti-PDE10A antibody (clone 3G9; A-F) or a commercial non-immune mouse IgG at the same concentration (G-I). Panels A, D & G show the positive immunofluorescent signal obtained using a DyLight 594-conjugated secondary antibody; Panels E & H show the presence/absence of the acrosome (PNA-FITC labeling) on spermatozoa from the same field of D & G, respectively. Panels B, F & I are Phase contrast image of the same respective field (A, D & G). Panel C shows the superposition of Panels A & B. Magnification is 1000X. Note that PDE10A is detected in the acrosomal region of acrosome intact (AI) but not of acrosome reacted (AR) sperm (D-F). Some non-specific staining is observed in the midpiece of both AI and AR sperm incubated with non-immune mouse IgG (G-I). Identical PDE10A localization is obtained when the immunodetection experiment is performed in the absence (A) or presence (D) of PNA-FITC, or using a FITC-conjugated secondary antibody (not shown).</p

PCR amplification of cDNA encoding PDE10A variants X3 and X5 in bovine testis and spermatogenic germ cells.

<p>RNA isolated from bovine testis, testicular tetraploid (spermatocytes) and haploid (spermatids) germ cells was reverse transcribed and the transcripts encoding PDE10A variant X3 and X5 were amplified by PCR using specific primers. Amplification products obtained with spermatid RNA from 2 different bulls are shown. Similar results were obtained with RNA isolated from the testis, spermatocytes and spermatids from another bull. The expected size of the amplicons is 2367 bp and 2349 bp for variants X3 and X5, respectively. The position of molecular weight standards (bp) is shown on the left.</p