Dicarbonyl L-Xylulose Reductase (DCXR), a “Moonlighting Protein” in the Bovine Epididymis

<div><p>During maturation and the acquisition of their fertilization potential, male germ cells are subjected to various sequential modifications that occur in the epididymis. Protein addition, reorganization or withdrawal, comprise some of these modifications. Dicarbonyl L-xylulose reductase (DCXR), a multifunctional protein involved in various enzymatic and protein interaction processes in different physiological systems, is one of the proteins added to spermatozoa in the epididymis. DCXR is a well-conserved protein with multiple characteristics including enzymatic activities and mediation of cell-cell interaction. In this study, we characterized the <i>DCXR</i> gene and protein expression in the bovine epididymis. Dicarbonyl L-xylulose reductase mRNA is differentially expressed in the caput, corpus, and cauda epididymide epithelial cells with a higher level observed in the cauda region. Tissue protein expression follows the same pattern as the corresponding mRNA expression with a cytoplasmic and apical distribution in the corpus and cauda epithelial cells, respectively. The protein can also be found with a nuclear localization in cauda epididymidis epithelial cells. Dicarbonyl L-xylulose reductase is secreted in the epididymis luminal compartment in the soluble fraction and is associated with microvesicular elements named epididymosomes. In spermatozoa, the DCXR protein was found in the cytoplasmic and membranous fractions. Expression of the DCXR protein is higher on caput spermatozoa but finally shows a weak detection in semen. These data describe <i>DCXR</i> in the bovine epididymis and reveal that its behavior differs from that found in humans. It seems that, in this model, the DCXR protein might have a questionable involvement in the fertilization process.</p></div

Immunohistochemical localization of bovine DCXR protein along the bovine epididymis.

<p>Caput (A), corpus (B and B*) and cauda (C) epididymidis using anti-DCXR antiserum (A, B and C). A pre-immune rabbit serum was used as a negative control (B*). Arrows indicate DCXR detected as a brown-red staining. The sections were counterstained in blue with Harris hematoxylin. LU = lumen; EP = epithelium; IT = interstitial tissue.</p

DCXR protein levels in the epididymal fluid.

<p>(A) Western blot analysis of DCXR protein expression in the caput and cauda epididymal fluid. Tot = total fluid 10 μg; soluble = equivalent volume of ultracentrifuged fluid; pellet = equivalent amount of epididymosomes. (B) DCXR detection in 10 μg of SDS extracts of epididymosomes from caput (Ca) and cauda (Cd) epididymidis. Detected with a rabbit anti-DCXR antiserum). Molecular weight is indicated on left (31 kDa).</p

Mammalian DCXR sequence homology.

<p>(A) Homology among four different mammalian DCXR protein sequences (percentage). UniProtKB identification number: <i>Homo sapiens</i>: Q7Z4W1, <i>Bos taurus</i>: Q1JP75, <i>Mus musculus</i>: Q91X52, <i>Mesocricetus auratus</i>: Q91XV4. (B) DCXR protein sequence alignment between human and bull. Only sequence differences are indicated. The alignment was made with <a href="http://multalin.toulouse.inra.fr/multalin/" target="_blank">http://multalin.toulouse.inra.fr/multalin/</a> online software.</p

DCXR protein levels associated with spermatozoa.

<p>(A) Western blot analysis on SDS protein extract from an increasing amount of caput, cauda, and ejaculated spermatozoa pellets (a = 5 × 10⁶ b = 10 × 10⁶ c = 20 × 10⁶). (B) DCXR detection on total caput and cauda spermatozoa (Tot) and on subcellular fractions: 20 μg of cytoplasmic (cyto); membrane (mem) and sonicated remaining spermatozoa (Tot soni). Molecular weight is indicated on left (31 kDa).</p

<i>DCXR</i> mRNA expression along the epididymis.

<p>(A) QPCR quantification on nine (9) different bovine epididymal sections from the caput to cauda (0 to 8). Inner plate is a schematic representation of the different bovine epididymal segments analyzed. (B) QPCR quantification on the three major sections (caput, corpus, and cauda) of the epididymis. Analysis made on five (5) different bovine epididymides. <i>DCXR</i> expression levels are normalized as a ratio of alpha-tubulin mRNA levels. ** = Significant difference by student’s t-test, t < 0.006.</p

<i>In situ</i> hybridization localization of bovine <i>DCXR</i> mRNA on tissue cryosections of bovine caput (A), corpus (B, D), and cauda (C) epididymidis.

<p>Arrows indicate <i>DCXR</i> mRNA staining in blue with the DIG-labeled antisense probe (A, B, C) detected using an anti-DIG antibody coupled to alkaline phosphatase followed by incubation with NBT-BCIP substrate. D panel: corpus section probed with the negative control sense cRNA probes. Counterstaining with neutral red. LU = lumen; EP = epithelium; IT = interstitial tissue.</p

DCXR protein levels along the bovine epididymis.

<p>(A) Western-blot analysis on SDS protein extracts from nine (9) bovine epididymis segments, as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120869#pone.0120869.g002" target="_blank">Fig. 2A</a>, 20 μg per lane. (B) Western-blot analysis of the three major sections caput, corpus, and cauda epididymidis probed with anti-bovine recombinant DCXR antiserum. Results are representative of five independent experiments. An anti-tubulin antiserum was used as an internal loading control.</p

Polymerase chain reaction primers sequences used for this study.

<p>The underlined sequence contains the restriction enzyme sites added for cloning purposes. Primers were designed using NCBI Primer-BLAST online tool (<a href="http://www.ncbi.nlm.nih.gov/tools/primer-blast" target="_blank">http://www.ncbi.nlm.nih.gov/tools/primer-blast</a>).</p><p>Polymerase chain reaction primers sequences used for this study.</p

Trypsin digestion of cauda epididymidal fluid before ultracentrifugation (Total); after ultracentrifugation (soluble fraction); epididymosomes, and bovine recombinant DCXR protein.

<p>Western blot detection of DCXR (32 kDa), MIF (12 kDa), AKR1B1 (35 kDa) or P25b (28 kDa) protein from fluid; epididymosomes or recombinant bovine-DCXR (recDCXR) protein treated (+) or not (−) with trypsin (25 μg/ml). Proteins were detected with a rabbit anti-DCXR antiserum, a rabbit anti-P26h/P25b antiserum [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120869#pone.0120869.ref011" target="_blank">11</a>] for P25b, a rabbit anti-MIF antiserum [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120869#pone.0120869.ref012" target="_blank">12</a>], and a rabbit anti-aldose reductase (AKR1B1) antiserum [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120869#pone.0120869.ref012" target="_blank">12</a>]. The results are representative of three independent experiments. Molecular weight standards (kDa) are indicated on left.</p