Fig 1.
Mammalian DCXR sequence homology.
(A) Homology among four different mammalian DCXR protein sequences (percentage). UniProtKB identification number: Homo sapiens: Q7Z4W1, Bos taurus: Q1JP75, Mus musculus: Q91X52, Mesocricetus auratus: Q91XV4. (B) DCXR protein sequence alignment between human and bull. Only sequence differences are indicated. The alignment was made with http://multalin.toulouse.inra.fr/multalin/ online software.
Fig 2.
DCXR mRNA expression along the epididymis.
(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. DCXR expression levels are normalized as a ratio of alpha-tubulin mRNA levels. ** = Significant difference by student’s t-test, t < 0.006.
Fig 3.
In situ hybridization localization of bovine DCXR mRNA on tissue cryosections of bovine caput (A), corpus (B, D), and cauda (C) epididymidis.
Arrows indicate DCXR 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.
Fig 4.
DCXR protein levels along the bovine epididymis.
(A) Western-blot analysis on SDS protein extracts from nine (9) bovine epididymis segments, as illustrated in Fig. 2A, 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.
Fig 5.
Immunohistochemical localization of bovine DCXR protein along the bovine epididymis.
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.
Fig 6.
DCXR protein levels associated with spermatozoa.
(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).
Fig 7.
DCXR protein levels in the epididymal fluid.
(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).
Fig 8.
Trypsin digestion of cauda epididymidal fluid before ultracentrifugation (Total); after ultracentrifugation (soluble fraction); epididymosomes, and bovine recombinant DCXR protein.
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 [11] for P25b, a rabbit anti-MIF antiserum [12], and a rabbit anti-aldose reductase (AKR1B1) antiserum [12]. The results are representative of three independent experiments. Molecular weight standards (kDa) are indicated on left.
Table 1.
Polymerase chain reaction primers sequences used for this study.