Characterization of carp seminal plasma Wap65-2 and its participation in the testicular immune response and temperature acclimation

Abstract

Two functionally distinct isoforms of warm-temperature acclimation related 65-kDa protein (Wap65-1 and Wap65-2) with a role in the immune response are present in fish. To our knowledge, contrary to Wap65-1, Wap65-2 has neither been isolated nor functionally characterized in carp especially in reproductive system. The aim of this study was to characterize Wap65-2 and ascertain its functions in immune response and temperature acclimation within reproductive system. Wap65-2 corresponded to one of the most abundant proteins in carp seminal plasma, with a high immunologic similarity to their counterparts in seminal plasma of other fish species and a wide tissue distribution, with predominant expression in the liver. The immunohistochemical localization of Wap65-2 to spermatogonia, Leydig cells, and the epithelium of blood vessels within the testis suggests its role in iron metabolism during spermatogenesis and maintenance of blood-testis barrier integrity. Wap65-2 secretion by the epithelial cells of the spermatic duct and its presence around spermatozoa suggests its involvement in the protection of spermatozoa against damage caused by heme released from erythrocytes following hemorrhage and inflammation. Our results revealed an isoform-specific response of Wap65 to temperature acclimation and Aeromonas salmonicida infection which alters blood-testis barrier integrity. Wap65-2 seems to be related to the immune response against bacteria, while Wap65-1 seems to be involved in temperature acclimation. This study expands the understanding of the mechanism of carp testicular immunity against bacterial challenge and temperature changes, in which Wap65-2 seems to be involved and highlights their potential usefulness as biomarkers of inflammation and temperature acclimation.

Keywords: Wap65; acclimation; cDNA; fish; hemopexin; infection; reproductive system; semen.

Figure 1

Purification of the Wap65-2 from carp seminal plasma. Lines: seminal plasma (SP) hydrophobic interaction (HIC) fraction; ion exchange chromatography (IEC) fraction, and fraction eluted after preparative electrophoresis (PE). Protein samples were analyzed by PAGE (A) and SDS-PAGE (B). Proteoforms of isolated Wap65-2a and Wap65-2b after 2-DE (C). Detection of Wap65-2 in carp seminal plasma after SDS-PAGE, native PAGE, and 2-DE using anti-Wap65-2 antibodies (D). Additional file 4 indicates which part of the corresponding 2-DE of seminal plasma is presented on the blot (D).

Figure 2

cDNA and deduced amino acid sequence of carp Wap65-2 (A) and predicted tertiary structure of carp Wap65-2 with rabbit hemopexin (B). The signal peptide sequence is shaded in dark grey and the six hemopexin-like repeats in the sequence are shaded in light grey. The inverted dark triangles indicate metal ion binding sites. Potential N-linked glycosylation sites are boxed, and predicted phosphorylation sites are denoted with stars. Peptides identified by mass spectrometry are underlined. Peptides identified after N-glycosylation are double underlined in red. Wap65-2 is shown as a colored cartoon, while the structural analog (rabbit hemopexin) is displayed using a backbone trace.

Figure 3

Multiple amino acid alignment comparing Wap65-2 sequences from carp with other vertebrates. Asterisks mark the identical amino acids in all sequences. Conserved histidine residues (His260 and His292), crucial for heme binding, are highlighted in dark shadowed boxes; conserved 10 cysteine residues, essential for structural integrity forming disulfide bridges, are presented in light shadowed boxes. Metal ion binding sites are marked with black triangles (NCBI GenBank accession numbers of the utilized sequences are listed in Additional file 5).

Figure 4

Phylogenetic analysis of the complete amino acid sequences of carp Wap65-2 and different species. A phylogenetic analysis was performed using a PhyML 3.0 Approximate Likelihood Ratio Test: SH-like with tree rendering with TreeDyn 198.3. (NCBI GenBank accession numbers of the utilized sequences are listed in Additional file 5).

Figure 5

Interaction with lectins (A, B) and deglycosylation of carp seminal plasma Wap65-2a and Wap65-2b (C, D). N-Glycosidase F (PNGase F) (+N) and O-glycosidase (+O). Staining for positive Datura stramonium agglutinin (DSA; A) and Maackia amurensis agglutinin (MAA; B) affinities of Wap65-2 (I—fetuin, II—carboxypeptidase Y, III—transferrin, and IV—asialofetuin). Fluorescent staining for glycoproteins using Pro-Q Emerald 300 (C) and for proteins using SYPRO Ruby (D). Lane identities: S—molecular mass marker, 1—Wap65-2a (control), 2—Wap65-2a incubated with O-glycosidase, 3—Wap65-2a incubated with N-glycosidase, 4—Wap65-2b (control), 5—Wap65-2b incubated with O-glycosidase, and 6—Wap65-2b incubated with N-glycosidase.

Figure 6

Western blot analysis of Wap65-2 in seminal plasma of fish species. 1—carp, 2—barbel, 3—dace, 4—chub, 5—burbot, 6—grayling, 7—rainbow trout, 8—ide, 9—asp, and 10—sturgeon. Seminal plasma proteins were separated using 1D-SDS-PAGE.

Figure 7

Immunohistochemical localization of Wap65-2 in carp testis (A), spermatic duct (B), and liver (C). Bars = 10 μm. A Strong signal for Wap65-2 is present in some cysts that contain spermatogonia A (SgA) and spermatogonia B (SgB), as well as in Leydig cells (arrowheads). At higher magnification (bottom image), note the strong signal around the epithelium of blood vessels (Bv). No immunopositive staining is observed in primary (Spc1) and secondary spermatocytes (Spc2), spermatids (Spt), and Sertoli cells (arrows). Immunonegative spermatozoa (Sz) are clearly visible. B In the spermatic duct, there is weak to moderate signal for Wap65-2 in columnar secretory cells (black arrows) and stromal cells (asterisks). Positive staining from luminal epithelium is visible near spermatozoa. C In the liver cells, there is a positive moderate signal for Wap65-2 localized to hepatocytes. Note the perinuclearly located signals (open arrows). Insets in A–C controls of testicular, spermatic duct, and liver cells when the primary antibody is omitted respectively.

Figure 8

Expression of carp Wap65-2 and Wap65-1 mRNA in carp tissues (n = 4). The results are presented as normalized copies per 100 000 copies of reference genes 40S ribosomal protein S11 (40S) and elongation factor 1 alpha. Stars indicate differences between Wap65-1 and Wap65-2.

Figure 9

mRNA expression of Wap65-1 and Wap65-2 in carp reproductive system [testis (A) and spermatic duct (B)] after temperature acclimation (n = 6 per condition). The results are presented as normalized copies per 100 000 copies of reference genes 40S ribosomal protein S11 and elongation factor 1 alpha. The bars represent means ± standard deviation. Different letters indicate significant differences between groups.

Figure 10

Modulation of mRNA expression of Wap65-2 (A) and Wap65-1 (B) in tissues during bacterial challenge with A. salmonicida (n = 6 per condition). The results are presented as normalized copies per 100 000 copies of reference genes 40S ribosomal protein S11 and elongation factor 1 alpha. The bars represent means ± standard deviation. Stars indicate significant differences between the control and treated group.

Figure 11

Expression of immune-related genes and 16S rRNA in carp tissue after bacterial challenge with A. salmonicida. IL-1βl—interleukin 1β (A), iNOS—inducible nitric oxide synthase (B). Quantification of A. salmonicida in tissues using 16S rRNA gene expression (C) (n = 6). The results are presented as normalized copies per 100 000 copies of reference genes 40S ribosomal protein S11 and elongation factor 1 alpha. The bars represent means ± standard deviation. Stars indicate significant differences between the control and treated group (A, B) while different letters indicate differences between tissues (C).