Sperm-Coating Beta-Defensin 126 Is a Dissociation-Resistant Dimer Produced by Epididymal Epithelium in the Bovine Reproductive Tract

Abstract

Beta-defensins are innate immune molecules, often described as antimicrobial peptides because of their bactericidal activity and are now known to have diverse additional functions, including cell signaling, chemoattraction, immunoregulation, and reproduction. In humans and primates, beta-defensin 126 has been shown to regulate the ability of sperm to swim through cervical mucus and to protect sperm from attack by the female immune system during transit toward the oviduct. Bovine beta-defensin 126 (BBD126) is the ortholog of human defensin 126, and computational analysis here revealed significant conservation between BBD126 and other mammalian orthologs at the N-terminus, although extensive sequence differences were detected at the C-terminus, implying possible species-specific roles for this beta-defensin in reproduction. We had previously demonstrated preferential expression of this and related beta-defensin genes in the bovine male reproductive tract, but no studies of bovine beta-defensin proteins have been performed to date. Here, we analyzed BBD126 protein using a monoclonal antibody (a-BBD126) generated against a 14 amino acid peptide sequence from the secreted fragment of BBD126. The specificity of a-BBD126 was validated by testing against the native form of the peptide recovered from bovine caudal epididymal fluid and recombinant BBD126 generated using a prokaryotic expression system. Western blot analysis of the native and recombinant forms showed that BBD126 exists as a dimer that was highly resistant to standard methods of dissociation. Immunohistochemical staining using a-BBD126 demonstrated BBD126 protein expression by epithelial cells of the caudal epididymis and vas deferens from both mature and immature bulls. BBD126 could also be seen (by confocal microscopy) to coat caudal sperm, with staining concentrated on the tail of the sperm cells. This study is the first to demonstrate beta-defensin 126 protein expression in the bovine reproductive tract and on bull sperm. Its dissociation-resistant dimeric structure is likely to have important functional implications for the role of BBD126 in bovine reproduction.

Keywords: bovine; epididymis; β-defensin 126.

FIG. 1

a) Multiple sequence alignment of BD126 peptide sequences from species across the animal kingdom. The degree of sequence conservation is shown below the alignment, and the conserved cysteines, including the seventh cysteine, are marked in red. b) Phylogenetic tree showing evolutionary relationship between BD126 peptide sequences for 15 species, including primates, domestic animals, and model species.

FIG. 2

Immunohistochemical localization of BBD126 in the bovine epididymis. Tissue sections from both immature (A–C) and mature (D–G) bulls were stained for the presence of BBD126. Tissue sections were stained with a mouse isotype control (A, cauda) or with anti-BBD126 antibody (B–G). Brown coloration indicative of positive staining was found in the caudal epididymis (B) and vas deferens (C) of the immature bull as well as in the caudal epididymis (D) and vas deferens (E) of the mature bull. Caput and corpal sections of the mature bull epididymis did not stain positive for BBD126 (F and G, respectively). Bar = 25 μm. Representative images of sperm and tissue sections from n = 3 bulls are shown.

FIG. 3

Immunocytofluorescence localization of BBD126 on sperm cells. Live sperm cells extracted from both testis and various regions of the epididymis (caput, corpus, and cauda) stained with a-BBD126 antibody. 4′,6-Diamidino-2-phenylindole (nuclear stain) is shown in column 1, a-BBD126 is shown in column 2, anti-SPAM control antibody is shown in column 3, and merging of both a-BBD126 and anti-SPAM labeled sperm is shown in column 4. An isotype control antibody staining is also shown (column 5). Representative images of sperm from n = 3 bulls are shown.

FIG. 4

Analysis of BBD126 expression on sperm by Western blot analysis. a) Sperm cells lysate extracted from testis (Tes), caput (Ca), corpus (Co), and cauda (Cd) of the epididymis stained with a-BBD126. b) Sperm cell lysate (lane 1) and seminal plasma (lane 2) from a normal bull; centrifuged seminal plasma (lane 3), and seminal plasma (lane 4) from a vasectomized bull. c) The predominant form of BBD126 is the dimer (14.2 kDa) but monomer is also present in sperm lysate, with a stronger band when 40 μg of sperm lysate was used. d) Sperm cell lysate (20 μg of total protein) was incubated at 95°C in sample buffer with 50 mM DTT for 5 min (lanes 1) and with 100 mM DTT (lane 2), but only boiling for 24 h with DTT led to the almost complete dissociation of the BBD126 dimer. Representative images of sperm from n = 3 bulls are shown.

FIG. 5

a) Recombinant BBD126 purification by gel filtration column in a fast protein liquid chromatography system. Two distinctive peaks B and C were observed plus a shoulder on the main peak, A. These were analyzed by SDS-PAGE and analyzed for total protein using Coomassie gel stain (b) or Western blot for BBD126 (c). The composition of peaks A and B is identical where most of the protein is found as the Trx-BBD126 form, 23.4 kDa. However, we can observe the presence of a 46.8 kDa band that seems to correspond to a dimer of the recombinant BBD126 as is detected by a-BBD126 in the Western blot analysis. In contrast C only contains the monomeric Trx-BBD126. Enterokinase digestion of the Trx-BBD126 fusion protein yields both forms of the rBBD126 peptide (d).