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.

Cauda Epididymis-Specific Beta-Defensin 126 Promotes Sperm Motility but Not Fertilizing Ability in Cattle

“…A custom monoclonal antibody specific for BBD126 (α-BBD126 antibody [Ab]) was ordered from GeneScript, and generated as described by Narciandi et al [ 29 ]. Briefly, five BALB/c mice were inoculated with a 14-amino acid chemically produced peptide (RNGERVINPPTGMC).…”

“…Cattle represent an excellent model in which to study issues of male fertility due to the large national databases available, the high number of progeny per sire, and the consequent high confidence associated with fertility estimates. BBD126 has been identified as an epididymal-specific protein that is secreted in the cauda epididymis of the bull [ 29 ]. However, neither its distribution on sperm nor its potential reproductive role has been studied.…”

Review: Understanding the causes of variation in reproductive wastage among bulls

“…In men, variation in the bd126 sequence contributes to subfertility (Tollner et al, 2011) while the BD126 peptide has been reported to mediate sperm binding to the oviductal epithelium (Tollner et al, 2008). Our group has reported bovine beta-defensin 126 (BBD126) to be extensively expressed in the reproductive tract of the bull with preferential protein expression in the cauda epididymis (Narciandi et al, 2011) and on sperm (Narciandi et al, 2016) with similar binding patterns on the sperm surface to macaque. Beta-defensin 126 increases the net negative charge on sperm (Tollner et al, 2012), increases sperm motility, mucus penetration in vitro (Fernandez-Fuertes et al, 2016) as well as sperm binding to oviductal epithelium in vitro (Lyons et al, 2018).…”

Immune regulatory molecules as modifiers of semen and fertility: A review

“…Of note, except for HBD2 and 3, HBD1 is expressed in the human testicular germ‐line in a consistent manner (Com et al, ). In bovine caudal epididymal and vas deferens epithelial cells, defensin β 126 (DEFB126) is associated with antimicrobial peptides involved in sperm motility (Fernandez‐Fuertes et al, ), cell signaling, chemoattraction, immunoregulation (Narciandi et al, ), and sperm binding to the oviductal epithelium (Lyons et al, ). In human studies, incubation of sperm with defensin improved sperm motility and pregnancy rate (Zupin et al, ).…”