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Primate β-defensin 126 regulates the ability of spermatozoa to bind to oviductal epithelial cells invitro. Bovine β-defensin 126 (BBD126) exhibits preferential expression in the cauda epididymis of the bull, but there have been few studies on its functional role in cattle. The aim of the present study was to examine the role of BBD126 in bull sperm binding to bovine oviductal epithelial cell (BOEC) explants. BBD126 has been shown to be highly resistant to the standard methods of dissociation used in other species and, as a result, corpus epididymal spermatozoa, which have not been exposed to the protein, were used to study the functional role of BBD126. Corpus epididymal spermatozoa were incubated with recombinant (r) BBD126 in the absence or presence of anti-BBD126 antibody. Addition of rBBD126 significantly enhanced the ability of epididymal spermatozoa to bind to BOEC explants (P<0.05). Anti-BBD126 antibody blocked the BBD126-mediated increase in sperm binding capacity. Ejaculated spermatozoa, which are coated with native BBD126 protein but also a large number of seminal plasma proteins invivo, were incubated with rBBD126 in the absence or presence of the anti-BBD126 antibody. Addition of rBBD126 significantly enhanced the ability of ejaculated spermatozoa to bind to BOEC explants (P<0.05), whereas rBBD126 also reduced corpus sperm agglutination (P<0.05). These results suggest that, similar to the role of its analogue in the macaque, spermatozoa with more BBD126 in their acrosome may represent spermatozoa with more oviduct binding capacity.

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., (© 2016 by the Society for the Study of Reproduction, Inc.)

Bovine beta-defensin 126 (BBD126) exhibits preferential expression for the cauda epididymis of males, where it is absorbed onto the tail and postacrosomal region of the sperm. The aim of this study was to examine the role of BBD126 in bull sperm function. Fresh and frozen-thawed semen were incubated in the presence of different capacitating agents as well as with phosphatidylinositol-specific phospholipase C. These treatments, which have been successful in releasing beta-defensin 126 from macaque sperm, proved to be ineffective in bull sperm. This finding suggests that the protein behaves in a different manner in the bovine. The lack of success in removing BBD126 led us to use corpus epididymis sperm, a model in which the protein is not present, to study its functional role. Corpus sperm were incubated with cauda epididymal fluid (CEF) in the absence or presence of BBD126 antibody or with recombinant BBD126 (rBBD126). Confocal microscopy revealed that rBBD126 binds to corpus sperm with the same pattern observed for BBD126 in cauda sperm, whereas an aberrant binding pattern is observed when sperm are subject to CEF incubation. Addition of CEF increased motility as well as the number of corpus sperm migrating through cervical mucus from estrus cows. However, it decreased the ability of sperm to fertilize in vitro matured oocytes. The presence of the antibody failed to abrogate these effects. Furthermore, when rBBD126 was added in the absence of other factors and proteins from the CEF, an increase in motility was also observed and no negative effects in fertility were seen. These results suggest that BBD126 plays a key role in the acquisition of sperm motility in the epididymis., (© 2016 by the Society for the Study of Reproduction, Inc.)