β-defensin 126 and sperm function in cattle

β-defensins are antimicrobial peptides also thought to have a role in sperm function. In cattle, β-defensin 126 (BD126) has been only detected in the male reproductive tract, with preferentially in the epididymis (Narciandi et al., Immunogenetics 63, 641–651, 2011). The macaque ortholog has been shown to enhance the ability of sperm to migrate through cervical mucus (Tollner et al., Hum. Reprod. 23, 2523–2534, 2008). A mutation in the BD126 gene has been linked to subfertility in men, only explained by reduced ability to penetrate through mucus in vitro (Tollner et al., Sci. Transl. Med. 3, 92ra6, 2011). The aim of this study was to examine the role of bovine BD126 in sperm function. Western blot (WB) analysis with a BD126 specific monoclonal antibody demonstrated significant BD126 on bovine sperm which previously published methods for macaque sperm failed to remove. WB analysis also revealed that while BD126 is present on sperm and in seminal plasma from intact bulls, it is undetectable in the ejaculate of vasectomised animals, indicating that it does not originate in the accessory glands. Further analysis demonstrated that the peptide is uniquely present in the cauda epididymis and is absent from sperm recovered from other epididymal regions, thus providing a model to study its function. Confocal analysis revealed immunofluorescent labelling of BD126 specific to the tail and acrosomal region in cauda sperm only, suggesting a role in sperm motility. We therefore hypothesized that addition of cauda fluid to corpus sperm would improve motility and ability to penetrate cervical mucus in vitro, and that this may be due to the activity of BD126. Testes were collected from adult bulls at an abattoir and sperm from the corpus and cauda epididymis, as well as cauda epididymal fluid (CEF), were recovered. Corpus sperm were incubated for 1 h with CEF in the absence or presence of BD126 antibody (Ab); untreated corpus and cauda sperm were used as controls. A higher number of cauda than corpus sperm migrated through cervical mucus (P<0.001) and addition of CEF increased the number of corpus sperm migrating through this matrix (P<0.05). The presence of the BD126 Ab failed to abrogate this effect. Analysis of motility using a computer assisted sperm analysis system indicated higher total and progressive motility in caudal sperm when compared with sperm from the cauda (P<0.001); again, addition of CEF increased progressive motility (P<0.05). In conclusion, we have characterised the expression of bovine BD126 as a protein in the cauda epididymis. Incubation of sperm from the corpus epididymis (which lack BD126) with CEF from the cauda (which contains BD126) resulted in enhanced sperm migration through cervical mucus, and higher motility. Further work will clarify the role of BBD126 and related β-defensins in mediating bovine sperm function.