As immature sperm migrate through the epididymis, they are bathed in region-specific epididymal fluid, which leads to a sequential addition, deletion, and modification of their surface proteins. These changes ultimately result in the acquisition of motility and fertilising abilities. Among the hundreds of proteins secreted by the epididymis, several β-defensins have been identified and correlated with male fertility in multiple species. In cattle, β-defensin 126 (BD126) is exclusively detected in the reproductive tract of pubertal males, with preferential mRNA expression in the epididymis. Both the macaque and human orthologs have been shown to play a role in the ability of sperm to migrate through cervical mucus. The aim of this study was to examine the role of bovine BD126 in sperm function. Western blot revealed that the peptide is uniquely present in both the cauda epididymis sperm and fluid and is absent from sperm recovered from other proximal epididymal regions, or the ejaculate of vasectomised animals. Confocal analysis showed immunofluorescent labelling of BD126 specific to the tail and acrosomal region in cauda sperm only, suggesting a role in motility. We hypothesised that addition of cauda epididymal fluid (CEF) or recombinant BD126 (rBD126) to immature corpus sperm would improve ability to penetrate cervical mucus. Testes from adult bulls were collected at an abattoir, and sperm from the corpus and cauda epididymis, as well as CEF, were recovered. Corpus sperm were incubated for 1 h with CEF in the absence or presence of BD126 antibody, or with different rBD126 concentrations (30 or 60 μg mL–1); untreated corpus and cauda sperm were used as controls. A higher number of cauda than corpus sperm migrated through cervical mucus from oestrus cows (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 antibody in CEF failed to abrogate this effect. Western blot analysis of the sperm samples revealed the antibody was not successful in blocking BD126 from binding onto the sperm surface, which would explain the lack of differences observed. Furthermore, the addition of rBD126 did not increase corpus sperm migration through mucus. In conclusion, we have characterised the expression of bovine BD126 protein in the bovine testis and epididymis. Incubation of sperm from the corpus with CEF from the cauda resulted in enhanced sperm migration through cervical mucus. However, incubation of sperm with rBD126 in the absence of other factors and proteins from the CEF failed to produce the same effect. These results suggest that the role of BD126 in cattle is different from that observed in primates. We are currently investigating other roles of BD126 and related β-defensins in mediating bovine sperm function.
This work was supported by a grant from the Irish Department of Agriculture, Food and The Marine under the Research Stimulus Programme (Grant No. 11S 104).
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