The role of β-defensin 126 on the ability of bull sperm to bind oviductal epithelium

Previous studies in primates have shown that β-defensin 126 regulates the ability of sperm to bind to oviductal epithelial cells, whilst in humans; mutations in the β-defensin 126 gene have been linked to male subfertility. Bovine β-defensin 126 (BBD126) exhibits preferential expression for the caudal epididymis of the bull but there have been no studies on its functional role in cattle. The aim of this study was to examine the role of BBD126 and BBD126 genetic variation on sperm motility, bovine oviductal epithelial cell (BOEC) binding ability and on sperm agglutination. In a previous study carried out by a member of our research group, Dr Emma Finlay, adjusted bull fertility phenotypes (based on a minimum of 1000 inseminations) for 7000 AI bulls were used to identify bulls of high and low fertility. The most divergent bulls (n=150) were selected for targeted sequencing of β-defensin genes and an association study was performed to identify genetic regions associated with sire conception rate. The most significantly associated single nucleotide polymorphisms (SNPs) were located in a haplotype consisting of 94 SNPs over 138kb, which included the β-defensin 126 gene, found only in bulls of high fertility. In this study, to examine the effect of the haplotype on sperm function, frozen-thawed sperm from high fertility bulls with (H+ive; n=4) and without (H-ive; n=4) the haplotype as well low fertility bulls without the haplotype (L-ive; n=4) were assessed for post-thaw motility using computer aided sperm analysis (CASA) and assessed for binding ability using in vitro BOEC binding assays (both monolayers and explants). BBD126 haplotype was found to have no effect on post-thaw sperm motility, however, bulls of high fertility with the haplotype (H+ive) had an increased ability to bind BOEC explants in comparison to bulls of high (H-ive) and low fertility (L-ive) without the haplotype (P<0.05). BBD126 has been shown to be highly resistant to methods of dissociation used in other species and, as a result, corpus epididymis sperm, a model in which the protein is not present, was used to study the functional role of BBD126 in sperm binding and agglutination. Corpus sperm were incubated with recombinant BBD126 (rBBD126) in the absence or presence of BBD126 antibody. Addition of rBBD126 enhanced the ability of sperm to bind BOEC and reduced sperm agglutination (P<0.05). The presence of the antibody inhibited the increase in sperm binding ability, however, it failed to abrogate the effect of the protein on sperm agglutination. These findings indicate that BBD126 and BBD126 haplotype plays a role in bovine sperm binding and that BBD126 protein has a non-specific effect on sperm agglutination.