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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.

The mechanism that causes the detachment of spermatozoa from the oviductal reservoir around the time of ovulation remains to be elucidated. Because the cumulus cells of the bovine oocyte are known to secrete progesterone (P4), and P4 has been shown to act upon cation channels of spermatozoa (CatSper) in human spermatozoa, it was hypothesised that P4 could induce hyperactivation due to an influx of extracellular calcium, and this would facilitate detachment of spermatozoa from oviductal epithelial cells. Therefore, this study aimed to investigate the role and mechanism of action of P4 in the release of spermatozoa from bovine oviduct epithelial cells (BOEC). Initial dose–response assessments on sperm hyperactivation determined the optimum concentration of P4 (10 nM), mibefradil (a non-specific Ca2+ channel antagonist; 5µM), NNC 55-0396 dihydrochloride (NNC; a CatSper antagonist; 2µM), mifepristone (a classical and membrane P4 receptor antagonist; 400nM) and AG205 (a membrane P4 receptor antagonist; 10μM). BOEC explants were incubated with frozen–thawed bovine spermatozoa for 30min, following which loosely bound spermatozoa were removed. Two experiments were completed. In Experiment 1, BOECs were treated for 30min with either no treatment, P4, NNC, mibefradil, P4+mibefradil, P4+NNC, P4+mibefradil+NNC or P4+EGTA. In Experiment 2, BOECs were treated for 30min with either no treatment, P4, mifepristone, AG205, mifepristone+AG205, P4+mifepristone, P4+AG205 or P4+mifepristone+AG205. The number of spermatozoa remaining bound per millimetre squared of BOEC explant was determined. Progesterone stimulated the release of bound spermatozoa from BOEC explants, whereas NNC, mibefradil and EGTA inhibited this release. The release of spermatozoa by P4 was inhibited in the presence of both mifepristone and AG205, whereas the combination of both had the greatest inhibitory action on P4 release of spermatozoa. These findings suggest the presence of a P4 membrane receptor on bovine spermatozoa and that P4-induced release of spermatozoa from BOECs is likely mediated by extracellular Ca2+.

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