This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We previously reported that DEFB126 (formerly ESP13.2) coats the entire surface of macaque sperm and remains until sperm become capacitated. The release of DEFB126 from sperm during capacitation is required for sperm recognition and binding of the zona pellucida, suggesting that DEFB126 masks zona pellucida ligands on the sperm surface. The observation that ESP13.2 potentially masks sperm cell surface components prompted us to examine the potential role of this protein as a means of protection from immune recognition. Cynomolgus macaque sperm were exposed to a number of treatments that are known to alter sperm surface coats, including capacitation. We utilized a novel in vivo assay to determine immune recognition: aldehyde-fixed whole sperm injections into rabbits. Following booster injections, immunoblot analyses of whole sperm prepared in various manners was conducted. On days 60 and 80 post initial immunization, the antisera showed a remarkably strong reaction to a single 34-36 kDa protein, which was shown to be DEFB126. Sera from rabbits that were immunized with sperm washed more rigorously using Percoll gradients showed an increase in the number and intensity of proteins recognized on whole sperm western blots, although the immune response to DEFB126 was dominant. When capacitated sperm, from which most of the DEFB126 had been released, were used as the immunogen, there was a dramatic increase in the immune recognition to a variety of protein bands. Sperm treated with neuraminidase to remove sialic acid on DEFB126 prior to fixation were shown to still possess DEFB126, but lacked the sialic acid component of the glycoprotein. These sperm were as immunogenic as capacitated sperm even though the desialylated DEFB126 still covered the entire cell surface. These sperm lost their highly negative charge (isoelectric point of DEFB126 shifted from pI 3.0 to pI 6.4). Experiments using different sperm plasma membrane protein-specific Igs showed that recognition did not occur when DEFB126 was present, but following capacitation these Igs readily recognized the exposed sperm membrane. We further characterized the carbohydrate groups of DEFB126 with lectin binding. Sperm exposed to fluorescein conjugated L-poly-lysine or alexa-488 conjugated histone showed a very uniform fluorescent labeling pattern over the entire sperm surface similar to that seen with anti-DEFB126 Ig. Sperm surface components that were released following treatment with caffeine/cAMP were blotted and probed with three different lectins which are known to recognize terminal sialic acid residues, and all three recognized the 35kDa DEFB126 band. Neuraminidase treatment of sperm shifted the molecular weight of DEFB126 from 34-36kDa to approximately 38-40kDa and removed or greatly inhibited sialic acid specific lectin recognition. O-glycanase treatment alone was ineffective at removal of the oligosaccharides, but prior treatment with neuraminidase was sufficient to enable the o-glycanase to effectively change the molecular weight to 10kDa, the polypeptide weight after removal of the carbohydrates, confirming that 70% of the molecule mass is associated with the carbohydrate portion. Western blots of neuraminidase-treated DEFB126 showed strong recognition with a number of lectins that recognize beta-galactose and also some lectins that recognize the n-acetyl galactosamine-serine/threonine, which is the proposed linkage of o-linked oligosaccharides. All of the lectins that showed recognition of DEFB126 on a western blot were also used to fluourescently probe sperm. The fluorescent patterns were identical to those seen with L-poly-lysine, sialic acid specific and galactose specific lectins and anti- DEFB126 Ig, which showed an even distribution along the entire sperm surface.
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