Localization of lactoferrin binding sites on equine spermatozoa

Seminal plasma (SP) contains proteins important to many physiological and immunological functions related to fertility and uterine defense mechanisms. We have previously found that CRISP-3 inhibits neutrophil binding to live spermatozoa and that lactoferrin (LF) promotes neutrophil binding to dead spermatozoa. However, the mechanism of this function has not been determined. The objective of this study was to localize LF binding sites on equine spermatozoa. Biotinylated equine LF was used as a probe for the localization of its binding sites, and biotin was visualized with fluorescent-NeutrAvidin. A total of 5 normal stallions were used for this study. An equal number of spermatozoa from 2 stallions were pooled in each experiment, which was replicated three times with different stallions. Gel-free semen with at least 70% motility was diluted 1:4 with Lactated Ringer’s Solution (LRS) immediately after collection, centrifuged at 400xg/10 min, washed oncewith 30mL of LRS and used within 2-3 h from collection. Spermatozoa (33 million/300 mL) were incubated at room temperature for 1 hour in the following reactions: 1) LRS (negative control); 2) LRS with fluorescent NeutrAvidin (control for endogenous biotin); 3) Biotinylated LF (100 mg/mL); and 4) Biotinylated BSA (100 mg/mL; control for non-specific binding). All treatments contained non-biotinylated BSA (100 mg/mL) to block any non-specific binding sites. At the end of the reaction, samples were centrifuged, and the spermatozoa were washed once in 600 mL of LRS and resuspended in 100 mL of LRS. Fluorescent NeutrAvidin was added at 1:100 dilution, andwetmountswere examined by a fluorescent microscopy at 630x magnification. Post incubation motility was less than 20% (8-18%) and this was true for all treatments as determined with light