The role of equine seminal plasma derived cysteine rich secretory protein 3 (CRISP3) in the interaction between polymorphonuclear neutrophils (PMNs) and populations of viable or dead spermatozoa, and bacteria.

Breeding-induced endometritis is a physiological reaction to clear the uterus from excess spermatozoa and bacteria after breeding. Cysteine rich secretory protein 3 in seminal plasma (spCRISP3) protects spermatozoa from binding and destruction by uterine PMNs, but it is not clear if this involves all sperm and bacteria, or if it is selective to a sub-population of live sperm. The objective of this report was to determine if spCRISP3 (1) is selective in its suppression of PMN-binding to sperm based on viability of spermatozoa, (2) protects bacteria from binding to PMNs, and (3) to determine the localization pattern of spCRISP3 on viable and dead sperm. Semen was collected from five stallions and each ejaculate was divided into (1) live and (2) snap frozen (dead) sperm. Two distinct sperm populations were confirmed by DNA fragmentation and membrane integrity assays. CRISP3 was purified from pooled seminal plasma, and binding of PMNs (isolated from peripheral blood) to the two sperm populations and E. coli was evaluated with flow cytometry in the presence of spCRISP3. In addition, localization of spCRISP3 on live and dead spermatozoa was determined by immunocytochemistry. Comparisons between treatments were analyzed using a one-way-ANOVA and Bonferroni's comparison test, or Kruskal-Wallis ANOVA if not normally distributed. spCRISP3 significantly suppressed binding of PMNs to live spermatozoa (p < 0.0001) but had no effect on dead sperm or bacteria (p > 0.05). Immunocytochemistry confirmed binding of spCRISP3 to live, but not dead spermatozoa. It was concluded that a selective interaction between spCRISP3 and live spermatozoa may be part of a biological mechanism that allows safe transport of viable spermatozoa to the oviducts, while enabling dead spermatozoa and bacteria to be eliminated in a timely fashion after breeding.