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9 March 2011 Equine CRISP3 Modulates Interaction Between Spermatozoa and Polymorphonuclear Neutrophils
A. Doty, W.C. Buhi, S. Benson, K.E. Scoggin, M. Pozor, M. Macpherson, M. Mutz, M.H.T. Troedsson
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Equine spermatozoa induce a uterine inflammatory response characterized by a rapid, transient influx of polymorphonuclear neutrophils (PMNs). Seminal plasma proteins have been shown to modulate the interaction between spermatozoa and PMNs, but a specific protein responsible for this function has not been identified. The objective of this study was to isolate and identify a protein in equine seminal plasma that suppresses binding between spermatozoa and PMNs. Seminal plasma was pooled from five stallions, and proteins were precipitated in 60% (w/v) ammonium sulfate and dialyzed (3500 MW cutoff). Proteins were submitted to a Sephacryl S200 column, and fractions were pooled based on the fraction pattern. Each pool was analyzed for protein concentration and tested for its suppressive effect on PMN/sperm binding. Protein pools with biological activity were submitted to ion-exchange chromatography (diethylaminoethyl [DEAE] Sephadex column) with equilibration buffers containing 0.1–0.5M NaCl. Eluants were pooled, analyzed for protein concentration, and tested for suppressive effects on PMN/sperm binding. Protein distribution and purity were determined by one- and two-dimensional SDS-PAGE, and the purified protein was submitted for sequence analysis and identification. This protein was identified as equine CRISP3 and was confirmed by Western blotting. Suppression of PMN/sperm binding by CRISP3 and seminal plasma was confirmed by flow cytometry (22.08% ± 3.05% vs. 2.06% ± 2.02% vs. 63.09% ± 8.67 for equine seminal plasma, CRISP3, and media, respectively; P < 0.0001). It was concluded that CRISP3 in seminal plasma suppresses PMNs/sperm binding, suggesting that CRISP3 regulates sperm elimination from the female reproductive tract.

A. Doty, W.C. Buhi, S. Benson, K.E. Scoggin, M. Pozor, M. Macpherson, M. Mutz, and M.H.T. Troedsson "Equine CRISP3 Modulates Interaction Between Spermatozoa and Polymorphonuclear Neutrophils," Biology of Reproduction 85(1), 157-164, (9 March 2011).
Received: 1 March 2010; Accepted: 1 February 2011; Published: 9 March 2011

polymorphonuclear neutrophils
seminal plasma
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