Equine CRISP3 Modulates Interaction Between Spermatozoa and Polymorphonuclear Neutrophils

• Published in: Biology of reproduction Vol. 85; no. 1; pp. 157 - 164
• Main Authors: DOTY, A, BUHI, W. C, BENSON, S, SCOGGIN, K. E, POZOR, M, MACPHERSON, M, MUTZ, M, TROEDSSON, M. H. T
• Format: Journal Article
• Language: English
• Published: Madison, WI Society for the Study of Reproduction 01-07-2011
• Subjects: Amino Acid Sequence; Ammonium Sulfate; Animals; Biological and medical sciences; Blotting, Western; Chromatography, Gel; Chromatography, Ion Exchange; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Horses - metabolism; Male; Molecular Sequence Data; Neutrophils - metabolism; Seminal Plasma Proteins - metabolism; Sequence Analysis, Protein; Spermatozoa - metabolism; Vertebrates: reproduction; Spermatozoa; Semen; Interaction; CRISP3; Germinal cell; Vertebrata; Reproduction; Mammalia; Animal; Horse; Perissodactyla; Seminal plasma; Neutrophil; polymorphonuclear neutrophils; Ungulata; equine
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod.110.084491

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.