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

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

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 oneand 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% 6 3.05% vs. 2.06% 6 2.02% vs. 63.09% 6 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.

Original languageEnglish
Pages (from-to)157-164
Number of pages8
JournalBiology of Reproduction
Volume85
Issue number1
DOIs
StatePublished - Jul 1 2011

Keywords

  • CRISP3
  • Equine
  • Polymorphonuclear neutrophils
  • Seminal plasma
  • Spermatozoa

ASJC Scopus subject areas

  • Reproductive Medicine

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