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Equine CRISP3 modulates interaction between spermatozoa and polymorphonuclear neutrophils
1Department of Animal Sciences, College of Agriculture and Life Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.
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 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.
Insights
Equine seminal plasma contains a protein, CRISP3, that suppresses the binding of sperm to neutrophils. This finding suggests CRISP3 regulates sperm removal from the mare's reproductive tract.
Area of Science:
- Reproductive immunology
- Protein biochemistry
Background:
- Equine spermatozoa trigger a uterine inflammatory response involving neutrophils.
- Seminal plasma proteins influence sperm-neutrophil interactions.
- The specific protein responsible for modulating this interaction remained unidentified.
Purpose of the Study:
- To isolate and identify a protein in equine seminal plasma that inhibits sperm-neutrophil binding.
- To elucidate the role of this protein in regulating the inflammatory response in the mare's reproductive tract.
Main Methods:
- Proteins from pooled stallion seminal plasma were precipitated and fractionated using ammonium sulfate precipitation and Sephacryl S200 chromatography.
- Active fractions were further purified by ion-exchange chromatography (DEAE Sephadex).
- Protein identification was achieved through SDS-PAGE, Western blotting, and sequence analysis; functional assays used flow cytometry to quantify PMN-sperm binding.
Main Results:
- A protein suppressing polymorphonuclear neutrophil (PMN)/sperm binding was isolated and identified as equine CRISP3.
- Equine seminal plasma and purified CRISP3 significantly reduced PMN/sperm binding compared to control media (63.09% ± 8.67% vs. 2.06% ± 2.02%, P < 0.0001).
- Flow cytometry confirmed the suppressive effect of CRISP3 on PMN-sperm interactions.
Conclusions:
- Equine seminal plasma contains CRISP3, a protein that effectively suppresses the binding of neutrophils to spermatozoa.
- CRISP3 likely plays a crucial role in regulating the clearance of spermatozoa from the female reproductive tract, modulating the post-mating inflammatory response.
