Equine CRISP3 Modulates Interaction Between Spermatozoa and Polymorphonuclear Neutrophils1

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.

Identification of Distinct Populations of Prostasomes That Differentially Express Prostate Stem Cell Antigen, Annexin A1, and GLIPR2 in Humans1

“…Consistent with a cytosolic orientation [54,55], we found GLIPR2 to be protected from protease treatment. CRISPs are particularly highly expressed in the male reproductive tract and have been implicated in sperm development, capacitation, motility, and fertilization [56] as well as in protection against phagocytosis by neutrophils [57]. Seminal plasma is known to modulate both innate and acquired immune responses locally within the female reproductive tract to prevent immune-mediated destruction of healthy sperm cells [19].…”

Seminal Fluid Signalling in the Female Reproductive Tract: Implications for Reproductive Success and Offspring Health

“…Treatment of human endometrial epithelial cells in vitro with recombinant IL8 at physiological levels can induce IL1B , IL6 , and LIF (Gutsche et al 2003 ). Outside of mice and humans, other novel seminal fl uid signalling molecules that have been examined are porcine sperm adhesion proteins (PSP)1 and PSP2, which contribute in part to the infl ux of neutrophils in pigs and can preserve sperm viability, motility, and mitochondrial activity (Rodriguez-Martinez et al 2010 ;Caballero et al 2006 ), and cysteine-rich secretory protein-3 (CRISP3) in horses, which mediates the interaction between sperm and neutrophils (Doty et al 2011 ). Positive correlations between specifi c seminal plasma proteins and fertility have been observed in the horse (Brandon et al 1999 ) and bull (Killian et al 1993 ).…”

Dose-dependent effects of homologous seminal plasma on motility and kinematic characteristics of post-thaw stallion epididymal spermatozoa

“…540 Andrology, 2015, 3, 536-543 may have different effects on spermatozoa: protection, activation, influence on motility and membrane integrity, capacitation and acrosome reaction (Stout et al, 2000;Sostaric et al, 2010;Doty et al, 2011;Barrier-Battut et al, 2013). Time of SP exposure and concentration of SP seem to be of major importance concerning sperm motility, membrane integrity, viability and fertility (Bergeron & Manjunath, 2006).…”

“…Addition of SP to preserved semen immediately before insemination may be beneficial for some reasons: SP modulates the immune response in the female genital tract to protect spermatozoa (Troedsson et al, 2000(Troedsson et al, , 2005Dahms & Troedsson, 2002;Alghamdi et al, 2004;Doty et al, 2011) and promotes elimination of non-viable spermatozoa (Troedsson et al, 2005); SP is able to increase immediate motility of fresh and frozen-thawed epididymal and ejaculated spermatozoa (Braun et al, 1994;Stout et al, 2000;Heise et al, 2011;Neuhauser et al, 2013;Morrell et al, 2014); SP seems to be important for sperm maturation, plasma membrane remodelling, capacitation, acrosome reaction and fertilization (T€ opfer-Petersen et al, 2005;Rodr ıguez-Mart ınez et al, 2011;Caballero et al, 2012;Barrier-Battut et al, 2013) and could therefore increase fertility of epididymal spermatozoa. Although adding SP to the flushing medium did not improve post-thaw motility of epididymal spermatozoa in all stallions (Heise et al, 2011), there was a significant improvement in motility when added after the freeze-thaw process (Neuhauser et al, 2013).…”