An influx of polymorphonuclear neutrophils (PMNs) into the uterine lumen is a natural mechanism to remove excess spermatozoa and bacterial contamination after breeding [[1], [2], [3], [4]]. A timely resolution of the transient inflammation is necessary for embryo survival when it descends from the oviduct into the uterus approximately 6 days after fertilization [3]. We have previously demonstrated that seminal plasma derived cysteine rich secretory protein 3 (spCRISP3), at least in part, mitigates this mechanism by protecting equine spermatozoa from binding to uterine PMNs [5]. CRISP3 is a major protein found in equine seminal plasma, constituting 0.3–1.3 mg/mL, located on the sperm surface, and has been positively correlated with fertility in stallions [[6], [7], [8], [9]]. CRISP3 has also been suggested to play a role in human reproduction as well as in innate immunity [10,11]. The protein is produced by B cells and is found in neutrophils, where it is located in the secretory granules but the exact role of CRISP3 in the immune system is not fully understood [12]. Udby et al. (2002) proposed that CRISP3 may participate in the protection against microorganisms, based on its diverse distribution in exocrine gland secretions and its presence in granulocytes [10]. The CRISP family of proteins is characterized by 16 conserved cysteine residues that fold into two domains: a N-terminal CRISP with a CAP domain that contains six conserved cysteine residues, and the smaller C-terminal CRISP that contains 10 conserved cysteine residues [12]. Three different CRISP proteins (1, 2 and 3) have been found in humans and horses, as well as in rats (1, 2 and 4), while all four members of the CRISP family are present in mice [12]. Transcripts for the CRISP proteins are present throughout the reproductive tract of both mice and humans, with CRISP1 in the entire epididymis, CRISP2 in the testis, epididymis, and seminal vesicles, and CRISP3 in the ampulla of the vas deferens [13]. In the horse, CRISP3 is located in the glandular portion of the ampulla of the vas deference, and to a lesser degree in the seminal vesicles [14]. Equine CRISP3 mRNA sequence demonstrates 82% homology with human CRISP2, 78% homology to guinea pig CRISP2, and 77% homology to human CRISP3 [6].

PMNs have been shown to bind to equine spermatozoa in vivo and in vitro, and they can form large clusters that reduce the motility of spermatozoa [15,16]. Alghamdi and Foster suggested that the formation of these clusters is due to interaction with neutrophil extracellular traps (NETs) [17]. NETs are web-like structures composed of chromatin that are released from PMNs in a special cell death pathway, first described as a mechanism to contain pathogens [18,19]. We have previously reported that spCRISP3 protects spermatozoa from binding to PMNs, but it was not clear if suppression of binding between PMNs and spermatozoa is a selective process involving a subset of viable and normal spermatozoa, or if the process is non-selective for all populations of spermatozoa [5]. We recently observed that a protein complex of tightly bound lactoferrin (LF) and superoxide dismutase (SOD-3) promotes binding between PMNs and dead, but not viable spermatozoa [20]. Furthermore, it is not clear if spCRISP3 also protects bacteria from binding and phagocytosis by PMNs. A selective mechanism for transport of viable sperm to the oviduct, while eliminating dead sperm and bacteria would be beneficial in species with intrauterine deposition of semen. The objectives of this study were therefore, 1) to determine if CRISP3 is selective in its suppression of PMN-binding to sperm based on viability of spermatozoa, 2) to determine if CRISP3 protects bacteria from being bound by PMNs, and 3) to determine the localization pattern of CRISP3 on viable and dead/damaged spermatozoa.