Equine seminal plasma derived lactoferrin regulates binding of polymorphonuclear neutrophils (PMNs) to spermatozoa

Abstract

Lactoferrin is an iron binding glycoprotein (w 80 kDa), which is present in equine seminal plasma and in the endometrium. It has important roles in enhancing cell-tocell interaction and in immune functions. Based on results from previous studies on PMNsperm binding and of lactoferrin in the host defense mechanism, we hypothesized that lactoferrin modulates the interaction of PMNs with spermatozoa in the uterus. The objectives of this study were to examine the role of lactoferrin in PMN-sperm binding and to characterize mRNA and protein expression of lactoferrin in the testis and epididymis. Lactoferrin was purified from equine seminal plasma by HPLC and identity of the protein was confirmed by western blot. Semen was collected and pooled from four stallions, washed twice in LRS at 600 x g and freeze-killed in liquid N2 followed by thawing at room temperature. Blood derived PMNs (3x105/ mL; pooled from 2 mares) were incubated 30 min with spermatozoa (4.2x106/mL) stained with propidium iodide (PI). PMNs and spermatozoa were incubated in the presence of Hank’s balanced salt solution, seminal plasma (pooled from 4 stallions), or lactoferrin (150 ug/mL). PMNsperm binding was determined using flow cytometry; PMNs were identified based upon FSC-SSC, and spermatozoa based upon PI labeling. A total of three experiments were performed in triplicates. Expression of lactoferrin in the testis and epididymis (caput, corpus and cauda) of four stallions was characterized by qPCR, immunohistochemistry and western blotting. Data were tested for normal distribution, and ANOVA was used for statistical comparisons. Both lactoferrin 62.5 3.3% and seminal plasma 76.5 0.3% significantly enhanced binding of PMNs to dead spermatozoa when compared to control media treatment 42.9 1.6% (p<0.05). mRNA for lactoferrin was highly expressed in the corpus epididymis, followed by the cauda and the caput, but was absent in the testis. Results were confirmed by immunohistochemistry with highly stained cells in the luminal epithelium of the corpus epididymis, followed by cauda and caput, but was absent in the testis. Western blots revealed presence of lactoferrin in the cauda and corpus with no visible bands in the caput or testis. Lactoferrin is a seminal plasma protein that is mainly expressed in the corpus and cauda epididymis of stallions. Seminal plasma enhances binding of PMNs to dead spermatozoa in vitro and purified lactoferrin was able to reproduce this effect. These findings suggest that one of the roles of lactoferrin in the reproductive tract of the mare is enhancing cell-to-cell interaction between PMNs and spermatozoa, which might facilitate elimination of dead sperm from the uterus in vivo. We have previously demonstrated a role for another seminal plasma protein (CRISP-3), in reducing binding between live spermatozoa and PMNs, allowing transport of this population of spermatozoa to the oviduct in an inflamed uterine environment.