부산대학교 도서관

Simple Summary: Lactoferrin is found in equine seminal plasma and has been suggested to mediate binding between sperm and polymorphonuclear neutrophils (PMNs) in the female reproductive tract. The overall objective of this study was to determine if lactoferrin binds preferentially to non-viable equine sperm. Seminal plasma lactoferrin was consistently bound to superoxide dismutase (SOD-3), forming a LF/SOD-3 protein complex. Various methods of biotinylation were evaluated to optimize the method and determine binding sites of LF/SOD-3 on equine spermatozoa. The LF/SOD-3 complex bound to equine sperm with preference to non-viable spermatozoa, possibly facilitating elimination of this sperm population through PMN-phagocytosis. Sperm-neutrophil binding is an important facet of breeding and significantly impacts fertility. While a specific seminal plasma protein has been found to reduce this binding and improve fertility (CRISP-3), additional molecule(s) appear to promote binding between defective sperm and neutrophils. Recent work has suggested one of these proteins is lactoferrin (LF), an 80 kDa iron-binding protein found throughout the body, but the purity of the protein was not confirmed. It is unknown if LF binds to sperm selectively based on viability, and if receptors for LF are located on equine sperm. To evaluate this, we attempted to purify equine seminal LF from five stallions (n = 5), biotinylate LF, and evaluate potential binding site(s) on spermatozoa. LF was consistently associated with superoxide dismutase (SOD-3), and all attempts to separate the two proteins were unsuccessful. Flow cytometric and microscopic analyses were used to compare LF/SOD-3 binding to viable and nonviable spermatozoa. Additionally, various methods of biotinylation were assessed to optimize this methodology. Biotinylation of seminal plasma protein was an effective and efficient method to study seminal plasma protein properties, and the binding site for LF/SOD-3 was found to be broadly localized to the entire sperm cell surface as well as selective towards nonviable/defective sperm. Although we were not able to determine if the binding to equine spermatozoa was through LF or SOD-3, we can conclude that equine seminal LF is tightly bound to SOD-3 and this protein complex binds selectively to nonviable spermatozoa, possibly to mark them for elimination by neutrophil phagocytosis. [ABSTRACT FROM AUTHOR]