Recombinant Human Seminal DNase.

Abstract

Seminal plasma deposited simultaneously with extended semen in the presence of neutrophils led to higher fertility in pigs and horses. The mechanism by which fertility was improved is not known, however, data suggest that seminal plasma conveys DNase activity which inhibits negative effects of neutrophils. Seminal protein extracts reduced sperm-neutrophil binding in vitro in a dose-dependent manner by hydrolyzing DNA extruded from activated neutrophils. DNase activity exists in semen from several mammalian species, and inhibits formation of sperm-neutrophil complexes. Seminal fertility-associated antigen (FAA) is a basic 31 kDa non-glycosylated heparin binding protein homologous to the family of DNase-I-like proteins (bovine FAA shares 88% identity to human DNase1L3). It is secreted by accessory sex glands and binds to sperm at ejaculation. FAA possesses two DNase-I-like signature motifs, and recombinant bovine FAA hydrolyzed DNA in vitro. FAA is a protein marker of fertility in bulls; sperm extracts containing detectable FAA indicated higher bull fertility compared to extracts lacking FAA. The objective of this study was to clone the human FAA homolog, determine whether it displayed biological activity previously described in seminal plasma, and investigate the hypothesis that human FAA modulates fertility. To that end, the human homolog to bovine FAA was cloned and expressed as a recombinant N-terminal his-tagged fusion protein. Gene-specific primers successfully amplified a 411 bp product of the human FAA gene spanning the DNase-I-like motifs. PCR product was cloned into a TOPO expression vector and transformed into BL21(DE3) cells. Expression of recombinant was induced by IPTG (4h, 37C). Optimal protein induction and solubility was determined by extraction in non-denaturing or denaturing buffers. Bacterial cell pellet was lysed and clarified extracts were purified by metal affinity chromatography. Purity was assessed by SDS-PAGE and verified by Western blotting. Blots were probed with anti-HisG-HRP conjugated antibody and with anti-bovine FAA monoclonal antibody to verify identity of the human homolog. Blots probed with anti-bovine FAA were incubated with goat anti-mouse HRP secondary antibody and developed by incubation in HRP substrate. Purified rFAA was renatured by multi-step dialysis with insoluble aggregates removed by centrifugation. DNA activity exhibited by human seminal plasma and purified recombinant human FAA was analyzed by degradation of calf thymus DNA using agarose gel electrophoresis. Seminal plasma from 12 patients displayed variation in amount of DNase activity detected. Marked degradation occurred in one-third, intermediate degradation in another third, with little to no activity in remaining samples. The expressed recombinant shared 90% nucleotide identity with bovine recombinant FAA (390 bp). Affinity-purified recombinant displayed a single band of 19.6 kDa using an anti-HisG and anti-FAA antibody. Human recombinant FAA displayed DNase activity in a dose response manner above 50 µg/ml. These data demonstrate high homology between bovine and human FAA and indicate that FAA is a primary source of intrinsic DNase activity in human semen. The recombinant human homolog to FAA can now be exploited (1) to evaluate if it modulates sperm/neutrophil binding, (2) as an antigen to develop antibodies for cytological studies, and (3) to use those antibodies to quantify FAA in semen in relation to fertility of patients.