Characterization Of Mannose Receptor On Human Sperm

Objective: Using different methods to judge the existence of mannose receptor onhuman sperm, and utilizing affinity chromatography technology to separate and purifythe receptor. Meanwhile, to discuss its relationship with Serum amyloidP-component(SAP)、 HSP70family molecules(HSP70、HSPA2、HSC70), and humanmacrophage mannose receptor (CD206), aims to determine the molecular character ofthe receptor, so as to establish the foundation for study on its roles in humanfertilization.Methods: Collection of normal human sperm samples, total protein was extractedfrom human sperm; then utilized DMA as a probe, Dot-Blot and Western Blotexperiments were carried out on human sperm crude protein, simultaneouslycombined the human sperm protein with mannose-Sepharose4B co-incubation andco-binding, DMA-FITC was used as a probe for immunofluorescence experiments.Affinity chromatography was used to separate and purify the human sperm mannosereceptor. Human sperm protein was incubated with mannose-Sepharose4B, using theDMA-FITC and anti-HSP70, anti-HSPA2, anti-HSC70, and anti-CD206-Biotinantibody respectively as a probe to carry out immunofluorescence experiments. Afterthat, for different stage sperm (fresh sperm、capacitation sperm and acrosomalreaction sperm), using the DMA-FITC and anti-HSP70, anti-HSPA2, anti-HSC70antibody respectively as a probe to carry out immunofluorescence experiments.Results: Dot-Blot and Western Blot, and the DM-beads which after binding to humansperm protein labeled with DMA-FITC all shows that hsMR exist in human spermprotein, and it could bind with DMA specifically. Western Blot shows that the MW ofhsMR existed in human capacitated sperm protein are about72KDa. After binding to human sperm protein, take some affinity chromatography column packing to carry outSDS-PAGE, many fine bands were seen when silver staining the PAGE. It suggestedthat not only one molecule can bind with the mannose. After binding to the proteinextracted from human washing sperm and capacitated sperm, DM-beads labeled withdifferent probes, results shows that HSP70、HSPA2and HSC70existed in humansperm protein, and they all have mannose-binding activity. Furthermore, binding tothe protein extracted from human washing sperm, green fluorescence was seen on theDM-beads labeled with anti-SAP; but not when binding with the protein extractedfrom human capacitated sperm. DM-beads binding to the protein extracted fromhuman washing sperm and capacitated sperm, no green fluorescence were seen. Itsuggested that CD206isn’t existed in human sperm protein. For different stage sperm(fresh sperm、capacitation sperm and acrosomal reaction sperm) labeled with differentprobes, results show that SAP exists on the surface of fresh sperm, located on the tails;HSP70mainly seen on the surface of acrosomal reaction sperm, located on the neckand middle; HSPA2was seen on the neck and middle of capacitation sperm, afteracrosomal reaction, green fluorescence mainly seen on the equatorial segment andpost-acrosomal region, and the typical bar-like structure visibled, on the middlealmost invisible; HSC70mainly seen on the equatorial segment and post-acrosomalregion, and the typical bar-like structure visibled, on the middle almost invisible, andsometimes fluorescence were seen on the whole sperm head.Conclusion: Mannose receptor of human sperm (hsMR) is a protein existed in humansperm, which have the ability of binding with mannose, the receptor include twokinds of molecules at least:(1) Serum amyloid P-component (SAP);(2) Heat shockprotein70family molecules (HSP70、HSPA2and HSC70). hsMR located on the tailsof fresh ejaculated sperm main component is SAP. In the process of spermcapacitation, SAP is lost, so it’s believed that SAP existed on the surface of spermtails. hsMR located on the head and middle of the tails main component is Heat shockprotein70family molecules (HSP70、HSPA2and HSC70). What’s more, HSP70mainly seen on the surface of acrosomal reaction sperm, located on the neck andmiddle; HSPA2was seen on the neck and middle of capacitation sperm, after acrosomal reaction, mainly located on the equatorial segment and post-acrosomalregion; HSC70mainly seen on the equatorial segment and post-acrosomal region. Allabout these suggested that HSPA2and HSC70might play important roles in humanfertilization. There are no CD206protein (human macrophage mannose receptor,hmMR) existed in human sperm. This shows that although hsMR and hmMR bothhave mannose-binding ability, they are not the same molecule. In this study, It’s forthe first time reported the relationship between hsMR and HSP70family molecules.