The Establishment Of Lectin Binding Profile Of Sperm And The Expression And Functional Studies Of Epididymal β-defensins

(3-defensins are important innate immune molecules that provide protective barriers between host and environment. We have reported that Binlb, a rat epididymis-specific β-defensin, beyond its bacteria killing activity, initiated sperm motility, which, for the first time, implies that some epididymis-specific β-defensin molecules may participate in sperm maturation. In macaque, a highly glycosylated β-defensin 126 (DEFB126) protein secreted from corpus of epididymis covers the entire sperm surface, which contributes substantially to the sperm glycocalyx. DEFB126 is essential for facilitating sperm penetration of cervical mucus, protecting sperm from immune recognition and immune surveillance by the female immune system, and mediating attachment of sperm to oviductal epithelia. Recent investigations have shown that about 20% men carry DEFB126 gene with a two-nucleotides deletion in the coding region, and sperm from men with homozygous variants of DEFB126 gene shows significantly reduced fertility. Based on previous studies, mRNA expression of DEFB126 reduced in del/del males and their sperm showed lower Agaricus bisporus (ABA) lectin binding ability and poor penetration of hyaluronic acid (a substitute of cervical mucus). So the researchers suggested that these phenomena were due to the changed glycoproteins on sperm surface in del/del genotype males, but the evidences are very inadequate. Lectin microarray is a novel and effective tool for the study of cell surface carbohydrate structures. But the sperm glycan profiling studied by this technology is completely blank. In this study, we attempt to clarify the mechanism of sperm fertility reduction caused by DEFB126 gene micro-deletion and find potential biomarkers that characterize the sperm surface glycomes of men with different DEFB126 genotypes.By optimizing the preservation and staining conditions of human sperm, we fixed sperm with 2% paraformaldehyde/0.2% glutaraldehyde and stained it with propidium iodide (PI) for the subsequent experiments and profiled the lectin binding profile of human sperm surface for the first time. Moreover, in order to demonstrate the glycocalyx changes of sperm of men with del/del genotype, lectin microarrays were employed to analyze and compare the lectin binding spectrum of sperm from men with wt/wt, wt/del, or del/del genotypes. Six lectins displayed statistically significant differences and one of them (MPL) could be a potential biomarker to identify sperms from homozygous wild-type donors and the donors carrying homozygous deletion variant. This study will be of great significance about understanding, clinical diagnosis and treatment of male subfertility.In the second part, we explored the expression and functions of epididymis-specific Defb42. Defb42 can bind to the acrosomal region and Apical Plasma Membrane (APM) of sperm from epididymal initial segment. We screened a specific siRNA downregulated Defb42, and packaged it into lentivirus of high titer to knockdown Defb42 expression in vivo. However, inflammatory response caused by injection at initial segments leaded to significant reduction of Defb42 protein, and the expression of Defb42 was negatively correlated with inflammatory cytokines IL-1β and IL-6. In addition, Defb42 showed the antimicrobial activity against E. coli K.12D31 and S. aureu, but not C. albicans and it was successfully expressed in 293T cell with pCMV-Tag4 system.In the third part, by using a promising intein-mediated fusion expression system, the recombinant DEFB106 was expressed and purified (yield:3-5 mg/L) under optimized conditions. The purified protein was characterized with Mass spectrometry (MS) and circular dichroism (CD) spectroscopy. The measured molecular weight was consistent with its theoretical value and the major secondary structure was β-sheet, which is the common structure of P-defensin family members. The purified DEFB106 showed antimicrobial activity against S. aureus beside E. coli and C. albicans. In addition, DEFB106 displayed high affinity for heparin and lipopolysaccharide (LPS). Besides, the localization of native DEFB106 was determined in epididymis, bone marrow and skin with antibody which showed that DEFB106 was mainly distributed in the basal cell nuclei of corpus and cauda regions. These observations paved the way for ascertaining the physiological and pathological functions of DEFB106 in the future.