The roles of sialyl-Lewis(x)-binding protein and sialidase on spermatozoa-zona pellucida interaction in humans

Abstract

A spermatozoon cannot fertilize an oocyte immediately after ejaculation, but acquire the fertilization capacity within the female reproductive tract by a process termed capacitation. This process involves relocalization of molecules in the plasma membrane of the sperm head resulting in acquisition of the binding capacity of the spermatozoa to the outer coat (zona pellucida, ZP) of the oocyte. Capacitated spermatozoa initiate fertilization by binding to the carbohydrate sequences on the ZP, which induces acrosome reaction of the ZP-bound spermatozoa. Defects in this binding are the major causes of reduced fertilization rates in assisted reproduction. Recently, by using ultrasensitive mass spectrometry, sialyl-Lewis(x) (SLeX) sequence has been identified as the most abundant terminal sequence on the glycans of human ZP. It is also the major carbohydrate ligand for human sperm-ZP binding. By using affinity chromatography followed by mass spectrometric analysis, chromosome 1 open reading frame 56 (C1orf56) was identified to be one of the SLeX-binding proteins in the membrane protein extracts of capacitated spermatozoa. The expression of C1orf56 was confirmed on both uncapacitated and capacitated spermatozoa by Western blotting and the protein quantity remains stable during this process. The surface expression on spermatozoa was also confirmed by flow cytometry. The acrosomal region of spermatozoa possessed C1orf56 immunoreactivity and its intensity increased after capacitation in vitro, suggesting translocation of C1orf56 to the region that responds to spermatozoa-ZP binding during capacitation. Anti-C1orf56 antibody did not affect sperm viability, motility and spontaneous acrosome reaction but inhibit spermatozoa-ZP binding and ZP-induced acrosome reaction dose-dependently. It even suppressed spermatozoa-SLeX binding significantly. Our data further demonstrated that capacitated human spermatozoa possess cell surface sialidase activity. Sialidase-1 and -3 immunoreactivities can be detected on the acrosomal region of spermatozoa. The surface expression of sialidase increased significantly after capacitation. Sialidase inhibitors can inhibit the sialidase activity significantly but have no effect on other sperm function parameters except ZP binding capacity and ZP-induced acrosome reaction. The involvement of sialidase in spermatozoa-ZP interaction was further demonstrated by the increased capacities of the ZP in binding to sperm and inducing acrosome reaction after sialidase treatment. A pilot clinical study was also performed to determine the possible relationship between sperm C1orf56 expression and the fertilization rates in clinical assisted reproduction. Our results showed that there was no direct relationship between C1orf56 expression and sperm motility or morphology. There was also no significant association between C1orf56 expression on capacitated spermatozoa and in vitro fertilization rate. For the acrosome-reacted spermatozoa, although there is a trend of lower C1orf56 expression in the low fertilization group when compared to the high fertilization rate group, results were not statistically significant (P=0.058). Therefore, this result needs to be confirmed in the future by study with larger sample size. To conclude, our studies provide evidence that both SLeX-binding protein C1orf56 and sialidase on spermatozoa play biological roles on human spermatozoa-ZP interaction. The outcome of this study provided a better understanding on spermatozoa-ZP interaction in humans.