The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding

Abstract

Fertilization is initiated by the binding of capacitated spermatozoa to the outer coat, zona pellucida (ZP), of the oocyte. Although the regulatory mechanisms of spermatozoa-ZP binding still remain elusive, accumulated evidence has suggested that spermatozoa-ZP receptor is a composite structure requiring coordinated action of different proteins assembled into a complex during capacitation. Up-regulation in sperm surface thiol content has been demonstrated by capacitation and thiol-disulphide exchange is crucial to protein structure, function, folding and localization. Protein disulphide isomerase (PDI) family has prominent effect in catalyzing thiol-disulphide modulation. ERp57, an enzyme known as PDI family A, member 3, has been shown to express in the human spermatozoa and to promote cell surface thiol content in other cell types. We therefore hypothesized that expression of ERp57 on sperm surface after capacitation modulates human spermatozoa-ZP binding via regulating thiol-disulphide exchange of the associated ZP receptor complex.

The first objective was to determine whether multimeric protein complexes on the plasma membrane of capacitated human spermatozoa are involved in spermatozoa-ZP binding. The extracted sperm plasma membrane proteins were resolved by blue native gel electrophoresis/sodium dodecyl sulphate polyacrylamide gel electrophoresis (BNG/SDS-PAGE). Subsequent mass spectrometric analysis identified two major human spermatozoa-ZP binding complexes. ERp57 was found to be a component of one of the complexes.

The second objective examined the relationship between capacitation, sperm surface thiol content and spermatozoa-ZP binding. It was found that capacitation induced sperm surface thiol content and ZP-binding capacity. And coherently, the ZP-binding capacity of uncapacitated spermatozoa was stimulated by in vitro up-regulation of sperm surface thiol content and promotion of ERp57 by thioredoxin and a phosphatase inhibitor calyculin A respectively. Results also indicated that sperm surface thiol was localized to the sperm head of capacitated spermatozoa, a region crucial for ZP binding.

The third objective investigated the correlation between sperm surface thiol status and ERp57 surface expression. Western blotting results confirmed the presence of ERp57 in human spermatozoa with its immunoreactivity on the acrosomal region significantly increased after in vitro capacitation. Sperm surface ERp57 expression was increased by capacitation and calyculin A treatments. Moreover, the treatment with functional blocking antibody against ERp57 significantly inhibited capacitated spermatozoa-ZP binding dose-dependently without affecting other sperm functions and suppressed the capacitation-induced sperm surface thiol up-regulation.

The fourth objective aimed to identify the ERp57-interacting proteins from the sperm membrane protein extracts. By applying co-immunoprecipitation and immunofluorescence staining respectively, calreticulin (CALR) was identified as an ERp57-interacting protein and detected on the capacitated sperm head surface. Blocking of CALR function by specific antibody significantly inhibited calcium ionophore A23187-induced acrosome reaction and spermatozoa-ZP binding without affecting other sperm functions. ERp57 was found to be involved in mediating the thiol groups on CALR.

In conclusion, this thesis illustrated that capacitation enables the translocation of ERp57 to the sperm surface, which then modulates spermatozoa-ZP binding via promoting the thiol content of the interacting protein. Results from this study provide clinical significance of thiol-disulphide modulation and/or ERp57 in assessing and enhancing the fertilizing potential of human spermatozoa for assisted reproductive technology treatment.