Electron-microscopic localisation of thiol and disulphide groups by direct monomaleimido-nanogold labelling in the spermatozoa of a marsupial, the tammar wallaby (Macropus eugenii)

Abstract

This study utilised a commercially available monomaleimido-nanogold reagent to directly label cellular thiol groups (SH) of marsupial (tammar wallaby) spermatozoa before and after reduction of disulphides (S-S) with mercaptoethylamine hydrochloride (MEA). The sperm surface, mitochondrial membranes, axoneme and tail fibres were all labelled with gold particles before MEA treatment and the label intensity was increased after S-S reduction. The acrosomal membranes and matrix of spermatozoa contained no detectable SH prior to MEA treatment. However, after moderate MEA treatment (1 mg/ml) gold label was associated with the acrosomal membrane and invaginated acrosomal membrane within the acrosomal matrix. After exposure to 5 and 10 mg/ml MEA, gold particles heavily labelled the acrosomal matrix. Thus, the acrosomal membranes and matrix of tammar wallaby spermatozoa both contain S-S cross-linked structures, and this may contribute to the unusual stability of the marsupial acrosome. Under all treatment conditions the nucleus remained unlabelled. This is consistent with early studies which indicated that cysteine was absent from the nuclear protamines. The study also demonstrated that monomaleimido-nanogold can be used to resolve SH- and S-S-rich cellular structures directly, in addition to its use to label antibodies and Fab fragments for immunochemical localisation.