Histochemical characterization of anionic constituents in oocyte-cumulus complex of rats

Fertilization is a process that involves the recognition and adhesion of negatively charged spermatozoa to the oocyte investments. It is not known, however, if charge properties of the interacting gametes play a role in fertilization. The present study evaluates the content and distribution of anionic constituents in the oocyte-cumulus complex of rats. Polycationic colloidal gold (PCG), ruthenium red (RR) and wheat germ agglutinin (WGA) were used as cytochemical markers of anionic sites at the light (LM) and electron microscopical (EM) levels. Isolated oocyte-cumulus complexes were fixed with glutaraldehyde (GA) and OsO₄ containing RR, or with GA without RR, and embedded in araldite or LR-gold. For LM, deresined, semi-thin, araldite-embedded sections were labelled with PCG intensified by silver, or with biotinylated lectins visualized by avidinperoxidase. For EM, thin LR-gold sections were labelled with PCG, whereas RR labelling was examined in araldite sections. The zona pellucida (ZP) failed to bind any of the polycationic markers used, but intensely bound neutralized WGA. In contrast, cumulus cell membranes bound PCG but not RR, whereas the oolemma bound RR but not PCG. The results indicate that the ZP is practically devoid of negatively charged constituents, and tends to repel positively charged ligands possibly due to the presence of cationic determinants. The binding of PCG to cumulus cells probably reflects a high content of membrane-bound heparan sulphate, whereas the binding of RR to the oolema indicates the presence of membrane sialoglycoconjugates. We suggest that during sperm-egg interaction the negatively charged spermatozoon may be electrostatically repelled by the cumulus cells, yet at the same time attracted by the ZP.