Homogeneity in the distribution of matrix components in the hamster zona pellucida as revealed by backscattered electron imaging fracture-label

Abstract

Background: Backscattered electron imaging fracture-label (BEI-FL), an adaptation of the fracture-label method for scanning electron microscopy, offers the advantage of providing information about the distribution of antigenic and receptor sites with respect to the three-dimensional organization of tissues and cells over relatively large surfaces. Recently, using post-embedding cytochemistry on thin-sections of Lowicryl-embedded oocytes, a homogenous distribution of glycoproteins in the zona pellucida (ZP) was demonstrated (Kan et al., 1989. Biol. Reprod., 40:585-598, Anat. Rec., 226:37-47; Roux and Kan, 1991. Anat. Rec., 230:347-360). However, it can be argued that the chemical nature of resins and the physical conditions of tissue processing required for post-embedding cytochemistry may introduce changes in the tissue components and result in altered distribution of components. On the other hand, freeze-fracture exposes constituents in a minimally denaturing manner and, since no embedding media are used, binding sites are sterically available to the probe. We have, therefore, applied BEI-FL to examine the distribution of matrix glycoproteins in the ZP of hamster oocytes.

Methods: Ovaries and cumulus masses obtained from superovulated female golden hamsters were fixed by immersion in 2.5% glutaraldehyde and processed for fracture-label. Tissues were labeled, respectively, with Wheat germ agglutinin (WGA) followed by ovomucoid-colloidal gold, Ricinus communis agglutinin I (RCA I)-colloidal gold or a monoclonal antibody against Hamster Oviductin-1 followed by protein A-gold, and then examined in the scanning electron microscope.

Results: Backscattered electron imaging revealed a homogenous distribution of WGA and RCA I binding sites throughout the cross-fractured matrix of the ZP of ovarian and postovulatory oocytes. Hamster Oviductin-1, an oviductal glycoprotein which is transferred to the ZP of oocytes during oviductal transit, was also found to be uniformly distributed throughout the ZP of postovulatory oocytes.

Conclusions: Our results indicate that BEI-FL can be advantageously used to examine extracellular matrices and are consistent with the concept that glycoproteins are uniformly distributed throughout the ZP of the hamster oocyte.