Abstract. A simple method is described for highresolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.F LUORESCENT labeling techniques have become increasingly popular in many areas of cell biology, particularly due to the expanded use of laser scanning confocal microscopy. Immunofluorescent localization of proteins and fluorescence-based in situ hybridization detection of specific nucleic acid sequences in cells and tissues have several advantages over other labeling procedures including relative simplicity, shorter processing times, high sensitivity, and good spatial resolution. One of the major disadvantages of fluorescent labeling has been the inability to use this technique for correlated light and electron microscopic studies in cases requiring better than light microscopic resolution. Several methods have been introduced in an attempt to overcome this limitation. These include the use of anti-fluorophore antibodies, e.g., anti-fluorescein, conjugated to peroxidase (Peters et al., 1990) and the process of fluorescence photooxidation of diaminobenzidine (DAB)? This latter method, first described by Maranto (1982), involves the use of fluorescent dyes to oxidize DAB into an insoluble reaction product.Photooxidation represents a relatively simple method to render fluorescently labeled preparations suitable for elec- tron nficroscopic examination. Briefly, a fluorescent dye is excited in the presence of DAB and gradually an oxidized DAB reaction product forms. The reaction product is highly insoluble and easily visible with ordinary transmitted light microscopy. Since the DAB reaction product is osmiophilic, this technique can be used for correlative light and electron microscopic studies using a variety of fluorescent compounds including Lucifer yellow, DiI, Bodipy ceramide, fluorogold, and fluororuby (Pagano et al., 1989;Bentivoglio and Su, 1990;yon Bartheld et al., 1990;Balercia et al., 1992;Lubke, 1993;Papadopoulos and Dori, 1993;Schmued and Snavely, 1993;Takizawa et al., 1993). A poten...

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The zona pellucida is the spherical glycoprotein shell that surrounds the mammalian oocyte at fertilization. Penetration of this shell by spermatozoa plays a crucial role in mammalian fertilization and any inability of spermatozoa to penetrate the zona pellucida inevitably leads to infertility. The purpose of this review is to summarize what we know or can infer about the three-dimensional structure of the zona pellucida, its construction and its properties as a polymer. Because the focus of the review is on three-dimensional structure, it relies heavily on data from mice. Features of the zona pellucida are discussed and two models for its construction are considered. The cortical reaction and its possible effects are discussed and avenues for future experimental work are identified.

Mammalian sperm undergo discharge of a single, anterior secretory granule following their attachment to the zona pellucida surrounding the oocyte. This secretory discharge is known for historical reasons as the acrosome reaction. It fulfils a number of purposes and without it, sperm are unable to penetrate the zona pellucida and fuse with the oocyte. In this review, we focus on the role of the acrosome reaction in the development of fusion competence in sperm. Any naturally occurring membrane fusion has two major sequential steps: a docking or adhesion step, in which two membranes adhere, and a fusion step, in which their lipid bilayers are destabilized and merged and a cellular compartment is either created or destroyed. Recent evidence suggests that there is an important role for oocyte integrins and sperm-bound disintegrins in mammalian sperm/oocyte adhesion and fusion. The fusion mechanism employed by sperm remains poorly understood, however, and circumstantial evidence suggests it is more complex than the interaction between a single protein species and its target. Sperm/oocyte fusion is probably the most accessible eukaryotic model for intercellular fusion currently available, partly because it is temporally separated from gene expression. Elucidation of the mechanism of sperm/oocyte fusion may throw light on the mechanism of other intercellular fusions such as myoblast fusion, and the evolutionary relationship of intercellular membrane fusion to intracellular membrane fusion.

Mouse preimplantation development represents a tightly controlled programme of gene expression and cell division, which starts with the fertilized egg and ends with implantation of the blastocyst approximately 4.5 days later. Spatial and temporal differences in gene expression underpin establishment of axes at the two-cell stage and development of the trophectoderm and inner cell mass after embryo compaction at the eight-cell stage. Approximately 15 700 mouse genes expressed during preimplantation development have been identified from cDNA sequences deposited in the UniGene database of the National Institutes of Health. This inventory of preimplantation genes is the starting point for identifying signalling modules that function in preimplantation development.