Novel cytoskeletal elements in mammalian eggs are composed of a unique arrangement of intermediate filaments

Mammalian eggs and embryos contain a major network of specialized cytoskeletal components known as 'sheets' that have not been identified in any other cell type. Although eggs from at least seven different mammalian species have been shown to contain these cytoskeletal structures, embedment-free electron microscopic analysis of these eggs revealed that two basic forms of cytoskeletal sheets exist, a solid, planar type of sheet typical of hamster and rat eggs and a fibrous sheet typical of mouse, porcine, bovine, canine, and human eggs. In this study we have investigated the structural composition of the fibrous type of sheet in mouse eggs by employing biochemical approaches as well as two forms of ultrastructural analyses including: (1) analysis of thick, resin-embedded specimens using an intermediate voltage electron microscope (IVEM); (2) analysis of replicas from quick-frozen, deep-etched specimens. Our results indicate that the sheets of mouse eggs and preimplantation embryos are composed of cylindrical bundles of 10-11 nm filaments, with each of these filaments held in register by periodically arranged crossbridges spaced 23-25 nm apart. This sheet substructure of filaments and crossbridges is covered by a particulate material which can be removed by non-ionic detergent. Immunoelectron microscopic analysis of mouse eggs demonstrates that sheets bind antibodies to keratin and to a small extent, actin, but do not bind antibodies to vimentin or tubulin. Confirmation that keratin exists in these eggs was obtained by electrophoretic separation and one- and two-dimensional Western blot analysis demonstrating the existence of keratin types 5, 6, 8, 16, and type Z. The low abundancy of keratin type 8 compared to other keratin types explains the difficulties other investigators have had identifying intermediate filaments in mammalian embryos since most investigators have used antibodies directed specifically against keratin type 8 or its pair keratin type 18. Examination of compacted mouse embryos reveals that the filamentous framework of sheets disassembled and established close contact with the basolateral plasma membrane and the nucleus. However, sheets at the apical plasma membrane of blastomeres attach to the membrane but remain intact. Based on our biochemical and ultrastructural data, the fibrous sheets of mouse eggs appear to be cytoskeletal structures comparable to the solid, planar sheets of the Syrian hamster egg and probably serve similar function(s) in eggs and embryos of several mammalian species.