Cytoskeletal sheets of mammalian eggs and embryos: A lattice‐like network of intermediate filaments

Abstract

Mammalian eggs and embryos possess a major cytoskeletal network composed of large planar "sheets" distributed throughout the cytoplasm. Cytoskeletal sheets are found neither in mammalian somatic cells nor in eggs or embryos of non-mammals. In this study, we have investigated the structural composition of the sheets in eggs and embryos of the golden Syrian hamster by (1) analysis of replicas from quick-frozen, deep-etched specimens, (2) analysis of thick, resin-embedded specimens using an intermediate voltage electron microscope (IVEM), (3) laser diffraction of EM images, (4) differential extraction with detergents, and (5) immunocytochemistry. Our results indicate that each sheet is composed of two closely apposed arrays of 10-nm filaments. Each filament within an array is held in register with its neighbor by lateral cross-bridges and the two parallel arrays of filaments are interconnected by periodic cross-bridges about 20 nm in length. Laser diffraction of negatives from IVEM images indicates that each array is composed of fibers that form a square lattice, and the two arrays are positioned in register by cross-bridges forming a single sheet. This lattice forms the skeleton of the sheets which is covered with a tightly packed layer of particulate material. By incubation in media containing different ratios of mixed-micelle detergents, it is possible to remove components sequentially from the sheets and to extract the particulate material. Immunocytochemical localization demonstrates that the sheets bind antibodies to keratin, and to a small extent actin, but do not bind antibodies to vimentin or tubulin. Examination of sheets within embryos at the time of embryonic compaction demonstrates that the sheets begin to fragment and disassemble in regions of blastomeres where desmosomes form, but undergo no structural alterations in interior and basal surfaces of the blastomeres. In regions of blastomere-blastomere contact the sheets fragment and associate with granules resembling keratohyalin granules found in keratinocytes.

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

“…This is a particular problem when imaging events that occur over periods of time ranging from hours to days, such as embryonic development. For this reason, much of our current understanding of subcellular morphological changes during mammalian embryonic development is based on images of fixed or static specimens at different developmental stages [2][3][4][5][6] . Thus, it can be difficult to interpret dynamic processes accurately, because the continuity of events must be inferred.…”

The subcortical maternal complex: multiple functions for one biological structure?

“…CPLs are highly abundant in the oocytes and have long been predicted to function as a storage form for the maternal contribution of ribosomes to the early embryo [29]. Additional work confirmed their involvement in ribosomal storage in oocytes [28] and showed that PADI6, NLRP5/MATER [32], and FLOPED/OOEP co-localize at the CPLs and are required for their formation.…”

“…1 Schematic representation of the hypothesized structure of the SCMC and localization within the MII oocyte Interestingly, PADI6 was seen to localize to [26] and be essential for the formation of the oocyte cytoplasmic lattices (CPLs), a fibrillar matrix composed of a proteinaceous component and RNA [27,28]. The CLPs contain five to seven parallel fibers with repeating units of about 20 nm [29]. The bundled fibers are first observed at early stages of oocyte growth (30-40 μm) [30] and persist in the early embryo until the blastocyst stage [31].…”

PKC Regulation of Gametogenesis and Early Development

“…Trim the block face to produce a square. Blocks are sectioned on an ultramicrotome with a knife angle adjusted to10° (Capco et al, 1993). 200 nm sections are collected on formvar-coated, carbon-stabilized grids precoated with poly-L-lysine and subsequently dried overnight in a vacuum desiccator.…”