Membrane organization in the preimplantation mouse embryo

HESTER P. M. PRATT 0 0 Department of Anatomy , Downing Street, Cambridge, CB2 3DY, U.K The preimplantation mouse blastocyst consists of two differentiated tissues, the trophectoderm (a structurally and functionally polarized epithelium) and the inner cell mass. The divergence of these two cell types can be traced back to a contact dependent polarization of the surface and cytoplasm at the 8-cell stage. Membrane/cyi:ocortical organization during this preimplantation period has been studied using freeze fracture in conjunction with the sterolbinding antibiotic filipin in an attempt to discern the molecular basis and origin of these surface asymmetries. The distribution of filipin reactivity within the different membrane domains showed that the surface polarity exhibited by trophectoderm and by blastomeres of the 8-cell stage is underlain by a heterogeneity in molecular organization of the membrane/cytocortex which may originate prior to the appearance of any overt surface polarity. The results are discussed in terms of the likely basis of this membrane/cytocortical asymmetry, its probable origins and the use of the preimplantation mouse embryo as a model system for studying the assembly of a polarized epithelium. - Dragsten, Blumenthal & Handler, 1981; Reggio Coudrier & Louvard, 1982; Van Meer & Simons, 1982; Sabatini et al. 1983). Cellular asymmetries are not only important in fully differentiated tissues but may also play a significant role in early development when differential inheritance of asymmetrically localized components can initiate or reinforce cell divergence (Johnson & Pratt, 1983). One particularly accessible example of this is the preimplantation mouse embryo where a contact-induced asymmetry of membrane and cytoplasm at the 8-cell stage is distributed between two daughter 16-cell-stage blastomeres to generate two phenotypically distinct subpopulations. There is good evidence to suggest that in the undisturbed embryo these two subpopulations form the foundation of the two primary tissues of the blastocyst, the ICM (inner cell mass) and the trophectoderm, a structurally and functionally polarized epithelium (Johnson, 1985a). The preimplantation mouse embryo therefore provides a system in which the development of two cell types (one polarized and the other apolar) can be traced in their entirety through six cell cycles in vitro and the organization of the intervening cell types studied. I chose to study this process at the level of the surface membrane since the cell surface is known to play a crucial role in early development (Johnson, 19856) and the main pathways for membrane biogenesis and recycling in epithelia have been outlined (Reggio et al. 1982; Van Meer & Simons, 1982). The application of freeze fracture replication of membranes to embryos provides approximately ten-fold higher resolution than scanning electron microscopy and I have used this technique in conjunction with the cholesterol-binding polyene antibiotic filipin (Severs & Robenek, 1983) to infer changes in the molecular organization of the membrane and cytocortex during cleavage. The main questions I have posed are the following: are the polarized membrane morphologies present at the 8-cell stage and in trophectoderm underlain by heterogeneity at the molecular level and if so does this heterogeneity increase in complexity as trophectoderm develops? Does molecular reorganization precede the overt morphological surface changes at any stage? The results of the freeze fracture cytochemical analysis are discussed in terms of these embryological questions as well as in the wider context of assembly and maintenance of asymmetry in other cell types. 1. Recovery of eggs and embryos Female HC-CFLP mice (Hacking & Churchill Ltd) aged 3-5 weeks were superovulated by intraperitoneal injections of 5 i.u. PMS (Folligon) followed 48 h later with 5 i.u. hCG (Chorulon) and caged overnight with HC-CFLP males if fertilized eggs or embryos were to be obtained. The presence of a vaginal plug the following morning was taken as an indication of successful mating. Eggs (or oocytes from non-mated females) were removed from oviducts at approximately 20 h post hCG and freed of cumulus cells by brief exposure to 0-1 M-hyaluronidase (Sigma) followed by a rinse in medium 2 containing 4mgml"1 BSA (M2+BSA) (Fulton & Whittingham, 1978). Fertilized eggs with two pronuclei and oocytes without pronuclei (i.e. that had not been activated) were selected for further analysis. Cleavage-stage embryos and blastocysts were flushed from oviducts or uteri using M2+BSA at the times (hours post hCG) indicated. ICMs were isolated from expanding blastocysts using immunosurgery as described by Johnson & Ziomek (1982). Mural trophectoderm was obtained by removing ICM and polar trophectoderm from 'giant' blastocysts which were generated by aggregating several 8- and 16-cell embryos together. The zonae were removed from the 8- to 16-cell embryos by brief exposure to acid Tyrode's solution (Nicolson, Yanagimachi & Yanagimachi, 1975), followed by a rinse in M2+BSA. They were then aggregated together in groups of ten to twelve using a 1:20 dilution of stock phytohaemagglutin (PHA, Gibco Laboratories, Grand Island Biological Co., Grand Island, N.Y.) and cultured to the expanded blastocyst stage in medium 16 plus 4mgml"1 BSA (M16+BSA, Whittingham, 1971) at 37C in 5 % CO2 in air. Microsurgery was conducted under a binocular dissecting microscope using a scalpel blade. The role of the cytoskeleton was examined by incubating embryos in cytochalasin D (O-SiUgmP1), colcemid (5 jug ml"1) or low calcium medium (Pratt, Ziomek, Reeve & Johnson, 1982) for 4 to 6 h prior to fixation. Embryos (with intact zonae) were fixed for freeze fracture in duplicate groups of 30-40 embryos in either the presence or absence of filipin. Early experiments also included a control containing the appropriate concentration of dimethylsulphoxide (DMSO). A 30mM solution of filipin (19-6 mgml"1 U-5956 (complex) Reference 8393-DEG-11-8, a gift from Dr Joseph E. Grady, The Upjohn Company, Kalamazoo, Michigan 49001, U.S.A.) was made up in DMSO and immediately diluted 1:100 to 300 jUM into a fresh solution of 3 % glutaraldehyde in 0-1 Msodium cacodylate pH7-3. Control samples were fixed in buffered glutaraldehyde alone or 1 % DMSO in glutaraldehyde. Samples were fixed overnight at room temperature in the dark. Tomatin (Serva Heidelberg, Germany) was used in an identical manner to filipin at a final concentration of lSOjUgml"1. Following fixation embryos were washed in 0-1 M-sodium cacodylate (pH7-3) and stored in the same buffer at 4C. 2. Freeze fracture replication of embryos In order to ensure that the maximum surface area was exposed to the freeze cleaving knife the embryos were embedded in gelatine on the flat, square bases of Beem capsules. This was achieved under a dissecting microscope by pipetting the fixed embryos (30 to 40) into a group on the base of the B (...truncated)