Field emission scanning electron microscopy and transmission electron microscopy studies of the chorion, plasma membrane and syncytial layers of the gastrula-stage embryo of the zebrafish Brachydanio rerio: a consideration of the structural and functional relationships with respect to cryoprotectant penetration

The structure of the chorion and plasma membranes of gastrula-stage zebrafish Brachydanio rerio embryos were studied using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM). These studies confirm the outer chorion membrane complex to be 1.5-2.5 mu m in thickness and to consist of three layers, electron-dense outer and innermost layers (0.2-0.3 and 1.0-1.6 mu m in thickness respectively) separated by an electron-lucent middle layer (0.3-0.6 mu m in thickness). The middle and inner layers are pierced by pore canals. A granular to farinaceous nature of the thin outer surface of the outer layer of the chorion has been revealed for the first time. The study provides original TEM images of the plasma membrane structures of gastrula-stage embryos, and FE-SEM and TEM images showing the plasma membrane to have three morpohologically distinct regions, being prominently ridged and folded at the surface of the blastoderm, smooth over the syncytial layer at the vegetal pole and with an intermediate region between the animal and vegetal pole where folding develops in advance of the expanding blastodermal disc of cells. FE-SEM and TEM studies reveal details of the syncytial layer (1-4 mu m thick) beneath the smooth plasma membrane at the vegetal pole, containing cytoplasmic organelles and small yolk globules. The significance of the structural detail shown in these studies is considered in the light of the difficulties experienced in cryopreservation of the embryo resulting from the inability of achieving cryoprotectant penetration of the yolk mass.