Ultrastructure of the vitelline coat in the ascidians Phallusia mammillata , Ascidia mentula and Ciona intestinalis : new aspects revealed by freeze-substitution and deep-etching

Abstract

The microstructure of the vitelline coat (VC) of the three ascidian species Phallusia mammillata, Ascidia mentula and Ciona intestinalis was analyzed by transmission electron microscopy (TEM) using freeze-substitution and deep-etching. In addition, eggs were examined by light microscopy and prepared by conventional chemical fixation methods for TEM in order to illustrate the advantages of cryopreparation for the preservation of the VC ultrastructure. In conventionally fixed eggs of P. mammillata and A. mentula the VC appeared as a tripartite structure, consisting of a thin outer fibrous layer (OFL), a central dense layer (CDL) and a relatively wide inner fibrous layer (IFL). In C. intestinalis a sparse OFL is made up by fine tufts of filaments but an IFL is lacking. In all three species the test cells were located in the area between the inner VC boundary and the egg cell membrane. Using our modified freeze-substitution method a clear improvement in preservation of the VC matrix was obtained. In all three species the OFL as well as the IFL in P. mammillata and A. mentula appeared as extended fibrous meshworks. Furthermore, the area of the perivitelline space, empty after conventional TEM preparation, was found to be filled by an additional matrix, designated here as perivitelline fibrous matrix (PVFM). Quick-freeze/deep-etch/rotary-shadowing enabled us to confirm the overall architecture of the egg matrix and allowed a further detailed investigation of the VC fine structure in P. mammillata. Accordingly, the vitelline coat of P. mammillata and A. mentula is a multilayered matrix composed of four distinct subunits (OFL, CDL, IFL, PVFM), whereas in C. intestinalis an IFL is lacking.