Ultrastructural morphology of the envelope of Dover sole Solea solea eggs from fertilization until hatching with emphasis on sample preparation.

This study is the first to describe the ultrastructural morphology of the envelope of Solea solea eggs from fertilisation until hatching. Defining the ultrastructural morphology of fish eggs is important for species identification and may assist in predicting the effect of external influences on these early life stages. In first instance, various fixation and embedding protocols were assessed to explore the morphology of the egg envelope, whereby the encountered difficulties were highlighted. The successful protocol for SEM proved to be combined fixation with 4% glutaraldehyde in 0.1M cacodylate buffer for minimum 4h with post-fixation of 2h with 1% OsO4 in 0.1M cacodylate buffer. For TEM, puncturing the egg envelope during the first steps of the fixation protocol was necessary to allow the embedding medium to penetrate through the egg envelope. Based on both scanning and transmission electron microscopical examination, three distinct layers were discerned in the egg envelope. During the development of the fish embryo, a change in the outer structure of the egg was observed. Scanning electron microscopical examination of one day post-fertilisation eggs (DPF) revealed a homogeneous outer layer, displaying a large number of pores uniformly distributed on the surface of the egg envelope. Starting from 2 DPF parts of the outermost layer or two outer layers peeled off. The second deeper layer showed larger pores, with less defined edges. In the third innermost layer irregular indentations were noted. On transmission electron microscopy the first outermost layer of 1 DPF eggs clearly folded into the pores. The second layer was more electron dense, had a uniform appearance and did not cover the surface of the pores. The third innermost layer was much thicker and possessed indentations. A total number of 12 undulating zones were discriminated based on different degrees of electron density. Prior to hatching, the compact structure of the innermost layer was distorted by dispersed holes and tears.