Effect of Osmolarity of the Fixative on the Ultrastructure of Preimplantation Rabbit Embryos

Abstract

In an attempt to clarify whether or not glutaraldehyde molecules contribute to the effective osmotic pressure of the fixative solution the ultrastructure of noncultured and in vitro-cultured day 1 3 and 4 rabbit embryos was evaluated. Total osmolarity of the fixative solution (200–800 mosm) was only varied by changing the aldehyde concentration, whereas the vehicle osmolarity (145 mosm) remained unchanged. Optimum preservation in all embryonic stages was obtained when total osmolarity of the fixative solution was 285–340 mosm. Higher (480–800 mosm) or lower (250 mosm) osmolarities of the fixative solution led to alterations mainly in mitochondria and smooth-surfaced endoplasmic reticulum. Shrinkage of cells and condensation of the cytoplasm occurred only occasionally. Compared with early cleavage stage embryos blastocysts were generally more susceptible to hyperton and hypoton fixative solutions. In vitro culture for 24 h per se did not have any influence on the fixation.

Handling difficult cryo-ET samples: A study with primary neurons fromDrosophila melanogaster

“…We examined the 'clearing' artifacts more closely and wondered if this may be caused by imbalances in osmotic pressure while the neurons were fixed. Classical electron microscopy studies have shown that ultrastructural changes can occur in a variety of cells and tissues if the fixative has osmolarities/osmolalities that are non-isotonic (Hegele-Hartung et al, 1989, Bozzola & Russell, 1999. Therefore, we measured the osmolalities of all the solutions that the Drosophila neurons were cultured or incubated in using freezing point depression (Table 4).…”

“…Based on osmolality measurements (Table 4), it seems that such patches could be due to excessive osmotic pressure from highly tonic solutions inducing plasmolysis. Chemical fixatives, especially paraformaldehyde, have a much higher osmotic pressure compared to physiological fluids (Bone & Denton, 1971, Hegele-Hartung et al, 1989, and an improper balance between the fixative and the cytoplasm can cause ultrastructural alterations where the intercellular space will be exaggerated due to hyper-osmolar fixatives or swelling of cells and organelles by hypo-osmolar fixatives (Bozzola & Russell, 1999). It may be possible that Drosophila neurons are more sensitive to osmotic pressures compared to other cellular targets, which could create 'empty' patches from cell lysis.…”

“…It is not well known if other artifacts, such as the disappearance/perturbation of microtubules, could arise due to the osmotic pressure imbalance between the fixative and the cytoplasm, or the chemical reactions of the fixatives themselves. Most past studies examining the effects of osmotic pressure and/or fixation on cellular ultrastructure have been done at a classical electron microscopy level (Bone & Denton, 1971, Bozzola & Russell, 1999, Hegele-Hartung et al, 1989. Such studies have many other steps postfixation that could occlude observations, such as further fixation with osmium tetroxide, dehydration, resin-embedding, sectioning, and metal-staining.…”

Enhancement of preimplantation mouse embryo development with optimized in vitro culture dish via stabilization of medium osmolarity

“…For instance, when preserving rabbit embryos, it was found that maintaining osmolarity within a specific range (285 to 340 mOsm) was crucial for effective preservation. Any deviation from this range resulted in structural changes to the embryos [ 32 ]. In the context of embryo culture, adjustments to osmolarity can aid in overcoming the two-cell block [ 33 ].…”