High‐pressure freezing of spermiogenic nuclei supports a dynamic chromatin model for the histone‐to‐protamine transition

Abstract

In this study, we present for the first time a description of the dynamic chromatin changes that occur during spermiogenesis in the internally fertilizing caenogastropod mollusc Nucella lamellosa. Chromatin condensation in developing sperm cells in some animals, such as the model biological system used here, involves the histone-to-protamine transition and proceeds through a patterning stage from granules to fibers to lamellae. This may be due to the physicochemical phenomenon of phase separation by spinodal decomposition, a dynamic mechanism known to generate pattern. This hypothesis is based entirely on published transmission electron microscopy photomicrographs using conventional fixation technology. We now report that spermatid nuclear patterning and subsequent condensation in testis of Nucella lamellosa fixed by high-pressure freezing and freeze substitution (HPF/FS) is similar to that in glutaraldehyde-fixed testis, and can be related to the processing of sperm nuclear basic proteins (SNBPs).

Comparison of methods of high-pressure freezing and automated freeze-substitution of suspension cells combined with LR White embedding

“…The most remarkable improvement was displayed by excellently preserved heterochromatin regions in nuclei as well nucleolar components (Fig. 4a, b), both of which being the most difficult structures to vitrify as was previously shown (Buser and Walther 2008;Martens et al 2009;McDonald 2007;Morphew and McIntosh 2003;Wild et al 2001). The samples exhibited slightly superior overall visibility and the clear contrast of all cellular structures (Fig.…”

A method for preserving ultrastructural properties of mitotic cells for subsequent immunogold labeling using low-temperature embedding in LR White resin

“…: moderate, ?/-: low. The optimal conditions for HPF, automated FS, LR White embedding and the best resulting quality of the specimen are given in bold for cryofixation (Buser and Walther 2008;Morphew and McIntosh 2003;Wild et al 2001), and chromatin is one of the most difficult structures to vitrify (Martens et al 2009;Morphew and McIntosh 2003) most probably because of being highly charged and thus binding many molecules of water (McDonald 2007). In the course of optimizing the protocol for HPF and automated FS of mitotic cells, we tested various protocols and conditions (summarized in Table 1).…”

Recent progress in histochemistry and cell biology

“…For interphase cells, reliable procedures of cryoimmobilization by high-pressure freezing (HPF) followed by freeze-substitution (FS) have been established, whereas such approaches have not been established for mitotic cells, as such cells are more fragile and sensitive to treatments. The cell nucleus in general is the most sensitive organelle for cryofixation (Buser and Walther 2008;Wild et al 2001) and chromatin one of the most difficult structures to vitrify (Martens et al 2009). Therefore, focusing on mitotic cells, Sobol et al (2011) successfully set out to optimize the approach of HPF/automated FS and LR White embedding of mitotic cells in order to optimize preservation of the ultrastructure and antigenicity, i.e., to make samples serviceable to subsequent immunolabeling protocols.…”