Allan L. Allenspach

Ultrastructural evidence of contractile systems in mouse palates prior to rotation.

Abstract Previous studies have shown that the palatal shelves of mouse embryos synthesize the contractile proteins actin and myosin at a rate equal to that of tongue just prior to shelf movement (day 14.5). The purpose of this study was to examine the morphology of the palatal shelves for evidence of a contractile system. Myosin ATPase histochemistry was performed on frozen sections of day-14.5 fetal mouse heads. Three areas of the palatal shelves gave a positive reaction: 1) A reaction product typical of skeletal muscle on the oral side of the posterior palate (region 1); 2) a “heavy-diffuse” reaction product on the tongue side extending from the top mid-palate to the posterior end (region 2); and 3) a “light-diffuse” reaction product along the oral epithelium in the mid-palate (region 3). Electron microscopy of excised day-14.5 palates was carried out after fixation in glutaraldehyde or an acrolein-dichromate solution. Region 1 contained a large area of developing and adultlike skeletal muscle. In the area of region 2 a large population of filamentous-rich mesenchymal cells was observed. In addition, large neurons coursing through both contractile systems were noted. Preliminary observations in region 3 indicated the possibility of a primitive (nonmuscle) contractile system in that area. The contractile and nervous systems in the palate, prior to rotation, indicate the possibility that an innervated embryonic muscle system may provide the “intrinsic shelf force” to rotate the shelves.

Comparative ultrastructure of fat body cells of freeze-susceptible and freeze-tolerant Eurosta solidaginis larvae after chemical fixation and high pressure freezing

Abstract In response to low environmental temperatures third instar larvae of the goldenrod gall fly, Eurosta solidaginis, undergo the process of cold-hardening in which they acquire freeze-tolerance. Cold-hardening includes the elevation of the supercooling point, the temperature at which body water spontaneously freezes, and the accumulation of the low-molecular-mass cryoprotectants, glycerol, sorbitol and trehalose. Although it is generally believed that freezing survival is only possible if the ice lattice is restricted to the extracellular space, the larval fat body cells survive intracellular ice formation. Fat body cells of freeze-susceptible (September-collected) and freeze-tolerant (January-collected) third instar larvae were prepared for transmission electron microscopic analysis using both conventional chemical fixation and high pressure freezing with freeze substitution. High pressure cryofixation procedures were superior to conventional fixation in both freeze-susceptible and freeze-tolerant cells. Endogenous cryoprotectants in freeze-tolerant larvae contributed to the superior ultrastructure of cytoplasmic organelles and nucleoplasm in high-pressure frozen fat body cells, while freeze-susceptible larvae, which lack high levels of cryoprotectants, contained fat body cells which sustained ice crystal damage following cryofixation. Mitochondria in fat body cells prepared using high pressure freezing/freeze substitution were characterized by smooth outer membranes and uniformly electron dense mitochondrial matrix. Conventionally prepared samples exhibited crenated cytoplasmic organelles with undulating membrane profiles; smooth profiles characterized high-pressure frozen structures. Lipid droplets were abundant in all fat body cells, however glycogen, the energy source of cryoprotectants, was variable in both freeze-susceptible and freeze-tolerant cells. Coalescence of lipid droplets, induced by intracellular freezing at progressively lower temperatures, caused displacement of interior cytoplasmic organelles including the nucleus to the cell cortex. This shift in cytoplasm had no affect on survival of the cells.

The role of acid phosphatase and β-glucuronidase in reopening and remodeling of the developing chick esophagus

Abstract Biochemical and ultrastructural methods have been employed to determine the relationship of the lysosomal system to reopening and remodeling of the developing chick esophagus. Biochemical data indicate an increase in both acid phosphatase and β-glucuronidase activity during esophageal organogenesis. The appearance of these hydrolase enzyme activities appears to be independently regulated. The increase in acid hydrolase activities parallels the increase in total protein at a time when the esophagus is greatly enlarging. The greatest activity of acid phosphatase was localized in the anterior occluded segment of the esophagus. With reformation of the lumen, the distribution of acid phosphatase becomes uniform along the organ. Increases in hydrolase activities correlate closely with cell degeneration and the differentiation of the epithelium. Acid phosphatase is identified cytochemically in the Golgi complex and lysosomes of embryonic epithelial cells. Surrounding mesenchyme contains only modest cytochemical activity. Diffuse Golgi complexes and primary lysosomes predominate during early stages studied. Secondary lysosomes and condensed Golgi complexes are characteristic of differentiating epithelium at later stages. Acid phosphatase activity appears selectively localized in vital epithelial cells, while degenerating and moribund cells, whose appearances are obligatory to the reopening process, fail to demonstrate enzymatic activity. The temporal association of increased hydrolase activity and cytochemical maturation of a lyosomal system suggests an active involvement of the system in esophageal organogenesis.

Liver Tissue Preparation Using a Modified Cryoultramicrotomy Kit

The great potential of cryoultramicrotomy is that it is possible to observe the morphology of a sample in the electron microscope while at the same time analyze for its elemental composition using x-ray microanalysis. There are other methods using indirect means of studying the chemical composition of cell organelles, such as digestion and centrifugation, but cryoultramicrotomy is the only direct method. This unique ability will make cryoultramicrotomy a vital tool in the field of cell biology and pathology in the near future. Our interest is the quantitative analyses of diffusible elements in the mitochondria of rat liver before and after exposure to toxins both singularly and in mixtures. To obtain reliable and reproducible data it is critical that each step in the technique be carried out correctly. Any deviation in any of the steps will leave the final results in doubt.