Kazuo Yamashita

An electron microscopic study on the possible site of production of ACTH in the anterior pituitary of mice.

SummaryAn ultrastructural study has been made of a new cell type different from five known cell types in the anterior pituitary of normal mice. This new cell type is characterized by the special morphology of secretory granules. The immature form of the granules with high electron density in and around the Golgi area becomes vesiculated in the cell periphery. With glutaraldehyde-osmium fixation, however, the content of such vesiculated granules acquires electron density.In attempts to certify the independence of this cell type not related to the others and to elucidate its physiological function, a series of experiments has been made.After adrenalectomy the cell type exhibits a characteristic response represented by hypertrophy of the Golgi apparatus and prominent other cell organelles. Under the same condition the FSH-producing cell to be differentiated from the new cell type does not undergo any cytological changes. Instead, all FSH-producing cells show typical alterations at 100th day after orchidectomy, when the cell in question remains intact. Adrenalectomy following 100 day-orchidectomy causes a stimulative effect upon the new cell type. Furthermore, administration of hydrocortisone results in a hypofunctional state of this cell type.All these findings indicate that the new cell type identified in the present study is the site of ACTH production.

Fine structure of the mouse anterior pituitary maintained in a short-term incubation system

SummaryAnterior pituitaries of mice were incubated for periods up to four hours in Krebs-Ringer bicarbonate glucose gassed with 95% O2∶5% CO2. The incubated explants survived and retained a fine structure that approximated the condition in situ. The few necrotic cells were sharply localized, and were found to be due to initial mechanical damage to the tissue.Some cells of the six granulated types exhibited slight but significant changes attributable to the liberation from the hypothalamic control: in LTH cells there was a release of preexisting granules and a development of cell organelles, whereas in other cell types there was an inhibition of release of granules and an enhanced digestion of the accumulated granules by the lysosomal system.Follicular cells responded uniquely to the changed environment by hypertrophy of the cytoplasm and were found to phagocytize cell debris. A part of non-epithelial elements of the gland showed a tendency to modulate cytologically.

A Simple and Rapid Method of Dissociating Hepatocytes from Fixed Liver of the Mouse

A simple and rapid method of dissociating hepatocytes of fixed liver tissue is described. Mouse liver was fixed by vascular perfusion with sodium phosphate buffered 2% formaldehyde-2% glutaraldehyde solution containing 0.02% picric acid and then osmicated in 2% OsO4 in phosphate buffer by immersion. Hepatocytes are easily dissociated by tapping the fixed tissue blocks in distilled water with a glass rod or by ultrasonics. This method results in very low cell fragility and a high yield of well preserved hepatocytes in suspension. For light microscopic examination the separated cells may be uniformly spread on a slide glass coated with Mayers egg albumen and stained. Electron microscopic evaluation of the dispersed cells indicated that they have intact cell membranes and retain the integrity of their cytoplasm and nuclei well. This method is most suitable for accurate determination of the nuclear content and size of individual liver cells, as well as of the number of mitotic cells, and is potentially useful for gathering other information on the morphometric cytology of the liver.

A simple method for the cultivation of anterior pituitary tissues: Electron microscopical evaluation of the cultured tissues

SummarySuitability of an ordinary incubation system for the culture of anterior pituitary tissues of mice was examined by electron microscopy. It was found that this system has many advantages over Trowells standard technique for tissue culture and is particularly suitable for the short-term culture.In this system the pituitary tissue cultures were maintained well for at least two days in a chemically defined tissue culture medium (TC 199) without any additives. Addition of 20% calf serum to the medium extended the period to up to five days. Any attempts to prolong it further, however, failed because of a massive necrosis and a great loss of the histological integrity.In the cultured tissues there an enhancement of the LTH cells and a suppression of the other granulated types of cells were observed. The follicular cells were found to hypertrophy and to actively participate in phagocytosis of cell debris.

An improved technique for dissociating hepatocytes from fixed mouse liver for cytochemistry.

A refined version of a method described previously for dissociating hepatocytes from fixed liver is described. The objective was to develop a procedure that would dispense with the postosmication of previously fixed tissue. In the new procedure fixed liver blocks are wrapped with a quadruple layer of nylon cloth, and, by squeezing them in a buffer solution, individually dissociated cells pass through the cloth without significant damage. The procedure makes it possible to dissociate liver tissue fixed with modified Karnovskys fixative, Zambonis fixative or cacodylate buffered glutaraldehyde. The subsequent compatibility of the single cells obtained with many cytochemical procedures has been confirmed.