Abstract
The present study was undertaken with a purpose to find out any fixing and staining method which demonstrates fibrous nature of the mitotic spindle using cultured rat and mice fibroblasts as material, and if possible, to obtain some information on the morphological structure of the spindle. After several trials with current methods available for demonsartion of the spindle structure, it was found that La Cour's method in combination with Lewitsky's fixative and aniline blue-orange G stains has been proved to be the most satisfactory one for the subject in question in the course of spindle formation in prophase through metaphase. Examination of the material treated with the La Cour's method has revealed that the formation of the spindle initiated by the appearance of fibrillar bundles running through the center of the nucleus and then they converged to at the centrosomes locating one each at the opposite sides of the nucleus. The fibrillar bundles showed a gradual development leading to the formation of the spindle. Surrounding the spindle thus formed there occurs a membranous structure which delimits the spindle from the cytoplasm. Remarkable fact is the presence of a clear zone between the membranous structure and the spindle surface. The clear zone is obviously discernible by its weak stainability from other parts of the cytoplasm. No chromosomes were found lying outside the clear zone. By several other methods the existence of the clear zone was also proved, though not very clear. The phase microscopy observations of living fibroblasts in vitro were also succeseful in demonstrating the occurrence of the clear zone by its low refractive index. The clear zone contains no visible granule. The membranous structure seems to serve to prevent cytoplasmic inclusions from entering into the clear zone. The structure is rather clearly visible at prophase and metaphase, while indistinct at ana and telophase. It remains unknown whether the membranous structure is of nuclear origin, or not.
The treatment of living fibroblasts with dilute acetic acid resulted in a gradual coagulation of the protoplasm, while the spindle fibres and clear zone become visible for a moment. This fact seems to support the evidence obtained in fixed and living materials for the existence of clear zone.
The in vitro observations of dividing spermatocytes of the grasshopper, Chrysochraon japonicus, seem to show that the metaphase spindle consists of two components, one being of cytoplasmic origin and the other of nuclear origin. The clear zone is not evident in the spindle of grasshopper spermatocytes, but the nuclear membrane was found to exist throughout the whole process of spindle formation in prophase and prometaphase.
