Centrosome structure and function is altered by chloral hydrate and diazepam during the first reproductive cell cycles in sea urchin eggs

Abstract

This paper explores the mode of action of the tranquillizers chloral hydrate and diazepam during fertilization and mitosis of the first reproductive cell cycles in sea urchin eggs. Most striking effects of these drugs are the alteration of centrosomal material and the abnormal microtubule configurations during exposure and after recovery from the drugs. This finding is utilized to study the mechanisms of centrosome compaction and decompaction and the dynamic configurational changes of centrosomal material and its interactions with microtubules. When 0.1% chloral hydrate or 350-750 microM diazepam is applied at specific phases during the first cell cycle of sea urchin eggs, expanded centrosomal material compacts at distinct regions and super-compacts into dense spheres while microtubules disassemble. When eggs are treated before pronuclear fusion, centrosomal material aggregates around each of the two pronuclei while microtubules disappear. Upon recovery, atypical asters oftentimes with multiple foci are formed from centrosomal material surrounding the pronuclei which indicates that the drugs have affected centrosomal material and prevent it from functioning normally. Electron microscopy and immunofluorescence studies with antibodies that routinely stain centrosomes in sea urchin eggs (4D2; and Ah-6) depict centrosomal material that is altered when compared to control cells. This centrosomal material is not able to reform normal microtubule patterns upon recovery but will form multiple asters around the two pronuclei. When cells are treated with 0.1% chloral hydrate or 350-750 microM diazepam during mitosis, the bipolar centrosomal material becomes compacted and aggregates into multiple dense spheres while spindle and polar microtubules disassemble. With increased incubation time, the smaller dense centrosome particles aggregate into bigger and fewer spheres. Upon recovery, unusual irregular microtubule configurations are formed from centrosomes that have lost their ability to reform normal mitotic figures. These results indicate that chloral hydrate and diazepam affect centrosome structure which results in the inability to reform normal microtubule formations and causes abnormal fertilization and mitosis.