Kenji Watanabe

Effects of intracellular ph on the mitotic apparatus and mitotic stage in the sand dollar egg

The effect of change in intracellular pH (pHi) on mitosis was investigated in the sand dollar egg. The pHi in the fertilized egg of Scaphechinus mirabilis and Clypeaster japonicus, which was 7.34 and 7.31, respectively, changed by means of treating the egg at nuclear envelope breakdown with sea water containing acetate and/or ammonia at various values of pH. The mitotic apparatus at pHi 6.70 became larger than that of normal fertilized eggs; that is, the mitotic spindle had the maximal size, especially in length at pHi 6.70. The spindle length linearly decreased when pHi increased from 6.70 to 7.84. By polarization microscopy, the increase in birefringence retardation was detected at slightly acidic pHi, suggesting that the increase in size of the spindle is caused by the increase in the amount of microtubules in the spindle. At pHi 6.30, the organization of the mitotic apparatus was inhibited. Furthermore, slightly acidic pHi caused cleavage retardation or inhibition. By counting the number of the eggs at various mitotic stages with time after treating them with the media, it is found that metaphase was persistent and most of the S. mirabilis eggs were arrested at metaphase under the condition of pHi 6.70. It is concluded that at slightly acidic pH, the microtubules in the spindle are stabilized and more microtubules assembled than those in the normal eggs.

Mitotic Asters Separate, although Chromosomes Do Not Separate at Slightly Acidic pHi in the Fertilized Egg of the Sea Urchin, Hemicentrotus pulcherrimus.

Abstract The effect of slightly acidic intracellular pH (pHi) on the development of the sea urchin, Hemicentrotus pulcherrimus was investigated. At first cleavage, the fertilized eggs were treated with artificial sea water containing sodium acetate (Ac-pHSW) at pH 6.8 or 7.0 at the onset of nuclear envelope breakdown, and their pHi decreased from 7.30 to 6.68 or 6.78, respectively. When the eggs were observed after fixation by indirect immunofluorescence and differential interference contrast microscopy, the mitotic stage of the treated eggs was arrested at metaphase and the mitotic apparatus was maintained until more than 50 min after the treatment, although it was smaller in size than that of non-treated eggs. On the other hand, the number of the mitotic asters increased from 2 to 3-4, and further to 6-8 following prolonged exposure, suggesting that the centrosomes had divided and replicated. These results suggest that the centrosome cycle advanced at slightly acidic pHi, even when the mitotic cycle did not advance beyond metaphase.