THE EFFECT OF LOW TEMPERATURE AND OF HYPOTONICITY ON THE MORPHOLOGY OF THE CLEAVAGE FURROW IN ARBACIA EGGS * ALLAN SCOTT Department of Biology, Union College, Schenectady, N. Y. and The Marine Biological Laboratory, Woods Hole, Massachusetts When Arbacia punctulata eggs are exposed to low temperature during the first cleavage, a pronounced stalk develops between the daughter blastomeres. A stalk-also develops at room temperature if the eggs are made to divide in hypotonic sea water or in sea water lacking calcium ion. The development of a conspicuous cleavage stalk is not a normal feature of the first cleavage in Arbacia, although it does occur regularly in some cells ; for example, when fibroblasts divide. The object of the work reported here was to examine the conditions under which the stalk is formed in Arbacia and to relate these facts to current theories of the mech-anism of cleavage. These particular experimental treatments were used because they were found to affect the appearance of the cleavage stalk. METHODS Eggs of Arbacia punctulata in the first cleavage served as experimental material. Ovulation was induced by the removal of the oral half of the test; eggs emerging from the genital pores were collected in a dish of sea water. The eggs were al-lowed to settle and the sea water was decanted after which fresh sea water was added. Two such washings were carried out to minimize contamination by coelomic fluid. Fertilization was effected by the use of diluted "dry" sperm, and the sperm were never more than one hour old. The fertilization membranes were removed by shaking. A heavy suspension of eggs was placed in a five-inch test tube, one-half full of the suspension, and shaken rapidly thirty times. Eggs so treated cleave in time with the controls. The best time for treatment is at 2% minutes after fertilization, for if shaken earlier, many exovates are formed, and if shaken later, many eggs retain the fertilization membrane. The alternative method of removing the fertilization membrane by treatment with the hatching enzyme (Ishida, 1936) was not attempted. The hyaline layer was removed in a few experiments by washing the eggs in calcium-free artificial sea water. This was accomplished by several decantations and additions of the calcium-free mixture. It was found that the hyaline layer regenerates somewhat if the eggs are returned to a solution possessing calcium ions ; hence if eggs are to lack the hyaline layer, they must be allowed to cleave in the calcium-free mixture. This study was largely accomplished by photographic means. Photomicro-1 This investigation was aided by a Grant-in-Aid from the Sigma Xi Alumni Research Fund. 272
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