THE MICROMANIPULATION OF LIVING CELLS 



25 



this layer becomes negligible and the coa- 

 lescency rises to a value actually one- 

 hundredfold that of the unfertilized eggs 

 possessing a vitelline membrane. 



Unfertilized eggs also give relatively high 

 values when they have been immersed for 

 about two minutes in a 1 i¥ urea solution, 

 which dissolves the investing vitelline mem- 

 brane, or when the eggs are churned with 

 microneedles. The churning process rup- 

 tures the vitelline membrane and makes it 

 discontinuous. 



Aging also has a decided effect; the coa- 

 lescency value of eggs, standing in sea 

 water for 8 to 12 hours, rises to 2.5 and 5. 

 For eggs remaining in sea water for 20 

 hours the value obtained has been as high 

 as 100, which is equal to that of eggs within 

 two minutes after insemination and with 

 their fertilization membranes removed (in 

 solutions of potassium chloride). Increas- 

 ing coalescency with age suggests that the 

 vitelline membrane gradually disintegrates 

 with time. 



Interesting data have also been obtained 

 on the development of the hyaline layer in 

 Arhacia eggs after fertilization. As shown 

 above, freshly fertilized eggs (in sea water) 

 with their fertilization membranes removed 

 have more than a tenfold greater coales- 

 cency than unfertilized eggs which still pos- 

 sess vitelline membranes. However, the 

 coalescencies of the fertilized eggs (in sea 

 water) decrease steadily and become zero 

 after 15 or more minutes. The increased 

 magnitude of the potential hill, as calcu- 

 lated from the size of the coalescing drops, 

 corresponds closely with the formation and 

 gradual thickening of the hyaline layer on 

 the surface of the egg. Such data suggest 

 that the presence of a hyaline layer in- 

 creases the tangential rigidity at the cell 

 surface. 



This hyaline layer is unstable in the ab- 

 sence of calcium in the external medium 

 and is readily dispersed in isosmotic solu- 

 tions of acidified potassium chloride. Even 

 when the layer is fully formed a sojourn 

 of the fertilized eggs in potassium chloride 

 causes the layer to become dissipated. The 

 coalescency value may rise one-hundredfold 

 over that of the unfertilized egg in sea 

 water. 



A reasonable interpretation of these ob- 

 servations is that the eggs with the lowest 

 potential hills (namely, highest coales- 

 cencies) are eggs which have the minimum 

 of extraneous coatings. It is entirely prob- 

 able that fertilized Arhacia eggs immersed 

 in potassium chloride offer the closest ap- 

 proach thus far to exposing the actual 

 protoplasmic surface layer or plasma mem- 

 brane of the eggs. 



It was of interest to find whether gelation 

 phenomena within the Echinoderm egg may 

 also serve as a barrier to the coalescence 

 reaction. The conclusion has been verified 

 after much experimentation that the naked 

 surface of the egg consists of material 

 which can be made to flow without losing 

 its integrity. Normally, there is an under- 

 lying and appreciably thick gelated cortex. 

 Moreover, in the dividing egg it has been 

 found that the consistency of the cortex in 

 the furrow region of a dividing egg is 

 greater than that of the poles. 



The degree of coalescence^ in regions of 

 varj^ing cortical consistency was deter- 

 mined on Lytechinus eggs after the fertili- 

 zation membranes had been removed and 

 the hyaline layer dispersed by immersing 

 the eggs from 10 to 20 minutes in isosmotic 

 potassium chloride solution ( Chambers and 

 Kopac 1937). In these experiments two 

 oils were used, mineral oil and oleic acid. 

 The sample of mineral oil used here had an 

 interfacial tension of 40 dynes per cm 

 against sea water, while the oleic acid under 

 similar conditions had an interfacial ten- 

 sion of 10 dynes per cm. The experiments 

 were done with oil drops of different sizes, 

 and mostly on eggs with the cleavage fur- 

 row differentiated. No difference in the 

 size of the oil drop at the time of coales- 

 cence was found between the polar and 

 equatorial regions of the egg. If increased 

 consistency lowered the coalescency, one 

 would expect larger drops to coalesce at the 

 furrow zone. This we did not find. These 

 results indicate that the consistency of the 

 cytoplasm is of minor importance in in- 

 hibiting coalescence. The tangential rigid- 

 ity or lack of it in the cell surface appears 

 to be the prime factor in controlling coa- 

 lescence. 



An interesting object is the aplanospore 



