thing in sea urchin eggs under more trying 

 biological circumstances. If radioactive RNA 

 precursors are injected into a gravid female 

 sea urchin, no radioactivity is found in the 

 mature eggs. This indicates that mature eggs 

 are finished and no longer making any RNA 

 and none can be forced in when the female is 

 ready to spawn. An alternative is to make a 

 female spawn and then let her carry out oogen- 

 esis, making a new crop of eggs in the presence 

 of radioactivity. This is not a very practical 

 procedure, at least with the species available 

 to us, because it means having animals in 

 large tanks of sea water, containing high levels 

 of radiophosphate circulating in the sea water 

 for weeks. However, there is a simple trick 

 that can be done. This is to make a female 

 spawn partially at the height of the normal 

 reproductive season and then to place her in 

 a tank that contains radioactive precursors for 

 about a week. Under those conditions (2), a few 

 of the oocytes complete their maturation to 

 replace the ones lost in the partial spawning. 

 We collect the mature eggs. Some of them are 

 highly radioactive, as I'll show you, and in 

 those the distribution of radioactive RNA can 

 be studied. 



Figure 6 is a section of an ovary of a sea 

 urchin. This is a highly lobulate organ, whose 

 walls contain an epithelium that gives rise to 

 the ootids. There are oocytes in this wall in all 

 stages of development, and an oocyte is identi- 

 fiable by its large germinal vesicle nucleus. 

 In some cases, you can see a nucleolus. This 

 is prominent because the oocytes are growing 

 and making ribosomes very rapidly. The ulti- 

 mate product of the differentiation - and I use 



I-ig. t). 



that word advisedly - of an oocyte into an egg 

 is an ootid. It is recognizable here by its small 

 pronucleus. These ootids fill the central parts 

 of the lumina of the lobes. When the animals 

 spawn, there is a highly stretched muscle in 

 the outer layer of the ovary that contracts and 

 fully mature ootids are extruded while the small 

 immature eggs remain inside. 



Now, if you perform the trick that I have 

 described - partial spawning and labeling for 

 a week - you find that it is possible to force 

 radioactivity into the cells as represented in 

 the autoradiogram shown in Fig. 7. This shows 

 a region near the wall. The wall consists of 

 three layers, an outer and an inner one, and 

 a muscle layer. The oocyte layer is next to 

 the wall. You see all of these cells are highly 

 labeled, both in nuclei and in the cytoplasm, 

 after a week. At the top of the figure is the 

 luminad region with the cells getting larger. 

 Everything in the region of the wall is radio- 

 active, except for one cell that happens to be 

 outside the wall and was fixed. 



There is an interesting progression, as 

 shown in Fig. 8. Close to the region shown in 

 Fig. 7 about three-fourths of the cells are 

 labeled, indicated by the left part of Fig. 8. 

 When they are, the number of silver grains 

 over each one is about the same. Those that 

 are not labeled have no counts above back- 

 ground. There seem to be very few interme- 

 diate conditions of radioactivity between cells 

 that have been making RNA at some constant 

 rate during the time of exposure and the 

 unlabeled ones that have finished before the 

 radioactivity was supplied. Moving toward the 

 lumen (left to right in Fig. 8), the number of 

 labeled cells becomes smaller until finally in 

 the central lumen, where the oldest eggs are, 

 there is no label at all. This fact suggests 

 that we are causing a few eggs to complete 

 their maturation and labeling them while this 

 is in progress. Silver grains represent counts 

 in RNA, because all these sections are DNAse 

 treated. 



We can extract and purify the labeled RNA. 

 The pattern obtained is shown in Fig. 9. One 

 thing is at once apparent. During the time that 

 labeling took place, these eggs were making 

 all the bulk kinds of RNA. Both ribosomal 

 species and 4S become radioactive and the radio- 

 activity (faint solid line) and bulk patterns 

 (dotted line) are superficially coincident. There- 

 fore, these eggs, during the late stages of their 

 maturation, are still making ribosomal RNA 

 and presumably ribosomal proteins as well. 



