76 MARY ELMORE SAUER AND DONALD DUNCAN 



Embryos received either 200 or 500 r of x-rays through the unopened 

 shell and were returned to the incubator. Control embryos were subjected 

 to all treatment given the others, except the actual irradiation. Individual 

 embryos were fixed at frequent intervals following irradiation, ranging from 

 1 to 76 hours. The following factors were employed in the irradiation: 250 

 kv, 30 ma, added filter 0.25 mm Cu plus 1.0 mm Al. 50 cm FOD, average 

 intensity of 145 r/min. 



Other embryos were labeled with tritium ( Sauer and Walker, 1961). 

 Some received only thymidine-H'*, usually 50 mc per embryo, specific activ- 

 ity 1.6 c/mmole. Others had received 200 r x-rays immediately preceding 

 treatment with thymidine-H'\ 



The embryos were fi.xed in Newcomer's (1953), Carnoy's. or Serra's 

 fixative, or in 10 or 50 Cf neutral formalin, embedded in paraffin and sec- 

 tioned at 3 to 6 IX. Stains used included hematoxylin and eosin, toluidine 

 blue O, May-Griinwald and Giemsa, Feulgen's, and methyl green-pyronin. 

 The May-Griinwald and Giemsa stain following Newcomer fixation was 

 preferred for general cytologic study. Kurnick's ( 1955a,b) procedure was 

 followed for DNase and RNase. The enzymes were purchased from Worth- 

 ington Biochemical Corporation, Freehold, New Jersey. For electron micro- 

 scopy, pieces of embryos were fixed in Dalton's osmic-dichromate mixture, 

 embedded in methacrylate, and sectioned with a Porter-Blum microtome. 



Results 



Numerous cells in the irradiated embryos have a striking appearance in 

 that one or more bodies approximately 2-5 /x in diameter lie within their 

 cytoplasm (Figs. 2-12). Such bodies occur in small numbers in normal chick 

 embryos but greatly increase in number following irradiation. In 2- to 3-day- 

 old chick embryos, they may begin to appear in the 2nd hour after exposure. 

 In the early hours after treatment the cells containing the inclusions may be 

 few or many, depending on the susceptibility of the particular embryo. The 

 distribution of the afTected cells at this time is wide but irregular: while 

 some fields show many of the bodies, extensive regions may contain none or 

 few. The bodies appear most constantly in the neural tube, especially in the 

 brain. Their number increases with time, so that a 60 hour embryo that has 

 received 200 r will 9 hours later contain large numbers of the inclusions in 

 every system (Figs. 3-6). The body lies within the cytoplasm of an appar- 

 ently normal cell. Its position adjacent to the nucleus is characteristic (Fig. 

 6) and it often flattens or indents the nucleus at the area of contact. 



In the series receiving 200 r, the inclusions are \ery numerous in embryos 

 fixed at 9 to 22 hours following irradiation. The number of inclusions then 

 decreases rapidly. Only small numbers of the cytoplasmic bodies remain at 



