48 BULLETIN OF THE 



d. Origin of the Zona Radiata and the Villous Layer. 



The youngest ovarian eggs in which either of the egg membranes has 

 been observed were about 430 p, in diameter, and the ovaries to which 

 they belonged were preserved just before the period of spawning began. 

 Sections of such an egg are shown in Plate VIII. Figs. 1 and 2. Tan- 

 gential sections (Fig. 2) show that the egg is enveloped in a layer of 

 polygonal granulosa cells whose boundaries are exceedingly faint, and 

 whose nuclei have very irregular outlines, being lobed or deeply incised, 

 in some cases almost to complete division. The nuclei contain one, and 

 frequently two small nucleoli, but otherwise appear homogeneous, and 

 are uniformly stained. Upon focusing just below this layer of granulosa 

 cells, one sees the surface of the yolk covered with innumerable fine, 

 close-set points, which are evenly distributed. 1 



Radial sections (Plate VIII. Fig. 1) supplement the surface views, and 

 show that the granulosa cells are relatively thin, and easily separable 

 from the underlying structures. Their protoplasm is finely granular, 

 and their boundaries are not distinguishable ; neither do their deep 

 surfaces appear to be defined by any membrane. Their nuclei are 

 considerably flattened, and irregular in outline. 



Immediately beneath the granulosa the surface of the yolk exhibits 

 fine radial, nearly parallel markings, which are close together and very 

 short. They are so intimately joined to the yolk that they seem to form 

 an integral part of it, and nowhere show the least tendency to become 

 detached from it. With high powers one can recognize a very thin corti- 

 cal portion of the yolk (membrane V), with which they seem to be contin- 

 uous. It is very difficult to ascertain the distance between the markings, 

 but about 21 of them may be counted in the space of 17 /x, so that the 

 average distance is not far from 0.8 /x. The length of each is about 0.5 /x. 



It would not be easy to determine from this stage alone whether the 

 markings indicate the beginnings of the formation of the zona radiata or 

 the villous layer. But even in this early condition the punctate mark- 

 ings of tangential sections appear brighter rather than darker when one 

 focuses high, so that the inference must be that they are due to minute 

 bodies which are more highly refractive than the surrounding substance. 



This conclusion is abundantly confirmed by the study of somewhat 

 larger ova. These bodies seem to increase in length with considerable 



1 In Fig. 2 (Plate VIII.) these punctations appear much too scattered in the 

 middle of the area which shows them. They are better represented toward the 

 margin of the area. 



