338 GERMINAL ORGANIZATION INDUCTION PHENOMENA 4 



over short distances. Pasteels could establish a relationship between the amount of a- and 

 ^-granules (Fig. 25), and he has good reason to believe that the cations of the basic 

 metachromatic dyes are at first combined with the a-granules and then detach from these, 

 taking along some part of them in their migration towards the fi-granules, upon the surface 

 of which they become concentrated. If stained eggs are deprived of Oj, they discolour by 

 liberating the dye cations. Vercauteren could show, on [^-granules isolated in vitro (although 

 not yet purified), that this was due to an oxydo-reduction process. It is also interesting to 

 quote that by centrifugation of clam eggs, blastomeres can be obtained which are apparently 

 free of granulations ; however these granulations are seen to reappear somewhat later. 



The p-granules of sea-urchins and Spiralia eggs are lighter than the yolk and than the 

 a's. (Fig. 24; 27, c, d; 28, a). In fixed eggs, they are PAS positive, even after enzymatic 

 digestion, stain directly by Alcian blue at a low pH (Fig. 28, a) and are apparently not rich 

 in RNA, whereas they exhibit a strong acid phosphatase activity. Thus, they appear as 

 mainly consisting of frankly acid mucopolysaccharides, with a protidic part acting as a 

 phosphomonoesterase in an acidic medium. 



It has not yet been settled whether the metachromatic granules encountered in ascidians 

 and mammals eggs behave exactly in the way just described. In Ascidiella, two categories 

 probably exist, and the secondary one contains acidic phosphatase, but it seems to consist of 

 minute bodies, and to be directly stainable. In any event, it is a fascinating picture to observe 

 the purplish a-granules (also stainable by neutral red, as in the case of Para-centrotiis) 

 mostly associated with the yellow myogenous plasm, surround and penetrate the aster rays 

 (Fig. 29). In mammals, only one category of granules could be detected so far. They stain 

 directly and have not as yet been followed in successive stages, probably owing to a reduction 

 process. But it was just established that they belong to a more complex system, formed of 

 heavier and lighter elements impregnable by osmium, thus most probably of Golgian nature. 



A third manifestation of vital metachromasy has been observed in two instances. In 

 Ascidiella scabra, a diffuse plasm has been detected in the subequatorial region of the recently 

 fertilized egg, pervading the region where the a-granules gather above the myogenous 

 plasm. This new component could be traced into the caudal part of the larval enteron 

 (Fig. 29; Dalcq, unpublished). In Chaetopterus, Mulnard also observed a rather continuous 

 area, his x-component (Fig. 28, d), which is probably microgranular and could be an 

 intermediate between the a- and [i-granules. 



What kind of organelles are the metachromatic granules? At first sight, they 

 were supposed to form a special category of mitochondria, but this initial inter- 

 pretation of mine lost more and more ground. As mentioned above, in rat and 

 mouse eggs it could be shown that we are dealing with corpuscules which are 

 neither Nadi-positive nor stainable by Janus green. In sea-urchin eggs, neither 

 the a- nor the [3-granules settle out on the same level as the mitochondria. Electron- 

 microscopic studies performed by Pasteels, Castiaux and Vandermeerssche (1958a, 

 b) have not revealed structures corresponding to the sought after [^-granules'. At 

 the level where these granules settle in centrifuged eggs, there is an abundance of 

 whole of Golgi vesicles, and it could well be that these granules are a part of the 

 Golgi apparatus^. The a-granules seem to have the same E.M. aspect as small yolk 

 platelets. Also, perhaps, the a- and [^-granules are not properly fixed by the buflfered 

 OSO4 mixture used. In the eggs of another bivalve, Spisida, Rebhun (1957) 



1 This enquiry had shown that a large amount of cytoplasmic RNA, which litterally fills, 

 mixed with mitochondria, the centrifugal cap of the centrifuged eggs (Fig. 27, a, b, c) is 

 certainly not bound to ultramicrosomes (Palade bodies). A similar conclusion has been 

 reached by Ruthmann (1958) in a E.M. study of crayfish spermatocytes. Thus, it might 

 well turn out that in gametes a large part of RNA is not included in the ultramicrosomes. 



2 In 1959, hypothesis received full confirmation. 



