Figure 299. — Diagram of successive stages of spermatogenesis in O. liirida. A — two indifferent germ cells on the wall 

 of the gonad; B — small group of spermatogonia, with reticular chromatin and conspicuous nucleoli; C — small group 

 of secondary spermatogonia; D — division of secondary spermatogonia to form spermatocytes; E — primary spermato- 

 cytes with slender chromosomes; F to K — division of primary spermatocytes; L to R — division of secondary spermato- 

 cyte; S to T — transformation of spermatid into the mature spermatozoon. Redrawn from Coe, 1931, fig. 2. 



gametogenesis in the gastropods Helix, Limnaea, 

 and Patella, identify the protein granules as 

 "mitochondria" and restrict the term yolk to fatty 

 inclusions. 



The role of the mitochondria in the formation of 

 yolk has not been fully resolved. According to 

 Rai (1930), the fatty yolk in the eggs of C. cucullata 

 is formed directly from the Golgi vesicles as it is 

 in ascidians, Helix, and other invertebrates; 

 mitochondria do not participate in the vitellogen- 

 esis, and albuminous yolk is absent in the egg of 

 the Indian oyster {('. cucullata). This view is in 

 agreement with the conclusion of Worley (1944), 

 who found no protein yolk in the eggs of Alijtilus 

 and Ostrea. The question is not settled because 

 apparently the cytologists have no clear agreement 

 on the difference between the protein yolk and 

 mitochondria. 



A study of cytoplasmic inclusions was made by 

 Cleland (1947, 1951), who separated the granules 

 found in egg cytoplasm of the oyster by diiferen- 

 tial centrifugation following homogenization. 

 Mature eggs were suspended in a solution of 

 0.2 M potassium chloride and 0.02 n sodium citrate 



buffered to pH 7.5. Homogenates were obtained 

 by blending the suspension in an electric blender 

 surrounded by an ice jacket. By centrifuging 

 the samples of homogenates at different speeds the 

 following types of granules were obtained: P gran- 

 ules or protein yolk; L granides or lipid yolk; 

 M granules or mitochondria; and S or submicro- 

 scopic granules. The P granules obtained by 

 Cleland's technique are spherical and can be 

 stained by Janus green B in the test tube. In the 

 living egg these granules are located along the 

 periphery and absorb Nile blue stain. After 

 being centrifuged at 5,000 times gravity for 5 

 minutes they form a thick centrifugal layer with 

 a sharp upper boundary. Alpha or lipid yolk 

 granules are also spherical. They occupy the 

 central part of the living egg. In the centrifuged 

 egg they form a centripetal layer with a sharp 

 lower boundary. Alpha granides of phospholipid 

 and neutral fat can be recovered from the super- 

 natants of homogenate suspensions. M graimles 

 or mitochondria in tlie live centrifuged egg form 

 a thin, rather loose layei' above the P granules 

 and stain both with Janus green B and Xile blue. 



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FISH AND WILDLIFE SERVICE 



