Cytochemical study on the development of chum salmon egg

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(1)Title. Cytochemical study on the development of chum salmon egg. Author(s). 西田, 秀夫. Citation. 北海道学芸大学紀要. 第二部. B, 生物学,地学,農学編, 12(2): 1-55. Issue Date. 1961-12. URL. http://s-ir.sap.hokkyodai.ac.jp/dspace/handle/123456789/5742. Rights. Hokkaido University of Education.

(2) Joural of Hokkaido Gakugei University. Vol. 12, No. 2 B. Dec., 1961. Cytochemical Study on the Development of Chum Salmon Egg. H idea NI.SHIDA Biological Laboratory, Iwamizawa Branch,. Holikaido Gakugei University. Contents I. Introduction'. 7. 8 to 16-cell stage. •II. K. Material and Methods'. 8. 32 to 64-cell stage.. •13. ffl. Results. 9. Later segmentation stage'. 1. Unfertilized eggs but not activated in water" 2. Unfertilized eggs activated in water" 3. Fertilized eggs activated in water ••. 11. Early gastrula-. •16. 12. Five somites stage'. •l9. V. Summary and Conclusion"""-"-"-""----31. fertilization-5. Formation of blastodisc-. •14. IV. Discussion-.........................................21. 4. Developing egg at 4 hours after. 6. 2 to 4-cell stage.. •13. 10. Early and late blastula'. VI. Literature cited .................................33 VII. Explanation of Plates ........................36. 10 I. Introduction. Since the latter half of the nineteenth century, there have been a good number of reports published on the development of the teleost fishes, likewise of other vertebrates. In those reports, the workers have dealt mainly with the morphological changes concerned through the development from cleavage to organogenesis. For example, there are three descriptions on the development of the chum salmon, Oncorhynchus keta. (Walbaum) which has been dealt with in the present study (Saito, '50, Mahon and Hoar '56, and Dispel '57). In recent years, Yamamoto ('58) has reviewed the whole. of the physiological studies on the fertilization and activation of fish eggs, and Kusa ('58) has studied on the cortical alveoli in some teleostean eggs including chum salmon. Thanks to those researches, there has been added to the classical descriptions of the development of the fish, a new information concerning detailed changes occur at the beginning of the development.. The latest addition to the informations above mentioned is the contribution to the knowledge of the excess deoxyribonucleic acid (excess DNA) of the animal eggs, acquired by the biochemical analysis. Thus it has been an interesting problem to be settled how the excess DNA plays its role in the course of development. The most important 1—.

(3) Hideo Nishida problem treated in this study is to solve the cytological and cytochemicl nature of the egg plasm and the yolk and the existing relation with each other throughout the early development. Especially, the synthesis of the excess DNA has been examined in view of the relationship between the blastoderm and the yolk.. II. Material and Methods The eggs of the chum salmon, Oncorhynchus keta (Walbaum), were obtained in December, 1957, and from November to December in 1958 and 1959 at Nishikoshi on the Chitose River in Hokkaido, Japan. The eggs in a jar and the sperm in a glass tube cooled with ice were transferred to the laboratory of the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo. It took about two hours to transfer from the locality to the institute. At each time of the collection one thousand eggs were artificially fertillized and incubated at the laboratory in a glass vat, about 40cm in diameter, of running spring water of which temperature ranged usually between 11° and 12°C. Besides some eggs were experimentally fertilized and incubated in l/io mol water solution of calcium chloride for six hours at room temperature, about 16°C. For the morphological and cytochemical studies, the eggs were taken out at random from the glass vat in which the fertilized eggs were incubated. The stages examined in the present study were of early development from fertilization to early gastrulation. The eggs were examined at 4 and 12 hnurs after the fertilization, in the former no typical blastodisc was formed while in the latter the typical one was formed. The 2, 4 and 8-cell stages were subsequently examined, and after them, some stages from 16-cell stage to later segmentation were also examined. Next, examinations were carried out on the stages from early blastula to early gastrula. These eggs were fixed in Bouin's, in Hamazaki s and in Helly s fluids. The actual number of eggs fixed is from 10 to 20 for one kind of fixative. After 24 hours, the eggs fixed in Bouin's fluid were transferred into 809o alcohol. The eggs were allowed to rest in Hamazaki's and in Helly's fluids for 36 hours respectively. Then they were washed with running water for 36 hours and subsequently dehydrated. Prior to embedding, the egg membrane of fixed egg was carefully removed so that the materials existed in the perivitelline space might not been lost. Then two thirds of the yolk in which there was no blastoderm or embryo was cut off by the knife. These operations were carried out in S0% alcohol, in the case of Bouin's fluid and in 50% alcohol, Hamazaki's and Helly's fluids. The latter eggs which were fixed in Hamazaki's or Helly's fluids were dehydrated within three days. The materials mentioned above were cleared in alcoholxylol and xylol, then embedded in paraffin. Sections were cut to 10 micra in thickness. The eggs fixed in Bouin's fixative were almost stained with Delafiedl's hematoyylin and eosin, the rest of them were stained with 0. \% water solution of toluidine blue and of methylene blue. Besides those staining the P'eulgen method was employed. Although there are four kinds of fluid in Hamazaki's fixative, two of them were employed in the present 2—.

(4) Cytochemical Study on the Development of Chum Salmon Egg. study : The Hamazaki's A fixative and Hamazaki's D. (Okamoto and others, '55).. A D KsfcO,. ....................................2.5g.. ». Na2SO.r-.-----.-----------.--------1.0g. v Hga. ........................................ -. sg.. Glacial acetic acid ..................... gml. // Distiled ^^^-••••••••••••••••••••••••••••••lOOml. //. (Glacial acetic acid is added prior to each time of fixation) The materials fixed in Hamazaki's fluids were siained with Hamazaki's carbolic fuchsin-iodine procedure (KFJ method, Hamazaki, '54) and some of them were also stained with Delafield's hematoxylin for the nucleus staining, moreover, the preparations treated with Hamazaki's fixatives were applied Feulgen reaction. In Feulgen reaction. the hydrolysis was carried out with 1 N hydrochloric acid at 60°C for three, four and six minutes. On some preparations, Feulgen reaction was carried out without hydrolysis so as to distinguish between deoxydbonucleic acid or its derivatives and plasmal or pseudoplasmal. In KFJ method and Feulgen reaction, prior to the staining some. preparations were allowed to rest within 0. 25^ barium hydroxide distilled water solution for 15 hours. Some other materials fixed in Hamazaki's fluids were stained with 0. \% water solution of toluidine blue and of methylene blue. In these cases, preparations were allowed to rest in the staining solutions overnight and then were differentiated in 70% alcohol in a short time, as the preparation was hardly to be stained and the dyes tended to fall off rapidly in the course of differentiation in alcohol. The McManus's. periodic acid Schiff reaction was employed for the materials fixed in Helly's fluid. In this case the control preparations were made with the exception of hydrolysis by the periodic acid. On the other hand, several examinations were conducted on some particular cases to observe the changes at the beginning of development. The first examination was made on the case where the eggs were unfertilized and laid in an isotonic Ringer's solution, the second was on the case where the eggs were unfertilized and laid in water, the third on the case where they were fertilized and laid in an isotonic Ringer's solution, and the fourth on the case where they were fertilized and laid in water, in which case the eggs were capable of normal development. In all these cases the eggs were fixed with Hamazaki's A fluid at ten minutes after the treatment. After fixation, the ooplasm of the eggs which were unfertilized and laid in an isotonic Ringer's solution tended to easily separate from the yolk and adhere to the egg membrane. The ooplasm with the egg membrane of the animal region separated from yolk was dehydrated and embedded. All the preparations were stained with KFJ method.. III. Results 1. Unfertilized eggs but not activated in water. Ooplasm. When the egg of the 3—.

(5) Hideo Nishida chum salmon is spawned, the yolk-free cytoplasm is somewhat more abundant in the region where the egg nuclear material is situated. This concentration of the peripheral ooplasm. at the nuclear pole (animal pole) cannot be observed in living egg owing to its opaque egg membrane. The area of nuclear residence is situated near the micropyle, and it has to be mainly conjectured in the only fixed egg. The plasm is 90,u in thickness in the nuclear pole and gradually gets thinner far away from the pole. At the thinnest part of the ooplasm covering almost the whole of the surface of the yolk, it is about 12. 5/< thickness. There is a great number of cortical alveoli in the cortex of ooplasm which is of one or two layers (fig. 10). The alveolus is about 25/< in diameter which has included cylindrical material, its diameter is 5-7. , /.i and its length 20,-' (fig. 12). Thecylindrical material is stained in the same way as the plasm is, and its base adheres to the periphery of the alveolus, on the other hand its upper part separates from the periphery. The plasm contains granular or globular elements and large or small vacuoles. Among these elements, yolk granules and globules are of distinct nature varing from 5/-( to 10/-( in diameter, and are stained in reddish purple by KFJ method, and scattered throughout the plasm. There is a space noticed between yolk granule or globule and plasm, and this space is about 1 '2/< wide, in section. Besides the yolk granule or globule there isa few of ketoenol grannies (Hamazaki, '54) about 2. 'oil. in size. They are stained in violet. The ground structure of the plasm is very fine and somewhat net-worked, and is very basiphil (Baker, '58) and particularly well stined with hematoxylin. The Baker's term, basiphil, is generally used as basophilic or basophil. Base of ooplasm. This is the border between ooplasm and yolk mass, that is, thh junction of the plasm and yolk mass. It is very irregular in form. There are some elements between them. One of these elements is the ketoenol secreting vacuole which was named for vacuole secreting ketoenol substance by the present author ('58) (figs.. 10, 11). This element is large and numerously distributed through the peripheral ooplasm at the nuclear pole, but on the other hand, is small and few in number according to the distance from the nuclear pole. The large one of the elements, being dumb-bell in shape, is 40," in height and 20/-< in width (figs. 12, 13). The smaller one is 15/< in height., 7. 5/-t in width. The upper half of these elements, being empty or including some colloidal substance, is embedded in the plasm and the lower half including dense colloidal substance is embedded in the yolk mass. In a few of them,. the upper half divides into two portions (fig. 13). The colloidal substance mentioned above is well stained in purple by KFJ method, nevertheless, it can hardly be recognized in the preparations treated with other fixatives and stains. There are aggregations of the ketoenol grannies in the border region, and the granules composed of this aggregation are varied from 0.5 to 2. 5/< in size. Sometimes, the aggregation of the granules included a vacuole.. Yolk mass. Under the base of the ooplasm is the yolk mass in which are distributed. 4—.

(6) Cytochemical Study on the Development of Chum Salmon Egg. numerous oil drops, which are abundant in the animal half of the egg. The yolk mass. of animal half is faintly stained in violet with KFJ method, while, that of vegetative half becomes gradually to be stain in dense reddish purple. However, some of yolk. globules which existed in the animal half are stained in reddish purple as well as the yolk mass of vegetative half. 2. Unfertilized eggs activated in water. The unfertilized eggs have been examined at ten minutes after the activation in water.. Ooplasm. In these eggs the concentration of ooplasm at the nuclear pole is distinct,. but in living egg it is hardly visible from the outside of egg membrane. The region of concentration of the plasm is, in sagittal section, about 0.19mm at the thickest part and 2mm in breadth. The margin of the plasm concentration is extended somewhat to the peripheral plasm covering the yolk mass. In theese experimentally activated eggs, the cortical alveoli disappear and then the surface of the plasm becomes smooth, and. it is especially noticed that very small granules faintly stained in red by KFJ method appear in the cortex of ooplasm instead of the cortical alveoli. Each of these small granules is 0. 8/.( in diameter, and the granules have been made up a layer 7. 5,u to 12. 'op. in thickness in the cortex of the region of concentrating ooplasm. These small granules are not stained with hematoxylin and are not positive to Feulgen reaction. It is clearly to be seen that a large number of the ketoenol granules are distributed throughout the region of the concentration of ooplasm (figs. 14, 16). The ketoenol granule stained in violet is about 2. 5,u in diameter. These granules are agglutinated each other. In the agglutination of these granules, a translucent part is detected. The yolk granules and globules varying 5/( to 12.5/u in diameter are distributed as in the unfertilized eggs but not in water. The periphery of the yolk granule and globule has a thin translucent space. Base of ooplasm. The base of ooplasm, i. e. the boundary between ooplasm and yolk mass, is very irregular in form just as in the intact egg. The ketoenol secreting vacuoles are laid across the border. They are found as of the same appearance as those of an intact egg. It is noticed, however, that the vacuoles are distributed not under the region of concentrating ooplasm, but in its margin and in the peripheral plasm covering the yolk mass. There are agglutinations of the ketoenol grannies which is basiphil here, and the granules composed of the agglutination are varied from 1.2/< to 2. 5/-< in diameter. The agglutination sometimes includes a vacuole. These agglutinations frequently appear in the region of concentrating ooplasm and they are about 30/-< in diameter. These appearances of the vacuoles coincide with the vacuoles detected by Mahon and Hoar ('56). There are two more elements besides those mentioned above, which exist in the base of ooplasm. One is a gathering of small yolk granules, being from 1.2,u to 2.5^ in diameter, stained in reddish purpule. These yolk granules seem to have. 5.

(7) Hideo Nishida resulted from the breaking down of the yolk mass. The other element is a colloidal ketoenol substance, whose outline is very indefinite. The elements are distributed in the peripheral ooplasm covering the yolk mass. The yolk mass appears to be of the same aspect as the intact egg. 3. Fertilized eggs activated in water. The fertilized eggs have been examined at ten minutes after the activation in water. Ooplasm. The concentration of the ooplasm at the nuclear pole is distinct, while it is hardly visible, in living eggs, from outside the egg membrane just as in the egg unfertilized and in water. The region of concentration of the ooplasm is, in section, about 0.2mm at the thickest part, and 1.8mm in diameter. The margin of the plasm concentration extends to the peripheral plasm covering the yolk mass. In these eggs which are activated initiate the normal course of a development, the cortical alveoli disappear and then the surface of a central area of the plasm of the concentrating region seems to be irregularly waved (fig. 15). Furthermore, it is noticed that there is a superficial membrane, 0.7/-< in thickness, being well refractive. The small granules, each of them 0. 8,'.< in diameter, gathering in the cortex of the region of the concentrated plasm are also found in the thickness of 7. 5/-< to 12.5/-( just as in the experimentally activated egg. The ketoenol granules are distributed throughout the plasm, and are varying in form and in agglutinations of themselves, and violet in color. A single granule is spherical in shape, while, the agglutinated granules have changed into irregular forms and have included a small vacuole colored in pink. The size of the agglutination is almost 5/(. These ketoenol granules and their agglutinations are more distinct than that of the experimentally activated eggs. The yolk granules and globules distributed in the plasm are from 5/< to 12. 5/-( in diameter and are very faintly stained in violet. There is also a clear space between yolk granules or globules and plasm. The ground structure of the plasm is granulous and is composed of basiphil granules, each of which is 0.7'p. in size. This structure often appears to be net-worked in consequence of a ranging of the basiphil granules. Base of ooplasm. The base of the plasm is irregular in form and the large yolk. globules stained faintly in violet adhere to the beneath of the plasm (fig. 17). The ketoenol secreting vacuoles are not distributed in the base of the central region of concentration of the plasm, but are mainly distributed in the area of marginal region. of the plasm, and they are frequently distributed in the animal half of the peripheral plasm covering the yolk mass, though, very small (fig. 18). These vacuoles are dumb-bell shape and laid across between plasm and yolk mass. Their lower half including dense colloidal ketoenol substance, is found to be just the same as in the experimentally activated eggs. Also the colloidal ketoenol substance is frequently orientated around a large yolk globule. The appearance of the ketoenol secreting vacuoles which exist in this area is almost the same as that of the experimentally activated eggs. The yolk granules or globules with the exception of those which exist.

(8) Cytochemical Study on the Development of Chum Salmon Egg. in the plasm are numerously distributed in the border area, In this condition of the egg, it is especially noticed that the large yolk globule, 25^< in diameter, orientated in this area is somewhat faint in color by KFJ method and in itself changes to granular or alveolar appeaarnce (figs. 19-23). In the peripheral plasm covering the yolk mass, with the exception of the small granules found in the cortex, the elements distributed in concentrated region of the plasm and border region are at random in distribution. In this region, all yolk globules are stained in reddish purple in the preparations treated with KFJ method. The yolk mass is just the same in appearance as that of the experimentally activated eggs. 4. Developing egg at 4 hours after fertilization. Ooplasm. Although, in this developmental stage of eggs, the region of concentration of the plasm has not yet been so elevated as in typical blastodisc, it is more concentrated than that of the eggs described in previous stage, and is 0.15mm thick and 1.5mm in diameter (fig. 24). The region of concentration of the plasm can be visible from the outside of the egg membrane in living eggs, and the thin peripheral plasm covering the yolk mass is extended from the region to other part of egg. The surface of plasm of concentration region is delicatlly and h-regulariy waved, and its superficial membrane as observed in previous eggs appears refractive and faintly blue in color, but the membrane appears to be often invisible in some parts (figs. 25, 26). And also it is found that a number of granulous or colloidal substance stained in reddish purple has adhered on the surface of the plasm (fig. 25). The small granules are distributed in the depth of 25;-t in the cortex of the region of concentration of the plasm, and they are abundant in the central cortex of the plasm. A single small granule is about 1.3,u in diameter and is stained in red, and also the granules are agglutinated to each other (figs. 25, 28). The granules have a tendency to increase in number according to the successive development. The granules resemble in appearance to the ketoenol granules, but they differ from the latter in the natures of stain and structure, that is, the former is stained in red while the latter is quite violet. The ketoenol granules are much more detectable in the preparations treated with Hamazaki's D fixative than A fixative which is lacking corrosive sublimate. The ground structure of the plasm is the same as in the previous stage. The yolk granules and yolk globules are often found in the plasm, all of which have a space around themselves keeping a distance from the plasm, and some of them are composed of a great number of bright small granules stained in reddish purple. There are large yolk globules, 2(V< in diameter, in the thin peripheral pasm covering the yolk mass, and they are stained in reddish purple or in faint violet. by KFJ method. When fixed in Bouin's fluid, the globule which is stained in reddish purple is not stained with hematoxylin, but the globale which is stained in faint violet is stained with hematoxylin. Base of plasm. Under the region of the concentration, the basement membrane separates the plasm proper from the yolk mass, and the peripheral region of the yolk.

(9) Hideo Nishida mass next to the basement membrane is granulous in structure. There are vacuoles on the basement membrane or across it, where the membrane is not detectable. The vacuoles mentioned above are 20/z to 30,u in diameter, and appear as hollow sphere. But some of them have included a colloidal ketoenol substance or have the ketoenol granules adhereing around the vacuole. The agglutinations of the ketoenol granules are also detected on the basement membrane at various parts. The ketoenol secreting vacua les are found at the base of plasm almost in the area of the margin of the plasm concentration, they are 40/t in height and 22. 5,u in width and are stained in purple (fig. 29). Although the appearance of them is almost the same in feature as in the previous stage, there is a certain number of projection into the perivitelline space through the thin plasm layer. It is recognized as important that there is a difference in nature between the part beneath the plasm of the region of the concentration and that under the peripheral plasm covering the yolk mass. Beneath the plasm of the region of the concentration, the basemet membrane is lined with granulous layer, 15,u thick. The granulous layer is stained in reddish violet. The above mentioned granulous layer has already been observed by Mahon and Hoar ('56) . Under the granulous layer there is a great number of gatherings of the yolk granules, each of them is about 5/-< or smaller. On the other hand, the granulous layer is lacking under the peripheral plasm covering the yolk mass. 5. Formation of blastodisc. In the developmental stage of eggs at 12 hours after the fertilization and activation, the ooplasm of the concentration in the animal region has been elevated and formed. into a typical blastodisc. Blastodisc. The blastodisc is now well rounded as the button stage which has been termed by Mahon and Hear ('56), therefore it can be clearly visible from the outside of egg membrane in living state. The blastodisc is 0. 27mm in height and 0. 95mm in diameter. The surface of blastodisc is somewhat waved and delicately serrated (figs. 40, 41), and also has covered itself with the superficial membrane, refractive and faintly blue in color. However the membrane is invisible in some places. The granulous or colloidal substance stained in faintly reddish purple have adhered on the surface of blastodisc. The small granules distributed in the cortex of blastodisc have increased in number and been formed into a layer, 2Q/J- to 50/-< thick (figs. 38-40). The granule is about 1.3/-t in diameter, and the granules sometimes have agglutinated each other as blocks. These granules are not basiphil and are not positive to Feulgen reaction. The melanin granules are also found as brightly brown grannies in the cortex of blastodisc, they are agglutinated as blocks and the blocks are situated just near the surface of the disc. The agglutination of the melanin granules is 5/-< in size. The ketoenol granules are seldom found in the disc in the preparations treated with Hamazaki's A fixative, but they are more abundant when treated with D fixative. A few of yolk granules or globules are distributed in the disc, they have a space keeping.

(10) Cytochemical Study on the Development of Chum Salmon Egg. a distance from the plasm around themselves. The vacuoles are distributed in the disc. Some of them have the ketoenol granules adhered on the surface, other vacuoles, sometimes, have included several granulous materials. The ketoenol granules are positive to Feulgen reaction, and it is clearly detected in the preparations treated with Hamazaki's D fixative and Feulgen method that there are numerous Feulgen reaction positive granules, 2. 5/-< in diameter, in the disc (fig. 30). These granules are negative. to Feulgen reaction both in the preparation which is carried out without hydrolysis (fig. 33) and in hydrolysis for six minutes (fig. 35). They are also negative in the preparation allowed to rest within the baryta water for fifteen hours prior to the reaction (fig. 34). These Feulgen positive granules are not detected in the preparations treated with Bouin's and Hamazaki's A fixative. The plasm of blastodisc is found to generally granulous, but it has net-worked near the nucleus. The basement membrane is clearly detected under the disc plasm as refractive and faintly blue membrane, and it has a watch glass like appearance as a whole. There is the transferring region to the yolk mass under the basement membrane and the region is granulous or fibrous. In this region there are numerous vacuoles and oil drope. Adhereing to the basement membrane the ketoenol granules and the yolk granules are found in the plasm of the disc. The transferring region to the yolk mass i.s composed of aggregations of granulous yolk which is positive to Feulgen reaction in the preparations treated with Hamazald's D fixative. Also these aggregations are. positive in Feulgen reaction in only the preparation which carried out hydrolysis for three or four minutes. These aggregations are mainly located under the disc in 15/-<. thick (figs. 30-32). Peripheral plasm. The base of the peripheral plasm covering the yolk mass is not lined with the basement membrane, and under the plasm is directly joined to the yolk mass. In the peripheral plasm a number of elements which exist in the disc are distributed in mixed state, whereas, the small granules found in the cortex of the disc and distinct Feulgen positive granules are not noticed. On the contrary, it is noticed in the marginal area of the disc that there are numerous ketoenol secreting vacuoles. A number of vacuoles have been changed in form, differently expressed. The colloidal substance of its lower half being almost transferred to the upper part, as a result of which the upper part embedded in the plasm, has swollen up as a large spheroid which is 30,-' in diameter (fig. 42). A certain vacuoles mentioned above have projected into the perivitelline space. The substance included in the vacuoles appears to be sometimes positive to Feulgen reaction, however, it is also positive in the preparations without hydrolysis and is negative in the preparations allowed to rest in the baryta water.. Yolk mass. Under the disc, tendency of granulation of the yolk mass is extended of wide range as 0. 15mm in depth and the granulated yolk has made numerous blocks.. But in the yolk mass situated under the peripheral plasm the teudency is less 9—.

(11) Hideo Nishida pronounced. Some of the yolk globules and the yolk mass are faintly stained with toluidine blue, methylene blue, methylene blue and hematoxylin. 6. 2 to 4-cell stage. Blastoderm. The blastoderm is 0.3mm in height and 0.95mm in diameter and its surface is somewhat waved and delicately serrated, and is covered with the superficial membrane which is not continuous in several loci. The membrane is refractive and. faintly blue in color (figs. 47-49). The small granules distributed in the cortex of blastoderm have incrased in number and have been formed into a layer, about 0.1mm thick, and also they are agglutinated as blocks, each block is about 7. 5/-< in size (figs. 43-49). In this stage the granules are also found around the nucleus, and a few of them are recognized in the jnnction of blasiomeres and in the mitotic apparatus. The granules located in these reegions are not agglutinated and each granule is 1.3/< in diameter. Generally speaking, the small granules are abundant in the cortex of blasoderm and are not positive to Feulgen reaction. The melanin granules, agglutinated as a block being 10/< X 2. 5/^< in size, are situated just near the surface of blasoderm. The ketoenol granules are few in the preparations treated with Hamazaki's A fixative, while in those treated with D fixative, they are numerous and are positive to Feulgen reaction. The yolk granules found in the blastoderm are somewhat spherical in shape.. each of which is about 5/< in diameter (fig. 49), are stained in reddish purple or faintly violet. Each of the yolk granules has a clear zone which separates the surface of the granules from the plasm in general. But this clear zone is not seen in the case of the smaller yolk granules. Three types of the vacuoles are noticed in the blastoderm ; the first one is a empty vacuole, the sscond is a vacuole agglutinating the ketoenol granules around itself, and the third one is a vacuole which has included colloidal ketoenol substance and also has the ketoenol granules around itself. In this stage, one more element is found in the blastoderm, mainly near the surface ; it is vacuolar and. has always included a solid body which is well stained with hematoxylin (fig. 44) and it is also recognized in the preparations treated with Bouin's fixative and stained Delafield's hematoxylin. The ground structure of blastoderm is composed of fine basiphil granules and net-worked fibrils. It is especially fibrous in the jointing area of two blastomeres and near the mitotic apparatus. The base of blastoderm appears to be flattened and the basement membrane is also clearly detected. In this region the vacuoles as were mentioned previously are particularly numerous. On the basement membrane there are the ketoenol granulea which often agglutinated as blocks and large yolk globules in which are often included small vacuoles or bright granules, and also blocks of melanin granules. Under the basement membrane is the transferring region to the yolk mass; upper area of this region is finely granulous or fibrous, and there are numerous aggregations of granulous yolk in the lower area. The nature of these aggregations of granulous yolk is the same as in the previous stage. Peripheral plasm. The peripheral plasm covering the yolk mass is about 10/-< — 10 —.

(12) Cytochemical Study on the Development of Chum Salmon Egg thick. In the peripheral plasm, the small granules in the cortex of blastoderm can not be recognized. The ketoenol secreting vacuoles which are not to be detected in the blastoderm are situated almost in the peripheral plasm near around the blastoderm, and they have tendency to gush out their colloidal substance, as a result they seem to be projecting into the perivitelline space (figs. 46, 48, 50). The substance in vacuoles is stained in purple in the preparations treated with KFJ method, and not stained with hematoxylin. Whereas, the substance which has been gushed out from the vacuoles into the perivitelline space is well stained with hematoxylin. The vacuoles are about 40/-< in hegiht and 25/-( in diameter. In the perivitelline space, numerous granulous substance are recognized in the preparations treated with KFJ method and well stained with hematoxylin. However, it is very difficult to recognize these substance in the preparations treated with other fixatives. Yolk mass. Under the blastoderm the yolk mass is granulated in 0. 3mm depth, and the granulated yolk has made numerous blocks. The yolk mass situated beneath the peripheral plasm shows lesser granulation. The yolk mass is stained in reddish purple. with KFJ method, though hardly stained with hematoxylin. 7. 8 to 16-cell stage. The blastoderm is 0.4mm in height and 0.93mm in diameter and its surface is somewhat waved and delicately serrated, and are almost covered with the superficial membrane (fig. 65). However, near the edge of blastoderm the membrane seems often to disappear. It is noticed in the surface of blastoderm that there are some depressions in which the granulous substance found in the perivitelline space are to be detected, and that the vacuolar elements which have included a solid body stained with hemaoxylin are also found (figs. 61, 62). These vacuolar elements have a tendency to increase in number, as a result, they become detectable between the blastomeres (fig. 63). Such small granules in the cortex of blastoderm as have been observed in previous stages are also distributed in the upper bla",tomeres, however, they are seldom in lower blastomeres. They are formed into a layer, about 40/-< thick, and are agglutinated near the surface of blastomere as blocks, each block being about 10/-< in size. Their nature in stain is the same as that of previous stage (figs. 51, 52, 54, 59). The melanin granules are also located just near the surface of blastoderm as blocks, each block being about 7/< in size. The ketoenol granules are seldom in the preparations treated with Hamazaki's A fixative (fig. 54). Their nature in stain is the same as that of previous stage. The yolk granules are distributed in the plasm of blastoderm, they have varied in size from 2. 5/-< to 12/-<. They are stained in reddish purple of faintly in violet and some of them have included the very small bright granules numerously. The vacuoles situated in the plasm of blastoderm are seldom seen in this stage. The cleavage furrow of some lower blastomeres is to be found inperfect, but the border of blastomeres is different from the other part of plasm. The ground structure of the blastoderm appears to granulous or fibrous and has a tendency to be fibrous at the border of blastomeres — 11 —.

(13) Hideo Nishida (figs. 60, 64). The structure is generally basiphil. However, the ground structure of blastoderm seems to have some different appearance depending on the fixatives. Indeed, it is very fibrous in the preparations which are fixed in Bouin's fluid and stained with hematoxylin, whereas, it seems to have a tendency to be granulous in the preparations treated with Hamazaki's A fixative and KFJ method. Moreover, it seems to be very granulous in the preparations treated with D fixative. The outer part of the blastoderm is more basiphil than the inner part on account of a great abundance of the basiphil granules. The nucleus of blastomeres is about 20/-< in diameter and its chroma.tin is. well stained with hematoxylin, but it is faintly positive to Feulgen reaction (figs. 65, 69, 70). There are some mitotic figures (figs. 71-73). The nucleus and aster are also surrounded by the small granules which are well found in the cortex of blastoderm. The base of the blastoderm appears to flattened and the basement membrane is clearly found except at the loci of the vacuoles varying in size from 10/-< to 50/-<. On the basement membrane, the aggregations of ketoenol granules such as mentioned in the previous stage are detected (fig. 57). Beneath the basement membrane is mainly occupied by the aggregations of granulous yolk which are positive to Feulgen reaction after three or four minutes hydrolysis in the preparations treated with Hamazaki's D fixative. The plasm-like and fibrous structures are detected at this region. Under the granulous yolk, the yolk often reacts to Schiff s reagent without hydrolysis, however, its reaction becomes of invisible state by the treatment of the baryta water. Under the blastoderm the yolk mass is granulated in 0.4mm depth, and the granulated yolk mass has often made numerous blocks. The yolk mass can be stained in reddish purple with KFJ method. The granulous substance which are positive to PAS reaction are. found in the oil drops (figs. 66, 67). Peripheral plasm. The peripheral plasm is about 10/.< thick. The small granules found in the cortex of blastoderm are not recognized. The ketoenol secreting vacuoles are so numerously distributed as to extend over 0.6mm in the distance from the blastoderm (figs. 55-54, 58). In this stage the vacuoles seem to be actively secreted their colloidal substance. The substance included in the vacuoles is stained in purple and not stained with hematoxylin, however, when secreted into the perivitelline space it is well stained with hematoxylin. Both substances in the vacuoles and in the perivitelline space are negative to PAS reaction of McManus method (fig. 68), and they are faintly positive to Feulgen reaction. On the other hand, the blastoderm, peripheral plasm and the yolk are positive to PAS reaction. The yolk mass situated beneath the peripheral plasm shows lesser granulation than the yolk mass of under the blastoderm.. Experimental egg. Six hours after fertilization, the egg sincubated in 1/10 mol water solution of calcium chloride (16°C), have reached to 8-cell stage. In these experimental eggs, the cortical alveoli are still remained. The blastoderm of these eggs is different from the normal one in the following points. The granulous yolk situated beneath the.

(14) Cytochemical Study on the Development of Chum Salmon Egg. blastoderm is faintly positive to Feulgen reaction. Sometimes the inclusion of ketoenol secreting vacuoles reacts to Schiff's reagent without hydrolysis or with hydrolysis of six minutes in the preparations with Hamazaki's D fixative. The colloidal substance reacting to Schiff's reagent without hydrolysis is found between yolk mass and nil drop, Mitotic figures are recognized in the blastoderm (fig. 74). Interphase nuclei being about 16/-< in diameter have numerous chromatins which are distinctly positive to Feulgen reaction. And also around the centrosome there is a basiphil ring of 5/-< width, diameter of which is 20'', 8. 32 to 64-cell stage. Blastoderm. The blastoderm is 0.45nnn in height and 1.15mm in diameter, and is composed of blastomsres of which each size is about 0.2mm in diameter (fig. 76). The surface of the blastoderm is somewhat waved and delicately serrated, and is almost covered with the superficial membrane. And also it is well recognized that the granulous substance found in the perivitelline apace is agglutinated on the surface of blastoderm. The cell membrane of the blastomere is often indistinct at the part facing intercellular space (figs. 81, 82). In the blastomere there are some vacuolar elements. which have included solid body stained with hematoxylin (fig. 75). Peripheral plasm. Although, generally speaking, the structure and nature of the peripheral plasm seems to be similar to that of previous stage, the activity of ketoenol secreting vacuoles is very noticeable comparing with that of the previous stage (fig. 77). The vacuoles have become to be 90/-< in height. The lower part of them is hollow, about 20/<, and is in the plasm. The upper part is as a vesicular form projecting to the perivitelline space. Furthermore, sometimes the upper part of the vacuole has directly adhered to the surface of blastoderm. The granulous substance sscreted from the vacuole are found numerously around the blastoderm in the perivitelline space, and they are well stained with hematoxylin. 9. Later segmentation stage. Blastoderm. The blastoderm is 0. 53mm in height and 1mm in diameter. As cleavages proceeding the blastomeres have become small in size. They are spheric and each of them is about 55/.( in diameter except those of the outer layer of the blastoderm. The cells of outer layer have come to be cylindrical form, and their nuclei are situated at the upper side of the cells. It is often found that those blastomeres are mitotically divided. In most of the blastomeres, superficial membranes are clearly detected, however, some of them are not distinct, and the granulous substances found in the perivitelline space are agglutinated to their surface. The cytoplasm of blastomeres appears to be fibrous and to be radially arranged. The nucleus is about 15/< in diameter, being a center of the an-angment. Through the cytoplasm there are ketoenol granules, each of them is 1.2,u in diameter, but they are few in the cortex of blastomeres. The yolk globule, about 12n in diameter, is often included in the blastomere. The central region of the blastomere has a tendency to be well stained — 13 —.

(15) Hideo Nishida with dyes. Among blastomeres there is a few indeterminate forms of cells of which nucleus is not detectable. Base of blastoderm. Some of the blastoderm cells located at the base are often united to a fibrous structure of the transferring region to the yolk mass, and the cells. of the margin of the blastoderm continue to the peripheral plasm covering the yolk mass. Under the basement membrane the blastoderm is lined with the aggragations of granulous yolk. The aggregations have a strong affinity to the dyes and are fadeless and positive to Feulgen reaction only in three or four minutes hydrolysis in the preparations treated with Hamazaki's D flxative. The agglutinations of ketoenol granules are also found in the base. Perepheral plasm. In the later segmentation stage the activity of the ketoenol secreting vacuoles is found to be somewhat declined. The size of the vacuoles is about 15.", and two thirds of the vacuole has projected to the perivitelline space. The yolk mass seems to be the same as the previous stage in the structure and nature. 10. Early and late blastula.. Early blastula. The blastoderm is 30/< in height and 1.2mm in diameter (fig. 87). The outer layer of cells of blastoderm (fig. 88) is composed of cylindrical cells, each of which is about 20/-t in diameter and 20 p. high. The nuclei of these cylindrical cells are located at the upper side of the cell. The cytoplasm of these cells appears as granulous or fibrous, and there are basiphil granules, being 2. 5/< in diameter. There is often the yolk granule in the cytoplasm of the blastoderm. Inner region of the blastoderm is composed of spherical cells, each of them being about 25,^ in size, and there are intercellular spaces among these cells. The cytoplasm around the nucleus is basiphil and granulous, but peripheral part is fibrous and transparent. The mitotic figures of the blastoderm cells are often found. It is noticed that some of the blastoderm cells have often included a vacuole (fig. 89). Moreover, sometimes the cell is filled with a large vacuole, its cytoplasm appears very thin. The vacuole has often contained some yolk granules. It is also detected, in the preparations without. hydrolysis for the control of Feulgen reaction, that almost all the cells in the blastoderm have adhered at their surface several granules colored in pink. The large one of such pink colored granules is about 5<" in diameter. Furthermore, they are well stained in. violet by the PAS reaction of McManus method. In this stage, the periblast has been formed as syncytium under the blastoderm and in the marginal region of the blastoderm. The periblast is mostly thick, about 40,u, under the edge of blastoderm, and becomes thin in proportion to its distance from the edge and continues to the peripheral plasm covering the yolk mass. The nuclei of the periblast are spherical in shape, and the nucleus is about 18,u in diameter. There are some giant nucleus in the periblast, which are 30,u in diameter, and lobed in outline. Besides, it is noticeably observed that there are some blocks of chromosomes (figs. 90-98). Sometimes they seem to be divided into two or three. Then each part is bridged by sticky chromosome. Under blastoderm — 14 —.

(16) Cytochemical Study on the Development of Chum Salmon Egg. edge, the periblast has a distinct superficial membrane which appears smooth, while on the other hand, in the central area of the periblast its surface seems to diffuse and the superficial membrane is invisible. There are numerous fine granules on the. surface of the central periblast. They are faintly stained in violet in the preparations treated with KFJ method, and also they are positive to PAS reaction of McManus method. It is recognized in the periblast that there is a few of vesicles of including a solid and bacilliform body (fig. 95). The ground structure of the periblast plasm is more basiphil than that of the blastoderm. Under the periblast, embedded in the yolk mass, it is composed of the aggregations of granulous yolk which is positive to Feulgen reaction of three or four minutes hydrolysis in the preparations treated with Hamazaki's D fixative. The ketoenol secreting vacuoles, found in the peripheral plasm are not so active as in the previous stage. The granules which are positive to PAS reaction of McManus method are also detected around some oil drops found in the yolk mass. Late blastula. The blastoderm is O.Snun in height and 1.5mm in diameter. At this. stage the subgerminal cavity is distinctly found between blastoderm and periblast (fig. 99). The outer layer cells of blastoderm are varied from 20/u to 8u in height, and the higher cells are cylindrical, and the lower cells are somewhat flattened in form. The granulous substance found in the perivitelline space have adhered to the surface of the. blastoderm (fig. 107). The ketoenol granules have been included through the blastoderm, and some of the outer layer cells appear particularly basiphil owing to their inclusion of numerous ketoenol granules. In outer layer of blastoderm, the blastomeres come into close contact with each other, while on the other hand, the cells in other parts have no such close contact, and the intercellular spaces between them is distinctly recognized. In the cells of the inner region of the blastoderm, the differentiation of cytoplasm is recognized, that is, there are two parts in the cytoplasm of which one part is as endoplasm, being abundant of the ketoenol granules around the nucleus, and the other is as ectoplasm being transparent and fibrous (figs. 102, 110-114). It is. evident in the blastoderm that some of cells divides mitotically and their chromosomes are distinctly positive to Feulgen reaction (figs. 108, 109). Furthermore, a certain number of giant cells, about 70," in diameter, are noticed at the lower layer of blastoderm (figs. 100, 101). In the subgerminal cavity, the ketoenol substance are often detected by KFJ method. The ketoenol'substance have a tendency to react to Schiff's reagent in the preparations without hydrolysis, however, it disappears in the preparations treated with the baryta water. The marginal edge of blastoderm comes into contact. with the periblast somewhat bending inward toward the central part of the blastoderm (figs. 105, 106). The periblast is the thickest under the edge of the blastoderm and becomes thin in proportion to its distance from there, and it is very thin at the central area. The surface of the periblast is much diffused at the central area. There are some ketoenol secreting vacuoles located in the central area of periblast which become — 15 —.

(17) Hideo Nishida more active in discharging their inclusions into the subgerminal cavity in comparison with earlier blastula (fig. 104). In the periblast around the blastoderm edge, numerous small granales, each of which about 1.2/-< in diameter, are detected (fig. 103). The small granules are red in color and are negative to Feulgen reaction, they are well recognized in the preparations treated with Hamazaki's A fixative and KFJ method, but are not distinct in the preparations treated with D fixative. There is a certain number of vesicles, about 25/-< in diameter, including a solid and bacilliform body, in the periblast. The nuclei of periblast are abundant in the marginal region, and they have a tendency to grow larger and to become irregular in form. The apparance of the nucleus membrane is various in structure. Hence, although there is a number of nuclei of which membrane are distinct, in some of nuclei their membranes seem to be lacking either at all parts or at least at a certain part. Particularly the nucleus. membrane is hardly detectable at the part adhereing to yolk globule. The mitotic divisions of the periblast nuclei are hardly detected in this stage. The ground structure of the periblast plasm is more basiphil than that of the blastoderm. Under the periblast, located on the yolk mass, it is composed of the aggregations of granulous. yolk being positive to Feulgen reaction just as in the early blastula (fig. 115). Moreover the aggregations are growing in number, in this stage. In the preparations treated with Hamazaki's D fixative, the nuclei of blastoderm and periblast do not become negative to Feulgen reaction with the treatment of the baryta water, but the cytoplasm of them become negative after the treatment. The ketoenol secreting vacuoles are found in both the periblast and the peripheral plasm. The ketoenol secreting vacuoles located in the peripheral plasm are not so active as that of the central region of. periblast Distinct basement membrane for dividing the plasm and the yolk mass is hardly detectable (fig. 116). The cortex of yolk mass is much varied in structure and staining, and in this region a great number of yolk globules are embdded. These yolk globules are varied in size, the larger one is about 40/-f in diameter and the smaller one is about 5/-t. Also there are differences among those globules in staining, some of the yolk globules have the complex nature of basiphil and not basiphil parts. The colloidal ketoenol substances stained in violet are found between yolk globules and plasm of the periblast, and also grannies stained in violet with KFJ method are noticed around the oil drops. Furfchermore it is for the first time to be detected that a certain number of basket-worked bodies are embedded in the yolk mass near the periblast. (fig. 116). They are not stained with KFJ method, but somewhat stained with hematoxylin, and the larger one of them is 50,u in diameter. Also a spherical body is sometimes recognized as being hollow in the basket-worked body. 11. Early gastrula. Blastoderm. The blastoderm is 0. 1mm in height at the central part and 0. 2mm at. the beginning of embryonic shield. The diameter of the flattend blastoderm is 2. 5mm (figs. 117, 118). The epiblast is four cell layer being about 50/< thick in the central — 16 —.

(18) Cytochemical Study on the Development of Chum Salmon Egg. region. The uppermost layer, i. e. periderm is much flattened with the exception of the cells situated in marginal region of blastoderm. Among these periderm cells there are some of very basiphil cells. The periderm has extended outwardly over the marginal edge of the blastoderm. The granulous substance found in the perivitelline space are agglutinated on the surface of the blastoderm. The beinning of hypoblast found inner side of the blastopore is a wedge-shaped cell mass. The cells of epiblast are somewhat spherical and their cytoplasm are basiphil and granulous. They have included the ketoenol granules, 1.2/-< to 2.5u in size, moreover the larger one has included a small vesicle among granules. And also these cells have often included a vacuole, 12. 5/-< in diameter, containing several purple granules. The epiblast is somewhat bent inward at the edge of the blastoderm. A certain number of cells are found in the subgerminal cavity in which granulous substance is to be detected (fig. 128). These cells are ameboid in form. The ketoenol granules are abundantly found around nucleus in the endoplasm of these cells. Although the periderm cells are in immediate contact with the periblast in a plane, the epiblast cells, on the other hand, are not so closely in contact with the periblast (fig. 122). The blastoderm is ten cells. thick at the level of the hypoblast near the blastoporal lip (fig. 119). The prostomial thickening can be recognized. The mitotic figures of tfie blastoderm cells are found. Periblast. In general, the surface of the periblast of the early gastrula is found to be smooth. The surface of the periblast is much smooth under the blastopore where the superficial membrane of the psriblast plasm is well recognized. In proportion to the distance from there to the anterior, the membrane becomes invisible and the surface of the plasm is diffuse, and also the hypoblast cells have a tendency to adhere on the periblast. The wandering cells are often aggluttnated each other and seem adherent to the surface of the periblast (figs. 127, 128). The area of a perfect free surface of. the central periblast is limited to the peripheral region near the germ ring (fig. 129). Owing to the projecting of the vacules being about 50/-( in diameter situated in the periblast, the periblast surface on the vacuoles seem to be pushed into the segmentation cavity at some parts. In case of the blastoderm cell attaching to the surface of the periblast, sometimes the cytoplasm of the blastoderm cell is continued with the plasm of penblast. The blastoderm cells attached to the periblast are few at the surface of the peripheral area of the central periblast. Under the germ ring, the surface is much flattened as clear plane and the superficial membrane has separated the periblasfc plasm from the blastoderm. It is detected in the whole area of the surface of the, periblast that the ketoenol secreting vacuoles are actively discharging their inclusions and that the yolk globules, varied in size from 5/( to 30/-<, are pushed into the ^subgerminal cavity. The periblast plasm is very thin in the central part and thick in the marginal, particularly it is very thick, about 100/j., under the germ ring. There are numerous samall granules (figs. 121-123), each of which about 1.2,.' in diameter, in the periblast at the contact with the blastoderm edge, and they are abundant in the cortex and have.

(19) Hideo Nishida a tendency to extend to the other part of the periblast. They are red in color and negative to Feulgen reaction, and also they are well recognized in the preparations. treated with Hamazaki's A fixative and stained with KFJ method. They may not detectable in the preparations treated with D fixative and are not detected in the preparations treated with Bouin's fixative and stained with Delafield's hematoxylin. There is a certain number of vesicles (fig. 120), about 25/< in diameter, including a. solid and bacilliform body in the periblast, and they have a tendency to increase in number. Besides, the hollow vesicles are found in the periblast near its surface. The nuclei of the periblast are abundant in the marginal part, and they increase in number so that they become to be found also in the centra.l part, but the nuclei found in the central part are small and somewhat flattened. The appearance of the periblast nuclei seems just as in the previous stage. The ground structure of the periblast plasm is more basiphil than that of the blastoderm, and seems fibrous in the preparations treated with Bouin's fixative subsequently stained with hematoxylin, however, it seems fibrous and granulous in those preparations treated with Hamazaki s A fixative subsequently stained with KFJ method, moreover, it seems granulous in those treated with D fixative. There is no basement membrane which divides the plasm and the yolk mass. In the preparation treated with Hamazaki s D fixative, the nuclei of blastoderm and periblast are also positive to Feulgen reaction with the treatment of the baryta water, but the cytoplaem of the blastoderm and the periblast is shown to negative to reaction. Under the periblast, located on the yolk mass, it is composed of the aggregations of grand ous yolk, being positive to Feulgen reaction. However, these aggregations are positive to the reaction after the three or four minutes hydrolysis in the preparations treated with Hamazaki's D fixative. In the yolk mass there are numerous oil drops, and granules which are positive to PAS reaction are found around the oil drops. A certain number of basketworked bodies are also embedded in the yolk mass, and they have a tendency to increase in number. The cortex of the yolk mass has granulated not only under the periblast but also under the peripheral plasm. The granulated region is about 0. 5mm thick. The suface of the peripheral plasm appears to be smooth and there is a superficial membrane on it with the exception of the parts in which the vacuoles or the yolk globules are located. The ketoenol secreting vacuoles have a decrease in number in here. The vacuoles are found in both the periblast and the peripheral plasm covering the yolk mass, but the vacuoles located in the latter are not so active as the former and are few in number. There are some of the blocks of melanin granules, and they have often included a small vacuole. The yolk globules situated in and beneath the periblast are varied in size being from 5/( to 40/<, and in regard to staining there are some differences among those globules. Some of the yolk globules have a complex nature, partly basiphil. The colloidenol substance stained in violet is often found between yolk globules and periblast plasm. — 18 —.

(20) Cytochemical Study on the Development of Chum Salmon Egg 12. Five somites stage. The germinal area is 4. 3 mm in diameter, and the germ ring at the opposite side of the blastopore is 40;( thick, and also at the extraembryonic region the blastoderm is 10/-< thick. The embryonic blastoderm is 0. 35mm thick at the head region of the embryo which is 1. 95mm in length. The notochord is found in the embryonic region, and at both sides of the notochord five pairs of somites are recognized. Also the Kupffer's vesicle has been formed under the posterior part of notochord. Embryonic blastoderm. There is a thin cell layer, periderm, at the out side of the neural ectoderm, it is composed of very flat cells, each of which is 55/-t in diameter and 2,u thick. In this cell layer there are a few of embryonic cells which are spherical and about 10,^ in diameter. However, the cells of this layer become rhombic in shape and are in immediate contact with the periclast. The periderm and the upper part of the neural ectoderm is basiphil. The neural ectoderm of the embryonic axis just behind head to the blastopore is about 30/-( thick and it communicates with the entoderm at the anterior of the blastopore. Also there is a communication of the neural ectoderm and the mesoderm near the head region. Anteriorly to the head, the neural ectoderm is extended forward and communicates directly with the periblast. At this part there is no boundary between neural ectoderm and periblast. The head is composed of spherical or rhombic form of cells, each of cells is about 12/-(, and each of their nuclei is about 6/-(, The notochord communicates with the entoderm and at more posterior part it communicates with the neural ectoderm. The more anterior part of the notochord is clearly differentiated from other part as the tissue being 45/-< in diameter. Each of their nuclei is spheical in shape and 12. 5/( in diameter. The entoderm is very thin under the notochord and is arranged in strait. Its cells are somewhat flattened. However, the entoderm spreads out under the anterior and the lateral side of the neural ectoderm where it is composed of two or three cells layer. At the anterior of the embryonic region, the differentiation of the entoderm and the mesoderm is not distinct.. And also, at more anterior region it is difificult to distinguish the periblast from the entoderm on account of the absence of dividing structure between them. The germ ring is composed of less than three cells layer. There is a flattened cavity in the extraembryonic region between embryonic region and germ ring, and the cavity appears to be a space existing between periderm and periblast, in which some granulous materials are to be found. Periblast. Generally speaking, the surface of periblast is much varied in nature and structure depending on the region situated in the germinal area. Thus, the surface of periblast is irregular and its superficial membrane is undetectable beneath the germ ring of the opposite side of the blastopore. The surface of the periblast beneath the germ ring of the lateral sides is smooth and there is a superficial membrane on the surface. And also, in the embryonic area, the surface is smooth at both side of embryo, however it is irregular near the dorsal Iip of blastopore, and beneath the proper embryonic blastoderm. It is found in the cortex of periblast that the small grannies are stained — 19 —.

(21) Hideo Nishida in red in the preparation treated with Hamazaki's A fixative subsequently stained with. KFJ method. The periblast is much varied in depth depending on the region of lining the germinal area and it is thicker under the proper embryonic blastoderm. There are numerous nuclei through the whole area of the periblast, and the nuclei are very much varied in size and form. Under the embryonic blastoderm the nuclei are especially abundant. A certain number of large nuclei situated beneath the head are found to be directly communicated with the blastoderm. The membrane of the large nuclei located in the inner part of the periblast are found to be lacking in some places. And the contents is to be seen as if they burst into the plasm of the periblast. The large nuclei, each of which about 30,-', are sometimes lobed, furrows and notches extend at various depths into their substance ; owing to this phenomenon, they appear to have included several small nuclei in themselves. The nuclei are somewhat flattened at thin periblast of extraembryonic area, and they are often found to be constricted. The small nuclei which resemble embryonic blood cells in shape are found in the periblast beneath the blastoderm, they are about 5/-< in diameter and their shape is quite circular, their nnclear membrane is distinct, and about two chromatin grannies are contained in themselves. They have a resemblance to the small wandering cell described by Fischer ('46) from embryonic chicken spleen. In the deep part of the periblast beneath the embryonic region, there are small bodies which are composed of Feulgen positive grannies, lacking a distinct limiting membrane. There is a great number of yolk globules which have a peripheral space in which the colloidal ketoenol substance is often found. The yolk globules are varied in size, form and structure. Some of them appear homogeneous, others are partially granulous or completely granulous. The yolk globules are difficult to stain, but some of them are faintly basiphil in part or completely basiphil. Although the yolk globules are negative to Feulgen reaction in the preparations treated with Hamazaki's A fixative, Feulgen positive part is detected in some of yolk globules in the preparations treated with D fixative. There is a number of vacuoles including colloidal ketoenol substance or agglutinated ketoenol granules in the periblast. Besides, there is a number of vesicles, about 25.". in diameter, including a solid body. The ketoenol secreting vacuoles are found at the surface of the periblast. It is noticed in this stage that the aggregations of granulous yolk are found to be rather limited to the anterior part of the head, they are the same as those found in previous stages. The periblast plasm is more basiphil than the cytoplasm of blastoderm and becomes more fibrous than the plasm of previous stage. There is no line of boundary between periblast and yolk mass. Yolk mass. The cortex of yolk mass has granulated not only under the periblast but also under the peripheral plasm. The granulated region is about 0. 5mm thick. At a number of parts under periblast, the yolk mass becomes so finely granulous and fibrous that it seems to be plasm of the periblast. The yolk mass is faintly stained with both acid and basic dyes. The yolk mass is covered, in the extragerminal area,. — 20 —.

(22) Cytochemical Study on the Development of Chum Salmon Egg. with a thin plasm containing several elements which are the yolk globules and the vacuoles including colloidal ketoenol substance. There is a great number of the basket-worked bodies embedded in the yolk mass, Besides, a great number of oil drops are located.. IV. Discussion The early development of Oncorhynchus keta has been made cytochemically in the present study. Not olny the development of germinal region but also the extragerminal. region including the yolk has been studied in detail. In the discussion about the results it is considered to be convenient that important points should be selected. Fixation and staining. The microscopic techniques which have been dealt with in a number of previous works on the development of teleost fishes, were those designed to establish the histogenesis and morphogenesis of the primitive embryonic form and the organ systems. Recently, it has come to be carried out in the embryology to examine the localization of particlar chemical constituent of each embryonic cell, or cytologiclly defined parts of the cell. Notwithstanding those progresses, there are few contributions to the cytoembryological knowledge on the teleost egg. It may be imperatively necessary to re-examine the microscopic techniques which have usually been employed in the study of the development of 0. keta. As a procedure in routine histology, Bouin's fixative and Delafield's hematoxylin staining is well used in the studies of the development of 0. keta, and this method has also been used in the present study in comparison with the proper cytochemical procedures. In this indirect staining, hematoxylin is used in conjunction with lake, and then selective destaining is achieved by decreasing the pH of the destaining fluid, however, its staining mechanism is. principally the same as the direct staining of toluidine blue and of methylene blue which are used also in the present study.. According to Gersh ('59), it is considered that the term basiphil is only a relative one, for by controlling the various factors it is possible to reduce basiphil almost to extinction or to increase it up to a certain maximum. And also he has mentioned that when the staining of nucleic acids is suppressed and the avidity of the associated proteins for acid dyes is heightened it becomes possible to stain nucleoproteins with acid dyes,. and that the degree of basiphil varies, depending on the fixative, the pH of the staining solution, the nature of the dyes used in staining, and the concentration of the dyes, etc. Hence it would be obvious that neither direct nor indirect staining is reliable in cytochemical analyses, but is useful as a prior method of conjecturing, and that a progressive contribution on the embryology might have been expected from the results by those methods. There are certain attempts of cytochemical or histochemical analyses on the development of 0. Kefa, that is, Mahon and Hoar ('56) in early development, and Yamada, J. ('59). in salmon fry. Besides, Daniel ('46) and Smith ('52) have carried out histochemical.

(23) Hideo Nishida studies on the development of Salmo. Thanks to those researches, some knowledge on lipids and carbohydrates in the development of those species have been acquired. On the other hand, though the studies on the nucleic acids have made much progress in the development of other animals, particularly in amphibia, aves and echinoderms; such studies are very poor in teleost fishes. For this reason it is necessary in cytochemical aspect to ascertain how the nucleic acids participate during the development of the present species, The author has selected firstly deoxyribonucleic acid (DNA) for this purpose, and there are following two reasons why the acid has been selected here. Firstly, there is the Feulgen method .for DNA as chief staining method in use, and the present evidence is largely consistent in showing that the Feulgen method is specific for DNA; on the other hand, it is neither easy nor reliable to discriminate ribonucleic acid from DNA in the staining of nudeoproteins by basic dyes. Secondly, there is a number of biochemical evidences of cytoplasmic deoxyribonucleoside, excess DNA, in the eggs of echinoderms, insects, amphibians and birds ; and it has been considered that the excess. DNA may be precursors for the formation of nuclear nucleoprotein during cleavage. Subsequently, according to Ely and Ross C'49), as regards Feulgen method, special attention has to be added to the fact that some of the reactive maierial which combines with the leucofuchsin has been dissolved during fixation or in the various steps of the test, and that the reactive material is present in small amounts in submicroscopic structures and the color is too weak to see. According to Ichikawa (53), inconjunction with Feulgen method, Hamazaki's fixatives are the appropriate solution with the ability. to distinguish between depolymerized DNA and DNA, And he has shown the index of identification of the DNA and its derivatives. Hence, in the present study, Hamazaki's fxatives have been employed. Hamazaki's D fixative has a resemblance to Zenker's in reagents, but is different in formula, that is, more of acetic acid and less of corrosive sublimate. Next, according to Lison ('53) five minutes' degree is appropriate as. hydrolysis in employing Zenker's fixative, while Hamazaki ('52) has admitted that three minutes' degree of hydrolysis is particularly appropriate for the purpose of proving depolymerized DNA and that over four minutes' degree of hydrolysis tends to the negative result. In the present examinations three minutes' degree of hydrolysis has been particularly appropriate for the depolymerized DNA and six miuntes' degree has fallen into a flatly negative result. And also, the grannies found in the blastodisc or blastoderm and the aggrgations of granulous yolk stiuated beneath the blastoderm are negative to Feulgen reactin in the preparations treated with Bouin's fixative and Hamazaki's A, whereas, they are distinctly positive to the reaction dealing with about three minutes hydrolysis in the preparations treated with Hamazaki's D fixative. Those results above mentioned, would be discussed hereafter in connexion with the problem of the true and the pseudo plasmal reactions to Schiff's reagent. According to Lison, the true plasmal reacts positively to Schiff's reagent without hydrolysis in — 22 —.

(24) Cytochemical Study on the Development of Chum Salmon Egg. the oaly preparation treated with fixatives which contain corrosive sublimate. The preparation treated with Hamazaki's D fixative in the present study, reacts rapidly to Schiff's reagent without hydrolysis, and the yolk mass immediately causes the plasmal reaction of intermediate degree, however, the preparation treated with Bouin's fixative and also that treated with Hamazaki's A fixative result faintly. From all these considerations, it would seem that there are certain amounts of true and pseudo plasmals in the yolk mass. But, in accordance with Ichikawa's index of identification of DNA and its derivertives, the several granules found in the blastoderm and the aggreations of granulous yolk situated beneath blastoderm have contained deoxyribonucleoside.. The original KFJ methods of Hamazaki have been employed in the present study. Hamazaki C'54) has considered these methods as specific procedures of identification of depolymerized DNA, that is, poly-deoxyribonucleotide, mono-deoxyribonucleotide, monodeoxyribonucleoside and purine, However, out of them, bile acid and fatty acid are also stained with KFJ method, these are different in staining .color from the. depolymerized DNA, that is, bile acid and fatty acid is reddish purple whereas the depolymerized DNA is quite violte. The normal nuclei (DNA) are not stained with Hamazaki's KFJ method; Seki C'51), Ichikawa ('53), Okamoto and others ('55) and Ishikawa ('55). The existence of tremendous amount of excess DNA in the animal eggs has been proved as the result of biochemical analysis. The excess DNA in the eggs is indicated by the apparent existence of 20 times the haploid amounts of DNA in a sea urchin, 103 times in a cricket, 4 X 103 times in a frog and 5 x 107 times in a chick ; therefore it would seem possible that there is also a great amount of excess DNA in the chum salmon egg. As regards the DNA content found in the animal eggs, it may be appropriate to consider that the DNA content of the eggs is under a special condition. The existence of great deal of excess DNA in the animal eggs is recently proved depending on the progress of appropriate methods in the biochemistry, for example, the. microbiolgical assay by Sugino and Okazaki ('57). According to Sugino and Okazaki ('57) there are two types of DNA, in the eggs of Pseudocenlrotus depressus and Hemicentrotiis pulcherrimus one is normal DNA which is not solved in the acid and another is excess DNA which is dissociated in the acid, and two types of DNA show a ratio of about two in normal DNA to eight in excess DNA, and this proportion is also acquired the same in the egg of Bufo vulgarls. Then they have considered that a large percentage of excess DNA may be the DNA dissociating in the acid. Prior to their study, Hoff-J^rgensen and Zeuthen ('52) have presented evidence in the frog egg that excess DNA counts up to ninety percent of the whole, and Gregg and Uvtrup ('55) have also acquired the same result. As the result, Hoff-J((rgensen and Zeuthen have considered that the excess DNA has been stored already in egg and is successively involved in new nuclei during the development.. 23.

(25) Hideo Nishida On the other hand,. the original Feulgen reaction does not corroborate the high figure. for the excess DNA. According to Cohn and Volkin ('57) it would seem that the microbiological assay measures deoxyribonucleosidic material other than DNA. However, unfortunately, no such evidence has been examined as yet in the egg of the present species. On the other hand, Briggs and King ('59) have communicated that a more important question concerns the relationship of DNA and excess DNA to the morphogenetic region of the egg, and that about this nothing at all is know, so for as thay are aware. Howeyer, they have added to them that Bieber ('58), as yet unpublished, has carried out an observation indicating that two types of DNA are present in the frog embryo, and that the ratio of the two types changes during development. Hence, the author has attempted to express cytochemically the development of chum salmon in the aspect of excess DNA discussed already here above.. Activation and cleavage. Whether unfertilized or fertilized, the eggs activated in water have changed considerably in the plasm, and cortical alveoli have disappeared and the small grannies appear in the cortex of the plasm, then .the appearance of the small granules and the disappearance of cortical alveoli seem to be concordant phenomena.. The small granules are well stained with KFJ method in the preparations treated with Hamazald's A fixative, but they are not positive to Feulgen reaction. Although the small granule resembles the ketoenol granule, the former is different from the latter in the reaction of Feulgen method. Consequently it would seem that the small granules are different to the ketoenol granules. According to Runnstsom and his co-workers ('59), it is found that in the unfertilized egg of the sea urchin, the layer, in the egg. surface is stained by the acetic ferric method of Hale. And after fertilization it is apparently split up into granular units which stain more intensely with the Hale method than they did before fertilization. Thus, the appearnce of the small granules observed in the present study absorbs much interest in connexion with the report of Runnstrom and co-workers.. When activated, there is little to be discriminated between the surface of the ferdlized egg and that of the unfertilized one. but the former is, if anything, irregularly waved at. its plasm surface. In the stage of formation of the blastodisc, the aggregations of granulous yolk appear beneath the blastodisc. And the aggregations contain deoxyribonucleoside on account of the examinations discussed already. The ketoenol granules found through the blastodisc are positive to Feulgen reaction. They are numerous in the preparations treated with D fixative. These granules contain deoxyribonucleoside. The ketoenol secreting vacua les are situated not only beneath the peripheral plasm but also beneath the concentrating region of plasm, i. e. apical plasm, proir to formation of the blastodisc. When the blastodisc is formed the vacuoles become unrecognizable beneath the blastodisc. As a result, the activation in water causes the following changes: disappearance of cortical alveoli, appearance of small grannies found in cortex, formation of blastodisc, disappearance of ketoenol secreting vacuoles beneath blastodisc. — 24 ~.