Fish (Leiognathus argenteum).

Transcription

1 Cytochemical Studies on Polysaccharide and Protein of Ovarian Egg-Cells of an Osseous Fish (Leiognathus argenteum). By Mono Ihnuma and Tomi Tsukuda (Department of Anatomy, School of Medicine, Keio-Gijuku University, Tokyo) The developing ovarian egg-cells have been studied and numerous reports have been published on them since long ago. When egg-cells grow and increase in size, their inner structure also change. In eggcells of an osseous fish, Leiognathus argenteum, which were used in my study, remarkable changes in nucleoli (Ito, 1938), in mitochondria (Ihnuma, unpublished) and in ribonucleic acid (Ihnu ma, unpublished) were also observed. In this paper the authors report the results of their cytochemical studies on polysaccharide and protein. Material and Methods Ovaries of an osseous fish (Leiognathus argenteum) were fixed with Chamy's solution or Bouin's solution. Paraffin sections were made. Polysaccharide was demonstrated by the periodic acid Schiff's reagent method (H otc hkiss, 1946) and protein with ninh'drin Schiff's reagent procedure (Yasuma and ichik awa, 1951). The principle of the latter reaction was explained as follows : /\ c R CH COOH--o\/oliR C COOH C NH2 CM OH \/1 00/\OH NH.CO/ R C COOH It NH +H20 R CHO NH3+CO2 R CHO and Schiff's reagent produce a purple red coloration, and this color indicates the localization of amino acid. The procedure of this method is as follows : 1. Fixation : absolute alcohol, Schaudin's solution or Zenker-acetic acid solution etc.

2 50 M. Ihnuma and T. Tsukuda 2. Deparaffinated slides into the OA % solution of ninhydrin in absolute alcohol for 2 to 3 hoursat 37 C. 3. Rinse in running water one minute. 4. Schiff's reagent for 30 minutes. 5. Wash well with three changes of the sodium bisulfite solution, two minutes for each baths. 6. Wash well with water. 7. Dehydrate, clear, and mount in balsam. The purple color, which is produced by the reaction between ninhydrin and protein, does not mean the localization of protein itself. By this method, however, the protein becomes R CHO, and therefore the Schiff's reagent can be colored by this aldehyde, showing the presence of protein. The collection and the fixation of the material were done by Prof. T. Ito of Gunma University. The authors express their. heartiest thanks for his kind help. Observations Polysaccharide : In egg-cells of the stage ca. 30/4, (Fig. 1), the nucleolus is spherical in shape and only one in number. The zona pellucida is the most deeply stained : the cytoplasm, the nucleolus are lightly stained, and the nucleus except the nucleolus is hardly stained. The cytoplasm is stained uniformly. In egg-cells of the stage ca 100p, (Fig. 2), the zona pellucida becomes thicker, the number of nucleoli increases, but the stainability of the cell elements is quite similar to that of the 30/h egg-cells. In the Bouin-fixed sections the cytoplasm includes irregular, empty and clear spherical portions which remain unstained ; whereas in the Champy-fixed sections these portions are stained black. These spheres, therefore, are fat droplets, which look clear and empty when dissolved away. In egg-cells of the stage ca 150p (Fig. 3), fat droplets increase in number and become larger in size. In the peripheral parts of the cytoplasm, there appear deeply stained red granules. These granules are considered as yolk granules in an early stage of development. After the treatment of the sections with diastase, some of these granules weaken in their stainability by theperiodic acid Schiff's reagent techni- que. This shows that some of these yolk granules contain glycogen.

3 Cytochemical Studies on Polysaccharide and Protein of Ovarian Egg-Cells. 51 In egg-cells of the stage ca 200p, (Fig. 4), the yolk granules and the fat droplets increase in number and become larger, and occupy the largest part of the cytoplasm. The larger the size of yolk granules, the weaker the coloration, In the peripheral region of the cytoplasm in the Bouin-fixed eggcells of the stage ca 300k (Fig. 5), there are parts which are also deeply. stained. In the Champy-fixed material, such deep coloration of cytoplasm is not seen. In the Bouin-fixed sections after treatment with diastase, application of the periodic acid Schiff's reagent method can not produce a distinct color. Therefore this strong coloration probably indicates the presence of glycogen. The coloration of large yolk granules with periodic acid Schiff's method shows no difference before and after treatment with diastase. The zona pellucida of the egg-cells of this stage is considerably thick, and shows no thionin-metachromasy (I hnu ma, unpublished). According to Monne and Slauterbac k (1950), in the Bouin-fixed material almost all of the hyaluronic acid is lost. In fact, in the Bouin-fixed material the coloration of zona pellucida with the periodic acid Schiff's reagent technique shows no difference before and after treatment with hyalurodidase. From these results it is concluded that the coloration of the 'Bouin-fixed zona pellucida i caused by a polysaccharide other than hyaluronic acid or sulfated mucopolysaccharide. By means of the periodic acid 2, 4-dinitrophenylhydrazine Schiff's reagent technique by Monne and Slauterbac k (1950), the coloration of the egg-cells become weaker as a whole, but the enlarged yolk granules stain yellowish. In other words the larger yolk granules combine easily with 2, 4-dinitrophenylhydrazine. This fact shows, according to Monne and Slau t e rback, that the larger yolk granules contain aminopolysaccharide. Protein : In egg-cells of the stage ca 30/4, (Fig. 6), the color of the nucleous produced by the protein reaction is more intense than the color produced by the polysaccharide reaction : the cytoplasm is stained homogeneously, and the nucleus except the nucleolus is also stained as deeply as the cytoplasm, though it is hardly stained by the polysacharide reaction. The unstained portion within the nucleus is an artificial product. In egg-cells of the stage ca 150p, (Fig. 8), a number of fat droplets have appeared already. In egg-cells of this stage small yolk granules begin to be demonstrated by the polysaccharide reaction in the peri-

4 52 M. Ihnuma and T. Tsukuda pher. al parts of the cytoplasm as descrived above. On the other hand, however, the protein reaction can not demonstrate these granules. This result means that in an early stage of development the yolk granules contain much polysaccharide. In egg-cells of the stage ca 200p. (Fig. 9), yolk granules become larger and increase in number, and at the same time they are more deeply colored by the ninhydrin Schiff's reagent procedure than the cytoplasm. The yolk granules in an early stage, which exist in the peripheral region of the cytoplasm, can not be demonstrated by the protein reaction. In egg-cells of the stage ca 300p. (Fig. 10), yolk granules are much larger and the coloration by the protein reaction is deeper, even more intense than the coloration by the polysacchrride reaction. It is to be concluded that the larger the yolk granules become the more the protein content of the granules increases. From these results of our observations it is conceived that in an early stage of development the yolk granules of egg-cells are composed mainly of low-moleculed polysaccharides, and that in an advanced stage of development, on the other hand, they are composed of highmoleculed polysaccharides, and at the same time their protein content increases. As the zona pellucida shows a weak protein reaction, therefore it contains polysaccharide, which shows no metachromasy with thionin, and protein. Summary 1. The change of polysaccharide and protein of developing eggcells of an osseous fish (Leiognathus argenteum) was observed cytochemically. 2. Zona pellucida contains much polysaccharide, and has no relation to the progress of the stage of development'. 3. A mature egg-cell contains glycogen within cytoplasm. 4. The protein content is extremely high in nucleoli, and high also in yolk granules. 5. In an early stage of development yolk granules contain a lot of low-moleculed polysaccharides; and in an advanced stage they contain at the same time highly polymerized polysaccharides and much protein.

5 CYTOCHEMICAL STUDIES ON POLYSACCHARIDE AND PROTEIN PLATE I Fig. 1 Fig. 2 Fig. 3 Fig v Fig. 5

6 CYTOCHEMICAL STUDIES ON POLYSACCHARIDE AND PROTEIN PLATE II Fig. 6 Fig. 7 Fig. 8 Fig i Fig. 10

7 Cytochemical Studies on Polysaccharide and Protein of Ovarian Egg-Cells. 53 References Hotchkiss, R. D from Glick, D Techniques of Histo-and Cytochemistry. Ito, T Ober die Formver5nderung der Randnukleolen der wachsenden Oozyten bei einem Knochenfisch mit besonderer Beriicksichtingung auf die Frage iiber den Austritt der Nucleolarsubstanz ins Zytoplasma. Cytologia 9:283. Monné, L. and D. B. SI au terback 1950 Differential Staining of various Polysaccharides in Sea Urchin Eggs. Exp. Cell Res. 1: 477. Y a sum a, A. and T. Ichik a w a 1951 A new cytochemical staining of protein. Medicine and Biology 19: 197. Explanation of Plates Fig. 1-5 Bouin's solution fixation, periodic acid Schiff's reagent method of Hotchkiss Fig Bouin's solution fixation, ninhydrin Schiff's procedure of Yasu to a and Ichikawa