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Cytochemical studies on the oogenesis of Dytiscus marginalis L
Experimental
CYTOCHEMICAL
Cell Research 55 (1969) 243-247
STUDIES D YTISCUS
ON THE
MARGINALIS
OOGENESIS
OF
L
A. FICQ and E. URBANI Laboratoire de Morphologic animale, UniversitC Iibre de Bruxeiles, Belgium, and Istituto di Istologia ed Embriologia, Facoltd di Scienze, Universitd di Roma, Italy
SUMMARY In a brief description of the oogenesis of Dytiscus marginalis, attention is focused on the role and the significance of the Giardina body and the nurse cells, in this organism. A staining technique, with sH-actinomycin followed by autoradiography, very sensitive for detecting acid-labile DNA has confirmed, with more details, the observations made after Feulgen staining: 1. During the early stages, the Giardina body and the nuclei of the nurse and the follicle cells, bind SH-actinomycin intensely. 2. At more advanced stages, the nuclei of the nurse and of the follicle cells remain heavily labelled by 3H-actinomycin. Although these structures are the only ones to be Feulgen positive at that stage, after staining with *H-actinomycin, the germinal vesicle of the oocyte is also labelled, and more weakly, the ooplasma. 3. Feulgen positive granules are visible in the cytoplasm, surrounding the nuclear membrane. They are radioactive after staining with *H-actinomycin and electron microscopy suggests a passage of nuclear material through the nuclear membrane. These results are discussed in the light of our present knowledge on the role of extra chromosomal DNA and of cytoplasmic DNA.
During the oogonial mitoses which occur in the terminal chamber of Dytiscus marginalis ovarioles, a peculiar structure appears in the nucleus and remains in 1 of the 2 daughter cells only. This first differential division is followed by a second, a third and a fourth one; but that structure always remains in the same cell: the future oocyte; the other 15 cells become the nurse cells. This observation was first made by Giardina [18] and the extrachromosomal body has been called the Giardina body [2]. These observations have been confirmed by many authors [3, 11, 19, 21,231. The Giardina body may, therefore, be considered as a kind of germinal determinant which distinguishes the oocyte from the nurse cells [31]; the follicle cells which surround the oocyte and the nurse cells have a different origin than the oogonia. When it appears, during the differential mito-
ses and in the small oocytes, the Giardina body is Feulgen positive: it can be stained by methyl green; after staining with acridine orange, it shows a green yellowish fluorescence and it absorbs in the UV at 255 m,u. It presents a slight affinity for pyronin [27, 28, 291. It incorporates selectively 3H-thymidine. All these data demonstrate that this extrachromosomal structure contains DNA 1301. In the growing oocyte, the Giardina body undergoes radical morphological and chemical transformations: it loses its spherical form and, progressively, becomes pyroninophilic. It no longer incorporates radioactive thymidine, but, instead, uridine and cytidine. When the Giardina body disintegrates, before vitellogenesis, the germinal vesicle of the oocyte becomes completely Feulgen negative, but remains basophilic; at the same time, the nuclei of the follicle and the nurse Exptl Cell Res 55
244
A. Ficq & E. Urbani
cells remain Feulgen positive [30]. Nurse cells and oocytes are growing simultaneously; channels can be seen between nurse cells and oocytes; they might constitute means for transferring RNA between these cells [2]. Although cytochemical and autoradiography techniques do not demonstrate such a passage of RNA of DNA from nurse cells to the oocyte, electron microscopy has shown a flow of mitochondria passing from the cytoplasm of the nurse cells to the ooplasm, where they undergo deep transformations [26]. Feulgen positive granules are visible in the cytoplasm of the nurse cells; they appear, under the electron microscope, as surrounded by mitochondria [l, 261. These observations are in good agreement with those demonstrating the existence of cytoplasmic DNA in oocytes [4, 5,9, 13, 14, 221. A staining technique with radioactive actinomycin, followed by autoradiography, first described by Brachet & Ficq [6, 71 and later used by several authors [8, 12, 251 has been found more sensitive than the Feulgen reaction for the detection of DNA. In the present work, this technique has been applied to Dytiscus ovaries, in order to study the Giardina body and the nurse cells.
MATERIAL
AND
TECHNIQUES
The above described phenomena are easily visible in Dytiscus marginalis during the months of Sept. till Dec., when oogenesis starts. The ovaries were dissected and fixed in acetic alcohol (1: 3) embedded in paraffin and sectioned at 7-10 u thickness. The Feulaen reaction was performed in the classical way except thit the hydrolysis with 1 N HCl took nlace during 12-24 h at room temoerature [20, 301. The sections prepared for autoradiography have been treated with 3H-actinomycin (Schwarz, Spec. act. 2.97 c/mM at the concentration of 10 PC/ml in distilled water, during 30-60 min; this was followed by a bath in cold actinomycin (10 fig/ml) and washing for 20 h in tap water. The usual autoradiography technique was applied, using Ilford emulsion L4 in gel form 1151. The preparations were developed 5 to 10 days later. Before mounting, the sections were stained with light green or Unna. Controls were made with DNAse (Worthington Bioch. Corp.) 2 mg/ml during 1 h, at 37°C and pH 6.8. The pretreatment of the glass with cold actinomycin, suggested by Ebstein [12] and designed to reduce the background grains, was not applied since the background of our preparations was very low. Exptl
Cell
Res 55
RESULTS The results obtained on the Dytiscus ovaries are illustrated by the micrographs (figs 1 to 12). In the terminal chamber (fig. 1) three oocytes contain cap-like Giardina bodies (G) which are Feulgen positive. The nuclei of the nurse cells and the follicle cells which do not yet present the oriented aspect, characteristic of more advanced stagesof oogenesis, are also Feulgen positive. After 3H-actinomycin staining of the same region of the ovariole (fig. 2), 8 oocytes contain heavily labelled Giardina body (the arrows indicate this structure, which may be cap-like or spherical). The nuclei of the nurse cells or the follicle cells are also labelled; the localizations of DNA are identical with those observed after Feulgen staining. Figs 3 and 4 show the same thing, but at a higher magnification. More advanced stages of oogenesis are illustrated in figs 5 and 6, in which groups of nurse cells (N) and one oocyte (0) surrounded by follicle cells, are visible. The nuclei of the nurse cells and the follicle cells are heavily labelled by 3H-actinomycin. But, at that stage of development, at which the Giardina body disintegrates and disappears, only the nuclei of the nurse cells and of the follicleVcells are Feulgen positive; the germinal vesicle of the oocyte is Feulgen negative and pyroninophilic. The examination, at higher magnification of oocytes at that previtellogenetic stage shows that only the nuclei of the follicle cells are Feulgen positive (fig. 7F); but 3H-actinomycin stains the germinal vesicle, and, more weakly, the ooplasm (fig. 8). The nurse cells, which grow simultaneously with the oocyte, always contain a strongly Feulgen positive nucleus. The labelling with 3H-actinomycin is intense in the nucleus; some labelled granules can well be seenin the cytoplasm (fig. 9). Sometimes the cytoplasmic labelling is rather uniform around the nucleus; sometimes, it is strong in one region and nearly absent in another (arrow, fig. 10). The Feulgen reaction clearly shows the presencein the cytoplasm of the granules (fig. 11) which bind labelled actinomycin.
The oogenesis of Dytiscus
marginalis L
245
Fig. 1. Three oocytes containing the Giardina body (G) stained by Feulgen reaction. In each figure, the interval between- two black lines represents 10 p. Fig. 2. Autoradiograph of the same region of the ovariole as fig. 1. The Giardina bodies have bound BH-actinomycin. Fig. 3. As fig. 1. Higher magnification. Fig. 4. As fig. 2. Higher magnification. Figs 5 and6 Groups of nurse cells (N) and oocyte (0) surrounded by follicle cells (F). Nuclei heavily labclled with ‘H-actinomycin. At that stage, the Giardina body is disintegrated.
The preparations treated by DNAse, no longer show a binding with actinomycin. The ultrastructure of the nuclear membrane of the nurse cells has been studied by electron microscopy [1, 261: fig. 12 shows the passage of nuclear material into the cytoplasm, through the nuclear membrane. Moreover, in the cytoplasm, one can observe structures having the same density as chromatin; these structures are in contact with mitochondria which, often, surround them. DISCUSSION The results obtained by the staining technique with 3H-actinomycin require some comments. Regarding the Giardina body, they confirm that, 16 - 691819
in the initial stages of oogenesis, this structure contains large amounts of DNA: the autoradiographs are quite similar to the images obtained after the Feulgen reaction. When the Giardina body disintegrates, it is no longer Feulgen positive but it becomes pyroninophilic; at that time, incorporation of 3H-thymidine in the germinal vesicle can no longer be detected [30]. The binding of 3H-actinomycin, however, reveals the presence of DNA in the oocyte nucleus and, in lower concentration, in its cytoplasm. As was pointed out earlier [6, 71 this method is more sensitive than the Feulgen technique for detecting small amounts of DNA and preserves better acid labile DNA. Our present observations confirm previous reExpti Cell Res 55
246
A. Ficq & E. Urbani
i
ports about the existence in oocytes of different animal species, of cytoplasmic DNA, at least during certain stages of oogenesis. In the large oocytes of Dytiscus, during vitellogenesis, it is difficult to detect the presence of cytoplasmic DNA. Regarding the nurse cells, one may point out that “H-actinomycin is equally bound by all the nuclei (figs 5, 6); this is not the case after injection of 3H-thymidine which only labels the nuclei preparing for division. The possible transfer of DNA from the nucleus to the cytoplasm previously studied by the Feulgen method and the use of radioactive thymidine, seems particularly clear with the technique utilized in the present work [6, 71. Exptl Cell Res 55
Fig. 7. Oocyte in previtellogenesis: Feulgen staining. Fig. 8. As fig. 7 after labelling with 3H-actinomycin. Figs 9 and IO. Nurse cells having bound 3H-actinomycm. Nuclei heavily labelled. Radioactive granules in the cytoplasm. Fig. II. Nurse cell after Feulgen reaction (higher magnification). Feulgen positive granules in the cytoplasm. Fig. 12. Ultrastructure of the --.-_--I, -.--1--.-. ULubt: cc41 uuClcUs; n-----ratsigtz of nuclear material from the nucleus (up) towards the cytoplasm (down) through the nuclear membrane.
In the case of the extrachromosomal DNA of the Giardina body and in that of the cytoplasmic DNA present in the oocyte and the nurse cells, the question is if a distinction between genetic and metabolic DNA arises; one also wonders about the role and significance of the cytoplasmic DNA. Several papers have, recently, focused the attention on extra DNA appearing in early oogenesis and linked to the nucleolar organizer [IO, 17, 241. According to their authors, this DNA might represent extra copies of the genes coding for ribosomal RNA. One of us (AF) has, on the other hand, provided the autoradiographic evidence of a DNA synthesis in very young oocytes
The oogenesis of Dytiscus
of Xenopus laevis: the radioactivity due to % thymidine incorporation is condensed around the nucleoli, at this early stage of oocyte growth
F61. Perhaps, Dytiscus marginalis, even if its oogenesis still presents some obscure points, could provide a suitable material for an approach to these important problems. E. Urbani wishes to express his gratitude to the Consiglio Nazionale delle Ricerche and to the Shell International Research, for subsidizing his work at the University of Brussels. This work was done under U.L.B. 007-61-ABIB.
Euratom
contract
no.
REFERENCES 1. Bertolini, B & Urbani, E, Rend accad naz Lincei 36 (1964) 240. 2. Bier, K, Kunz, W & Ribbert, D, Chromosoma 23 (1967) 214. Bier, K & Ribbert, D, Naturwiss 53 (1966) 115. :: Brachet, J, Compt rend acad sci Paris 261 (1965) 1092.
- Protoplasma 63 (1967) 86. 6. Brachet, J & Ficq, A, Compt rend acad sci Paris 258 (1964) 6258. 7. - Exptl cell res 38 (1965) 153. 5.
marginalis L
247
$. &%ch&, J L Preumont, A M, Ann histochem 11 (1966) 157. 9. Brachet. J & Ouertier. J. Exntl cell rer 32 (1964) 410. :$ Brown, ‘D D & Dawid,‘I B; Science 160 (1968) 272. Debaisieux, P, La cellule 25 (1909) 207. 12: Ebstein, B S, Exptl cell res 35 (1968) 709. 13. Favard-SerBno, C & Durand, M, Develop biol 6 (1963) 184. 14. - Ibid. 6 (1963) 206. 15. Ficq, A, IISN monograph 9. Brussels (1961). 16. - Exptl cell res 53 (1968) 691. 17. Gall, J G, Proc natl acad sci 60 (1968) 553. 18. Giardina, A, Intern monatschr anat Physiol 18 (1901) 417. 19. Jacquiert, A, Bull histol appl 12 (1935) 281. z Jordanov. J. Acta histochem 15 (1963) 135. Lima-de-Faria, A & Moses, M T,\J cell biol30 (1966) 177. 22. Muckenthaler, F A & Mahowald, A P, J cell biol 28 (1966) 199. m3 Nusbaum-Hilarowicz, J, Z wiss Zoo1 117 (1917) 554. LJ. 24. Perkowska, E, Mac Gregor, H C & Bimstiel, M L, Nature 217 (1968) 649. 2.5. Plessman Camargo, E & Plaut, W, Exptl cell res 35 (1968) 713. 26. Steinert, G & Urbani, E, Proc IV Europ reg congr electron microscopy (Sept 1968). 27. Urbani, E, Rend accad naz Lincei 8 (1950) 153. 28. - Ibid 8 (1950) 504. 29. - Riv bio142 (1950) 413. 30. Urbani E & Russo Caia, S, Rend ist sci univ Camerino 5 (1964) 19. 31. Wilson, A B, The cell in development and heredity. Macmillan, New York (1925). Received October 10, 1968
Exptl
Cell
Res 55
CYTOCHEMICAL
Cell Research 55 (1969) 243-247
STUDIES D YTISCUS
ON THE
MARGINALIS
OOGENESIS
OF
L
A. FICQ and E. URBANI Laboratoire de Morphologic animale, UniversitC Iibre de Bruxeiles, Belgium, and Istituto di Istologia ed Embriologia, Facoltd di Scienze, Universitd di Roma, Italy
SUMMARY In a brief description of the oogenesis of Dytiscus marginalis, attention is focused on the role and the significance of the Giardina body and the nurse cells, in this organism. A staining technique, with sH-actinomycin followed by autoradiography, very sensitive for detecting acid-labile DNA has confirmed, with more details, the observations made after Feulgen staining: 1. During the early stages, the Giardina body and the nuclei of the nurse and the follicle cells, bind SH-actinomycin intensely. 2. At more advanced stages, the nuclei of the nurse and of the follicle cells remain heavily labelled by 3H-actinomycin. Although these structures are the only ones to be Feulgen positive at that stage, after staining with *H-actinomycin, the germinal vesicle of the oocyte is also labelled, and more weakly, the ooplasma. 3. Feulgen positive granules are visible in the cytoplasm, surrounding the nuclear membrane. They are radioactive after staining with *H-actinomycin and electron microscopy suggests a passage of nuclear material through the nuclear membrane. These results are discussed in the light of our present knowledge on the role of extra chromosomal DNA and of cytoplasmic DNA.
During the oogonial mitoses which occur in the terminal chamber of Dytiscus marginalis ovarioles, a peculiar structure appears in the nucleus and remains in 1 of the 2 daughter cells only. This first differential division is followed by a second, a third and a fourth one; but that structure always remains in the same cell: the future oocyte; the other 15 cells become the nurse cells. This observation was first made by Giardina [18] and the extrachromosomal body has been called the Giardina body [2]. These observations have been confirmed by many authors [3, 11, 19, 21,231. The Giardina body may, therefore, be considered as a kind of germinal determinant which distinguishes the oocyte from the nurse cells [31]; the follicle cells which surround the oocyte and the nurse cells have a different origin than the oogonia. When it appears, during the differential mito-
ses and in the small oocytes, the Giardina body is Feulgen positive: it can be stained by methyl green; after staining with acridine orange, it shows a green yellowish fluorescence and it absorbs in the UV at 255 m,u. It presents a slight affinity for pyronin [27, 28, 291. It incorporates selectively 3H-thymidine. All these data demonstrate that this extrachromosomal structure contains DNA 1301. In the growing oocyte, the Giardina body undergoes radical morphological and chemical transformations: it loses its spherical form and, progressively, becomes pyroninophilic. It no longer incorporates radioactive thymidine, but, instead, uridine and cytidine. When the Giardina body disintegrates, before vitellogenesis, the germinal vesicle of the oocyte becomes completely Feulgen negative, but remains basophilic; at the same time, the nuclei of the follicle and the nurse Exptl Cell Res 55
244
A. Ficq & E. Urbani
cells remain Feulgen positive [30]. Nurse cells and oocytes are growing simultaneously; channels can be seen between nurse cells and oocytes; they might constitute means for transferring RNA between these cells [2]. Although cytochemical and autoradiography techniques do not demonstrate such a passage of RNA of DNA from nurse cells to the oocyte, electron microscopy has shown a flow of mitochondria passing from the cytoplasm of the nurse cells to the ooplasm, where they undergo deep transformations [26]. Feulgen positive granules are visible in the cytoplasm of the nurse cells; they appear, under the electron microscope, as surrounded by mitochondria [l, 261. These observations are in good agreement with those demonstrating the existence of cytoplasmic DNA in oocytes [4, 5,9, 13, 14, 221. A staining technique with radioactive actinomycin, followed by autoradiography, first described by Brachet & Ficq [6, 71 and later used by several authors [8, 12, 251 has been found more sensitive than the Feulgen reaction for the detection of DNA. In the present work, this technique has been applied to Dytiscus ovaries, in order to study the Giardina body and the nurse cells.
MATERIAL
AND
TECHNIQUES
The above described phenomena are easily visible in Dytiscus marginalis during the months of Sept. till Dec., when oogenesis starts. The ovaries were dissected and fixed in acetic alcohol (1: 3) embedded in paraffin and sectioned at 7-10 u thickness. The Feulaen reaction was performed in the classical way except thit the hydrolysis with 1 N HCl took nlace during 12-24 h at room temoerature [20, 301. The sections prepared for autoradiography have been treated with 3H-actinomycin (Schwarz, Spec. act. 2.97 c/mM at the concentration of 10 PC/ml in distilled water, during 30-60 min; this was followed by a bath in cold actinomycin (10 fig/ml) and washing for 20 h in tap water. The usual autoradiography technique was applied, using Ilford emulsion L4 in gel form 1151. The preparations were developed 5 to 10 days later. Before mounting, the sections were stained with light green or Unna. Controls were made with DNAse (Worthington Bioch. Corp.) 2 mg/ml during 1 h, at 37°C and pH 6.8. The pretreatment of the glass with cold actinomycin, suggested by Ebstein [12] and designed to reduce the background grains, was not applied since the background of our preparations was very low. Exptl
Cell
Res 55
RESULTS The results obtained on the Dytiscus ovaries are illustrated by the micrographs (figs 1 to 12). In the terminal chamber (fig. 1) three oocytes contain cap-like Giardina bodies (G) which are Feulgen positive. The nuclei of the nurse cells and the follicle cells which do not yet present the oriented aspect, characteristic of more advanced stagesof oogenesis, are also Feulgen positive. After 3H-actinomycin staining of the same region of the ovariole (fig. 2), 8 oocytes contain heavily labelled Giardina body (the arrows indicate this structure, which may be cap-like or spherical). The nuclei of the nurse cells or the follicle cells are also labelled; the localizations of DNA are identical with those observed after Feulgen staining. Figs 3 and 4 show the same thing, but at a higher magnification. More advanced stages of oogenesis are illustrated in figs 5 and 6, in which groups of nurse cells (N) and one oocyte (0) surrounded by follicle cells, are visible. The nuclei of the nurse cells and the follicle cells are heavily labelled by 3H-actinomycin. But, at that stage of development, at which the Giardina body disintegrates and disappears, only the nuclei of the nurse cells and of the follicleVcells are Feulgen positive; the germinal vesicle of the oocyte is Feulgen negative and pyroninophilic. The examination, at higher magnification of oocytes at that previtellogenetic stage shows that only the nuclei of the follicle cells are Feulgen positive (fig. 7F); but 3H-actinomycin stains the germinal vesicle, and, more weakly, the ooplasm (fig. 8). The nurse cells, which grow simultaneously with the oocyte, always contain a strongly Feulgen positive nucleus. The labelling with 3H-actinomycin is intense in the nucleus; some labelled granules can well be seenin the cytoplasm (fig. 9). Sometimes the cytoplasmic labelling is rather uniform around the nucleus; sometimes, it is strong in one region and nearly absent in another (arrow, fig. 10). The Feulgen reaction clearly shows the presencein the cytoplasm of the granules (fig. 11) which bind labelled actinomycin.
The oogenesis of Dytiscus
marginalis L
245
Fig. 1. Three oocytes containing the Giardina body (G) stained by Feulgen reaction. In each figure, the interval between- two black lines represents 10 p. Fig. 2. Autoradiograph of the same region of the ovariole as fig. 1. The Giardina bodies have bound BH-actinomycin. Fig. 3. As fig. 1. Higher magnification. Fig. 4. As fig. 2. Higher magnification. Figs 5 and6 Groups of nurse cells (N) and oocyte (0) surrounded by follicle cells (F). Nuclei heavily labclled with ‘H-actinomycin. At that stage, the Giardina body is disintegrated.
The preparations treated by DNAse, no longer show a binding with actinomycin. The ultrastructure of the nuclear membrane of the nurse cells has been studied by electron microscopy [1, 261: fig. 12 shows the passage of nuclear material into the cytoplasm, through the nuclear membrane. Moreover, in the cytoplasm, one can observe structures having the same density as chromatin; these structures are in contact with mitochondria which, often, surround them. DISCUSSION The results obtained by the staining technique with 3H-actinomycin require some comments. Regarding the Giardina body, they confirm that, 16 - 691819
in the initial stages of oogenesis, this structure contains large amounts of DNA: the autoradiographs are quite similar to the images obtained after the Feulgen reaction. When the Giardina body disintegrates, it is no longer Feulgen positive but it becomes pyroninophilic; at that time, incorporation of 3H-thymidine in the germinal vesicle can no longer be detected [30]. The binding of 3H-actinomycin, however, reveals the presence of DNA in the oocyte nucleus and, in lower concentration, in its cytoplasm. As was pointed out earlier [6, 71 this method is more sensitive than the Feulgen technique for detecting small amounts of DNA and preserves better acid labile DNA. Our present observations confirm previous reExpti Cell Res 55
246
A. Ficq & E. Urbani
i
ports about the existence in oocytes of different animal species, of cytoplasmic DNA, at least during certain stages of oogenesis. In the large oocytes of Dytiscus, during vitellogenesis, it is difficult to detect the presence of cytoplasmic DNA. Regarding the nurse cells, one may point out that “H-actinomycin is equally bound by all the nuclei (figs 5, 6); this is not the case after injection of 3H-thymidine which only labels the nuclei preparing for division. The possible transfer of DNA from the nucleus to the cytoplasm previously studied by the Feulgen method and the use of radioactive thymidine, seems particularly clear with the technique utilized in the present work [6, 71. Exptl Cell Res 55
Fig. 7. Oocyte in previtellogenesis: Feulgen staining. Fig. 8. As fig. 7 after labelling with 3H-actinomycin. Figs 9 and IO. Nurse cells having bound 3H-actinomycm. Nuclei heavily labelled. Radioactive granules in the cytoplasm. Fig. II. Nurse cell after Feulgen reaction (higher magnification). Feulgen positive granules in the cytoplasm. Fig. 12. Ultrastructure of the --.-_--I, -.--1--.-. ULubt: cc41 uuClcUs; n-----ratsigtz of nuclear material from the nucleus (up) towards the cytoplasm (down) through the nuclear membrane.
In the case of the extrachromosomal DNA of the Giardina body and in that of the cytoplasmic DNA present in the oocyte and the nurse cells, the question is if a distinction between genetic and metabolic DNA arises; one also wonders about the role and significance of the cytoplasmic DNA. Several papers have, recently, focused the attention on extra DNA appearing in early oogenesis and linked to the nucleolar organizer [IO, 17, 241. According to their authors, this DNA might represent extra copies of the genes coding for ribosomal RNA. One of us (AF) has, on the other hand, provided the autoradiographic evidence of a DNA synthesis in very young oocytes
The oogenesis of Dytiscus
of Xenopus laevis: the radioactivity due to % thymidine incorporation is condensed around the nucleoli, at this early stage of oocyte growth
F61. Perhaps, Dytiscus marginalis, even if its oogenesis still presents some obscure points, could provide a suitable material for an approach to these important problems. E. Urbani wishes to express his gratitude to the Consiglio Nazionale delle Ricerche and to the Shell International Research, for subsidizing his work at the University of Brussels. This work was done under U.L.B. 007-61-ABIB.
Euratom
contract
no.
REFERENCES 1. Bertolini, B & Urbani, E, Rend accad naz Lincei 36 (1964) 240. 2. Bier, K, Kunz, W & Ribbert, D, Chromosoma 23 (1967) 214. Bier, K & Ribbert, D, Naturwiss 53 (1966) 115. :: Brachet, J, Compt rend acad sci Paris 261 (1965) 1092.
- Protoplasma 63 (1967) 86. 6. Brachet, J & Ficq, A, Compt rend acad sci Paris 258 (1964) 6258. 7. - Exptl cell res 38 (1965) 153. 5.
marginalis L
247
$. &%ch&, J L Preumont, A M, Ann histochem 11 (1966) 157. 9. Brachet. J & Ouertier. J. Exntl cell rer 32 (1964) 410. :$ Brown, ‘D D & Dawid,‘I B; Science 160 (1968) 272. Debaisieux, P, La cellule 25 (1909) 207. 12: Ebstein, B S, Exptl cell res 35 (1968) 709. 13. Favard-SerBno, C & Durand, M, Develop biol 6 (1963) 184. 14. - Ibid. 6 (1963) 206. 15. Ficq, A, IISN monograph 9. Brussels (1961). 16. - Exptl cell res 53 (1968) 691. 17. Gall, J G, Proc natl acad sci 60 (1968) 553. 18. Giardina, A, Intern monatschr anat Physiol 18 (1901) 417. 19. Jacquiert, A, Bull histol appl 12 (1935) 281. z Jordanov. J. Acta histochem 15 (1963) 135. Lima-de-Faria, A & Moses, M T,\J cell biol30 (1966) 177. 22. Muckenthaler, F A & Mahowald, A P, J cell biol 28 (1966) 199. m3 Nusbaum-Hilarowicz, J, Z wiss Zoo1 117 (1917) 554. LJ. 24. Perkowska, E, Mac Gregor, H C & Bimstiel, M L, Nature 217 (1968) 649. 2.5. Plessman Camargo, E & Plaut, W, Exptl cell res 35 (1968) 713. 26. Steinert, G & Urbani, E, Proc IV Europ reg congr electron microscopy (Sept 1968). 27. Urbani, E, Rend accad naz Lincei 8 (1950) 153. 28. - Ibid 8 (1950) 504. 29. - Riv bio142 (1950) 413. 30. Urbani E & Russo Caia, S, Rend ist sci univ Camerino 5 (1964) 19. 31. Wilson, A B, The cell in development and heredity. Macmillan, New York (1925). Received October 10, 1968
Exptl
Cell
Res 55