Local accumulation of Feulgen-positive granules in the egg cortex of Dentalium dentale L.

0 Authors' address: Zoological Laboratory , Janskerkhof 3, Utrecht , The Netherlands. 16-2 1 From the Zoological Laboratory, University of Utrecht and the Stazione Zoologica at Naples A special feature of eggs of several species of annelids and molluscs is the formation of a polar lobe at stage-specific intervals during the cleavage phase. In the scaphopod Dentalium, a polar lobe develops at the first, the second and the third cleavage (Wilson, 1904). The cytoplasm set apart in the polar lobes is ultimately confined to the D blastomere and its derivative cells. Wilson observed that after removal of the polar lobe at the first cleavage a larva develops which lacks an apical tuft and the post-trochal region. After removal of the polar lobe at the second cleavage a larva develops which lacks most of the post-trochal region but possesses an apical tuft. A similar result to the one following the removal of the first polar lobe is obtained by removal of the vegetal one-third of an unfertilized egg. These results indicate that morphogenetic determinants present in the first polar lobe are already set aside in the uncleaved and unfertilized eggs, and that the determinants for the apical tuft are no longer present in the second polar lobe. The study of maturation in amphibian oocytes has shown that a relationship exists between nuclear DNA and Feulgen-staining granules appearing in the cortex after breakdown of the germinal vesicle (Brchet 1965, 1967). As Brchet pointed out this cortical localization may be significant with respect to the importance of the dorsal cortex (grey crescent) in later development. Therefore, we have studied the distribution of DNA in the egg cells of Dentalium as far as it can be traced with the Feulgen method. - The experiments were carried out in May and June 1968, at the Zoological Station in Naples with eggs of Dentalium dentale L. The animals were kept in running sea water on a layer of sand. From the beginning of June spontaneously released eggs were obtained daily from animals brought in from the bay as well as from animals kept in the laboratory. Immediately after deposition the eggs 246 L. P . M. T I M M E R M A N S A N D O T H E R S are ellipsoidal but within 4-6 min they become spherical and can be fertilized. Eggs may be obtained artificially by breaking the shell and opening the ovaries. These eggs, however, take 15-20 min before they become rounded. The eggs were cultured at 22 C in Boveri dishes in filtered and boiled sea water. Fixa tions were made at 5 min intervals between oviposition and third cleavage. For cytological and histochemical observations the eggs were fixed in Zenker's fluid, sectioned, and stained either with Heidenhain's haematoxylin or with methyl green-pyronin. For the detection of DNA, whole eggs were fixed and stained with a Feulgen procedure. The eggs were fixed in a mixture of 80 % ethanol, 40 % formalin and acetic acid (85 cm + 5 cm3 +10 cm3) and after 60 min transferred to 96 % ethanol. 3 After hydrolysis in 5 N-HC1 at 25 C for 30 min, the eggs were stained with Feulgen's reagent for 60 min and mounted. Control eggs were treated for 12 and 24 h with DN-ase (Worthington, 0-2 mg/ml) at 37 C in tris buffer pH 7-4, to which 0003 M-MgS04 was added. Together with each DN-ase treatment a group of eggs of a comparable stage was treated with Tris buffer pH 7-4 at the same temperature and for the same period. RESULTS AND OBSERVATIONS 1. Maturation of unfertilized eggs (a) Eggs obtained by artificial means. Right after deposition the eggs are ellipsoidal and contain a large centrally placed germinal vesicle with a large nucleolus (30-50 [i in diameter). At this stage ten chromosomes in the stage of diakinesis are visible (Fig. 1 A, B). The nucleolus (Fig. 2) consists of a pyro EXPLANATION OF FIGURES 1. Vegetal pole 2. Nuclei of follicle cells. 3. Chromosomes. 4. Germinal vesicle. 5. Nucleolus. 6. Aster. 7. Hair-like protrusions at vegetal pole. 8. Animal pole 9. Polar bodies. Fig. 1 A, B. Feulgen staining of whole egg, x 560. Egg artificially released, seen from the vegetal pole. The nuclei of the follicle cells and the chromosomes of the germinal vesicle are stained. No Feulgen-positive granules at the vegetal pole. Fig. 2. Meridional section, methyl green-pyronin, x 560. The same stage as Fig. J. Egg obtained by artificial means, ellipsoidal; germinal vesicle intact, nucleolus with pyroninophilic peripheral part and unstained centre, follicle cells at the vegetal pole. Fig. 3. Oblique section, methyl green-pyronin, x 560. Egg 20 min after artificial release. The nuclear membrane has disappeared, the nucleolus is disintegrating, the chromosomes are visible in the cytoplasm. Fig. 4. Meridional section, iron haematoxylin, x 900. Hair-like protrusions at the vegetal pole. Feulgen-positive granules in egg cortex L. P. M. TIMMERMANS AND OTHERS 11 % ninophilic peripheral part, rich in RNA, which may contain several 'drops' or vacuoles and an unstained centre. The eggs are covered at the vegetal side by an annular layer of follicle cells (Figs. 1 and 2). At about 10 to 15 min after release of the eggs, the germinal vesicle opens and the nuclear membrane disappears. The nucleoplasm, the chromosomes and the nucleolus mix with the cytoplasm (Fig. 3). The nucleolus still may consist of a pyroninophilic peripheral part and an unstained centre. Fifteen to twenty minutes after release of the eggs, the follicle cells are stripped off. At the same time the eggs become spherical and are surrounded by a jelly coat. The chromosomes move towards the animal pole, together with the maturation spindle which has become visible in the meantime (Fig. 3). The nucleolus is still visible in the cytoplasm, but has started to disintegrate. (b) Eggs released spontaneously. The eggs are ellipsoidal upon deposition, with the animal and vegetal poles located at the ends of the short axis. At the vegetal side a radial pattern is observed in which three or four dark bands of granules alternate with light bands (Fig. 5). Usually a germinal vesicle is no longer present and the follicle cells have already been stripped off. The chromo somes are condensed and are situated close to each other near the animal pole. The nucleolus is still present in the cytoplasm, but has started to disintegrate. Within 4-6 min after deposition the eggs become spherical. 2. Maturation divisions and cleavage The first polar body is formed within 30 min after fertilization, the second polar body 30 min later. After another 30 min first cleavage starts. Second and third cleavage follow also after intervals of 30 min. The first polar lobe is formed at the first cleavage, the second and third polar lobes are formed at the second and third cleavages. At the vegetal pole hair-like protrusions are observed from oviposition until after the second maturation division (Fig. 4). During the maturation divisions a cytoplasmic protrusion is formed at this point (Fig. (...truncated)