The effect of removing the polar lobe in centrifuged eggs of Dentalium

0 Author's address: Zoological Laboratory , Janskerkhof 3, Utrecht , The Netherlands. 3 J EEM 19 1 From the Zoological Laboratory, University of Utrecht, and the Caribbean Marine Biological Institute , Curacao Classical evidence for the existence of morphogenetic substances was provided by experiments with spiralian eggs possessing a polar lobe: Ilyanassa (Crampton, 1896; Clement, 1952, 1956, 1962); Dentalium (Wilson, 1904); Chaetopterus (Tyler, 1930); Sabellaria (Hatt, 1932; Novikoff, 1938); and Mytilus (Rattenbury & Berg, 1954). Eggs from which the polar lobe had been removed developed into embryos with specific abnormalities. In Dentalium, after removal of the polar lobe at the trefoil stage, a trochophore larva is formed without post-trochal region and apical tuft. Removal of the polar lobe at second cleavage causes a larva without post-trochal region, but with an apical tuft. Wilson concluded that specific cytoplasmic materials essential to the formation of the apical tuft are contained in the first but no longer in the second polar lobe. Centrifuging the uncleaved egg just before first cleavage will disturb the normal distribution of substances. The morphogenetic substance for the apical tuft may be also displaced, so that at the formation of the first polar lobe this substance is not only present in the lobe, but also in the blastomeres. The object of the present investigation was to study the effect of removing the first polar lobe in eggs centrifuged immediately before the beginning of first cleavage. - brownish grey oocytes are packed as coins. The testes are milky-white tubules. As a result, it is possible to determine the sex of the living animals with a rather high degree of certainty. Oocytes and sperm may be obtained by breaking the shell with two pairs of forceps and opening the ovary or testes with a sharp needle. Another method is to push up the animal in its shell with a blunt needle. It then releases oocytes or sperm through the small opening at the rear side. Both methods have their disadvantages as the oocytes are easily destroyed. Ripe oocytes are biscuit-shaped with a diameter of 175-200 p. They are olivecoloured with a large bright centre nearly devoid of pigment. In histological sections this appears to be the germinal vesicle. The oocytes are surrounded by a membrane with a wrinkled appearance (Text-fig. 1, no. 1). About 20 min after release from the ovary, the germinal vesicle suddenly breaks down in some of the oocytes, which become spherical. The original membrane ruptures and the remains become attached to a new membrane, which is elevated from the egg surface. In this manner a clear capsule is formed around the egg, which then becomes 160/t in diameter (Text-fig. 1, no. 2). The eggs appeared to be sensitive to polyspermy, which causes abnormal cleavage. In order to avoid polyspermy as much as possible the following method was used. The sperm, which just after opening of the testes are immobile and start moving only after about 20 minutes, were collected in a dish with sea-water. With a braking pipette a very small quantity of motile sperm was sucked up and disseminated over the eggs. In this way nearly all eggs were fertilized at the same moment and polyspermy was practically avoided. Eggs ofDentalium antillarum appeared to be rather sensitive to centrifugation. When exposed to a centrifugal force of 400 g for 10 min, the eggs did not cleave or cleaved abnormally. In the present experiments a centrifugal force of 300 g for 10 min was applied, which causes a good stratification of the egg substance. This treatment is certainly not harmless to the eggs, but as for our purpose the effect of centrifugation on the egg substances was to be as great as possible, the consequences had to be accepted. Furthermore, all eggs showing an abnormal first or second cleavage were eliminated. By using a sharp tungsten needle, the polar lobe can be removed easily at first or second cleavage. The eggs were cultured in filtered and boiled sea-water in dishes with a cover-glass in an air-conditioned room at about 25 C. Eggs fixed in Bouin's fluid were sectioned at 5 /i and stained with Heidenhain's haematoxylin or Herlant's tetrachrome staining. Embryos fixed with Kleinenberg's fixative were stained with Delafield's haematoxylin. They were mounted between slide and coverglass, supported by a piece of paper. During the formation of the first and second polar bodies (15 and 35 min after fertilization) no polar lobe forms in Dentalium. About 55 min after fertilization a polar lobe appears at the vegetative pole of the egg, marking the beginning of first cleavage (Text-fig. 1, no. 3). While the lobe rounds off, a cleavage furrow is formed at the animal side of the egg and a little later also at the vegetative side. Text-fig. 1.1, Ripe oocyte after release; 2, egg, after throwing off the original membrane and the formation of the capsule; 3 and 4, formation of the 1st polar lobe; 5, trefoil stage with polar lobe (1.1); 6, fusion of the polar lobe with the CD cell; 7, 2-cell stage; 8 and 9, formation of the 2nd polar lobe (1.2); 10, 4-cell stage seen from the vegetative pole; 11, formation of the 3rd polar lobe (1.3); 12, 8-cell stage seen from the vegetative pole. When the egg reaches the trefoil stage, no connexion between the lobe and the blastomeres can be observed (Text-fig. 1, no. 5). After 4-5 min. a connexion between the polar lobe and one of the blastomeres appears, and subsequently the lobe fuses with the CD blastomere (Text-fig. 1, nos. 6 and 7). About 30 min after the beginning of first cleavage, a second polar lobe is formed, which is well rounded with respect to the four blastomeres (Text-fig. 1, nos. 8 and 9). Finally, at third cleavage, a polar lobe is present, which, however, does not separate completely from the D-blastomere. A first quartette of micromeres is split off at the animal side of the egg (Text-fig. 1, nos. 11 and 12). In the subsequent cleavages, which follow each other rapidly, a polar lobe is not formed. Gastrulation starts about 4 h after first cleavage; 3 h later trochophores with a well-developed apical tuft are swimming in the dish (Text-fig. 2, no. 1). These larvae are very delicate and difficult to rear. As for our purpose a study of the trochophore stage was most important, the embryos were studied immediately after they had left the capsules. Text-fig. 2. Normal trochopore of 7 h; 2, Normal trochophore of 24 h.; 3, larva of 7 h., after removal of thefirstpolar lobe; 4, larva of 7 h., after removal of the second polar lobe (a.t., apical tuft; m., mouth; sh., shell gland). The surviving larvae start metamorphosing at the end of the first day (Textfig. 2, no. 2). They can be found at the bottom of the dish, moving only sluggishly. After 48 h metamorphosis is complete and a young Dentalium is present, complete with foot and shell. (b) The structure of the egg In the ripe oocyte a conspicuously large germinal v (...truncated)