The structure of a morphogenetic cytoplasm, present in the polar lobe of Bithynia tentaculata (Gastropoda, Prosobranchia)

0 address: Zoology Laboratory , Janskerkhof 3, Utrecht , The Netherlands. 27 E MB 31 1 From the Zoological Laboratory, University of Utrecht , The Netherlands In the eggs of many annelids and molluscs the first cleavages are characterized by the appearance of polar lobes. The contents of these lobes are of great importance for further development, as is shown by experiments involving removal of the lobe or deletion of cells receiving the lobe contents. In Ilyanassa, for instance, lobe-dependent structures are: foot, eyes, operculum, statocysts, shell, heart and intestine (Crampton, 1896; Clement, 1952, 1956, 1962; Cather, 1967; Atkinson, 1971). The successively appearing lobes do not all have the same effect. In Dentalium (Wilson, 1904), Sabellaria (Hatt, 1932; NovikofT, 1938a, b) and Mytilus (Rattenbury & Berg, 1954) development of the apical tuft of the larva is dependent on the presence of the first polar lobe, but not on the second one. Attempts to identify polar lobe factors have given very poor results up till now. In many cases differences in composition can be demonstrated between the cytoplasm of the polar lobe and the rest of the egg (Pitotti, 1947; Clement & Lehmann, 1956; Pasteels & Mulnard, 1957; Reverberi, 1958, 1970; Berg & Kato, 1959; Collier, 1960a, b; Dalcq & Pasteels, 1963; Crowell, 1964). But centrifugation experiments (Clement, 1968; Verdonk, 1968) have established that in Ilyanassa and Dentalium the morphogenetic factors are not located in the displaceable components of the polar lobe cytoplasm. Unfortunately ultrastructural and histochemical studies on displacement of cytoplasmic components in polar lobes of centrifuged eggs are lacking. - SUMMARY In the first polar lobe of the egg of Bithynia tentaculata a cup-shaped mass of small vesicles is described, which fills the greater part of the lobe. It is named the 'vegetal body'. With methyl green-pyronin the vegetal body stains clearly, but after treatment with RNase no staining occurs, thus indicating the presence of RNA. The first polar lobe of Bithynia is of great importance for further development of the embryo and it is argued that the vegetal body could be a morphogenetic cytoplasm, responsible for the developmental effects of the polar lobe. Accumulation of morphogenetic substances in special areas of the egg and segregation of these accumulations into special blastomeres are not restricted to polar-lobe-forming organisms, but can be demonstrated in many other animals. In spite of such a widespread occurrence of the segregation phenomenon, identification of the morphogenetic substances in the segregated cytoplasm has not been successful. Even in the polar granules of insect eggs, 'the only case known where developmentally significant information is localized in organelles that can be seen and followed during development' (Mahowald, 1971), it is not known whether it is the protein or the RNA component which is developmentally significant. We describe a structure in the polar lobe of Bithynia tentaculata which could be a second case of an organelle storing morphogenetic substances. Bithynia tentaculata is a freshwater prosobranch snail which can be collected in ditches around Utrecht, Holland. In aquaria in the laboratory, egg masses are soon deposited on plant leaves. These egg masses consist of capsules which cannot be separated from each other. The tough capsule membrane is first perforated with a very sharp knife in order not to compress the egg. Then the egg can be removed from the capsule with a hair-loop and the viscous capsule fluid can be washed off in tap-water. Fixation and staining for light microscopy. Eggs fixed in Zenker's fluid were stained with Heidenhain's iron haematoxylin and eosin for general study or with methyl green-pyronin for nucleic acids (Brachet, 1942,1953). For the latter stain eggs usually were fixed in ethanol-acetic acid (3:1) for a more vivid stain. For the Feulgen method, eggs werefixedin ethanol-acetic acid (3:1) and either stained as sections or in toto according to the method of van den Biggelaar (1971). Fixation and staining for electron microscopy. Eggs were fixed for 3 h at 4 C in a mixture of equal parts 2 % glutaraldehyde and 2 % osmium tetroxide, both in 0-1 M Na-cacodylate buffer at ph 7-4. The eggs were then washed in buffer, oriented in agar, dehydrated in a graded series of ethanol, followed by propylene oxide, and embedded in Epon 812. Sections were stained for 10 min in a saturated solution of uranyl-acetate in 70 % methanol, followed by 1 min in a lead solution according to Reynolds (1963). I. Observations with the light microscope At the vegetal pole of freshly laid eggs of Bithynia, in which the germinal vesicle is still present, a densely staining cytoplasm is present. At this stage the dense plasm is intimately connected with the surface of the egg (Fig. 1). After the germinal vesicle has disappeared, the dense cytoplasm rises from the surface (Fig. 2) and soon assumes the shape of a cup with the open side towards the vegetal pole. Because of its position we call it the 'vegetal body'. It stays at the Figs. 1-5. Light micrographs of eggs of Bithynia tentaculata stained with iron haematoxylin and eosin. x 350. Fig. 1. Egg just after oviposition with the germinal vesicle still present. Arrow indicates dense cytoplasm at the vegetal pole. Fig. 3. Egg at first cleavage. Arrow indicates dense cytoplasm now present as cupshaped vegetal body in the polar lobe. Fig. 5. Section through the CD-blastomere at the beginning of second cleavage. Arrow indicates the second polar lobe, filled with clear cytoplasm but missing the vegetal body. Fig. 6. Light micrograph of an egg at first cleavage, stained with methyl greenpyronin. The vegetal body (arrow) is densely stained. Fig. 7. Electron micrograph of the first polar lobe with vegetal body. Arrow points to a dense body. AZ, Attachment zone; L, lipid; M, mitochondrion, x 9700. vegetal pole, surrounded by clear cytoplasm, until first cleavage, when a polar lobe is formed. Compared with other polar lobes, described in molluscs, this lobe is remarkable in several respects: it is extremely small (diameter 20-30 /an) and it is usually nearly free from yolk granules. The vegetal body is incorporated in the first polar lobe (Fig. 3). After first cleavage the vegetal body is transferred with the lobe to the CD-blastomere. During the whole 2-cell stage it remains at the vegetal side of the CD-blastomere (Fig. 4). At second cleavage the vegetal body suddenly disappears at about the start of anaphase. A second polar lobe is formed at this stage, which, however, is never as completely separated as the first polar lobe and remains broadly connected with the CD-blastomere. The second lobe contains a clear cytoplasm but the vegetal body is no longer present (Fig. 5). In order to study the chemical nature of the vegetal body, we stained it with the Feulgen method for the presence of DNA. Both in sections (...truncated)