Occurrence of intercellular bridges between follicle epithelial cells in the ovary of Apis mellifica queens

Intercellular cytoplasmic bridges (fusomes or ring canals) occur in the gonads of both sexes and have been described previously for a number of insect species (Maho-wald, 1972; Telfer, 1975). They arise during cystoblast formation and are especially known from the polytroph-meroistic ovaries of Dermaptera, Mecoptera, Coleóptera, Lepidoptera, Díptera and Hymenoptera, including the honeybee (Engels, 1968). From the cystocytes, the oocyte and trophocytes of a varied but species-specific number are differentiated later. Hirschler (1942) proposed that the intercellular bridges represent the spindle remnants of differential mitoses. Later, Fawcett, Ito & Slautterback (1959) found that incomplete cell divisions during the cystoblast-forming cytokinesis resulted in intercellular bridges. Recent electron-microscopic studies have confirmed this view, and demonstrated numerous microtubules in the intercellular bridges (Mahowald, 1971; Cassidy & King, 1972). The origin and distribution of intercellular bridges, as well as the intercellular transport mechanisms with particular reference to the polarity of oocytes and trophocytes, have been reviewed in great detail by Telfer (1975).

Hitherto, ovarian intercellular bridges were described only between oocytes and trophocytes and between trophocytes. The follicle epithelium which is believed to be of somatic origin, arises from repeated mitotic proliferation of the prefollicular tissue in the germarium. As an investing layer, it has to adapt itself continuously to the growing volume of the egg follicles. This is achieved both by cell proliferation and by structural transformations of its cells. It has not hitherto been shown that the follicle cells also remain connected with each other through fusomal intercellular bridges. In our electron-microscopic studies of Apis mellifica queen egg follicles, typical intercellular bridges in the follicle epithelial cells have been observed and these are described here.

Ovaries of the queens were fixed in 2·5 % glutaraldehyde in 0·2 M sodium cacodylate buffer and 0·1 M sucrose adjusted to pH 7·4 for 2 h. Following the buffer wash the materials were postfixed in 2 % osmium tetroxide and embedded in Epon. Thin sections were contrasted with uranyl acetate and lead citrate. Grids were examined under Siemens Elmiskop 102 at 60 kV. Light-microscopic observations were made on Bouin-fixed paraplast sections stained with iron haematoxylin-eosin.

The oocyte development in Apis has been divided into 6 stages of oogenesis (Engels, 1973). Mitotic activity in the follicle epithelium is found only up to stage 2. In stages 3 and 4, the follicle epithelial cells become endopolyploid, as evidenced by incorporation of [³H]thymidine.

The intercellular bridges as remnants from the previous mitoses persist in the differentiated epithelium. They appear as distinct membrane breaks of 0·5 μm diameter between 2 neighbouring cells and are situated close to the oocyte (Fig. 1). This position seems to be more or less constant. For the sake of comparison, an intercellular bridge between 2 trophocytes is represented at the same magnification (Fig. 2). The size and structural differences can be readily visualized. The inter-trophocytic ring canal measures about 3·5 μm. The cell membranes in its neighborhood show convolutions lacking in follicle epithelium. The sectioned lips of the nurse chamber intercellular bridge assume a characteristic figure-3 shape, while in the follicle epithelium the rims are evenly surfaced (Figs. 1, 6). As a consequence of the different diameters, cell organelles like mitochondria (Fig. 2) are seen to pass through the intercellular bridges between trophocytes, but only ribosomes (Figs. 1, 6, 7) are seen within the ring canals of the follicle cells.

The origin of the intercellular bridges found in the differentiated follicle epithelium of stages 3 and 4 can be visualized in young follicles. In stage 1, the intercellular bridges occur in various phases of incomplete cell divisions (Fig. 4A). As a result of this special mitoses, a single follicle cell may be simultaneously connected with 1 to even 4 neighbouring cells (Fig. 4B). Originating from a few prefollicular cells found in stage 0 a complete epithelium investing the whole follicle is formed.

This mode of follicle cell proliferation is still visible in stage 4. In surfacial sections of the follicle epithelium branching rows of cells connected by intercellular bridges can be recognized (Fig. 3). From such sections, the formation of the follicle epithelium could be reconstructed.

The intercellular bridges observed in stage 1 or 2 follicles often represent early or mid phases of cytokinesis. In such cases the cell furrow between the 2 sister follicle cells is still wide open (Figs. 4A, 5). The width of the intercellular bridge varies between 1·5 and 3·5 μm. The rims are recurved into a figure-3 shape. A heavy deposition of osmiophilic material is seen on their medial borders. Numerous microfilaments extend between them (Fig. 5). Obviously, these filaments are responsible for constricting the intercellular bridge into a narrow ring, as it is seen in the later stages of 3 and 4 (Figs. 1, 6, 7), and then disappears without leaving any trace.

The differentiated ring canal is reinforced with electron-dense stabilizing material with a layer of less-dense material both inside and outside (Figs. 6, 7). The ribosomes keep a certain distance away from the electron-dense lips.

In all stages the follicle epithelium investing the nurse chamber is an inconspicuous layer of elliptical cells. Intercellular bridges in this scattered network could not be detected.

The intercellular bridges between the follicle epithelial cells described here for the first time resemble entirely in their ultrastructure those between the trophocytes and the oocyte and trophocytes (Mahowald, 1972). In Apis mellifica, in the fully differentiated state, the only characteristic difference between the 2 types of intercellular bridges is in size. The diameter of the follicle cell ring canal is considerably smaller than that of the oocyte-trophocyte complex, which is reflected in the type of cell organelles that pass through them. Wide intertrophocytic ring canals are known from other species too (Ramamurty, 1964).

The follicle epithelial intercellular bridges result from incomplete cytokineses like those in the oocyte-trophocyte complex (Cassidy & King, 1969; Mahowald, 1971; Schulze, 1971), which arise during the formation of cystoblasts (Hirschler, 1945) in the posterior region of the germarium (King, 1970). The occurrence of probably contractile microfilaments, which were found only within intercellular bridges during the early stages of follicle epithelium formation, confirms this similar origin.

It has generally been assumed hitherto that intercellular bridges are formed only between differentiating cells of the germ line. They serve for the intercellular exchange of materials, especially the transport of macromolecules and cell organelles (Telfer, 1975; Ramamurty, 1976). It is also believed that the intercellular bridges represent a structural basis for the synchronization of the differentiation process during ontogenesis (Fawcett et al. 1959). Intercellular bridges were described in various Articulata, namely, Annelida (Fischer, 1975), Crustacea (Johannisson, 1971) besides Insecta (cf. Mahowald, 1972) as well as in the Vertebrata (Burgos & Fawcett, 1955; Fawcett, 1961; Norrevang, 1968). In the pseudopolytrophic nurse chamber of the Cecido-myidae (Díptera), by a single division, an oocyte of the first order and one trophocyte are formed. So, a primarily 2-celled cystoblast results. Later the trophocyte is joined syncytially to other cells (Madhavan, 1973; Mahowald & Stoiber, 1974) which, in their origin, are somatic follicle epithelial cells, but behave like the trophocytes by becoming endopolyploid (Matuszewski, 1968) and supply RNA to the oocyte (Kunz, Trepte & Bier, 1970). Thus there are exceptions to the general rule relating to the germline cystoblast origin of the trophocytes in the polytrophic ovarian follicles of insects.

The mode of origin of the trophocytes in Apis mellifica, as described by Paulcke (1901) and Hegner (1915), conforms to the general pattern, including the formation of intercellular bridges (Engels, 1968). The somatic origin of the follicle epithelium especially in Apis, or in the Hymenoptera in general, cannot be taken as well established yet, because detailed studies have not been made. Therefore, it is conceivable that here the follicle epithelial cells might arise, as an exception, from the germ line and as such could be capable of developing intercellular bridges. Otherwise, the fusomal phenomenon in the honeybee follicle epithelial cells described here would be rather unusual. In any case, further insect species should be looked into more carefully with the electron microscope to determine whether intercellular bridges occur in the follicle epithelium.

Apart from the fact that ribosomes can apparently pass the intercellular bridges between follicle cells nothing is known about their function. According to the electrophysiological studies of Woodruff & Telfer (1973, 1974), there exists a polarity directed towards the oocyte within the nurse chamber-oocyte complex. Most probably the transport of macromolecules and cell organelles is based on a mechanism depending upon the principle of electrophoresis. To date no intercellular transport phenomena in the follicle epithelium have been described.

The synchronizing function of the intercellular bridges as postulated especially by Fawcett (1961), has repeatedly been questioned (cf. Moens & Go, 1971). In the case of the follicle epithelium such an hypothesis is particularly illuminating. Contrary to the trophocytes and oocyte, the functions of the follicle epithelial cells are manifold; for example, soon after the vitellogenic phase, it secretes the chorion. Thus the follicle epithelium undergoes within a short time, quite a heterogeneous differentiation programme (Kafatos, 1975).

Perhaps the follicle epithelium could also play an active role in maintaining the potential differences within the follicle (Woodruff & Telfer, 1974). These authors have further envisaged the possibility that the fusomes, and not the membranes, are the actual structural factors for the battery effect. To clarify such hypotheses further experimental studies are necessary.

The authors wish to thank Dr Ch. F. Bárdele, Dr U. Mays and M. Lacombe for extending the electron-microscopic facilities, and P. Bdrsch, E. Hutter, M. Schürmann, E. Sonemann and A. Vees for technical assistance. P. S.R. acknowledges the award of a research fellowship of the Alexander von Humboldt-Stiftung, which made this work possible. It was also supported by the Deutsche Forschungsgemeinschaft.