Using an improved fixation method for electron microscopy, we have found germline granules in Caenorhabditis elegans embryos shortly after fertilization and prior to the first cleavage. They are localized in the egg cytoplasm which becomes segregated into the posterior blastomere at the first cleavage. In the following divisions, the granules continue this pattern of asymmetric segregation and are ultimately segregated into the germline precursor cell. The granules are then symmetrically segregated into the germline cells.

‘Germline granules’ have been observed by both light and electron microscopy in many organisms from hydra to man (Eddy, 1975). These have been called ‘polar granules’, ‘dense bodies’, and ‘nuages’. Such ‘granules’ have attracted attention because they may participate in the determination of cells to become the germline (Illmensee & Mahowald, 1974; Wakahara, 1977; Wakahara, 1978). In an electron microscopic study of the nematode Caenorhabditis elegans, Krieg et al. (1978) observed cytoplasmic structures characteristic of the germline cells in embryos as early as the 6-cell stage. We have further characterized these structures by electron microscopy, in order to find the earliest embryonic stage at which these structures could be identified and to see how they become segregated into the germline cells.

Nematodes

Wild-type C. elegans var. Bristol were grown at 20 °C on Petri dishes with E. coli 0P50 as a food source as described previously (Brenner, 1974; Hirsch, Oppenheim & Klass, 1976).

Preparation of embryos for electron microscopy

Gravid worms were washed off a Petri plate with distilled water and collected by centrifugation. A concentrated suspension of worms was put in a depression dish. Degassed 1 % NaOCl (Fisher Scientific Co.) with 0·25 M-KOH was added for 2-3 min until eggs were released from the partially lysed adults. The eggs were collected on a Nucleopore Corp. polycarbonate membrane filter (8·0 μm) and rinsed with M9 salt solution. The egg shell was removed from the embryos by digestion with chitinase (U.S. Biochem. Corp.) according to a procedure developed by Dr. Paulo Bazzicalupo. The chitinase was first dissolved at 20 mg/ ml in a salt solution of 50mM-NaCl, 70mM-KCl, 2·5mM-MgCl2,2·5mM-CaCl2, then centrifuged at 12 000 g. The supernatant fluid was added to the eggs at room temperature. The eggs were observed under a dissection microscope at ×25 magnification. As soon as the egg shell was seen to be removed, the eggs were transferred to fixative.

Eggs were fixed for 1-2 h at room temperature in 4% glutaraldehyde in 100 mM sodium phosphate buffer, pH 7·4; 2mM-MgCl2. Postfixation was done with 1% osmium tetroxide in 100mM-sodium phosphate buffer, pH 7·4; for 15 min. The eggs were encased in agar during the water rinse so that several eggs formed a group that could be sectioned together. Eggs were stained en bloc in 1 % uranyl acetate, dehydrated in ethanol, and embedded in Epon between two microscope slides.

Sectioning was done on a Porter-Blum MT-2 microtome and sections were stained with uranyl acetate and lead citrate and viewed in a Hitachi H-600 electron microscope.

Nomenclature

The nomenclature of the early embryonic cells lineages is that of Deppe et al. (1978).

Fertilization and the early cleavages of C. elegans have been described on a light microscope level (Schierenberg, 1978; von Ehrenstein & Schienenberg, 1980). We have found the germline granules in the earliest embryos we examined: the ‘pseudocleavage’ stage shortly after fertilization and prior to the first true cleavage. In Fig. 1, these granules can be seen near the posterior pole of the egg near the male pronucleus. Much vesicular material is present in the pseudocleavage egg but the darker staining granules can be distinguished. As the egg and sperm pronuclei move together and fuse to form the zygotic nucleus, the granules remain at the posterior pole. While they are distributed highly asymmetrically with respect to the anterior-posterior axis of the embryo at this stage, they do not show any apparent asymmetry along the left-right or dorsal-ventral axes. During the first cleavage, the granules remain at the posterior pole of the egg and are thus partitioned into the posterior blastomere, P1 (Fig. 2).

Fig. 1.

Pronuclear stage of C. elegans embryogenesis. (A) The pseudocleavage has occurred. The female pronucleus is in the anterior (left) end of the egg and the male pronucleus resides in the posterior (right) end of the egg. Arrows point to the germline granules. Mag. 2700 ×. (B) Higher magnification of the posterior end of the egg in A showing two granules near the male pronucleus. Mag. 8300×. (C) Migration of the pronuclei towards the middle of the egg. Arrows show the position of the germline granule. Mag. 2700×.

Fig. 1.

Pronuclear stage of C. elegans embryogenesis. (A) The pseudocleavage has occurred. The female pronucleus is in the anterior (left) end of the egg and the male pronucleus resides in the posterior (right) end of the egg. Arrows point to the germline granules. Mag. 2700 ×. (B) Higher magnification of the posterior end of the egg in A showing two granules near the male pronucleus. Mag. 8300×. (C) Migration of the pronuclei towards the middle of the egg. Arrows show the position of the germline granule. Mag. 2700×.

Fig. 2.

Germline granules in the 2-cell embryo of C. elegans. (A) The anterior AB blastomere is on the left and the posterior P1 blastomere on the right. The 2-cell embryo contains germline granules in the posterior end of the P1 blastomere, as designated by arrows. Mag. 2900×. (B) and (C) Higher magnifications of germline granules in the 2-cell embryo showing the granular morphology of the particles which are unbounded by membranes. Endoplasmic reticulum and several mitochondria are visible near the granules. (B) Mag. 23000×; (C) Mag. 33 000×.

Fig. 2.

Germline granules in the 2-cell embryo of C. elegans. (A) The anterior AB blastomere is on the left and the posterior P1 blastomere on the right. The 2-cell embryo contains germline granules in the posterior end of the P1 blastomere, as designated by arrows. Mag. 2900×. (B) and (C) Higher magnifications of germline granules in the 2-cell embryo showing the granular morphology of the particles which are unbounded by membranes. Endoplasmic reticulum and several mitochondria are visible near the granules. (B) Mag. 23000×; (C) Mag. 33 000×.

In the second cleavage of the living embryo, the P1 spindle initially is formed along the anterior-posterior axis of the egg. However, as cleavage begins, the spindle rotates in the dorsal-ventral plane so that one daughter, EMSt, is anterior-ventral and the other, P2, is posterior-dorsal. The germline granules appear to become localized in the dorsal sector of the P1 blastomere prior to the cleavage of this cell, as seen in Fig. 3. When P1 divides, they are partitioned into the P2 blastomere (Fig. 3B).

Fig. 3.

Segregation of the germline granules during the division of the P1 blastomere. (A) The P1 blastomere (right side of figure) is in anaphase. The granules (arrows) are concentrated in the region of the P1 blastomere that will become the P2 daughter blastomere. Mag. 2700×. (B) The P1 blastomere has reached telophase and the granules (arrows) remain segregated into the region of the cell that becomes the P2 daughter blastomere. Mag. 2800×.

Fig. 3.

Segregation of the germline granules during the division of the P1 blastomere. (A) The P1 blastomere (right side of figure) is in anaphase. The granules (arrows) are concentrated in the region of the P1 blastomere that will become the P2 daughter blastomere. Mag. 2700×. (B) The P1 blastomere has reached telophase and the granules (arrows) remain segregated into the region of the cell that becomes the P2 daughter blastomere. Mag. 2800×.

P2 will divide into blastomeres C and P3, and P3 will later divide into D and P4. The C and D blastomeres divide several times to form part of the somatic tissues of the animal, while P4 divides only once more during embryogenesis into Z2 and Z3, which form the germline (Kimble & Hirsh, 1979; J. Sulston, personal communication). The germline granules continue the pattern of asymmetric segregation described above, and are ultimately contained only in the P4 blastomere (Fig. 4). At the division of P4, they are segregated equally into both Z2 and Z3 (data not shown).

Fig. 4.

Germline granules in the P4 cell of a 24-cell-stage embryo of C. elegans. (A) The P4 cell, which is the lower right cell of the embryo contains the granules adjacent to its nucleus. Mag. 9000×. (B) Higher magnification of the P4-cell nucleus with its flanking granules. Mag. 17000×.

Fig. 4.

Germline granules in the P4 cell of a 24-cell-stage embryo of C. elegans. (A) The P4 cell, which is the lower right cell of the embryo contains the granules adjacent to its nucleus. Mag. 9000×. (B) Higher magnification of the P4-cell nucleus with its flanking granules. Mag. 17000×.

As the uncleaved egg, P0 divides and gives rise to the series of blastomeres P1, P2, P3 and P4, the granules become much larger in size and are found in closer proximity to the nucleus (compare Figs 3 and 4). Cytoplasmic granules can be seen in the P3 and P4 blastomeres, even in the living embryo with the light microscope, and are probably identical to the granules we see with the EM. The few large granules found in the P3 and P4 blastomeres could come from an aggregation of the numerous smaller granules we find in the earlier stages, or perhaps by the differential growth and attrition of certain granules.

The granules are often observed in close approximation to endoplasmic reticulum and mitochondria, and are not enclosed by membranes. We have not observed any striking association of the granules with microtubules or filaments that might suggest a mechanism for their movement.

In the study by Krieg et al. (1978), these granules were seen as ‘electron light cytoplasmic areas’. With our fixation methods, the granules appear as electron dense spherical bodies of an apparent fibrous nature, similar to the structure of germline particles described in other organisms (for review, see Beams & Kessel, 1974; and Eddy, 1975). The ultrastructure of the granules themselves offers no obvious suggestion of a possible function in the embryo.

These EM studies have demonstrated that the germline granules of C. elegans are present and asymmetrically distributed in embryos as early as the pronuclear stage of development. These granules continue to be segregated into the P-lineage throughout the early cleavages.

In Drosophila, germline-specific granules are also located at the posterior pole of the fertilized egg. The nuclei which migrate into this region at the first cytoplasmic division become the germline cells. In contrast, the germline granules of C. elegans must be segregated asymmetrically through four divisions before they are in the definitive germline cells. The granules of Drosophila have been implicated in determining the nuclei that they surround to become germ nuclei. If the C. elegans granules have a similar function, either they are not active in the P0, P1, P2 and P3 blastomeres, or these blastomeres are not competent to respond to the hypothetical ‘signal’. In this light, it is perhaps interesting that the granules only begin showing a strong association with the nucleus of the P4 blastomere, having been dispersed in the cytoplasm of the earlier stages.

Fuchs (1913) noticed granules he termed ‘ectosomes’ surrounding one of the asters in the first cleavage of the arthropod, Cyclops vividis. In successive divisions, these granules were asymmetrically segregated to only one of the daughter cells, and were finally localized in two of the primordial germ cells with a lineage pattern identical to that of C. elegans. The association of these granules with the asters of the cleaving blastomeres suggested an obvious mechanism for their segregation. As described above, the germline granules of C. elegans are asymmetrically distributed even before the first spindle is formed. Similarly, the prelocalization of the granules in the later blastomeres, prior to spindle formation, make an astral involvement in their specific segregation less likely in this animal. Still, a transient association of the granules with cytoplasmic microtubules or microfilaments could have been missed in our studies.

While germline specific granules have apparently not been observed in other species of nematodes, perhaps due to the difficulties of obtaining adequate fixation of the early stages, there are striking differences between cytoplasms of somatic and germline precursor cells during the embryogenesis of the nematode Ascaris megalocephala (Boveri, 1899). Shortly after the first cleavage of the fertilized Ascaris embryo, chromosomal diminution occurs in one of the daughter cells; chromosomal fragments are left in the cytoplasm and are subsequently lost. However, the sister blastomere, P1, which will ultimately produce the germline cells as in C. elegans, retains the full chromosomal complement. The potential not to undergo chromosomal diminution is segregated into P0, P1, P2, P3 and P4 in A. megalocephala just as the germline granules are in C. elegans. There is evidence that the chromosomal diminution is prevented by the cytoplasm the germline normally receives; if the first cleavage is altered such that this cytoplasm is divided between the first two sisters, diminution does not occur in either cell (Boveri, 1910; Hogue, 1910). Thus there is a clear difference in the properties of somatic precursor versus germline precursor cells. Though C. elegans does not appear to undergo chromosomal diminution (Emmons, Klass & Hirsh, 1979; Sulston & Brenner, 1974) the asymmetric segregation of granules we observed could conceivably be related to the same basic cytoplasmic difference between the somatic and germline precursor blastomeres.

Krieg et al. (1978) reported seeing ‘electron light cytoplasmic areas’ in germline precursors in C. elegans embryos from the 6-cell stage onward. These ‘areas’ were localized around the nuclei. Thus, the segregation pattern and intracellular localization of these ‘areas’ are the same as those of the granules we have observed. Although their morphology is quite different, presumably as a result of the different fixation procedures, it is likely that those ‘areas’ represent the same structures as the granules we have described.

Strome & Wood (1982) recently reported staining particulate cytoplasmic components of the germline cells and their precursors in C. elegans embryos using FITC-conjugated rabbit anti-mouse IgG. The location and segregation of the staining material appears to be identical to that of the granules reported here, suggesting that the antiserum may be recognizing some component of these structures. Unfortunately the antiserum does not bind to the particles after aldehyde fixation, preventing a direct comparison of the particles recognized by the antiserum and the granules described here (S. Strome, personal communication).

We thank J. Richard McIntosh for helping develop the improved fixation and for valuable discussions. This work was supported by Public Health Service Grant No. 19851.

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