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Keywords:

  • fertilization;
  • first cleavage mitosis;
  • gonomery;
  • insect;
  • mosaic

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. First cleavage mitosis in Bombyx mori
  5. First cleavage mitosis in Bombyx eggs treated by low temperature
  6. Gonomery in other insects and arthropods
  7. The sea urchin and Ascaris types of the first cleavage
  8. Conclusions
  9. Acknowledgements
  10. References

Fertilization in animals is now considered to be of the ‘sea urchin type’; that is, haploid male and female pronuclei completely fuse shortly after sperm entry into the egg, followed by the formation of a mitotic spindle to allow cleavage mitoses to proceed. However, two other patterns of fertilization and early embryonic mitosis in some animal species are known: an Ascaris type and a gonomeric type. The gonomeric type of fertilization in insects and other arthropods is not well known and is quite different from the sea urchin and Ascaris types. In the present article, the author examines the peculiar gonomeric fertilization, using mainly the silkworm as an example.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. First cleavage mitosis in Bombyx mori
  5. First cleavage mitosis in Bombyx eggs treated by low temperature
  6. Gonomery in other insects and arthropods
  7. The sea urchin and Ascaris types of the first cleavage
  8. Conclusions
  9. Acknowledgements
  10. References

Compared with other animal groups, the fertilization process in insects and in whole arthropods, including the union of gametes and subsequent mitosis, is scarcely known. The technical difficulties due to the thick egg chorion and abundant yolk mass in the eggs prevent observation of the process. However, a few examples were published with sketches, some elaborate and some rough, from the late 1800s to early 1900s (Wilson 1934) in copepod Cyclops by Rückert (1895) and in Drosophila melanogaster by Huettner (1924) and Sonnenblick (1950). The method of fertilization leading to early embryogenesis in these animals has been named gonomery. Gonomeric fertilization is quite different from the processes seen in the sea urchin, which are now broadly used for accounts of animal fertilization in textbooks. It is also different from the Ascaris type of fertilization observed by van Beneden in 1883 (ref. Wilson 1934), who showed for the first time the contribution of male and female pronuclei in fertilization. In gonomeric-type fertilization, two pronuclei do not fuse even after their union. They stay side by side, and each of the pronuclei independently forms a mitotic apparatus with a haploid chromosome group and separation of chromosomes occurs on each spindle. Karyogamy takes place at the end of the first cleavage in D. melanogaster and Bombyx mori (Kawamura 1978); in Cyclops double nuclei are observed even after the cleavage stage.

The aim of the present article is to reintroduce the observations of the early investigators on the gonomeric cleavage of insects and arthropods, because these have since been forgotten.

First cleavage mitosis in Bombyx mori

  1. Top of page
  2. Abstract
  3. Introduction
  4. First cleavage mitosis in Bombyx mori
  5. First cleavage mitosis in Bombyx eggs treated by low temperature
  6. Gonomery in other insects and arthropods
  7. The sea urchin and Ascaris types of the first cleavage
  8. Conclusions
  9. Acknowledgements
  10. References

Kawamura (1978) documented the first cleavage mitosis in the silkworm Bombyx mori. Union of sperm and egg pronuclei takes place at 120 min after deposition and the first cleavage mitosis occurs at 150 min (26°C). The egg nucleus that temporarily stops meiosis at metaphase I enters the meiotic process by the stimulation of sperm entrance into the egg (Rasmussen 1977). A large monaster is formed around the sperm pronucleus and then around the united egg and sperm pronuclei (Fig. 1a). Unlike in sea urchin-type fertilization, fusion of these pronuclei does not occur. Spindle fibers appear independently in each of the pronuclei and the two spindles are not always parallel (Fig. 1b,c). The spindles assume parallel orientation with their elongation. At metaphase (Fig. 1d) and anaphase (Fig. 1e), the paternal and maternal chromosome groups still remain separate on a fused spindle. Karyogamy occurs at telophase, when separate chromosome groups reach a united spindle pole (Fig. 1f). Afterwards, the mitotic divisions are as usual with diploid chromosomes.

image

Figure 1. The first cleavage mitosis in the silkworm. (a) Union of male and female pronuclei. (b) Prophase. Two small spindles in the paternal and maternal pronuclei take rectangular positions. Two serial sections are superimposed. (c) Late prometaphase. Two independent spindles from the two pronuclei are going to assume a parallel position. (d) Metaphase. Paternal and maternal chromosome groups are separate on a spindle. (e) Early anaphase. Two chromosome groups are still separate. (f) Telophase of the first cleavage. Chromosome groups are fused on the spindle poles (karyogamy). (a) Bar, 20 μm; (b–f) bar, 10 μm (from Kawamura, N., 1978; reprinted by permission of John Wiley & Sons Inc.).

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First cleavage mitosis in Bombyx eggs treated by low temperature

  1. Top of page
  2. Abstract
  3. Introduction
  4. First cleavage mitosis in Bombyx mori
  5. First cleavage mitosis in Bombyx eggs treated by low temperature
  6. Gonomery in other insects and arthropods
  7. The sea urchin and Ascaris types of the first cleavage
  8. Conclusions
  9. Acknowledgements
  10. References

Generally, low temperature disturbs the association of tubulin with microtubules in the spindle (Inoue et al. 1974). Mosaic silkworms are easily produced by applying a cold shock to the eggs at the time of the first cleavage and returning them to room temperature after 24 h (Kawamura 1979). When a female of the striped silkworm strain (black larva) and a male of the plain silkworm strain (white larva) are mated, the low temperature treatment induces various kinds of mosaic larvae, including bilaterally symmetrical mosaics with a black and white pattern (Fig. 2). In the egg at 1 h after return to room temperature, chromosome condensation is proceeding in two pronuclei that assume longitudinal position (Fig. 3a). Figure 3b shows two small spindles found in a cold-treated egg at 2 h after return to room temperature. The size and form of these small spindles, which have been named ‘compact spindles’ (Kawamura 1978), are different from the first cleavage spindles in untreated eggs. It is thought that these compact spindles come from the sperm and egg pronuclei that are prevented from union by the low temperature treatment. The compact spindles are capable of continuing mitosis (Fig. 3c). Mosaic silkworms seem to arise from such cold-treated eggs in which the two separated pronuclei independently repeat mitosis.

image

Figure 2. The fifth (final) instar larvae from the silkworm eggs treated by low temperature (– 10degC for 24 h). They display various mosaic patterns, including a bilaterally symmetrical mosaic.

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image

Figure 3. The first cleavage mitosis in cold-treated eggs. (a) One hour after return to room temperature. (b) Two hours after return to room temperature. Two compact spindles derived from paternal and maternal pronuclei are formed independently. (c) Three hours after return to room temperature. A compact spindle enters anaphase. Bars, 10 μm (from Kawamura, N., 1978; reprinted by permission of John Wiley & Sons Inc.).

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As to the formation of the compact spindles in B. mori eggs, the question arises as to whether each of the male and female pronuclei furnishes a functional microtubule organizing center (MTOC). Recent advanced techniques in immunofluorescence staining have allowed visualization of the MTOC in the sea urchin, mouse (Schatten et al. 1986) and D. melanogaster (Riparbelli et al. 1997). Riparbelli and her coworkers have reported that the male gamete does not supply either centrosomal protein or gamma- tubulin in the D. melanogaster MTOC, while the centrosomal components are present in the egg. These components nucleate microtubules in a monastral array after activation, but are unable to organize bipolar spindles. Drosophila melanogaster eggs have anastral meiotic spindles lacking centrioles. Hatsumi and Endow (1992) have described that in D. melanogaster chromatin plays an important role in spindle microtubule organization in absence of centrosomal material. If the data obtained in D. melanogaster are applicable to the silkworm, the microtubules of the compact spindle may be organized by some components derived from chromatin.

Gonomery in other insects and arthropods

  1. Top of page
  2. Abstract
  3. Introduction
  4. First cleavage mitosis in Bombyx mori
  5. First cleavage mitosis in Bombyx eggs treated by low temperature
  6. Gonomery in other insects and arthropods
  7. The sea urchin and Ascaris types of the first cleavage
  8. Conclusions
  9. Acknowledgements
  10. References

Silkworms generally deposit hibernating eggs that wait for natural hatching until the following spring. For artificial hatching, the eggs must be treated with hot (45–48°C) acid solution consisting of concentrated hydrochloric acid and 2% formaldehyde. Although treatment with the acid solution is violent enough to kill cells and tissues, silkworm eggs survive under the protection of the chorion and show almost 100% hatchability. This fact shows the severe difficulty in finding an adequate fixative for silkworm, or insect, eggs. Peeling the chorion without harming the egg membrane of a newly deposited egg is almost impossible in the living state. One additional difficulty is that the separate chromosome groups on a spindle without overlapping can only be displayed when the spindle is observed at the right angle. Thus, only a small number of reports on the gonomeric first cleavage mitosis in arthropods have been made in the past hundred years.

Some examples of gonomeric cleavage in insects were introduced by early authors. Henking’s observation (1892) on fertilization in the red fire (linden) bug Pyrrhocoris apterus (Hemiptera) may have been the first to show gonomeric cleavage mitosis. In his drawing (Fig. 4), two separate chromosome groups are located on an anaphase spindle. He described that ‘the first cleavage spindle is formed and the equatorial plate is divided but the chromosome groups which come from male and female nuclei are still visible.’Huettner (1924) and Sonnenblick (1950) showed the process of the first cleavage mitosis in D. melanogaster (Diptera) (Fig. 5) and Nachtsheim (1913) observed it in the honeybee Apis mellifica (Himenoptera). Gonomeric first cleavage has also been observed in the cricket Gryllus bimaculatus (Orthoptera) by Tanaka (1992; Fig. 6). Rückert’s (1895) observation on gonomeric cleavage mitoses in the copepod Cyclops, of Crustacea (Fig. 7), suggests that this type of fertilization is general not only in insects but also in other species of Arthropoda. The gonomeric type of fertilization is found only in species of the Arthropoda group, as far as I know.

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Figure 4. Anaphase figure of the first cleavage mitosis in the red fire bug Pyrrhocoris apterus (Hemiptera; after Henking 1892). RKI, first polar body; RKII, second polar body.

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image

Figure 5. Early stages of gonomeric first cleavage in Drosophila melanogaster. (a) Union of two pronuclei. (b) Spindle fibers appear in each of the pronuclei. (c) Two spindles assume parallel positions as they elongate at prometaphase. (d) Metaphase of the first cleavage mitosis. Paternal and maternal chromosome groups are separate on a spindle. (after Huettner 1924; reprinted by permission of John Wiley & Sons Inc.).

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image

Figure 6. Gonomeric fertilization and the first cleavage mitosis in the cricket (Gryllus bimaculatus). Indirect immunofluorescence staining for alpha-tubulin (green) and staining by Hoechst 33258 for chromosomes (blue). (a) Union of male and female pronuclei. (b,c) Prometaphase and metaphase. Spindles are formed in each of the pronuclei. (d) Telophase. Bars, 10 μm (figure courtesy of Mrs H. Tanaka).

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image

Figure 7. Gonomeric first and second cleavage mitosis in the copepod Cyclops (after Rückert 1895).

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There are some reports, although few, on laterally symmetrical mosaics with male and female characteristics in butterflies. As described in the previous section, these bilaterally symmetrical mosaics can be interpreted by the fact that the compact spindles, each of which comes from paternal or maternal pronucleus, continue embryonic mitosis independently.

The sea urchin and Ascaris types of the first cleavage

  1. Top of page
  2. Abstract
  3. Introduction
  4. First cleavage mitosis in Bombyx mori
  5. First cleavage mitosis in Bombyx eggs treated by low temperature
  6. Gonomery in other insects and arthropods
  7. The sea urchin and Ascaris types of the first cleavage
  8. Conclusions
  9. Acknowledgements
  10. References

The sea urchin type of fertilization is well known: pronuclei unite shortly after the entrance of the sperm and they fuse completely to form a fusion nucleus. The doubled central bodies are located on two ends of the fusion nucleus and the mitotic apparatus is formed between the central bodies. The paternal and maternal elements cannot be distinguished in the fused nucleus or on the spindle. There is another type of first cleavage mitosis, known as the Ascaris type, that was described by van Beneden in 1883 in his historic paper on the fertilization of Ascaris megalocephala (Nematoda; ref. Wilson 1934) and by Lillie (1901) in Unio (Bivalvia, Mollusca; Fig. 8). In the Ascaris type of fertilization, each of the egg and sperm pronuclei has already given rise to a chromosome group at the time of union. Karyogamy takes place after the mitotic apparatus is formed between the divided sperm aster and, therefore, a true fusion nucleus is not formed.

image

Figure 8. Ascaris-type fertilization in Unio. (a) Slight advance in aster and spindle formation after union of male and female pronuclei. (b) Origin of the central spindle. (c) Long chromosomes appearing. (d) The first cleavage spindle at metaphase (after Lillie 1901; reprinted by permission of John Wiley & Sons Inc.).

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Wilson (1934) has described that the difference between the two types of fertilization is determined mainly by the time element; that is, the length of the interval between the entrance of the sperm and the union of the pronuclei. He has shown experimentally that the sea urchin type of fertilization can be artificially altered to the Ascaris type by prolonging the time from sperm entrance to the union of the pronuclei in the etherized eggs of Toxopneustes (Echinoidea). In an ultrastructural study, Longo (1973) displayed electron micrographs of the syngamy process in the sea urchin Arbacia punctulata (Ehinoidea), the mussel Mytilus edulis (Bivalvia), the surf clam Spisula solidissima (Bivalvia) and the rabbit Oryctolagus cuniculus (Mammalia). He described that syngamy of the female and male pronuclei in M. edulis, S. solidissima, and the rabbit is of the Ascaris type, in which maternal and paternal chromosome groups are intermixed on the equatorial plate of the first mitotic spindle in absence of fusion nucleus formation. He stated that the sea urchin and Ascaris types of fertilization are related to the stage of meiosis in the egg and the time of insemination. Furthermore, many light and electron microscopic investigations have indicated that the eggs of most organisms exhibit an Ascaris type of fertilization (Longo 1973). The three types of fertilization are schematically summarized in Fig. 9.

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Figure 9. Schematic illustration comparing three types of fertilization: (i) the sea urchin type; (ii) the Ascaris type; and (iii) the gonomeric type. (A) Sea urchin type. Fusion of pronuclei, syngamy, occurs shortly after the union of the male and female pronuclei. (B) Ascaris type. A fused nucleus is not formed, but chromosomes from parents are intermixed on the spindle. (C) Gonomeric type. After the union of pronuclei, spindle fibers appear in each of the pronuclei independently. Paternal and maternal chromosome groups are separate on the spindle. Karyogamy occurs at the telophase of the first cleavage mitosis.

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. First cleavage mitosis in Bombyx mori
  5. First cleavage mitosis in Bombyx eggs treated by low temperature
  6. Gonomery in other insects and arthropods
  7. The sea urchin and Ascaris types of the first cleavage
  8. Conclusions
  9. Acknowledgements
  10. References

As shown in Figs 1,5, the type of fertilization seen in B. mori and D. melanogaster is distinct from the sea urchin and Ascaris types. In silkworm eggs, fusion of male and female pronuclei does not take place as seen in the sea urchin egg and, unlike in the Ascaris type of fertilization, the maternal and paternal chromosome groups are not intermixed on the equatorial plate of the first cleavage spindle. Definite differences are that a mitotic spindle is independently formed within each of the pronuclei, paternal and maternal choromsome groups are clearly distinguishable on a fused spindle at metaphase and anaphase and karyogamy occurs at telophase of the first cleavage mitosis. As previously described, female meiosis in the silkworm arrests at metaphase and starts the subsequent process at the insemination of sperm, just as for the fertilization process in the mussel (Bivalvia) shown by Longo (1973). Therefore, Wilson’s (1934) and Longo’s (1973) claims that the meiotic state of the egg at the time of insemination and the condition of the pronuclei at the time of their association are related to the type of fertilization are not consistent with gonomeric cleavage in arthropods. In B. mori and D. melanogaster eggs, polar bodies are not formed, but the polar body nuclei remain in the periplasma of the egg. This may be correlated with the special pattern of the first cleavage mitosis in insects.

It is well known that parthenogenesis is a general occurrence in some insects. There are two types of natural parthenogenesis with respect to the number of chromosomes: diploid and haploid. The two types are connected by transitional cases in which development begins with a haploid number of chromosomes but later becomes diploid (Wilson 1934). Haploid parthenogenesis is characteristic of many social insects, such as honeybees, ants and wasps (Himenoptera). In these insects, parthenogenesis without any process of syngamy is considered to be an effective way to produce successive generations for some periods of the year. In the insects of the gonomeric type, the ability to form a spindle from a female pronucleus and to continue subsequent mitoses makes haploid parthenogenesis possible. It is not clear, however, what stimulation other than sperm insemination induces mitosis of the female nucleus.

According to Hawksworth and Kalin-Arroyo’s survey (1995) on the number of animal species now known on the earth, arthropods comprise approximately 1 065 000 species of the total number of 4 615 000 animal species. The gonomeric type of fertilization may be a general occurrence in arthropod species and one-fourth of animal species may belong to this group. It seems that the gonomeric type of fertilization may be an extreme example of one branch, the sea urchin type of fertilization may be an extreme example of the other branch and the Ascaris type may cover the wide variety of intermediate cases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. First cleavage mitosis in Bombyx mori
  5. First cleavage mitosis in Bombyx eggs treated by low temperature
  6. Gonomery in other insects and arthropods
  7. The sea urchin and Ascaris types of the first cleavage
  8. Conclusions
  9. Acknowledgements
  10. References

The author thanks Dr Ken Sahara, Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan and Dr Naoko Yamashiki, Biology Laboratory, Rakuno Gakuen University, Ebetsu, Japan for their valuable comments on this article and Dr Motoaki Sato, Biology Laboratory, Rakuno Gakuen University for his help in drawing the schematic figure (Fig. 9). Dr Sahara kindly helped in reproducing the figures in this article. The author also thanks Mrs Hikaru Tanaka-Sato for permission to use the photos in Fig. 6 from her Masters thesis. The author appreciates the permission of the Copyright Permissions Department of John Wiley & Sons Inc. to use Figs 1, 3, 5 and 8 that were previously published in papers by Kawamura (1978), Huettner (1924) and Lillie (1901). Despite all efforts, the author was unable to locate the copyright holders of Figs 4,6. She will make contact with them to obtain permissions promptly when they can be located.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. First cleavage mitosis in Bombyx mori
  5. First cleavage mitosis in Bombyx eggs treated by low temperature
  6. Gonomery in other insects and arthropods
  7. The sea urchin and Ascaris types of the first cleavage
  8. Conclusions
  9. Acknowledgements
  10. References
  • Hatsumi, M. & Endow, S. A. 1992. Mutants of the microtubule motor protein, nonclaret disjunctional, affect spindle structure and chromosome movement in meiosis and mitosis. J. Cell Sci. 101, 547559.
  • Hawksworth, D. L. & Kalin-Arroyo, M. T. 1995. Magnitude and distribution of biodiversity. In Global Biodiversity Assessment (Ed. V. H. Heywood), pp. 107–191. Cambridge University Press, Cambridge.
  • Henking, H. 1892. Untersuchung über die ersten Entwicklungs vorgänge in den Eiern der Insekten. III. Zeitsch. F. Wiss. Zool. 54, 1274.
  • Huettner, A. F. 1924. Maturation and fertilization in Drosophila melanogaster. J. Morphol. 39, 249265.
  • Inoue, S., Borisy, G. G., Kiehart, D. P. 1974. Growth and lability of Chaetopterus oocyte mitotic spindles isolated in the presence of porcine brain tubulin. J. Cell Biol. 62, 175184.
  • Kawamura, N. 1978. The early embryonic mitosis in normal and cooled eggs of the silkworm, Bombyx mori. J. Morphol. 158, 5772.
  • Kawamura, N. 1979. Cytological studies on the mosaic silkworms induced by low temperature treatment. Chromosoma 74, 179188.
  • Lillie, F. R. 1901. The organization of the egg of Unio, based on a study of its maturation, fertilization, and cleavage. J. Morphol. 17, 227292.
  • Longo, F. J. 1973. Fertilization: A comparative ultrastructural Review. Biol. Reprod. 9, 140215.
  • Nachtsheim, H. 1913. Cytologische Studien über die Geschlechtsbestimmung bei der Honigbiene (Apis mellifica L.). Arch. F. Zellforsch. 11, 196241.
  • Riparbelli, M. G., Whitfield, W. G., Dallai, R., Callaini, G. 1997. Assembly of the zygotic centrosome in the fertilized Drosophila egg. Mech. Dev. 65, 135144.DOI: 10.1016/s0925-4773(97)00066-x
  • Rückert, J. 1895. Uber das Selbstandigbleiben der väterlichen und mütterlichen Kernsubstanz während der ersten Entwicklung des befluchteten Cyclops-Eies. Arch. Mikroskop. Anat.45, 3.
  • Rasmussen, S. W. 1977. The transformation of the synaptonemal complex into the ‘elimination chromatin’ in Bombyx mori oocytes. Chromosoma 60, 205221.
  • Schatten, H., Schatten, G., Mazia, D., Balczon, R., Simerly, C. 1986. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs. Proc. Natl Acad. Sci. USA 83, 105109.
  • Sonnenblick, B. P. 1950. The early embryology of Drosophila melanogaster. In Biology of Drosophila (Ed. M. Demerec), pp. 62–167. Hafner Publishing Co., New York.
  • Tanaka, H. 1992. Behavior of nucleus and mitotic apparatus during pronucleus formation, syngamy and cleavage in the egg of the cricket, Gryllus bimaculatus (De Geer). Master of Agriculture Thesis, Rakuno Gakuen University, Ebetsu, Japan (unpubl.).
  • Wilson, E. B. 1934. The cell in development and heredity, 3rd edn. Macmillan Co., New York.