Recent Advances in Insect Embryology in Japan and Poland
Edited by H. Ando and Cz. Jura
Arthropod. EmbryoL. Soc. Jpn.
(ISEBU Co. Ltd., Tsukuba) 1987

181

Structure of Egg Cortex Relating to
Presumptive Embryonic and Extraembryonic Regionsin Silkworm, Bombyx mori (Bombycidae: Lepidoptera)

Y oshihiro KO BAYASHI
andKeiichiro MIY A
Synopsis
In the egg cortex of the mature Bombyx egg, large pyronin-positive granules exist attachedto the periplasm. These granules are not distributed evenly, but are localized in the definite region. With the beginning of development, these granules break down and the regional differenceof the egg cortex disappears. However, this distribution pattern in the mature egg coincides withthe extent of the embryonic and extraembryonic regions at the early germ band stage, and thisfact suggests that the granules might play an important role for differentiation of both regions.

Introduction
Ooplasm of the insect egg usually consists of a cortical thin layer, the periplasm,
including no yolk spheres, and the reticuloplasm, a network containing yolk spheres in
its mesh. Of these two cytoplasmic structures, the periplasm seemed to contributechiefly to organization of the basic body pattern of the embryo and have been studied
from various 'viewpoints, resulting in establishment of fate map in some species (seeSander, 1976). However, from the morphological point of view, researches of structures
essential for embryonic pattern formation hardly succeeded, except for some structures,
one of which was the posterior polar plasm relating to the pole cell segregation. Especially in Drosophila, structure and function of the posterior polar plasm have been studied vigorously and many interesting results are reported (see Okada, 1986).
In Bombyx, Takami (1942, 1946) analyzed the embryonic development with cauter-182

Yoshihiro Kobayashi and K. Miya

ization method and made a rough fate map. Subsequently, Miya (1958) pursued the
problems on germ cell differentiation with the same method and suggested the region
and the time of germ cell segregation, but any characteristic structure similar to theposterior polar plasm could not been confirmed. Afterward, Suzuki (1969) found thatpyronin-positive granules distributed within the egg cortex in the newly-laid egg andobserved their change with the progress of development, and Miya (1978) studied theirultrastructure and obtained the conclusion that these granules were a kind of roughsurfaced endoplasmic reticulum, rER, constructing concentric whorl. Furthermore,
Yamauchi is studying their origin by electron microscopy (personal communication).
The granules are not distributed evenly within the egg cortex, but are localized at the
specific region. The present paper deals with the distribution pattern of these granules.

Material and Method
For light microscopic observation, non-diapause eggs of the strain "Daizo" wereused as material. The eggs were fixed with Carnoy's fluid and cut 6 - 15 f1 m thickwith the usual paraffin method. The sections were stained with Mayer's acid hemalumor Kurnick's methylgreen-pyronin. As the counterstaining, the sections were treatedin RNase solution diluted with buffer of five thousand times at 55°C for 1 hr. In orderto calculate the extent of the region where the pyronin-positive granules exist in themature egg and the germ anlage appears at the early germ band stage, the sections
were transcribed by a projector and measured by a curvimeter.
For electron microscopy, the same method as in the previous report was used
(Miya, 1978).

Results
1. Distribution of the pyronin-positive granules in mature egg
The mature egg is covered by the pyronin- and hematoxylin-negative vitellin
membrane lying just under the chorion. The egg cortex is composed of thehomogeneous layer stained lightly with pyronin and hematoxylin, and the region containing large pyronin- and hematoxylin-positive granules. These granules sometimes
coagulate together with the surrounding cytoplasmic inclusions to form very large
structures.
The granules are distributed unevenly within the egg cortex. In the cross section
the granules are distributed numerously in both lateral sides and only few and/or smaller granules are observed in the ventral side, but hardly any exist in the dorsal side ofthe egg (Figs. 1, 2, 6 B). In the frontal section the granules are observed in both lateralsides, within the range of about 10-90% of the egg length from the posterior pole, but
in the anterior and posterior regions granules hardly exist (Fig. 6 A).
In the anterior region a pyronin-positive discoidal structure is recognized and thisstructure is correspondent to the specialized arrangement of rER (Miya, 1978, 1984,1985). Figures 3 and 4 represent the ultrastructure of the pyronin-positive granules.

Differentiation of embryonic and extraembryonic regions in Bombyx

183

2
Fig. l. Cross section of the mature egg. Ds: dorsal side, Ls: lateral side, Vs: ventral side ofthe egg. X 60.
Fig. 2. Magnified figure of the lateral side of the egg shown in Fig. 1. Pg-, pyronin-positive
granule. X 240.
These granules are constructed with large concentric whorls of rER, in which no
mitochondria exist, but sometimes dense organelles connecting with rER are observed(Fig. 4, Ea). Figure 5 is an electron micrograph showing the enlarged structure of therER.
As shown in Fig. 6, when the extent of the egg cortex without pyronin-positive
granules is indicated as the ratio for the entire circuit, the following values are
obtained: 20.3 ± 1.7 % for the anterior polar region, 16.8 ± 0.8 % for the posteriorpolar region, and 15.6 ± 1.6 % for the dorsal side of the egg.

2. Structural change of the pyronin-positive granules with beginning of development
Electron-microscopically, the first structural change by entry of sperms into theegg appears in the vitellin membrane and a distinct fertilization membrane is formed(Miya, 1978, 1984). However, from light microscopical observation, resumption of thematuration division, which rests at the metaphase of the first division, is characteristic
and the direction of the spindle becomes from parallel to vertical to the egg surface.
Fifteen to 30 minutes after oviposition, the division comes at anaphase and then about
1 hour after the second division begins. At this stage the structure of egg cortex
changes remarkably; the vitellin membrane changes to become the fertilization membrane and the homogeneous layer stained lightly disappears, resulting in approach of

184

Y oshihiro Kobayashi and K. Miya

Figs. 3, 4. Electron micrographs representing egg cortex of the mature egg. L, lipid droplet; rER, rough-surfaced endoplasmic reticulum; Vm, vitellin membrane; Vs,
small yolk sphere. Scale: 1 f-L m.

Differentiation of embryonic and extraembryonic regions in Bombyx

Fig. 5.

185

Electron micrograph representing the magnified concentric whorl of rER. Scale: 1,urn.

the pyronin-positive granules just under the fertilization membrane. With the progressof development, the pyronin-positive granules disappear and a relatively homogeneous
layer stained with pyronin becomes recognizable under the fertilization membrane(Figs. 7, 8). Figures 9 and 10 are electron micrographs representing this process; thelarge whorl of rER attaching to the periplasm begins to break down to fuse together
with the compoments of the periplasm and at the same time the cisternae of rERchange from the lamellar to the tubular and/or vesicular structure.

3. Penetration of the cleavage nuclei into the periplasm and formation of the blastoderm and the germanlageAbout 12 hours after oviposItIOn, the cleavage nuclei enclosed with cytoplasmic
coat reach the egg cortex and penetrate into the periplasm, but this process does notoccur at the same time in all region of cortex as reported by Miya (1958). In the present observation the cleavage nuclei penetrate into the periplasm at the lateral sides
near the anterior pole at first, and then the penetration extends posteriorly and dorsally, and to the posterior pole at last. After penetration of the cleavage nuclei into theperiplasm, nuclear divisions parallel to the egg surface occur and the cell membrane isformed around each nucleus in the same order as of penetration into the periplasm. Figures 11 - 14 represent this situation in the cross section; formation of the cell membrane begins at the lateral side (Fig. 12), on the other hand, the penetrating nuclei areYoshihiro Kobayashi and K. Miya

186~: xI00=20.3:1:1.7 (oto)
Ls

l~ X
Ls

100 = 15.6 :t 1.6 (°/0)

A
Fig. 6.

Pp~: X 100= 16.8:t0.8 (0/0)

Diagrammatic figures of the frontal (A) and cross (B) sections representing distribution pattern of the pyronin-positive granules. Ap: anterior pole, Pp: posteriorpole, Ds: dorsal side, Ls: lateral side, Vs: ventral side of the egg, Nc, Lc: length ofthe entire circuit, mc, nc, le: extent of the"regions lacking pyronin-positive granules
at the anterior, posterior and lateral sides of the egg, respectively.

Fig. 7. Cross section of the 3-hours old egg. X 240.
Fig. 8. Cross section of the 7-hours old egg. Cn, cleavage nucleus. X 240.

Differentiation of embryonic and extraembryonic regions in Bombyx

187

Figs. 9, 10. Electron micrographs representing structural change of rER. Ch, chorion; Ea,
dense organelle connecting with rER; Fm, fertilization membrane; L, lipid droplet;rER, rough-surfaced endoplasmic reticulum; Vs, small yolk sphere. Scale: I fL m.

Yoshihiro Kobayashi and K. Miya

188

13
Figs. 11-14. Cross section (11) and its magnified figures at the lateral (12), ventral (13)and dorsal (14) sides of the 13-hours old egg. Ds: dorsal side, Ls: lateral side,
Vs: ventral side of the egg. Fig. 11, X 60; Figs. 12-14, X 120.

Differentiation of embryonic and extraembryonic regions in Bombyx

189

,;;,\
Vs:,

,~

~

,ILs

•r

Ls

J

','

I.

\ .~~
Os

Figs. 15 -18. Cross section (15) and its magnified figures at the lateral (16), ventral (17)and dorsal (18) sides of the 15-hours old egg. Ds: dorsal side, Ls: lateral side,
Vs: ventral side of the egg. Fig. 15, X 50; Figs. 16-18, X 120.

190

Yoshihiro Kobayashi and K. Miya

Figs. 19, 20. Electron micrographs representing the cell of the germ anlage (19) and thecell of the extraembryonic region (20). L, lipid droplet; Mit, mitochondria; N,
nucleus; Nu, nucleolus; Vc, vacuole; Vs, small yolk sphere. Scale: 1 J1 m.

Differentiation of embryonic and extraembryonic regions in Bombyx

191

few and formation of cell membrane does not occur yet at the dorsal side (Fig. 14).
During this process one or two nucleoli appear in the. nucleus, suggesting beginning ofactivation of genes originated from the zygote. In Bombyx the above process is not carried out by formation of typical cleavage furrow, but by protrusion of the cleavage nuclei beyond the initial level of the egg surface (Takesue et al., 1980; Takesue, 1985).
Through the above-mentioned processes the cellular blastoderm is completed, but
the structure of the blastoderm cells is different corresponding to the egg regions; thecells lying at the ventral and both lateral sides of the egg are smaller in size and are
distributed densely, while the cells at the dorsal side are larger and exist separately.
The former cells are the cells of the embryonic region, which form the germ anlage later, and the latter cells are the cells of the extraembryonic region, which develop theembryonic envelope (Figs. 15-18).
Figures 19 and 20 are electron micrographs representing the cell of the germanlage and that of the extraembryonic region, respectively. The latter is larger andcontains more numerous mitochondria, vacuoles, and other cytoplasmic inclusions than
the former one.
As shown in Fig. 21, when the extent of the extraembryonic region in the crossand frontal sections is indicated as the ratio for the entire circuit, the following values
are obtained: 21.8 ± 5. 7 % for the anterior region, 14.4 ± 1.6 % for the posterior
region, and 13. 7 ± 5.2 % for the dorsal side of the egg. When these values are compared with those of corresponding values shown in Fig. 6 and examined by Hest, in allcases any significant differences are not recognized. Consequently, the egg cortex inwhich pyronin-positive granules are distributed seems to pre-indicate structurally theembryonic region in the mature egg.

Discussion
The periplasm of the insect egg has been considered to play an important role forformation of the basic body pattern of the embryo and the fate map is described inseveral insect species (e. g. Drosophila, Poulson, 1950; Tenebrio, Anderson, 1972; Sander,1976). However, relation between the egg architecture and the basic body pattern has
not been clarified, except for the posterior polar plasm and the anterior polar plasm.
Especially, as to the function of the posterior polar plasm for formation of the polecells, many investigations have been carried out from the various points of view, andOkada (1986) reviewed recently many results on cytoplasmic function of the posteriorpolar plasm for segregation of germ line in Drosophila. On the other hand, as to theanterior polar plasm, relation between its architecture and function is not analyzed
enough. Zissler and Sander (1973, 1982) reported in Smittia egg that the periplasm
protrudes inward as a cone-shaped plug at the anterior pole, and Ripley and Kalthoff(1983) obtained from the temporal change of the ratio of the double cephalons, induced
by UV-irradiation, the following results; the cytoplasmic ribonucleoprotein particles existing in a yolk-free cytoplasmic cone observed behind the anterior pole of the newly
laid egg, diffused posteriorly with the progress of development, and then migrated into
the periplasm of the anterior part of the egg.
In Bombyx there is no specialized structure within the periplasm except the anter-Yoshihiro Kobayashi and K. Miya192

ior polar plasm, but it has been considered that the periplasm seemed to play an important role during the early embryogenesis, the same as in other insect. Takami(1942, 1946) analyzed the role of the periplasm by cauterization and suggested the existence of the regional difference within the periplasm, Miya (1958) obtained the similar possibility as to the germ cell differentiation by the same procedure, and Kobayashi(1980, 1981) suggested the significance of the periplasm for the early embryogenesis by
UV -irradia tion.
In the anterior polar plasm of Bombyx egg, arrangement of the various organelles,
especially rER, is different from that in the other region of the periplasm (Miya, 1978,
1984, 1985), but this specialized structure seems to relate to reception of sperms, andthe evidence concerning to determination of the longitudinal body pattern as in Smittia
was not obtained yet.
The pyronin-positive large granules, a characteristic structure seen only in the mature egg of Bombyx, are distributed widely, attached to the periplasm and dispersedwith activation of the egg by sperm entry (Suzuki, 1969). These granules constitutelarge concentric whorls or rER electron-microscopically, and the stacks of cisternaechange to the tubular and/or vesicular structure with entry of sperms. From the present results, these structures do not distribute evenly with the egg cortex, but seem tobe absent in the presumptive extraembryonic region lying at the anterior, posterior and

;:x

Ap

100=21.8:t5.7 (~O)

Ls...

.

Pp
Fig. 21.

Lb

Vs

..

Ls

l:

nbXI00=14.4:1: 1.6 (~o)
Nb

XI00=13.7:t5.2("'0)

Diagrammatic figures of the frontal (A) and cross (B) sections representing theembryonic and extraembryonic regions. Ap: anterior pole, Pp: posterior pole, Ds:
dorsal side, Ls: lateral side, Vs: ventral side of the egg, Nb, Lb: length of the entire circuit, mb, nb, Ib: extent of the extraembryonic region at the anterior, posterior and lateral sides of the egg, respectively.

Differentiation of embryonic and extraembryonic regions in Bombyx

193

dorsal side of the egg. Furthermore, the distribution pattern of these granules coincides
with that of the germ anlage and the presumptive embryonic envelope at the earlygerm band stage. Though future investigation will be necessary, the unequal distribution of rER is one of the regional differences of the periplasm and this architecture
might play an important role for formation of the basic body pattern of the embryo,through qualitative and/or quantitative differences of protein synthesis.

References
Anderson, D. T., 1972. The development of holometabolous insects. In S. J. Counce and C. H.
Waddington (eds. ), Developmental Systems: Insects, Vo!. I, 165-242, Academic Press, London, New York.
Kobayashi, Y., 1980. Effects of UV irradiation on the early embryonic development in the silkworm, Bombyx mori. I. Relation between development and stages or egg regions irradiated (inJapanese).]. Seric. Sci.jpn. 49: 197-204.
- - - - - , 1981. Effects of UV irradiation on the early embryonic development in the silkworm, Bombyx mori. 11. Development of irradiated eggs (in Japanese). ]. Seric. Sci. jpn. 50:
51-58.
Miya, K., 1958. Studies on the embryonic development of the gonad in the silkworm, Bombyx
mori L. Part I. Differentiation of germ cells.]. Fac. Agr., Iwate Univ. 3: 436-467.
- - - - - , 1978. Electron microscope studies on the early embryonic development of the silkworm, Bombyx mori. I. Architecture of the newly laid egg and the changes by sperm entry. ].
Fac. Agr., Iwate Univ. 3: 436-467.
- - - - - , 1984. Early embryogenesis of Bombyx mori. In R. C. King and H. Akai (eds. ),Insect Ultrastructure, Vo!. 2, 49-73. Plenum, New York.
- - - - - , 1985. Determination and formation of the basic body pattern in embryo of thedomesticated silkmoth, Bombyx mori (Lepidoptera, Bombycidae). In H. Ando and K. Miya(eds. ), Recent Advances in Insect Embryology in Japan, 107-123. Arthropod. Embryo!. Soc.
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Okada, M., 1986. Cytoplasmic function segregating germ line in Drosophila embryogenesis. Zool.
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Poulson, D. F., 1950. Histogenesis, organogenesis and differentiation in the embryo of Drosophilamelanogaster Meigen. In M. Demerec (ed. ), Biology of Drosophila, 168-274. Wiley, NewYork.
Ripley, S. and K. Kalthoff, 1983. Changes in the apparent localization of anterior determinants
during early embryogenesis (Smittia spec., Chironomidae, Diptera). Roux's Arch. Devl. Biol.
192: 353-361.
Sander, K., 1976. Specification of the basic body pattern in insect embryogenesis. Adv. Insect. Physiol. 12: 125-238.
Suzuki, K., 1969. Distribution and Change of Pyronin-positive Granules during the Early Development in the Silkworm, Bombyx mori (in Japanese). B. A. thesis, Fac. Agr., Iwate
Univ.
Takami, T., 1942. Experimental studies on the embryo formation in Bombyx mori. I(in Japanese).
Zool. Mag. 54: 337-343.
- - - - - " 1946. Experimental studies on the embryo formation in Bombyx mori. V. Presumptive mesodermal and neural regions of the egg (in Japanese). Seibutsu I: 208-211.
Takesue, S., 1985. Blastoderm formation in the silkworm, Bombyx mori (Lepidoptera, Bombycidae). In H. Ando and K. Miya (eds. ), Recent Advances in Insect Embryology in Japan,
125-129. Arthropod. Embryo!. Soc. Jpn. (ISEBU Co. Ltd., Tsukuba).
- - - - , H. Keino and K. Onitake, 1980. Blastoderm formation in the silkworm egg (Bombyx
mori L. ).]. Embryol. Exp. Morphol. 60: 117-124.

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Yoshihiro Kobayashi and K. Miya

Zissler, D. and K. Sander, 1973. The cytoplasmic architecture of the egg cell of Smittia spec. (Diptera, Chironomidae). I. Anterior and posterior pole regions. Roux Arch. EntwMech. Org. 172:
175-186.
- - - - - , and, 1982. The cytoplasmic architecture of the insect egg cell. In R.
C. King and H. Akai (eds. ), Insect Ultrastructure, Vol. I, 189-221. Plenum, New York.

Authors' addresses: Dr. Y. Kobayashi
Seikatsu-Gakuen
High
School,Mitake 2-13-1, Mrioka, Iwate 020,
Japan
Dr. K. Miya, Prof. Emeritus (Iwate
University)
Yamagishi 3-11-30, Morioka, Iwate
020,Japan