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

37

Oogenesis of Tetrodontophorabielanensis (Waga) (Collembola).
Preliminary Ultrastructural Studiesof the First Egg Envelope Formation*

Anna KRZYSZTOFOWICZ
andElzbieta KISIEL
Synopsis
Formation of the first egg envelope at final stage of oogenesis, by means of TEM was studied. The material for envelope accumulates in perioocytic space, between microvilli of oolemma.
At the same time, in peripheral layer of ooplasm of the oocyte numerous electron-dense vesicles
are observable. The formed first egg envelope is 4 p m thick and outer one 0.2 p m thick. The
outer layer is covered by concentrations, oval in shape, built of substance of very high electrondensity. Between these concentrations and follicular cell a structure is situated similar to the basal lamina. The ultrastructure of the first egg envelope, described in this paper, closely resembles
the ultrastructure of chorion described by W6jtowicz (1978).

Introduction
In the recent years, the formation and structure of the egg envelopes of Pterygota
have been subject of many studies (Furneaux and Machay, 1976; Hinton, 1981;
Margaritis, 1985; Regier and Kafatos, 1985), but the studies concerning Apterygota
are rather scanty.
According to Matsuzaki (1973) and Palevody (1976), the vitelline membrane in* Supported by Grant CPBP 04-0l-5-03.

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A. Krzysztofowicz and E. KisielCollembola is produced by oocyte, whether the chorion is produced by the cells of oviduct (Matsuzaki, 1973).
In this paper we describe the first egg envelope, produced by the oocyte at final
stage of oogenesis. Knowingly, we call it "first egg envelope", because at present it isdifficult to decide whether it represents vitelline membrane or chorion (see also Discussion).
.

Material and Methods
The specimens were collected in the vicinity of Krak6w. In the species studied,the process of oogenesis is synchronized in all egg chamber of the ovaries (Krzysztofowicz, 1971, 1975; Bilinski, 1976). The oogenesis begins in August and ends in Octoberor early November. The egg envelopes are formed by the end of oogenesis. In nature,
the temperature influences the oocyte maturation. The maturation takes place after
temperature dropping below - lOt, following 2 - 4 days with temperature above ot.
Such condition happens, however, at different time by the end of October or beginningof November, with fluctuation up to 14 days. This causes difficulty in collection of thematerial in proper phase of egg envelope formation and in understanding of their nature.
Adult females were decapitated and fixed in 2 % osmium tertoxide for 1.5 hr inphosphate buffer at pH 7.4 with saccharose (256 mg/lO ml of fixative). Following dehydration, in series of alcohol and aceton, the specimens were embedded in Epon 812.
Ultrathin sections were stained with· uranyl acetate followed by lead citrate, and examined in Tesla BS-500.

Results
By the end of oogenesis, the oocyte produces numerous microvilli on its surface.
Between microvilli a fibrous material appears of medium electron-density.
As the maturation of the oocyte progresses, the number of microvilli and amout offibrous material increase. At the same time, within the oocyte peripheral ooplasm
numerous vesicles form, filled with electron-dense 'substance. A part of these vesicles isconnected with oolemma of the oocyte, another part is lying deeper in periplasm. At
the level of connections with oolemma, the vesicles extrude the electron-dense substance outside of the oocyte into the perioocytic space (Figs. 1- 3). The extruded substance condenses and progressively fills the perioocytic space (Figs. 1-6).
The above described processes represent preparatory phase for the formation of
the first embryonic envelope.
During the preparatory phase, the follicular cells, surrounding the oocyte, themselves do not form microvilli, but their cytoplasm is rich in RER and electron-dense
granules. Similar granules are also observable in the perioocytic space. At the latephase of the first egg envelope formation (see below), the cytoplasm of the follicular
cells is poor in both RER and electron-dense granules (Figs. 5, 6), and finally thesestructures are not longer observable.

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39

Figs. 1. Successive stages of first egg envelope formation. (1 st of 6)
Fe, follicular cell; G, dense granule; MV, microvilli; 00, oocyte; PO, perioocytic
space. Note material for first egg envelope (asterisk) and vesicles with electrondense substance in peripheral ooplasm (arrows). X 49,000.

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A. Krzysztofowicz and E. KisielFigs. 2. Successive stages of first egg envelope formation. (2nd of 6)
BL, basal lamina; Fe, follicular cell; G, dense granule; MV, microvilli; 00,
oocyte; PO, perioocytic space; Y, yolk. X 39,000.

Oogenesis of Tetrodontophora bielanensis

Figs. 3. Successive stages of first egg envelope formation. (3rd of 6)EV, first egg envelope in formation phase; Fe, follicular cell; G, dense granule;00, oocyte; PO, perioocytic space. X 32,000.

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A. Krzysztofowicz and E. KisielFigs. 4. Successive stages of first egg envelope formation. (4 th of 6)EV, first egg envelope in formation phase; Fe, follicular cell; 00, oocyte; PO,perioocytic space; Y, yolk. X 30,000.

Oogenesis of Tetrodontophora bielanensis

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Figs. 5. Successive stages of first egg envelope formation. (5 th of 6)
BL, basal lamina; EV, first egg envelope; FB, fat body; Fe, follicular cell; MV,microvilli; 00, oocyte. X 30,000.

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A. Krzysztofowicz and E. KisielFigs. 6. Successive stages of first egg envelope formation. (6 th of 6)
BL, basal lamina; EV, first egg envelope; Fe, follicular cell; MV, microvilli;
00, oocyte. Note secretion from ooplasm into perioocytic space (arrows).
X 30,000.

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During the preparatory phase, the layer of material destined for the first egg envelope reaches the thickness of about 0.5 fL m. Above this layer upper portions of microvilli of the oocyte are observable, arranged in a chaotic pattern. Tips of some microvilli join together and form compact aggregations (Fig. 6).
The middle phase of the first egg envelope formation was not observed in thisstudy. The final phase is illustrated in Figs. 7 and 8.
The first egg envelope, at its final phase of formation, is about 4 fL m thick and iscomposed of two layers. The inner layer, neighbouring the oolemma of the oocyte, isabout 3.8 fL m thick and shows a high electron-density. The second layer, bordering
the perioocytic space, is only 0.2 fL m thick and shows much less electron-density. At
this final stage of first egg envelope formation, the oolemma develops numerous evaginations filled with peripheral ooplasm. The width of the perioocytic space measures
now about 0.6 fL m, and is enclosed by the element similar in structure to the basal
lamina, 0.4 fL m in thickness. Within the perioocytic space aggregations of heterogenic
material occurs, showing high electron-density (Fig. 7). Between these aggregations theflocculent material is loosely dispersed (Figs. 7, 8). Above the material, similar in
structure to the basal lamina, the oocyte is surrounded by the follicular cells showing
now signs of degeneration. The basal lamina of the follicular cells, at this time, is very
thin.

Discussion
Matsuzaki (1973) and Palevody (1976) studied the first egg envelope formation inseveral collembolan species and named it the "vitelline membrane" . According tothem, the collembolan eggs are covered also by the second envelope the chorion.
However, they did not studied the formation of the second envelope, only Palevody
speculated that chorion is produced in oviduct.
The egg of Tetrodontophora bielanensis is covered by two envelopes (W6jtowicz,1978): the vitelline membrane and chorion. The structure of the first egg envelope, described in this paper, is very similar to the chorion described by W6jtowicz. The only
difference concerns the absence of the pits on the surface of the first egg envelope described by W6jtowicz, but the oval structures shown in this paper in Fig. 7 may represent the pits in stage of formation.
The first egg envelope of T. bielanensis completely differs from the vitelline membrane described in the same species by Krzysztofowicz (1986a, b). It is therefore
reasonable to postulate that in the species studied, as the first egg envelope the chorion
is formed and then the vitelline membrane.
The process of formation of egg envelopes in other apterygotan orders than theCollembola is better known. Bitsch (1980) studied vitelline membrane and chorion
formation in Thysanura. According to him, both envelopes arise in the same manner as
in Pterygota. Protura were analysed by Bilinski and Klag· (1977) and Diplura by
Bilinski (1983), but only process of chorion formation was studies, which in both orders
is produced by follicular cells.
The process of the first egg envelope formation in T. bielanensis is similar to that
occuring in Myriapoda and Crustacea. Egg membrane formation of Myriapoda were46

A. Krzysztofowicz and E. KisielHE

Fig. 7.

Fragments of two egg chambers with first egg envelope. BLL, basal-like lamina;
EXL, external layer of first egg envelope; Fe, follicular cell; 1NL, Internal layer offirst egg envelope; Note oval heterogenic structures (asterisk) and between themflocculent material (arrows). X 18,000.

Oogenesis of Tetrodontophora bielanensis

Fig. 8.

47

Fragment of first oocyte envelope. BLL, basal like lamina; EXL, external layer offirst egg envelope; 1NL, internal layer of first egg envelope; 00, oocyte. Note invaginations of oolemma (arrows). X 18,000.

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A. Krzysztofowicz and E. Kisielstudied by Herbau (1974), however, the author named the first egg envelope as vitelline membrane, which according to him degenerates before chorion formation. The
chorion is formed by the epithelial cells of the ovary. According to Biliiiski (1981) in
Symphyla, precursors of chorion are most likely synthesized by follicle cells.
Crustacea were studied by Souty (1980), Zerbib (1980) and Talbot (1981).
However, both mentioned authors used very confusing nomenclature for naming egg
envelopes and because of this, at present any discussion concerning the analogy ofthese structures in Arthropoda is premature.

References
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- - - - - - , 1981. Ultrastructural studies on oogenesis in Symphyla. 11. Cortical granules andthe chorion. Cell Tiss. Res. 215: 431-436.
- - - - - - , 1983. Oogenesis in Campodea sp. (Insecta, Diplura). Chorion formation and theultrastructure of follicle cells. Cell Tiss. Res. 228: 165-170.
- - - - - - , and j. Klag, 1977. The oogenesis in Acerentomon gallicum jonescu (Protura). An
ultrastructural analysis of chorion formation. Acta BioI. Crac., Ser. Zool. 20: 101-106.
Bitsch, j., 1980. Ultrastructure de I'epithelium folliculaire et des enveloppes de l'ovocyte chez
Lepismachilis targionii (Grassi) (Thysanura: Machilidae). Int. j. Insect Morphol. Embryol. 9: 2540.
Furneaux, P. j. S. and A. L. Machay, 1976. The composition, structure and formation and thevitelline membrane of the insect egg-shell. In H. R. Herburn (ed. ), The Insect Integument,
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Herbau, C., 1974. Etude cytochemique et origine des envelop pes ovocytaires chez Lithobius forficantus (L. ) (Myriapode, Chilopode). Symp. Zool. Soc. Lond. 32: 237-247.
Hinton, H. E., 1981. Biology ofInsect Eggs. Vols. 1-3. Pergamon Press, Oxford, New York.
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Crac., Ser. Zool. 14: 299-305.
- - - - - - , 1975. Histochemical and ultrastructural analysis of blocking nurse cells in thefemale gonad of Tetrodontophora bielanensis (Waga) (Collembola). Acta BioI. Crac., Ser. Zool. 18:
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- - - - - - , 1986a. Ultrastructural studies on embryonic development of Tetrodontophorabielanensis (Waga) (Collembola). Formation of the 1st and 2nd blastodermal cuticles. Part I.
TEM studies. Acta BioI. Crac., Ser. Zool. 28: 19-26.
- - - - - - , 1986b. Ultrastructural studies on embryonic development of Tetrodontophorabielanensis (Waga) (Collembola). Formation of the 1st and 2nd blastodermal cuticles. Part II.
SEM studies. Acta BioI. Crac., Ser. Zool. 28: 27-31.
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Tomoceridae). Int. j. Insect Morphol. Embryol. 2: 335-349.
Palevody, C., 1976. L'Ovogenese chez les Collemboles Isotomides, Cytologie et Approche Physiologique. Doctoral thesis, Universite Paul Sabatier de Toulouse.
Regier, j. C. and F. C. Kafatos, 1985. Molecular aspect of chorion formation. In G. A. Kerkut
and L. I. Gilbert (eds. ), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 1, Embryogenesis and Reproduction, 113-151. Pergamon Press, Oxford, New York.
Souty, C., 1980. Electron microscopic study of follicle cell development during vitellogenesis in
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Talbot, P., 1981. The ovary of the lobster, Homarus americanus. 11. Structure of the mature follicle
and origin of the chorion. J. Ultrastruct. Res. 76: 249-262.
W6jtowicz, K., 1978. Fine structure of the egg chorion of Tetrodontophora bielanensis (Waga) (Collembola). Folia Bioi., Krakow 26: 323-327.
Zerbib, Ch., 1980. Ultrastructural observation of oogenesis in the Crustacea Amphipoda Orchestia
gammarellus (Pallas). Tiss. Cell 12: 47-62.

Authors'· address: Prof. A. Krzysztofowicz and MSc. E.
KisielDepartment of Systematic Zoology, Institute of Zoology, Jagiellonian University, ul. Karasia 6, 30-060 Krak6w,
Poland