/. Embryo/, exp. Morph. Vol. 38, pp. 49-62, 1977
Printed in Great Britain
49
Immunohistochemical studies
of vitellogenin during embryogenesis in the
cockroach Blattella germanica
ByAKIRA TANAKA1
From the Department of Biology, Faculty of Science,
Nara Women's University, Japan
SUMMARY
The fate of vitellogenin, vitellogenic female-specific protein, during embryogenesis in the
German cockroach was investigated by an immunofluorescent technique. The period of the
embryonic development of this insect is about 25 days at 25 ± 1 °C, and it has been divided
into 18 stages (Tanaka, 1976).
Vitellogenin-specificfluorescencewas evenly distributed among yolk spheres until stage 4.
The intensity of thefluorescenceof each yolk sphere, however, began to be uneven at stage
5, and the unevenness became more distinct in stages 6-7 to reach a mosaic appearance with
respect tofluorescence.After stage 8, the mosaic appearance disappeared, and the intensity
offluorescenceof the yolk region decreased on the whole as the stage advanced.
In vitellophages, no specificfluorescencewas observed until stage 6. Very strong fluorescence, however, became observable in some vitellophages at stage 7. All vitellophages became
fluorescent after stage 8. Some vitellophages, however, lost thefluorescenceafter stage 12,
though the strongfluorescencestill persisted in others until a fairly late stage when the cells
became shrunken.
The specificfluorescencewas observed in the yolk region only until stage 10. The nuclei of
the body region of the embryo, however, began to fluoresce very strongly at stage 11 when
dorsal closure had occurred. It persisted during stages 11-13, and abruptly declined after
stage 14. The cytoplasm of the body region also had comparatively weakfluorescenceat these
stages. In the appendageal region including the antennae and pleuropodia, the specific
fluorescence was not observed at all either in the nuclei or cytoplasm during development.
INTRODUCTION
Since the early publication of Telfer (1954) on the Cecropia silkworm, the
female-specific blood protein, vitellogenin, has been shown in many species of
insects (cf. Doane, 1973) including Blattella germanica (Tanaka, 1973), and
ample evidence has been accumulated on its fluctuation in blood, and sequestration by vitellogenic oocytes (cf. Telfer, 1965; Engelmann, 1970; Doane,
1973). The site of synthesis of vitellogenin has been shown to be the fat body
(Brookes, 1969; Engelmann, 1969; Pan, Bell & Telfer, 1969; Hagedorn
1
Author's address: Department of Biology, Faculty of Science, Nara Women's University, Nara 630, Japan.
50
A. TANAKA
& Judson, 1972), and the localization of vitellogenin in this organ was clearly
demonstrated immunohistochemically (Tanaka & Ishizaki, 1974).
Nothing is known, however, about the fate of vitellogenin during embryogenesis. Vitellogenin is presumed to be a reserve material which is utilized in
nutrition by the growing embryo, but this does not exclude other possible functions in embryogenesis.
It is of interest, therefore, to investigate the role of vitellogenin in the course of
the embryonic development. The present study forms the first step in revealing
what this role may possibly be by examining immunohistochemically the fate
of vitellogenin during embryonic development.
MATERIALS AND METHODS
Collection of oothecae
The cockroaches were reared at the temperature of 25 ± 1 °C with constant
supply of food and water. The rearing method was described in detail previously
(Tanaka, 1973). Oothecae of various developmental stages were removed carefully from their mothers and immersed in 90 % ethanol at - 80 °C. Only the
first ootheca in the reproductive cycles of each female was subjected to examination.
Fixation of embryos
The best method of fixation for usual histological studies was heating the
oothecae at 80 °C (Tanaka, 1976). For the present studies of immunohistochemical analysis, however, the heating method had to be avoided because this
destroyed the antigenicity of vitellogenin. The frozen oothecae in 90 % ethanol
at - 80 °C were brought under the dissecting microscope, and the oothecal capsule was carefully torn away with the aid of fine forceps. The batch of naked
embryos in the ethanol was stored at 4 °C. By so doing, the ethanol permeated
the tissue of the embryos as the temperature rose. The batch of the embryos
was then dehydrated and embedded in Tissuemat (melting point, 56-5 °C;
Fisher Scientific Co.). The blocks were stored at 4 °C. Sections were cut at 6 jum
and stained.
Preparation of anti-vitellogenin serum
Rabbit antiserum against the extract of mature ovaries of B. germanica was
prepared as described previously (Tanaka, 1973). Since this antiserum contained
antibodies against several antigens besides vitellogenin, it was absorbed with
acetone-dried powder prepared from whole male cockroaches which had been
starved for a day, to obtain vitellogenin-specific antiserum. The acetone
powder was mixed with 0-15 M saline and centrifuged to obtain a wet pellet. The
pellet was then mixed with the antiserum which had been diluted twice with 0-01
M phosphate-buffered saline, pH 7-1 (100 mg dry powder/ml antiserum). The
Immunohistochemistry of vitellogenin in Blattella
51
mixture was allowed to stand for 1 h and centrifuged. The above procedures
were repeated once more with fresh acetone powder. The immunological purity
of the anti-vitellogenin serum thus obtained was verified by immunoelectrophoresis (Tanaka & Ishizaki, 1974).
Preparation of FITC-conjugated antibody
An equal amount of saturated solution of ammonium sulphate was added to
the anti-vitellogenin serum. The precipitated globulin fraction was dissolved
with the saline at a concentration of about 10 mg/ml, and it was conjugated with
fluorescein isothiocyanate (FITC) (Baltimore Biological Laboratory, Inc.) in a
ratio of 100 mg globulin: 1 mg FITC by stirring in carbonate buffer, pH 9-5, for
4 h at 4 °C. Unreacted FITC was removed by Sephadex G-25 chromatography.
The conjugate was run through a DEAE-cellulose column in order to remove
antibodies with high negative charges. The molecular ratio of FITC to the
protein of the conjugate used for staining was 0-9-1-0.
Staining of the sections with FITC-conjugated antibody
The sections were deparaffinized and washed in three baths of 0-01 Mphosphatebuffered saline, pH 7-1. They were incubated at 37 °C in moist chambers with
the conjugate, which gave the best fluorescence when applied at a concentration
of about 1 mg/ml. The incubations lasted for 60 min at 37 °C and were followed
by three washings with the phosphate-buffered saline. The sections were mounted
in a mixture of the phosphate-buffered saline, glycerin, and polyvinyl-alcohol
(saponification value 86-89 mol. %) (Rodriguez & Deinhardt, 1960).
Observation and photography
The sections were examined in two different optical systems. One used an
ordinary fluorescence microscope equipped with a high-pressure mercury lamp,
and the other consisted of a conventional light microscope with a 40 W tungsten
lamp using FITC interference filter system (Tiyoda Optical Co. Ltd, Tokyo)
(Rygaard & 01 sen, 1971). Photographs appearing in this paper were taken
through the FITC interference filter system using Kodak Tri-X Pan Film.
Haematoxylin staining after immunohistochemical staining
After the immunohistochemical preparations were observed and photographed, the same sections were re-stained with haematoxylin to correlate the
observed fluorescence with structures in conventional histology. The sections
were first soaked in 0-01 M phosphate-buffered saline, pH 7-1, for a day. After
the cover-glass peeled off, the sections were carefully washed in three baths of
the phosphate-buffered saline and in water to remove the mounting medium,
and then stained with haematoxylin.
52
A.TANAKA
Control tests
In order to prove that the fluorescence observed after staining with labelled
antibody was due to an immunological reaction, the following control tests
were carried out. The intensity of the autofluorescence of embryonic tissue was
first examined at all stages of the development, and then the following control
staining tests were performed.
When the sections of representative stages, i.e. stage 6, 8, 11,14 and 16
embryos, were stained with simple FITC solution, the fluorescence was almost
at the level of the unstained controls in all stages examined. Therefore, the nonspecific staining with FITC itself was ruled out. Then absorption tests and blocking tests were carried out at stage 9 + and 11+ embryos. When the conjugated
antibody was absorbed with vitellogenic female blood before staining, the
intensity of fluorescence dropped almost to the level of the unstained control
(Fig. 4C). Treatment of the slides with unlabelled homologous antiserum prior
to staining greatly diminished the intensity of fluorescence. Further, when the
slides of stage 9 + and 11+ embryos were stained with FITC-conjugated antibody which was prepared against the antigen of non-cockroach origin, the
intensity of fluorescence was almost at the level of the unstained control. Thus,
the non-specific binding of immunoglobulin was ruled out. By the above-mentioned control tests, vitellogenin specificity of the fluorescence was confirmed,
and the following results were obtained.
RESULTS
The period of embryonic development of the German cockroach is about 25
days at 25 ± 1 °C. This period has been divided into 18 stages (Tanaka, 1976).
The fate of vitellogenin was investigated by an immunofluorescent technique
throughout these stages.
The eggs within the oothecal compartments assume the shape of half an elliptical disc, about 3 mm long, 1 mm wide and 0-3 mm thick. The orientation of
the embryo within the egg, which is shown in Fig. 1, does not change during
embryogenesis. The broken line indicates the plane of the cross-sections, and
the plane of the paper is parallel to that of the longitudinal sections.
Vitellogenin-specific fluorescence was evenly distributed among yolk spheres
from stage 1 to stage 4 (Fig. 2A). Yolk spheres, however, became somewhat
large and the intensity of fluorescence began to be uneven among spheres at
stage 5, when the segmentation of abdomen was completed; several spheres
having weak fluorescence appeared among other strongly-fluorescent spheres
(Fig. 2B).
A cross-section at the middle part of the embryo at stage 6 is shown in Fig.
2C. The embryo is observed at the top of the photograph and the remainder is
the yolk region. At this stage, the yolk spheres seemed to have coalesced to form
Immunohistochemistry of vitellognein in Blattella
53
Cephalic
Dorsal
Caudal
Lateral aspect of egg
Figure 1
fewer but larger yolk clumps. The weakly-fluorescent spheres increased in
number, and the strongly-fluorescent spheres decreased, to show a mosaic
appearance with respect to fluorescence.
A longitudinal section in the mid-dorsal part of the egg at stage 6+ is shown in
Fig. 2D and the same section after haematoxylin staining in Fig. 2E. As the
strongly-fluorescent spheres progressively decreased in number, the intensity of
the fluorescence was progressively increased. No difference was recognized between the weakly-fluorescent spheres and the strongly-fluorescent ones after
haematoxylin staining of the same section. Yitellophages which are identifiable
after haematoxylin staining had no fluorescence at this stage.
At stage 7, when the pleuropodia appeared, some vitellophages began to have
strong fluorescence (Fig. 3 A, B). At this time, the vitellophages in the ventral
yolk region tend to become fluorescent earlier than those in the dorsal yolk
region. A few yolk spheres in the dorsal region still had strong fluorescence.
At stage 8, all vitellophages came to have very strong fluorescence (Fig. 3C,
D). Meanwhile, the strong fluorescence observed in a few yolk spheres up to
stage 7 disappeared.
At stage 11, when the dorsal body wall had closed, the nuclei of the body region
of embryo began to shine strongly with vitellogenin-specific fluorescence (Fig.
54
A. TANAKA
Immunohistochemistry of vitellogenin in Blattella
55
4A, B). The yolk region is on the right, the body region of the embryo is at
centre, and the appendageal region of the embryo is on left in the photographs.
Very strong fluorescence was observed in the vitellophages in the yolk region
and in the numerous nuclei of the body region of embryo, and comparatively
weak fluorescence was observed in yolk spheres. No specific fluorescence was
observed in the appendageal region of embryo. A cross-section in the middle
part of embryo at stage 11 + is shown in Fig. 4D, E. Very strong fluorescence was
observed in the vitellophages and the nuclei of body region, but none in the
appendageal region.
At stage 12 some vitellophages began to lose their fluorescence (Fig 5 A, B),
those in the ventral region being affected sooner than those in the dorsal yolk
region. The nuclei of the body cells adjacent to the vitellophages, which had
lost their fluorescence, gave an intense reaction though their cytoplasm was only
weakly fluorescent (Fig. 5 A).
After stage 13, the fluorescence of body cell nuclei abruptly declined. A
cross-section in the middle part of the embryo at stage 14 is shown in Fig. 5C.
The fluorescence of the body cell nuclei has almost disappeared, and several
mycetocytes, which had somewhat strong autofluorescence, were observed in
the body of the embryo. The specific fluorescence was still observed in the vitellophages and yolk spheres which formed the contents of the midgut.
After stage 16, the strong fluorescence of vitellophages declined as they shrunk.
At stage 17, vitellophages had almost disappeared and the specific fluorescence
was observed in only a small part of the midgut (Fig. 5D). Many mycetocytes
were observed in the tissue of embryo.
The results of immunofluorescent staining in the various regions of embryo
are summarized in Table 1.
FIGURE 2
(A) Cross-section through the middle part of a stage-1 egg which was stained with
FITC-conjugated anti-vitellogenin serum (x 95). The upper end of the section is the
ventral end of the egg. Other photographs of the cross-section are similarly orientated.
(B) Cross-section of a stage-5 embryo (x95). Arrows indicate weakly-fluorescent
spheres.
(C) Cross-section of a stage-6 embryo (x 95).
(D) Longitudinal section in the mid-dorsal part of a stage-6+ egg (x 95).
(E) The same section as in photograph D stained with haematoxylin. Arrows
indicate vitellophages.
56
A. TANAKA
D
Fig. 3. (A) Cross-section in the middle part of a stage-7 embryo which was stained with
FITC- conjugated anti-vitellogenin serum (x 95).
(B) The same section as in photograph A stained with haematoxylin. Long arrows
indicate strongly-fluorescent vitellophages. Short arrows indicate negativelyfluorescent ones.
(C) Cross-section of a stage-8 embryo (x 95).
(D) The same section as in photograph C stained with haematoxylin.
Jmmunohistochemistry of vitellogenin in Blattella
Fig. 4. (A) Longitudinal section of the mid-ventral region of a stage-11 embryo
which was stained with FITC-conjugated anti-vitellogenin serum (x 95).
(B) The same section as in photograph A stained with haemotoxylin.
(C) Control staining. Cross-section of a stage-.ll+ embryo which was stained with
serum absorbed by vitellogenic female blood (x 95).
(D) Cross-section of a stage-ll+ embryo (x 95).
(E) The same section as in photograph D stained with haematoxylin. (a): appendage
region; (b): body region
57
58
A. TANAKA
(a)
A
Fig. 5. (A) Cross-section in the middle part of a stage-12 embryo which was stained
with FITC-conjugated anti-vitellogenin serum (x 95).
(B) The same section as in photograph A stained with haematoxylin.
(C) Cross-section of a stage-14 embryo (x 95).
(D) Cross-section of a stage-17 embryo (x95). (a): appendage region; (b): body
region.
...
0
1
3
4
5
6
7
8
9
10
12
13
14
16
17
20
21
11+ 12
12+ 13
14
14+ 16
16+
+
+
+
+
+
+
+
±
+ + + + + + + + + + + + + + +
+ + + + + +
+
±
±
+
+
+
+
—
_
—
—
_
_
_
_
_
_
_
_
_
11
The intensity of fluorescence was arbitrarily classified into four grades: —, negative; ± , weak; + , moderate; + + , strong.
Embryonic stage
1
1 + 2
3 + 5
6
7
8
9+11
Yolk
+ + + + + + + + + + + + + + +
+
+
Vitellophage
+ + + + + ' + +
Nucleus of body region
.
.
.
.
.
— + +
Cytoplasm of body region
.
.
.
.
.
—
—
_
—
+
Nucleus and cytoplasm o f .
.
.
.
_ _
_
_
_
_
appendageal region
Day at 25 °C
24
17
18
±
±
.
.
_ _
_
22
Table 1. Intensity of vitellogenin-specific fluorescence in the various regions of the embryo at different developmental stages
W
S'
s"
2
§.
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^
^
^
^
|
15-
©
60
A. TANAKA
DISCUSSION
Some interesting observations were made on the fate of vitellogenin during
embryogenesis. The first was that the vitellogenin-specific fluorescence,
which was distributed homogeneously in all yolk spheres at the beginning of
development, began to fade differentially among the spheres at stage 5, and the
differential fading proceeded to result in a mosaic appearance with respect to
fluorescence at stages 6 and 7. Histological differences, however, other than
intensity of fluorescence were not observed among the yolk spheres, at least
after haematoxylin staining.
The second observation of interest was that vitellophages became strongly
fluorescent after stage 7. This may indicate that the vitellophages, as their name
implies, sequester vitellogenin from the surrounding yolk spheres. When the
vitellophages began to fluoresce at stage 7, those in the ventral yolk region tended
to fluoresce earlier than those in the dorsal yolk region. A similar tendency
was also observed at stage 12 when some vitellophages lose their fluorescence.
Considering that the germ band originates in the ventral side of egg and the
body wall develops from there toward the dorsal side, these tendencies show that
the developmental sequence of the ventral region of egg precedes that of the
dorsal region during embryogenesis.
The third observation of interest was that strong fluorescence was observed in
the nuclei of the body region of the embryo in stages 11-13. Especially strong
fluorescence was observed in the nuclei near the vitellophages which lost their
fluorescence at stage 12. This fact suggests that the vitellogenin in the nuclei is
transported from the neighbouring vitellophages.
The author wishes to express his gratitude to Dr Hironori Ishizaki of Nagoya University
for critical reading of the manuscript, and to Professor Eiji Ohnishi of Nagoya University in
whose laboratory this work was performed.
REFERENCES
V. J. (1969). The induction of yolk protein synthesis in the fat body of an insect,
Leucophaea maderae, by an analog of the juvenile hormone. Devi Biol. 20, 459-471.
DOANE, W. W. (1973). Role of hormones in insect development. In Developmental Systems:
Insects, vol 2 (ed. S. J. Counce, & C. H. Waddington), pp. 291-497. London and New
York: Academic Press.
ENGELMANN, F. (1969). Female specific protein: biosynthesis controlled by corpus allatum in
Leucophaea maderae. Science, N. Y. 165, 407-409.
ENGELMANN, F. (1970). The Physiology of Insect Reproduction. Oxford and New York:
Pergamon Press.
HAGEDORN, H. H. & JUDSON, C. L. (1972). Purification and site of synthesis of Aedes aegypti
yolk proteins. /. exp. Zool. 182, 367-377.
PAN, M. L., BELL, W. J. & TELFER, W. H. (1969). Vitellogenic blood protein synthesis by insect
fat body. Science, N. Y. 165, 393-394.
RODRIGUEZ, J. & DEINHARDT, F. (1960). Preparation of a semipermanent mounting medium
for fluorescent antibody studies. Virology 12, 316-317.
RYGAARD, J. & OLSEN, W. (1971). Determination of characteristics of interference filters. Ann.
N. Y. Acad. Sci. Ill, 430-433.
BROOKES,
Immunohistochemistry of vitellogenin in Blattella
61
A. (1973). General accounts on the oocyte growth and the identification of vitellogenin by means of immunospecificity in the cockroach, Blattella germanica (L.). Devi
Growth & Differ. 15, 153-168.
TANAKA, A. (1976). Stages in the embryonic development of the German cockroach, Blattella
germanica (L.) (Blattaria: Blattellidae). Kontyu, Tokyo, 44, 512-525.
TANAKA, A. & ISHIZAKI, H. (1974). Immunohistochemical detection of vitellogenin in the
ovary and fat body during a reproductive cycle of the cockroach. Blattella germanica. Devi
Growth & Differ. 16, 247-255.
TELFER, W. H. (1954). Immunological studies of insect metamorphosis II. The role of a sexlimited blood protein in egg formation by the Cecropia silkworm. /. gen. Physiol. 37, 539558.
TELFER, W. H. (1965). The mechanism and control of yolk formation. Ann. rev. Entomol. 10,
161-184.
TANAKA,
(Received 20 April 1976)
EMB 38