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Hormonal control of ovarian development in the silkworm Bombyx mori.

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Archives of Insect Biochemistry and Physiology 5:167-177 (1987)
Hormonal Control of Ovarian Development in
the Silkworm, Bombyx mori
Kozo Tsuchida, Masao Nagata, and Akinori Suzuki
Departments of Agrobiology (K.T., M.N.) and of Agricultural Chemistry (A.S.),Faculty of
Agriculture, University of Tokyo, Bunkyo-Ku, Tokyo, Japan
Hormonal control of ovarian development was examined in Bombyx mori.
The weight of the ovary increased suddenly by 3 days after pupal ecdysis,
and vitellogenin could be immunologically detected in the ovary at that time.
The ecdysteroid titers during pupal-adult development, quantified by
radioimmunoassay, increased from day 0 to day 2. Ovarian development was
arrested for a long period in brainless pupae and isolated pupal abdomens.
injection of 20-hydroxyecdysone into such preparations stimulated development of t h e ovaries, and vitellogenin could be detected in ovaries 2 days
after injection. The results suggest that 20-hydroxyecdysone acts by
stimulating the growth of ovary.
Key words: vitellogenin, PTTH, ecdysteroid titer, ovarian development
The pharate pupa of the female silkworm Bombyx mori L., initiates the
production of eggs after somatic growth is completed. The production of
mature eggs involves the synthesis of a female-specific protein, vitellogenin,
in the fat body and its incorporation into the developing oocyte as vitellin.
Silkworm vitellin and vitellogenin have been identified by electrophoretic
and immunological techniques [1,2]. Although the vitellogenin may be transported by micropinocytosis [3-51, its uptake into the oocyte is highly selective
Hormonal control of oogenesis and vitellogenesis in insects has been
extensively reviewed by Hagedorn and Kunkel [7l and Engelmann [S]. In
Bombyx and saturniid silkworms, ovarian development occurs during the
pupal period and juvenile hormone is not involved, but ecdysteroids have
been shown to stimulate ovarian development [9]. Chatani and Ohnishi [lo]
reported that exogenous 20-hydroxyecdysone injected into the isolated pupal
Received September 4,1985; accepted January 22,1987.
Address reprint requests to Dr. Kozo Tsuchida, Department of Biochemistry, Bio Sci West,
University of Arizona, Tucson, AZ 85721.
0 1987 Alan R. Liss, Inc.
Tsuchida, Nagata, and Suzuki
abdomen also induced the initiation of ovarian development in B. mori. In
the early half of the pupal stage B. mori vitellogenin is detectable only in the
hemolymph, but in the latter half vitellin increases rapidly in the oocyte
while vitellogenin levels are maintained in the hemolymph [ll]. This suggests that ecdysteroids are involved in the uptake of vitellogenin into the
oocyte during pupal-adult development.
Because so little is known about the subject, this paper deals mainly with
the relationship of the endocrine system to ovarian development during
pupal-adult development of B. mori.
The bivoltine race of B. mori, Hosho, was employed. The eggs were
incubated at 25°C under LD*18:6, and newly hatched larvae were reared on
artificial diet at 25°C under LD 12:12. The pupae were kept at the same
conditions as the larval stages.
Surgical Techniques
Isolated abdomen. Fifth-instar mature larvae started spinning 8 days after
the last larval ecdysis and pupated 4 days later. From the third day of the
spinning period to the second day after pupation (day 1pupa), each female
was ligated between the thorax and abdomen by using a silk thread. The
insect was separated at the ligature and the cut surface was sealed with
melted wax. The isolated abdomens were kept at 25°C until needed.
Brain removal. From day 1 of the spinning period (ie, 3 days before
pupation) to within 12 h after pupation, brains were removed from diethylether-anaesthetized specimens. Larval brains were extirpated through a
small opening cut in the front of the head capsule. Pupal brains were
removed through a small opening cut between the bases of the antennae. A
few crystals of phenylthiourea were placed in the hemocoel, and the wound
was sealed with melted wax. The brainless pupae were kept at 25°C until
Implantation of brain and prothoracic gland. The recipients were anaesthetized-isolated abdomens and brainless pupae that had been prepared as
described above. The brains and prothoracic glands of female pharate pupae
were removed on day 2 of the spinning period, placed in Grace’s insect
saline, and then implanted into the abdominal segment of the anaesthetized
host female.
Into isolated abdomens 20-hydroxyecdysone (Rhoto Pharmaceutical Co.,
Osaka, Japan), dissolved in 2% ethanol (0.1, 1.0 or 10 pg/pl), was injected. A
*Abbreviations: DW = distilled water; LD = light/dark; PBS = phosphate-buffered saline
(NaCI 137 mM, Na2 HP04 3.22 mM, NaH2 PO4 1.28 rnM, pH 7.4); PlTH = prothoracicotropic
hormone; RIA = radioimmunoassay.
Hormonal Control of Ovarian Development
crude extract of PTTH was prepared as described by Suzuki et al. [12]. The
crude PTTH from either 2.5 heads or five heads of B. mori adult in 1pl of DW
was injected into debrained pupae. The crude extract of B. mori heads
contains two species-specific PTTHs, 4K-PTTH is active on Sumiu brainless
pupae and 22K-PTTH is active on Bombyx brainless pupae [13,14]. The crude
PTTH used in this experiment contained both species (5 unitslhead of 4KPTTH and 1unitlhead of 22K-PTTE-I).
Determination of Ovarian Development
Ovaries were collected every day from five female pupae. They were
rinsed in PBS and blotted on filter paper for several minutes, and their wet
weights determined.
Quantification of Ecdysteroids
The concentration of ecdysteroids in hernolymph was determined by RIA.
Antiserum against 20-hydroxyecdysone was prepared by the method of
DeReggi et al. [15] and [23,24-3H]ecdysone (New England Nuclear) was used
as the labeled ligand. The antiserum exhibited approximately equal affinity
for ecdysone and 20-hydoxyecdysone, but none for cholesterol. The assay
was conducted as follows: hernolymph (10 p1) was individually taken from
ten insects at 12-h intervals. Ecdysteroids were extracted with 300 pl of
methanol; the solvent was evaporated; and the residue was dissolved in 200
pl of DW. Fifty microliters of each sample was mixed with 50 pl of [23,24-3H]
ecdysone (about 5,000 cpm), and 100 p1 of antiserum solution, containing I/
500 the original concentration of the antiserum in borate buffer (0.1 M boric
acid, 0.05 M borax, 0.075 M NaCl, pH 8.4) was added. After reaction at 4°C
overnight, 200 pl of saturated ammonium sulfate was added and the mixture
was centrifuged. The precipitate was washed with 200 pl of 50% saturated
ammonium sulfate and then dissolved in 200 pl of DW and mixed with 2 ml
of scintillation cocktail (AmSCII, Amersham). Radioactivities were measured
for 10 min with a scintillation spectrophotometer; 20-hydroxyecdysone was
used as a standard and ecdysteroid amounts were expressed as 20-hydroxyecdysone equivalents.
Preparation of Protein Extracts for Electrophoresis and Immunodiffusion
The excised ovaries were homogenized with ten volumes (wlv) of PBS.
The ovary suspension was centrifuged at 7,000 g for 15 min at 5°C. Supernatants were stored at -80°C until time of analysis.
Immunodiffusion Analysis
Double-diffusion tests were performed according to the method of Ouchterlony [16] in 1.5% agar suspended in Veronal -NaC1 buffer (NaCl 145.3
mM, Veronal 3.1 mM, Veronal sodium 1.8 mM, pH 7.5). Antigen wells were
5 mm from each other. Diffusion was allowed to proceed overnight at 25°C.
The gels were then washed thoroughly in PBS.
Tsuchida, Nagata, and Suzuki
Acrylamide Gel Electrophoresis
Slab-electrophoresis of the protein samples were carried out in 7% polyacrylamide gel and 5 mM Tris-glycine buffer at pH 8.3. The gels were stained
with 0.5% of Coomassie brilliant blue and destained in 7% acetic acid.
Eedysteroid Titers
The ecdysteroid titers in the hemolymph of B. mori were determined by
RIA (Fig. 1).During pupal-adult development these titers changed significantly. They increased dramatically between day 0 (1,600 ngiml hemolymph)
and day 2 (2,810 t. 240nglml hemolymph). This result is essentially the same
as that obtained with the bioassay technique [17,18].
Ovarian Development of Brainless Pupae
Brain removal from female silkworm was performed each day from day 1
of the spinning period until shortly after pupal ecdysis. Almost all of the
pharate pupae which had the brain removed by day 2 of the spinning period
showed an arrested ovarian development. When the brain was removed on
day 3 of the spinning period, ovarian development started after pupation in
about 30% of the cases. At 10 days after pupation the mean ovarian weight
was 170 mglindividual, while the average weight in unoperated individuals
was approximately 500 mg. When the brain was removed within 2 h after
pupation, the ovaries began to undergo development in most cases, and by
10 days the mean ovarian weight increased to 210 mg. At 10 days after
pupation these brainless individuals showed apolysis and continuing development of the adult integument.
= 2
f l
days a f t s larval-pupal rtdyris
Fig. 1. Ecdysteroid titer (ng 20-hydroxyecdysone equivalent/ml) in the hemolymph during
female pupal-adult development. The data represent the mean f SD for ten animals at each
time point.
Hormonal Control of Ovarian Development
Three brains that had been removed from pharate pupae on day 2 of the
spinning stage were implanted into the abdomens of debrained pupae on 10
days after surgeon. The recipients developed and the weight of their ovaries
increased to a mean of ca 120 mg on the 10th day after implantation (Fig. 2).
Crude PTTH was injected into pupae that were in a state of arrested
ovarian development as a result of brain removal. As described in the Materials and Methods, the crude PTTH injection was carried out by using two
dosage levels: 2.5 headslF1 DW and 5 headslpl DW. The weight of ovaries of
the injected pupae started to increase at about 3 days after injection (Fig. 3).
In the case of the five-heads dose, the mean ovarian weight was about 100
mglpupa 10 days after injection. In the 2.5-heads dose, the ovaries developed
only to a mean weight of 35 mglpupa. Accordingly, it is apparent that the
m y s after implantation
Fig. 2. The effects of implanting of three brains ( V ) or one brain (B)
on ovarian development
in debrained female pupa. The open squares show the ovarian development of brainless
Days after injection
Fig. 3. Ovarian development obtained after injecting crude P T H extracts into brainless
abdomens of female pupae (CI,5 headslpl; V,2.5 heads/pI; 0 ,control).
Tsuchida, Nagata, and Suzuki
injection of crude P’ITH induces an ovarian development the degree of which
is dependent upon the dose injected.
Ovarian Development in Isolated Abdomens
Isolated abdomens were prepared by performing thorax-abdomen ligatures from day 3 of the larval spinning period to day 2 of the pupal stage. In
the case of ligatures performed up to day 0 of the pupal stage, no signs of
ovarian development were seen during the entire observation period (about
30 days). However, 70% of the isolated pupal abdomens ligatured on the
first day after pupation initiated ovarian development, and the ovarian weight
increased up to about 310 mglfemale by 10 days postpupation. In addition,
when the ligature was performed on day 2 of the pupal stage, almost all of
ovaries in the isolated abdomens showed growth, and continued to increase
in ovarian weight to a mean value of approximately 380 mglindividual on 10
days after pupation.
Isolated abdomens prepared by ligaturing within 12 h after pupation were
stored for 10 days at 25°C. Subsequently, three brains from day 2 spinningstage larvae were implanted into each isolated abdomen. The ovaries of the
isolated abdomens with implanted brains did not develop; the mean ovarian
weight was only 20 mglpupa, even on the 10th day after implantation.
However, when three brains and three prothoracic glands were implanted,
the weight of the ovaries increased, reaching a value of about 135 mglfemale
at 10 days after implantation (Fig. 4).
Isolated abdomens prepared by ligaturing within 12 h after pupation were
stored for 10 days and then injected with 1pl of solution containing 0.1, 1.0,
or 10 pg of 20-hydroxyecdysone. Regardless of the injected dose, the weight
of ovaries began to increase 2 days after injection. The development of
ovaries was positively correlated with the amount of hormone injected; at 10
days after injection the ovarian weight was 75 mglfemale in the 0.1-pg group,
120 mg in the 1.0-pg group, and 210 mg in the 10-pg group (Fig. 5).
Days after implantation
Fig. 4. Ovarian development in isolated abdomens of female pupae after implanting three
brains and three prothoracic glands ( 0 ), or three brains ( 0 ). The open triangles indicate
control values.
Hormonal Control of Ovarian Development
Days after injection
Fig. 5. Ovarian development obtained after injecting varying doses of 20-hydroxyecdysone
(0 ; 10 pg/ul, 0 ;1 pg/pI, V ;0.1 pglpl) into isolated abdomens of female pupa. When I pg/pl
of the juvenile hormone mimic ZR515 was injected (El), the ovary showed almost no
In contrast to these results, when 1pg/pl acetone of the juvenile hormone
mimic ZR515 was injected into each isolated abdomen, the ovaries showed
almost no development.
Effect of Crude PTTH and 20-Hydroxyecdysone on Ovarian Protein
The brain was removed on day 2 of the spinning stage. At 10 days after
this procedure, the debrained pupae were injected with crude PTTH (5
headslpl DW) or 20-hydroxyecdysone (1 pglpl 2% ethanol). Changes in
ovarian proteins after injections were monitored by polyacrylamide gel electrophoresis. In the case of the debrained pupae injected with the crude
PTTH, the amount of ovarian protein increased at 4 days after injection. In
regard to pupae injected with 20-hydroxyecdysone, the electrophoresis pattern was found to change more quickly than in pupae injected with PTTH
(Fig. 6 ) .
Hormonal Control of Incorporation of Vitellogenin Into the Ovaries
By using Ouchterlony’s agar-gel immunodiffusion [16], the relationship
between the ovarian incorporation of vitellogenin and 20-hydroxyecdysone
was investigated. In debrained pupae, no incorporation of vitellogenin was
detected even after several weeks. However, when crude MTH (5 headslpl
DW) or 20-hydroxyecdysone (1pg/pl2% ethanol) was injected into debrained
pupae, vitellogenin could be detected in the ovaries 2 or 3 days after injection
(Fig. 7).
In many insects, ovarian development is controlled by hormone(s) [S], and
juvenile hormone seems to be the primary hormone. However, in the case
Tsuchida, Nagata, and Suzuki
Days after injection
0 2 4 6 8 1012
0 2 4 6 8 1012
2 O - h ~ r o x ~ ~ y ~ ~
crude PT 1 H
Fig. 6. The effect of crude PTTH and 20-hydroxyecdysone on ovarian proteins of debrained
pupa. Pupae (ten days after debraining)were injected with crude PTTH or 20-hydroxyecdysone.
0 2 4
Days after injection
Fig. 7. Ouchterlony's immunodiffusion with ovary extract. Center well held vitellogenin
antiserum and both side wells hold ovary extract from debrained pupae. When 5 headslpl
crude PTTH extract (above) or 1 pglpl 20-hydroxyecdysone (below) were injected into the
debrained pupae, vitellogenin could be detected in the ovary 2 days after injection. Numbers
in parentheses show the days after debraining.
Hormonal Control of Ovarian Development
of the silkworm and other Lepidoptera, ecdysteroids have been reported to
play a central role in oQariandevelopment [9,18].
The present study revealed the following points: (1)In B. mori, the brain
secretes PTTH before pupation and thereby stimulates the prothoracic gland.
(2) The prothoracic gland secretes ecdysone; its concentration in the hemolymph reached a peak (ca 3 pglml) on day 2 of the pupal stage and then
decreased after day 4 to reach a low point (ca 5 nglml) on day 6 of the pupal
stage. (3) The weight of the ovary increases very suddenly after day 3 of the
pupal stage, and vitellin can be immunologically detected in the ovary on
this day.
It is known that if B. mori and other species of Lepidoptera are debrained
at a certain time during their development, adult differentiation is arrested,
and they become so-called "Dauer pupae" [19-211. According to Ishizaki
[21], the time of the critical period of MTH secretion for adult development
varies markedly among the races of B. mori, ranging from the feeding period
of the fifth-instar larva to shortly after larval-pupal ecdysis. In this study, the
Chinese race "Hosho" was employed, and if this race is debrained before
pupation, its adult development is totally arrested. In addition, if isolated
abdomens are prepared from the silkworm before pupation, adult development does not occur. Although ovarian development is arrested for a long
time in these debrained pupae and isolated abdomens, injection of 20-hydroxyecdysone enables the ovaries to develop. Moreover, the weight of the
ovary increases in direct proportion to the dose of 20-hydroxyecdysone
injected. This phenomenon is also related to an increase in number of mature
follicles; when the injected dose of 20-hydroxyecdysone was small, approximately half of the follicles in the ovariole were found to be absorbed. The
same results have been reported by Chatani and Ohnishi [lo], who also
observed that exogenous ecdysteroids are immediately inactivated following
injection into isolated abdomens, with no detectable activity on the second
day after the injection. On the basis of these findings it can be concluded
that ecdysteroids are essential for ovarian development in B. moui, and they
need to be secreted continously if ovarian development is to be completed.
This idea is supported by the ecdysteroid titers determined by RIA reported
here and elsewhere [El.
After the peak level of the ecdysteroids is achieved in the hemolymph, the
development of the ovary begins. At the same time, vitellogenin becomes
immunologically detectable in the ovary. Vitellogenin could not be found in
the ovaries of debrained pupae or isolated abdomens even 10 days after
operation, but there is immunological evidence for its presence in the ovary
on the second day after injection. This may mean that 20-hydroxyecdysone
acts on the follicular epithelial cells of the ovary to make them competent for
vitellogenin uptake.
In Drosophilu [23] and Sarcophuga [24] it is known that ecdysteroids stimulate the vitellogenin synthesis. Izumi and Tomino [25] reported that the
biosynthesis of vitellogenin in B. mori starts immediately after pupation, and
Ohno et al. [26] showed that ecdysteroid could stimulate the biosynthesis of
hemolymph proteins including female specific protein. While in B. mori it is
tempting to speculate that ecdysteroids may act on both the stimulation of
Tsuchida, Nagata, and Suzuki
vitellogenin biosynthesis by fat body and uptake of vitellogenin by ovary, we
have no evidence to support such a suggestion. However, it is clear that the
development of ovary in B. mori is induced by ecdysteroids, and the ovary
seems to undergo progressive development that accompanies vitellogenin
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