879 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 877–884
6-n-propyl-2-thiouracil PTU to inhibit deiodinases. The supernatant remaining after centrifuging at 1500g
was used for RIA. For more quantitative determinations, tissues or food were homogenised in 3 ml of phosphate
buffer containing PTU and 2.5 mg pronase. The homo- genate was incubated at 37
° C for 12 h, and then 1.5 ml
of NH
4
OH were added. The supernatant from centrifug-
ation at 1500g was evaporated to dryness and the residue taken up in 1 ml of distilled water for processing by
RIA. Haemolymph was not extracted prior to assay. RIA was performed using commercial kits Inter-med-
ico, Markham, Ont. for total T4 and T3. The standards supplied in the kit were used for most samples. Dilution
curves of gut contents and food were linear and parallel to the standard curves. For quantitation of haemolymph
T3, a standard was prepared based on locust haemo- lymph. Twenty millilitres of haemolymph were extracted
from locusts through a small incision in the cervical membrane. A small quantity a few crystals of phenyl
thiourea to inhibit darkening, and 0.1 sodium azide to inhibit bacterial growth were added. The haemolymph
was subjected to extraction with activated charcoal fol- lowed by centrifugation at 1550g to remove any T3, and
the resulting supernatant used as a solvent in preparing the standards of T3.
3. Results
3.1. The action of other thyroid hormone derivatives on cell volume
While T3 is active in inducing reduction in cell vol- ume, 3
9,59,3-triiodothyronine reverse T3, rT3 is devoid of activity at all concentrations tested Fig. 1. However,
T2 is extraordinarily active, causing significant P ,0.02
reductions in cell volume at concentrations as low as
Fig. 1. The effect of reverse T3 on the optical path difference here
expressed as degrees of rotation of the polarising analyser of follicle cells in vitro. Each bar represents the mean of determinations on 25
cells, and the vertical lines indicate the standard error of the mean. Fig. 2.
The effect of T2 3,5-diiodothyronine on the optical path difference of follicle cells in vitro. Data treated as in Fig. 1.
0.01 nM, with optimal activity appearing at 0.1–1.0 nM, two orders of magnitude lower than the optimum of 100
nM for JH III or T3 Fig. 2. Only two other diiodo derivatives are available. One of these, 3,3
9-diiodothy- ronine, is weakly active, producing a significant increase
in optical path difference only at 1.0 µ
M P ,0.01,
while the other derivative 3 9,59-diiodothyronine is
inactive Fig. 3. A precursor of T4, 3,5-diiodotyrosine, is also inactive data not shown.
3.2. The uptake of rho-T3 When follicle cells are exposed in vitro to rho-T3 at
1 µ
M, the cells contain fluorescent material when exam- ined after 1 h by epifluorescence microscopy. This flu-
orescence is too weak to photograph, but the fluor- escence can be detected by confocal microscopy, where
Fig. 3. The effect of other diiodothyronines on the optical path differ-
ence of follicle cells in vitro. Data treated as in Fig. 1.
880 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 877–884
Fig. 4. Confocal image of a vertical section derived from a z series
through the follicular epithelium of an ovariole exposed to 1 µ
M rho- T3 for 90 min. The basal haemocoel side of the epithelium is at the
top of the photograph. The clear spaces indicated by n represent the nuclei of the cells. The line represents 3
µ m.
the rho-T3 is seen to be in the form of vesicles Fig. 4. While the fluorescence in a conventional microscope is
weak, it can be detected by the fluorometer attached to the microscope. Fig. 5 depicts a typical result of an
experiment in which follicles were exposed to rho-T3 at room temperature and on ice, and were assessed 1 h
later. In the same experiment some follicles were pre-
Fig. 5. The fluorescence of follicular epithelium exposed in vitro to
1 µ
M rho-T3 for 60 min. C, control; ICE, exposed at 0 °
C; CHX, pre- incubated in 1 mM cycloheximide for 30 min before exposure to rho-
T3 at room temperature. The fluorescence units are arbitrary, with the settings on the photometer adjusted so as to give a zero reading for
epithelium not exposed to rho-T3. Each bar represents the means of determinations made on at least five follicles, and the vertical lines
indicate the standard error of the mean. Fig. 6.
The fluorescence of follicular epithelium exposed to 1 µ
M rho-T3 for 60 min control or pre-incubated in 100 nM JH III or JH
I. See Fig. 5 for details.
incubated for 30 min in 1 mM cycloheximide and then placed in cycloheximide and rho-T3 for 60 min. While
those follicles exposed at room temperature contained abundant fluorescence, those exposed at 0
° C showed
very little uptake. Incubation with cycloheximide also blocked uptake.
When follicles are exposed to rho-T3 in the presence of JH III at 100 nM, uptake is markedly inhibited
P ,0.01 but when JH I is included as the competing
ligand, there is no effect Fig. 6. When the follicles are pre-incubated in an antibody raised in rabbits against the
35 kDa membrane binding protein for JH III for 30 min before
adding rho-T3,
uptake is
markedly less
P ,0.001 than for follicles not exposed to the antibody
Fig. 7. Although non-immune serum was not included in the protocol of this experiment, separate experiments
using non-immune rabbit serum detected no effect on the uptake of rho-T3 or on the JH or T3 stimulation of
the OPD data not shown.
Fig. 7. The fluorescence of follicular epithelium exposed to 1
µ M
rho-T3 for 60 min control or pre-incubated for 30 min in an anti- serum 1:1000 against the putative membrane receptor for JH III
before exposure to rho-T3. See Fig. 5 for details.
881 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 877–884
Fig. 8. The effect of pre-incubation in 1
µ M 6-n-propyl-2-thiouracil
on the stimulation by T3 or T2 of the optical path difference of follicle cells in vitro. Data treated as in Fig. 1.
3.3. The effect of inhibitors of 5 9-deiodinase
When follicle cells are pre-incubated in 1 µ
M PTU for 30 min before being exposed to T3 or T2, the
capacity for T3 to induce a reduction in cell volume is eliminated, while T2 retains its capacity, albeit at a
reduced level, to cause a significant P ,0.01 increase
in OPD Fig. 8. Similarly, exposure of follicle cells to aurothioglucose at 100 nM inhibits the action of T3
P ,0.05, but not that of T2 Fig. 9.
3.4. The effect of time on the action of T3 and T2 We have not previously timed carefully the interval
between application of a ligand and the assessment of its effect on the optical path difference. When T3 is
added to a suspension of follicle cells and the effect mea- sured as quickly as possible within 10 min, its effect
on the optical path difference is small, whereas incu- bation for 60 or 120 min before the effect is assessed
Fig. 9. The effect of pre-incubation in 100 nM aurothioglucose on
the stimulation by T3 or T2 of the optical path difference of follicle cells in vitro. Data treated as in Fig. 1.
Fig. 10. The effect on the optical path difference of exposure of fol-
licle cells in vitro to 10 nM T3 or T2 for varying lengths of time indicated in min. Data treated as in Fig. 1.
produces a greater effect P ,0.01 for 10 min vs. 60
min. With T2, however, the effect after 10 min is not different from the effect after 60 min P
.0.1 Fig. 10. 3.5. The effect of cycloheximide on the action of T3
and T2 Because cycloheximide blocks the uptake of rho-T3,
we examined its effect on the action of T3 and JH III. The cells were pre-incubated for 30 min in 1 mM
cycloheximide before adding the hormones. The effect on the OPD was assessed 30 min later. The effect of T3
is abolished by the inhibitor, whereas the effect of JH III was undiminished Fig. 11.
3.6. The occurrence of immunoreactive T3 and T4 Extracts of ovaries, brains, thoracic muscles, fat bod-
ies and digestive system were tested by RIA for T4 and T3. Of these only gut particularly mid-gut yielded con-
Fig. 11. The effect of pre-incubating follicle cells in vitro in 1 mM
cycloheximide CHX for 30 min on the effect of 100 nM T3 or JH III on the optical path difference.
882 K.G. Davey Insect Biochemistry and Molecular Biology 30 2000 877–884
sistently positive results. In three separate quantitative determinations, there were 80–270 mean 163 pg of T3
equivalents per gut and 8–10.5 mean 9.0 µ
g T4 equiva- lents per gut.
When the gut tissue and the gut contents were assayed separately, only gut contents displayed any immunoreac-
tivity. When food was extracted, wheat shoots were found to contain 28 ng of T3 and 1.8
µ g T4 equivalent
activity per gram fresh weight, while bran contained 23 µ
g of T3 and 243 µ
g of T4. Only T3 immunoreactivity is detected in haemo-
lymph. Its titre varies to some degree with the gono- trophic cycle of the female Fig. 12.
4. Discussion
