close

Вход

Забыли?

вход по аккаунту

?

Manduca sexta prothoracicotropic hormoneevidence for a role beyond steroidogenesis.

код для вставкиСкачать
A r t i c l e
Manduca sexta
PROTHORACICOTROPIC
HORMONE: EVIDENCE FOR
A ROLE BEYOND
STEROIDOGENESIS
Robert Rybczynski, Chelsea A. Snyder, John Hartmann,
and Lawrence I. Gilbert
Department of Biology, CB 3280, University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina
Sho Sakurai
Department of Biology, Kanazawa University, Kanazawa, Japan
Prothoracicotropic hormone (PTTH) is a homodimeric brain peptide
hormone that positively regulates the production of ecdysteroids by the
prothoracic gland of Lepidoptera and probably other insects. PTTH was
first purified from heads of adult domestic silkworms, Bombyx mori.
Prothoracic glands of Bombyx and Manduca sexta undergo apoptosis
well before the adult stage is reached, raising the recurring question of
PTTH function at these later stages. Because Bombyx has been
domesticated for thousands of years, the possibility exists that the presence
of PTTH in adult animals is an accidental result of domestication for
silk production. In contrast, Manduca has been raised in the laboratory
for only five or six decades. The present study found that Manduca
brains contain PTTH at all stages examined post-prothoracic gland
apoptosis, i.e., pharate adult and adult life, and that PTTH-dependent
changes in protein phosphorylation and protein synthesis were observed
in several reproductive and reproduction-associated organs. The data
indicate that PTTH indeed plays a role in non-steroidogenic tissues and
suggest possible future avenues for determining which cellular processes
are being so regulated. & 2009 Wiley Periodicals, Inc.
Grant sponsor: NSF; Grant number: IOB 0516623.
Correspondence to: Lawrence I. Gilbert, Department of Biology, CB3280, University of North Carolina,
Chapel Hill, NC 27599-3280. E-mail: lgilbert@unc.edu
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 70, No. 4, 217–229 (2009)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20295
218
Archives of Insect Biochemistry and Physiology, April 2009
Keywords: PTTH; phosphorylation; translation; reproduction; accessory
glands; neuropepeptide
INTRODUCTION
Prothoracicotropic hormone (PTTH) is a homodimeric protein synthesized in the
brain of Lepidoptera, and presumably many other insects. PTTH stimulates
ecdysteroid biosynthesis by the prothoracic glands of larval and pupal animals
resulting in the initiation of the molting process. It was first purified from heads of the
adult silkworm Bombyx mori and tests in vitro demonstrated clearly an ecdysteroidogenic effect at nanomolar concentrations (Kataoka et al., 1987, 1991; see Rybczynski,
2005). Similar results were obtained following the cloning and expression of
recombinant PTTH (rPTTH) of the tobacco hornworm Manduca sexta (Gilbert et al.,
2000; Shionoya et al., 2003). In retrospect, finding PTTH in adult Bombyx was very
surprising. The only demonstrated function of PTTH is the stimulation of ecdysteroid
synthesis by the prothoracic glands, which undergo apoptosis during pharate adult
life, well before adult eclosion (Dai and Gilbert, 1997). Following the loss of the
prothoracic glands, circulating ecdysteroid levels eventually decline to very low levels
at the time of adult eclosion both in Bombyx and Manduca. However, in Bombyx at least,
hemolymph PTTH titers appear to plateau, or even increase, during the period
following gland apoptosis (Dai et al., 1995; Mizoguchi et al., 2001, 2002; see also
Rybczynski, 2005). Since Bombyx has been domesticated, i.e., inbred for thousands of
years, the possibility exists that the presence of PTTH after prothoracic gland
apoptosis represents an artifact of domestication. In contrast, laboratory colonies of
Manduca have been in existence for only about half a century. The present study was
undertaken to determine if Manduca brains contained PTTH past the period of
prothoracic gland apoptosis, and, if so, to examine potential tissue targets and possible
functions.
MATERIALS AND METHODS
Animal Rearing and Dissections
Manduca larvae were group-reared at 261C on an artificial diet and a 16:8
light:dark cycle (Rybczynski and Gilbert, 1994). Under these conditions, the fifth
(final) larval instar stage lasts E10 days and adults eclosed after 22 to 23
days of pharate adult development. Larvae were placed into wooden pupation blocks
when they ceased feeding and started wandering on day 5 of the fifth instar
(V5). Animals close to adult eclosion were placed in cages with net walls and a dish of
honey water for feeding. Only healthy adults were sampled, i.e., animals that had not
properly expanded their wings or showed other structural abnormalities were
discarded.
Larval, pupal, and young pharate adult prothoracic glands or other tissues were
extirpated and incubated with test materials as described previously (Rybczynski and
Gilbert, 1994). Adult heads were collected from animals that were immobilized under
CO2 after being placed at about 41C. For testing the PTTH extracts for their effect on
steroidogenesis and protein phosphorylation, paired larval prothoracic glands from
Archives of Insect Biochemistry and Physiology
PTTH: Evidence for Non-Steroidogenic Role
219
day 3 of the fifth instar (V3) were utilized. One gland served as a matched control for
the gland that received the PTTH extract. The paired ovaries were treated similarly.
Other tissues were sampled by cutting two similar pieces and treating one as the
control and the other as the experimental. A few tissues could not be sampled in a pairwise manner from the same animal and in those cases samples from different animals
were paired. Following dissection, a 30-min pre-incubation period was initiated, after
which the tissue samples were transferred rapidly to wells containing fresh Grace’s
medium7brain extracts. A dose of 0.25 brain equivalent of extract per 25 ml was used
for tests with V3 prothoracic glands, with a 1-h incubation period.
After the incubation, the glands or other tissues were flash frozen at 801C for
future SDS-PAGE immunoblot analyses, while the media samples were frozen at
201C with the addition of 100 ml of PBS, for later radioimmunoassay of ecdysteroids.
For experiments with tissues other than the prothoracic gland, the standard, wellcharacterized PTTH-containing brain extract was utilized (Gilbert et al., 2000). A
starting test dose of 0.25 brain equivalent and a 1-h incubation were used but higher
doses and other incubation times were also tested. Doses of PTTH brain extract
exposed to tissues never exceeded 0.75 brain equivalent (or 25% PTTH extract of total
volume), so that no experiments exceeded estimated physiological conditions. If
immunoblot blot analysis revealed changes in phosphorylation as detected by an antiphospho ERK and/or an antibody detecting proteins phosphorylated on serines by
PKC, the experiment was repeated using rPTTH (a gift of the Kataoka Laboratory) to
insure that the observed change was attributable to PTTH in the extract.
Protein synthesis was assessed using a modified version of the above-described in
vitro protocol. Tissues were transferred to methionine-free Grace’s medium for a
30-min preincubation, after which the tissues were transferred to fresh methioninefree Grace’s containing 25 mCi per 50 ml 35S-methionine7PTTH for a 1-h incubation.
Following this incubation, tissues were flash frozen until analyzed by SDS-PAGE. After
SDS-PAGE, gels were fixed and exposed to X-ray film as described previously
(Rybczynski and Gilbert, 1994). Developed films were analyzed with an Alpha
Innotech FluroChem 8800 Imaging System using AlphaEaseFC software (San
Leandro, CA).
Reagents
Grace’s Medium was obtained from GibcoBRL (Grand Island, NY). Partially purified
PTTH was extracted from brains as described previously (Rybczynski and Gilbert,
1995); this material has the same specificity and effects on prothoracic gland
ecdysteroidogenesis and signal transduction as pure Manduca rPTTH (Gilbert et al.,
2000; Rybczynski et al., 2001; Rybczynski and Gilbert, 2006). Partially purified PTTH
was extracted from Manduca heads using a modification of the technique used for
brains. Briefly, pupae were immobilized on ice and adults were immobilized with CO2,
following which heads were removed, flash-frozen, and stored at 801C. To extract
PTTH, the heads were ground with a small mortar and pestle that had been previously
chilled on dry ice. The resultant powder was transferred to a tube containing 2 ml
Grace’s medium diluted 1:1 with water and 1 mM PMSF and a small crystal of
phenylthiourea. The mixture was boiled for 5 min., and then centrifuged to remove
particulates, followed by ultrafiltration with 10-kDa Ultracon tubes until the retentate
was 100 ml or less (see Rybczynski and Gilbert, 1995).
Archives of Insect Biochemistry and Physiology
220
Archives of Insect Biochemistry and Physiology, April 2009
Recombinant PTTH was a generous gift of Drs. H. Kataoka and S. Nagata
(University of Tokyo, Japan). A monoclonal antibody against the dually-phosphorylated
Drosophila melanogaster ERK was from Sigma-Aldrich (St. Louis, MO). The antibody
recognizing PKC-phosphorylated substrate proteins was from Cell Signalling
Technology (Beverly, MA).
Immunoblots
SDS-PAGE and immunoblotting were performed as described previously (Rybczynski
and Gilbert, 2003). Immunoreactive proteins were visualized with chemiluminescence,
measured directly with the Alpha Innotech FluroChem 8800 Imaging System.
Radioimmuoassay (RIA) of Ecdysteroids
Thawed samples were vortexed, centrifuged at 15,000g for 5 min, and aliquots of the
supernatant/PBS mix were assayed in duplicate for ecdysteroid content as described
previously, using the SHO-3 antibody developed by one of us (SS) (Warren and
Gilbert, 1986, 1988; Kiriishi et al., 1990). The results of the RIA experiments are
expressed as the Ar (activation ratio), defined as the ratio of the ecdysteroid production
by a treated gland divided by the ecdysteroid production of the contralateral gland
(Rybczynski and Gilbert, 1994). This ratio corrects for inter-animal variation in basal
ecdysteroid production due to slight differences in developmental stage. RIA results
are presented as the mean7S.E.M.
RESULTS
As an initial test for the presence of PTTH in adult Manduca, brains were carefully
dissected from adult animals up to four days after eclosion and standard PTTH
extractions were performed. These PTTH preparations were incubated for 90 min
with V3 prothoracic glands. This particular stage was chosen because it has been the
most studied in regard to the glands’ response to PTTH (e.g., Gilbert et al., 2000). This
analysis revealed that adult brain extracts were capable of stimulating prothoracic
gland ecdysteroid synthesis about threefold relative to matched control glands (Ar 5 3;
data not shown). PTTH-containing extracts prepared from day-1 pupal (P1) brains
typically yield an Ar of 4–10 under the same conditions. The excision of brains from
adult and near adult animals is slow and labor intensive relative to that with P1 animals,
increasing the chance that PTTH may be released or lost from the brain during
dissection, and thus yielding an underestimate of the brain’s PTTH content. To avoid
this possible problem, an alternate method of preparing extracts was developed,
utilizing whole heads rather than excised brains. When PTTH was prepared from
adult (A1) brains and A1 heads, there was no difference in the ability of the resulting
extract to stimulate ecdysteroid synthesis when compared to extirpated brains. As a
result of this finding, a series of whole-head PTTH extracts was prepared covering the
period of pupal-adult development from P1 to day 6 after adult eclosion (A6) and was
assayed for PTTH activity. The results revealed that PTTH activity was present at all
stages tested (Fig. 1). The distinct peak on P7 (day 7 of pharate adult development)
coincides with a large peak in the ecdysteroid hemolymph titer observed previously
(Warren and Gilbert, 1986); There is also a suggestion of a minor peak occurring just
after adult eclosion and extending for approximately four days, followed by a
Archives of Insect Biochemistry and Physiology
PTTH: Evidence for Non-Steroidogenic Role
221
30
X
ECDYSTEROID PRODUCTION
(AR )
25
20
15
ADULT ECLOSION
X
10
X
5
X
X
X
X
X
X
X
X
X X
X
A7
P2
3
A0
A1
A2
A3
A4
0
P2
7
P1
4
P1
0
P1
P7
P4
P1
0
DEVELOPMENTAL STAGE
Figure 1. Relative brain PTTH content in pupal, pharate adult, and adult Manduca as estimated by the
ability of 0.25 brain equivalents of brain extract to elicit increased ecdysteroid synthesis, relative to
contralateral glands. Extracts were prepared from 4 to 8 heads, and were tested with one gland each from 6
to 9 pairs of glands. The activation ratio (AR) is the ratio of ecdysteroid production by a treated gland divided
by ecdysteroid production of the contralateral gland. Means are shown7SEM.
progressive decline. It is of some interest that the PTTH content of the adult brains
showed that extracts of female brains from A1 to A3 had slightly higher activity than
did male brains (data not shown).
The possibility exists that some or all of the ecdysteroidogenic effect of pharate
adult and adult head and brain extracts is due to the presence of one or more
heretofore undescribed ecdysteroidogenic factors in addition to, or instead of,
authentic PTTH. Since an antibody against Manduca PTTH was not available, an
indirect method was used to determine if the ‘‘real’’ PTTH was present in the brain
extracts. PTTH, both as present in P1 brain extracts and in pure recombinant form,
elicits rapid changes in the phosphorylation of the extracellular signal-regulated
kinase (ERK: Rybczynski et al., 2001) and of several proteins phosphorylated on
serines by protein kinase C (Rybczynski and Gilbert, 2006). These effects have been
characterized best with V3 prothoracic glands. Consequently, these glands were
challenged with brain extracts from pupal and adult stages and the effects of these
PTTH extracts on protein phosphorylation were analyzed by SDS-PAGE followed by
immunoblotting with antibodies that recognize dually-phosphorylated ERK (dpERK)
or PKC-phosphorylated proteins. The results indicate that the phosphorylation
patterns elicited by rPTTH treatment are the same as the results obtained from all
brain and head extracts tested (Fig. 2). Because it is unlikely that another factor would
stimulate exactly the same suite of phosphorylations, these results indicate the
presence of PTTH in all extracts examined, although they do not exclude
unequivocally the presence of as yet unreported ecdysteroidogenic factors.
Given the presence of functional PTTH in Manduca brains long after the
programmed cell death of the prothoracic glands, heretofore the only demonstrated
target of PTTH, an attempt was made to identify other tissues that might respond to
Archives of Insect Biochemistry and Physiology
222
Archives of Insect Biochemistry and Physiology, April 2009
A
Matching Lanes
A
B
C
PTTH
A
P14
B
rPTTH
C
A7
MW
(kDa)
66
50
43
35
B
Matching Lanes
PTTH
A
B
C
A
P14
B
rPTTH
C
A7
dpERK
Figure 2. Pupal and adult head PTTH preparations elicit the same phosphorylations in the prothoracic
gland as does rPTTH. A: Proteins phosphorylated by PKC. V3 prothoracic glands were incubated for
1 h7PTTH-containing extracts prepared from P14 or A7 heads, or rPTTH. Gland proteins were then
subject to SDS-PAGE followed by transfer and immunoblot analysis with an antibody recognizing proteins
phosphorylated by PKC on serine residues (PKCsub antibody). Proteins marked with arrows are consistently
phosphorylated by PKC in response to all PTTH preparations tested and these data are representative of all
such tests. Matching lanes indicate that the samples identically labeled came from paired glands of the same
animal. B: ERK phosphorylation. The immunoblot shown in A was stripped of the antibodies employed to
detect proteins phosphorylated by PKC and re-probed with an antibody against dually-phosphorylated ERK.
These data are representative of all such tests. Matching lanes indicate that the samples identically labeled
came from paired glands of the same animal.
PTTH. An obvious candidate was the ovary, since ovarian steroidogenesis has been
demonstrated in flies (Warren et al., 1996) and mosquitoes (Hagedorn et al., 1975)
although under the influence of a neuropeptide other than PTTH in the case of the
mosquito (Brown et al., 1998). Manduca ovaries at several developmental stages were
incubated with 3H-cholesterol and PTTH for varying amounts of time and both the
tissue and incubate analyzed for ecdysteroid synthesis (see Warren et al., 1996, for the
methodology used here). No conversion to ecdysone, or to ecdysone precursors, could
be detected (data not shown).
Since PTTH stimulates changes in the phosphorylation of several proteins in the
prothoracic gland (Rybczynski and Gilbert, 2006), the same experimental paradigm
used to study these changes in the gland was applied to a variety of other tissues
during the pupal, pharate adult, and adult stages in an attempt to identify a possible
target of PTTH and perhaps be able to explain the presence of PTTH in the adult and
pharate adult Manduca. No consistent changes in PTTH-elicited phosphorylation
patterns were observed in proteins extracted from antennae, ventral nerve
cord, brain, fore- and hind-wings, fat body, and fore-legs, surveyed at multiple
Archives of Insect Biochemistry and Physiology
PTTH: Evidence for Non-Steroidogenic Role
223
developmental stages using both the dpERK and PKC-substrate antibodies. However,
such phosphorylation changes were observed in extracts of ovary and male accessory
gland tubules of adult animals. These changes in PTTH-dependent phosphorylation
patterns were also seen in some samples from younger animals, although not
consistently. The changes in the ovarian proteins were further localized to the accessory
gland reservoirs using the dpERK and PKC substrate antibodies (Fig. 3). Aside from
ERK, the identity of these proteins is unknown. In the male accessory gland tubules,
ERK phosphorylation and PKC-dependent phosphorylations were also observed (Fig.
4). Note that the suite of phosphoproteins detected in both control and PTTH-treated
tissues is different than that observed in prothoracic glands, and, further, that the PKCphosphorylated proteins detected in female accessory gland reservoir extracts differ
from those found in extracts of male accessory gland tubules (Figs. 3 and 4).
PTTH stimulates increases in protein synthesis and/or accumulation in prothoracic glands in two ways, a general overall increase and the increased synthesis of
specific proteins (Rybczynski and Gilbert, 1994). The former can be quantified by
determining 35S-methionine incorporation into trichloroacetic acid-precipitable
proteins. This general or trophic response can also be readily detected following
Figure 3. A: PTTH-stimulated PKC-dependent phosphorylation changes in adult day 4 female accessory
gland reservoirs. Reservoirs were incubated for 1 h7PTTH extracts (prepared from P1 brains), then
subjected to SDS-PAGE/immunoblotting. Blots were probed with the PKCsub antibody. The approximate
MWs of proteins consistently detected in reservoirs challenged with PTTH is indicated on the right.
B: PTTH-stimulated ERK phosphorylation changes in female accessory gland reservoirs. The blot shown in
A was stripped and reprobed with the anti-diphospho-ERK antibody. The same samples that showed PTTHstimulated PKC-dependent phosphorylation reveal a PTTH-stimulated protein increase in ERK
phosphorylation.
Archives of Insect Biochemistry and Physiology
224
Archives of Insect Biochemistry and Physiology, April 2009
Figure 4. A: PTTH-stimulated PKC-dependent phosphorylation changes in adult day-4 male accessory
gland tubules. Samples were incubated for 30 min7PTTH-containing extracts (prepared from P1 brains),
then subjected to SDS-PAGE/immunoblotting. Blots were probed with the PKCsub antibody. The
approximate MWs of proteins consistently detected in reservoirs challenged with PTTH are indicated on
the right. B: PTTH-stimulated ERK phosphorylation changes in male accessory gland tubules. The blot
shown in A was stripped and reprobed with the anti-diphospho-ERK antibody. The same samples that
showed PTTH-stimulated PKC-dependent phosphorylation reveal a PTTH-stimulated protein increase in
ERK phosphorylation.
SDS-PAGE and autoradiography. A7 male accessory glands and seminal vesicles and
female accessory gland reservoirs were incubated with 35S-methionine7PTTH and
the tissues examined via SDS-PAGE and autoradiography. In addition, Malpighian
tubules and fat body were likewise examined; these two tissues had shown inconsistent
phosphorylation changes in response to PTTH in early experiments. In all five cases,
electrophoretic analysis revealed that PTTH caused a general decrease in protein
synthesis and/or accumulation (Figs. 5 and 6). Careful examination of the
autoradiographs produced by two different time exposures did not reveal any protein
band–specific changes (data not shown).
DISCUSSION
PTTH was first isolated in quantity from adult Bombyx heads, raising a long-standing
question about its presence and function following prothoracic gland apoptosis during
Archives of Insect Biochemistry and Physiology
PTTH: Evidence for Non-Steroidogenic Role
225
Figure 5. A: Decreased general protein synthesis in adult day-4 and -7 male accessory glands
during exposure to PTTH. Gels run with 35S-methionine radiolabelled samples were dried and then
exposed to X-ray film to detect overall and/or specific changes in radiolabelled protein accumulation.
B: Decreased general protein synthesis in adult day-4 seminal vesicles during exposure to PTTH.
Samples treated as in A.
Archives of Insect Biochemistry and Physiology
226
Archives of Insect Biochemistry and Physiology, April 2009
Malphighian
Tubules
Fat
Body
MW
(kDa)
200
116
97
66
55
45
36
PTTH
+
+
+
+
Figure 6. Decreased general protein synthesis in male adult day-5 Malpighian tubules and fat body during
exposure to PTTH. Gels run with 35S-methionine radiolabelled samples were dried and then exposed to
X-ray film to detect overall and/or specific changes in radiolabelled protein accumulation.
pharate adult development (see Rybczynski, 2005). Given the millennia of domestication of this species, one can argue that the presence of PTTH in adult animals is a nonfunctional relic due to selection by the silk industry. This possibility is supported by
analyses of Bombyx PTTH hemolymph titers showing different patterns of zeniths and
nadirs, depending on the silkworm strains analyzed (see Rybczynski, 2005). In
contrast, Manduca has been raised in laboratory colonies beginning in the midtwentieth century and there has been very little overt selection for specific traits. Thus,
Manduca may be a more evolutionarily relevant model for studying the presence and
function of PTTH in adult insects.
A survey of Manduca head extracts from just after the larval-pupal molt to a week
following adult eclosion revealed ecdysteroidogenic activity that is attributable to
PTTH. That it is the PTTH in these extracts eliciting ecdysteroidogenesis is supported
by the observation of prothoracic gland protein phosphorylation patterns identical to
those seen when pure rPTTH was used to stimulate glands (see also Gilbert et al.,
2000). Further, PTTH activity was demonstrated in the brains of all developmental
stages surveyed, including adults a week after eclosion. During the first half of pharate
adult development, the PTTH content of the brain varied as might be expected from
previous work on hemolymph ecdysteroid titers (Warren and Gilbert, 1986), i.e., a
large increase in PTTH content was detected at approximately the same time (V7) that
the large peak of circulating ecdysteroids occurs that elicits pupation. In contrast to the
ecdysteroid titer, however, head PTTH content does not continually decline after this
peak but reaches a fairly stable level that continues for up to a week after adult
eclosion. A small peak occurs just before adult eclosion followed by a second small peak
two days after eclosion, and it is only after five more days that a decline below pupal
values is noted. Since the large V7 peak in PTTH corresponds with a known
physiological role, these latter two peaks are suggestive of a role for PTTH regulating
pre- and post-adult eclosion physiological events.
In the prothoracic gland, PTTH stimulates the phosphorylation of ERK in a PKCdependent fashion as part of the transductory cascade leading to enhanced
ecdysteroidogenesis, as detected by a phosphorylation-specific antibody, as well as
Archives of Insect Biochemistry and Physiology
PTTH: Evidence for Non-Steroidogenic Role
227
proteins phosphorylated by PKC on serine residues as analyzed with a second antibody
(Rybczynski and Gilbert, 2006). These antibodies were used to survey a variety of
pupal, pharate adult, and adult tissues for responses to PTTH in an attempt to
elucidate a function for PTTH in animals devoid of prothoracic glands. The only
consistent changes, increases in protein phosphorylation, were detected in
structures of the adult reproductive systems, i.e., female accessory gland reservoirs
and the male accessory gland tubules. As noted previously, several lines of
evidence reveal that it is the PTTH in our extracts that elicit the effects observed;
future studies on the accessory glands should utilize rPTTH as well. These
two structures, along with male seminal vesicles, also displayed PTTH-dependent
overall decreases in protein synthesis/accumulation, as measured by 35S-methionine
incorporation into proteins. Experiments with ovaries failed to reveal PTTHdependent ecdysteroid synthesis at all stages examined. However, minute quantities
of de novo synthesized ecdysteroid from radiolabeled cholesterol may have been
masked by the endogenous content of non-labeled ecdysteroids and precursors
present in the organ. This possibility should be investigated in the future. Although
one could argue that the above results are artifacts of the in vitro conditions, this is
highly unlikely. Earlier studies with rPTTH indicate that the extracts used in
these experiments (0.25 to 0.75 brain equivalents of PTTH) contain physiologically
realistic amounts of PTTH, i.e., in the low nanogram to sub-nanogram range (Gilbert
et al., 2000).
The composite data present a picture of developmentally regulated PTTH content
in the brain of Manduca, long after the prothoracic glands have undergone
programmed cell death and hemolymph ecdysteroid levels have declined to extremely
low levels. The data also suggest that PTTH in adult Manduca may regulate the
function and/or development of the reproductive physiology of both sexes. The male
accessory glands (paragonia) contain a variety of products including defensive
molecules, modifiers of female reproductive physiology, mediators of sperm transfer,
pheromonostatic peptide, among others (see Wolfner et al., 2005) as well as a host of
diverse proteins (Acps) including peptides, prohormones, and glycoproteins
(Chapman, 2008). The knowledge of their roles has expanded greatly in recent years
and includes influences on sperm storage, decreased female receptivity, increased egg
production, increase in the synthesis of peptides with immune functions, increased
feeding of the female, and apparent modulation of juvenile production (see Chapman,
2008). Indeed, there appears to be up to 133 seminal fluid proteins transferred to the
female in the case of Drosophila (Findlay et al., 2008). In the case of the female,
the accessory glands (gonadal glands) are associated with the spermatotheca, supply
the glue for binding eggs to the substratum, e.g., leaves, and many other functions
(Wolfner et al., 2005).
The observation that protein synthesis in Malpighian tubules and fat body may
also be down-regulated by PTTH does not obviously support the hypothesis of a role
for PTTH in reproduction. However, it is possible that resources diverted from other
tissues and organs may be important for successful reproduction in the broadest sense
and down-regulation in many cases is as important as up-regulation in the control of
cellular processes.
With the complexity of molecules in the accessory glands and other constituents
of the reproductive system, our studies do not elucidate effects of PTTH at the
cellular or molecular levels. Although we can neither pinpoint the cellular site of
action nor the function of the phosphorylated proteins involved, we believe
Archives of Insect Biochemistry and Physiology
228
Archives of Insect Biochemistry and Physiology, April 2009
strongly that the data provide a base for further investigations of the role of
PTTH in adult lepidopterans using physiological, biochemical, and molecular
approaches.
ACKNOWLEDGMENTS
We thank Dr. James T. Warren of our laboratory for his expert help in the experiments
involving the possible incorporation of radiolabeled cholesterol into ecdysone or
intermediates in the ecdysone biosynthetic pathway.
LITERATURE CITED
Brown MR, Graf R, Swiderek KM, Fendley D, Stracker HT, Champagne DE, Lea AO. 1998.
Identification of a steroidogenic neurohormone in female mosquitoes. J Biol Chem
273:3967–3971.
Chapman T. 2008. The soup in my fly: evolution, form and function of seminal fluid proteins.
PLoS Biology 6:1379–1382.
Findlay GD, Yi X, MacCoss JJ, Swanson WJ. 2008. Proteomics reveals novel Drosophila seminal
fluid proteins transferred at mating. PLoS Biol e178.
Dai JD, Mizoguchi A, Satake S, Ishizaki H, Gilbert LI. 1995. Developmental changes in the
prothoracicotropic hormone content of the Bombyx mori brain-retrocerebral complex and
hemolymph: analysis by immunogold electron microscopy, quantitative image analysis, and
time-resolved fluoroimmunoassay. Dev Biol 171:212–223.
Dai JD, Gilbert LI. 1997. Programmed cell death of the prothoracic glands of Manduca sexta
during pupal-adult metamorphosis. Insect Biochem Mol Biol 27:69–78.
Gilbert LI, Rybczynski R, Song Q, Mizoguchi A, Morreale R, Smith WA, Matubayashi H,
Shionoya M, Nagata S, Kataoka H. 2000. Dynamic regulation of prothoracic gland
ecdysteroidogenesis: Manduca sexta recombinant prothoracicotropic hormone and brain
extracts have identical effects. Insect Biochem Mol Biol 30:1079–1089.
Hagedorn HH, O’Connor JD, Fuchs MS, Sage B, Schlaeger DA, Bohm MK. 1975. The ovary as
a source of alpha-ecdysone in an adult mosquito. Proc Natl Acad Sci USA 72:3255–3259.
Kataoka H, Nagasawa H, Isogai A, Tamura S, Mizoguchi A, Fujiwara Y, Suzuki C, Ishizaki H,
Suzuki A. 1987. Isolation and partial characterization of prothoracicotropic hormone of the
silkworm, Bombyx mori. Agric Biol Chem 51:1067–1076.
Kataoka H, Nagasawa H, Isogai A, Ishizaki H, Suzuki A. 1991. Prothoracicotropic hormone of
the silkworm, Bombyx mori: amino acid sequence and dimeric structure. Agric Biol Chem
55:73–86.
Kiriishi S, Rountree DB, Sakurai S, Gilbert LI. 1990. Prothoracic gland synthesis of
3-dehydroecdysone and its hemolymph 3 beta-reductase mediated conversion to ecdysone
in representative insects. Experientia 46:716–721.
Mizoguchi A, Ohashi Y, Hosoda K, Ishibashi J, Kataoka H. 2001. Developmental profile of the
changes in the prothoracicotropic hormone titer in hemolymph of the silkworm Bombyx
mori: correlation with ecdysteroid secretion. Insect Biochem Mol Biol 31:349–358.
Mizoguchi A, Dedos SG, Fugo H, Kataoka H. 2002. Basic pattern of fluctuation in hemolymph
PTTH titers during larval-pupal and pupal-adult development of the silkworm, Bombyx
mori. Gen Comp Endocrinol 127:181–189.
Rybczynski R. 2005. Prothoracicotropic hormone. In: Gilbert LI, Iatrou K, Gill SJ, editors.
Comprehensive molecular insect science, vol. 3. Oxford: Elsevier. p. 61–123.
Archives of Insect Biochemistry and Physiology
PTTH: Evidence for Non-Steroidogenic Role
229
Rybczynski R, Gilbert LI. 1994. Changes in general and specific protein synthesis that
accompany ecdysteroid synthesis in stimulated prothoracic glands of Manduca sexta. Insect
Biochem Mol Biol 24:175–189.
Rybczynski R, Gilbert LI. 1995. Prothoracicotropic hormone elicits a rapid, developmentally
specific synthesis of b tubulin in an insect endocrine gland. Dev Biol 169:15–28.
Rybczynski R, Gilbert LI. 2003. Prothoracicotropic hormone stimulated extracellular signalregulated kinase (ERK) activity: the changing roles of Ca21- and cAMP-dependent
mechanisms in the insect prothoracic glands during metamorphosis. Mol Cell Endocrinol
205:159–168.
Rybczynski R, Gilbert LI. 2006. Protein kinase C modulates ecdysteroidogenesis in the
prothoracic gland of the tobacco hornworm, Manduca sexta. Mol Cell Endocrinol 251:78–87.
Rybczynski R, Bell SC, Gilbert LI. 2001. Activation of an extracellular signal-regulated kinase
(ERK) by the insect prothoracicotropic hormone. Mol Cell Endocrinol 184:1–11.
Shionoya M, Matsubayashi H, Asahina M, Kuniyoshi H, Nagata S, Riddiford LM, Kataoka H.
2003. Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm,
Manduca sexta. Insect Biochem Mol Biol 33:795–801.
Warren JT, Gilbert LI. 1986. Ecdysone metabolism and distribution during the pupal-adult
development of Manduca sexta. Insect Biochem 16:65–82.
Warren JT, Gilbert LI. 1988. Radioimmunoassy: ecdysteroids. In: Gilbert LI, Miller TA, editors.
Immunological techniques in insect biology, New York: Springer. p. 181–214.
Warren JT, Bachmann JS, Dai JD, Gilbert LI. 1996. Differential incorporation of cholesterol and
cholesterol derivatives into ecdysteroids by the larval ring glands and adult ovaries of
Drosophila melanogaster: a putative explanation for the l(3)ecd1 mutation. Insect Biochem
Mol Biol 26:931–943.
Wolfner MF, Heifetz Y, Applebaum S. 2005. Gonadal glands and their gene products. In:
Gilbert LI, Iatrou K, Gill SJ, editors. Comprehensive molecular insect science, vol. 1.
Oxford: Elsevier. p. 179–212.
Archives of Insect Biochemistry and Physiology
Документ
Категория
Без категории
Просмотров
4
Размер файла
199 Кб
Теги
hormoneevidence, prothoracicotropic, beyond, role, sexta, manduca, steroidogenesis
1/--страниц
Пожаловаться на содержимое документа