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Effects of precocene II on female-specific hemolymph polypeptides in Oncopeltus fasciatus.

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Archives of Insect Biochemistry and Physiology 6:49-58 (1987)
Effects of Precocene I I on Female-Specific
Hemolymph Polypeptides in Oncopeltus
fasciatus
T. Martinez and M.D. Garcera
Departamento de Quimica Orgdnim Biolbgica, Centro de Investigaci6n y Desarrollo (C. S.J. C.),
Barcelona, and Departamento de Fisiologia Animal, Facultad de Ciencias Biol6gicas, Valencia,
Spain
Using polyacrylamide gel electrophoresis (PAGE) under denaturing conditions,
two major polypeptides of 200,000 and 170,000 daltons were detected i n the
hemolymph of mature female Oncopeltus fasciatus, but they were not found
in the hemolymph of males or newly emerged females. Those polypeptides
constituted the two major bands of early vitellogenic oocytes; however, they
were absent from the yolk of mature eggs. The slower-migrating band (200,000
daltons) appears to correspond to a vitellogenic protein already identified in
0. fasciatus, whose synthesis has been suggested to be independent of
juvenile hormone (JH).Treatment of newly emerged adult females with the
corpus allatum cytotoxin precocene II prevented the appearance of the
female-specific bands and induced an important accumulation of other
proteins in the hemolymph. Yolk deposition was also inhibited in those
animals. Topical application of JHto precocene-treated females restored the
appearance of the 200,000 and 170,000 dalton polypeptides in the hemolymph.
These results suggest that J His required for the synthesis of female-specific
polypeptides in 0. fasciatus.
Key words: juvenile hormone, vitellogenic oocytes, yolk proteins
INTRODUCTION
Juvenile hormone (JH) is necessary for egg maturation in most insects;
however, its involvement in reproduction seems to vary from species to
Acknowledgments: We thank Prof. W.S. Bowers for the generous gift of precocene [I, Dr. R.
Martinez-Pardo for his interest in this work, and Dr. J.A. Veenstra for critically reading the
manuscript. This study was supported by a grant from the Spanish Ministry of Education and
Science to T.M.
Received March 24,1986; accepted May 5,1987.
Address reprint requests to T. Martinez, Dept. Qufmica Organica Biologica (C.S.I.C.), J. Cirona
Salgado 18-26,08034 Barcelona, Spain.
0 1987 Alan R. Liss, Inc.
50
Martinez and Carcera
species, and different control mechanisms of oogenesis have been proposed
[l-31. In Heteroptera, evidence from experiments with allatectomized females treated with JH suggests that vitellogenin synthesis is regulated by JH
in Dysdercus intermedius [4] and Triatomu protructu [5]. Synthesis of vitellogenin
could also be stimulated in males of Rhodnius proEixus with the JH analog
methoprene [6]. JH appears to be required, at least partially, for yolk deposition in the species of this order examined [5,7l.
In Oncopeltus fusciatus, oviposition occurs at irregular intervals, but egg
development seems to be a continuous process [8]. Although this heteropteran species is also JH-dependent for vitellogenesis [8-lo], it is unusual in
that an antigenically incomplete vitellogenin (vitellogenin A) appears in the
hemolymph of diapausing females, before CA" activation [lo]. This protein
is also present in precocious adults, resulting from treatment of larvae with
precocene 11, animals in which the CA is supposed to be inactive [ll]. These
observations suggested that synthesis of vitellogenin A could be independent
of JH. Only after stimulation with JH did an antigenically complete vitellogenin (vitellogenin AB) appear in the hemolymph, probably resulting from
cleavage of the A form. It is thought that it is the mature vitellogenin (AB),
the protein incorporated by the oocytes with a further modification during
uptake or oocyte maturation [l0,12].
In the present study, we electrophoretically analyze the effects of CA
inactivation by precocene I1 [13] on hemolymph and ovarian polypeptides of
0. fasciutus and present some evidence suggesting an influence of JH on the
synthesis of major female-specific polypeptides, one of them putative vitellogenin A.
MATERIALS AND METHODS
Experimental Animals and Treatments
Oncopeltus fusciatus was reared in the laboratory at 29°C _+ 1°C at 60-65%
relative humidity, and 16 h photophase. Animals were fed on a mixed diet
of milkweed and sunflower seeds, and water was supplied ad libitum.
Precocene 11 Treatment
Newly emerged adult females (0-2-h-old) were treated with precocene I1
(6,7-dimethoxy-2,2-dimethylchromene;
a gift of W.S. Bowers) by the contact
method [14]. The animals were confined for 42 h in petri dishes pretreated
with an acetone solution of 500 pg precocene I1 (7.86 pg/cm2).After treatment,
females were transferred to normal rearing conditions, and males were
added the following day. Control animals were maintained in petri dishes
treated with acetone.
JH Treatment
To reverse the effects of precocene 11, JH I (R. Maag, Ltd., Dielsdorf,
Switzerland) was topically applied to precocene-treated animals on the ab*Abbreviations: CA = corpus allatum; JH = juvenile hormone; PAGE = polyacrylamide gel
electrophoresis; SDS = sodium dodecylsulfate.
Precocene Effects on Female-Specific Polypeptides
51
dominal sternites. Fifteen females were treated immediately after precocene
exposure; in 20 other females, JH application was initiated five days after
precocene treatment. In both groups, 5 pg of JH I (in 0.5 pl acetone) was
applied daily for four consecutive days. Control animals were treated with
acetone after exposure to precocene 11.
Sample Preparation
Hemolymph (4 pl vol) was collected from the stubs of sectioned legs in
glass capillary tubes coated with heparin, and it was diluted to a concentration of 10% with a solution containing 15 mh4 tricine, pH 7.4, 6.4% sucrose,
2 mM MgC1, and 20 mM KC1. Ovaries were dissected from adult females,
washed several times in insect saline, and homogenized in 60 p1 of the same
solution. Homogenates of different parts of the ovary were also prepared.
Chorionated eggs from mature females were opened and yolk was collected.
Samples were boiled for 4 min in 77 mM Tris HC1 pH 8.0, 1.8% SDS, 4.4%
2-mercaptoethanol, and 8.5% glycerol [15]. They were stored at -20°C until
use.
Individual animals were used in the present study to electrophoreticdy
analyze the protein content of hemolymph and ovary of the same female.
Sample collection was performed at 5 day intervals, starting at day 5 until
day 30 after precocene I1 treatment. Hemolymph and ovaries from five
controls and from seven precocene-treated females were analyzed in every
period (ie, 5, 10, 15, 20, 25, and 30 days after treatment). Collection of
hemolymph in those females treated with JH I (or acetone) was performed
five days after the initial dose (ie, 5 or 10 days after precocene treatment).
SDS-PAGE
Electrophoresis was performed under denaturing conditions at a constant
current of 20 mA and 4"C, using the discontinuous system described by
Conejero and Semancik [16]. The resolving gel contained 9% acrylamide.
Two microliters of 0.01% bromphenol blue was added to the samples before
electrophoresis. Gels were fixed in an aqueous solution of 12.5% trichloroacetic acid and 25% isopropanol and stained with 0.05% Coomassie brilliant
blue R 250. The molecular weight standards (Bio-Rad) used were: myosin
(M, = 200,000), P-galactosidase (M, = 116,250), phosphorylase B (M, =
92,500), bovine serum albumin (M, = 66,200), and ovalbumin (M, = 45,000).
RESULTS
With the present electrophoretic conditions, we could clearly visualize in
the hemolymph of mature 0. fasciatus females two major polypeptides with
M,s of 200,000 and 170,000. These polypeptides were not detected in the
hemolymph of adult males of aferent ages (Fig. 1).No appreciable concentrations were observed in the hemolymph either of fifth-instar nymphs or of
newly emerged females, which showed a hemolymph electrophoretic pattern
similar to that of males.
The other characteristic polypeptides of mature female hemolymph appeared to be common to both sexes. Some of these bands were present in
52
Martinez and Carcera
5
l9
10
d"?
6 '3
20
15
d"?
25
6' ' 9
d'
200 1
,
170-
Fig. 1. SDS-electrophoretic analysis of hemolymph of 0. fasciatus adult females and males.
Four microliters of hemolymph in 55 pI volume (see text) were loaded per well. Number of
days after the imaginal molt is indicated at top. Arrows show female-specific polypeptides
with approximate molecular weights of 200,000 and 170,000 daltons.
important concentrations in young mature females; however, they were
greatly reduced in the hemolymph of females older than two weeks (eg,
band 6 in Figs. 2 and 3). Other polypeptides appeared to be depleted from
the hemolymph of females of that age (eg, bands 9 and 10 in Figs. 2 and 3).
The electrophoresis of crude ovary extracts of reproductive females showed
two major bands with the same mobilities as the female-specific hemolymph
polypeptides (bands 2 and 3 in Fig. 2) suggesting that they result from the
incorporation of the hemolymph polypeptides into the oocytes. However,
the electrophoretic patterns of yolk from mature eggs or of whole eggs did
not show those polypeptides (Fig. 4). Different regions of well developed
ovaries were processed separately for electrophoresis in order to localize
those bands within the ovary; we found that they constituted the major
polypeptide bands of early vitellogenic oocytes (ca 0.5 mm in length). No
appreciable concentrations of the yolk polypeptides of mature eggs were
observed in those early follicles (Fig. 4).
Effects of Precocene I1
Comparison of hemolymph electrophoretic patterns of control animals
with those of females exposed to precocene I1 revealed important changes in
the latter. The female-specific polypeptides were not detected in the hemolymph of any of the precocene-treated animals examined 5 or 10 days after
exposure to precocene 11, whereas both polypeptide bands were always
present in the controls (Fig. 2). The other characteristic hemolymph bands of
young mature females were observed, however, in all experimental animals.
Females examined from day 15 after precocene treatment revealed a conspicuous accumulation of nonspecific hemolymph polypeptides (Fig. 3).
Some of these bands, which could hardly be detected in controls of the same
age, were present in considerable concentrations in precocene-treated fe-
Precocene Effects on Female-Specific Polypeptides
5
53
10
C
PI I
C
PI I
1
2
3
10
U
HF
OV
HF
OV
HF
HF
OV
HF
OV
Fig. 2. SDS-PAGE of hemolymph (HF) and crude ovary extracts (OV) of 0. fasciatus adult
females. Number of days after treatment i s indicated at top. The electrophoretic pattern of
hemolymph with characteristic polypeptide bands (1-10) i s shown to the left. Arrows indicate
the apparent absence of female-specific polypeptides (bands 2 and 3) from the hemolymph
of precocene Il-treated animals (PII). C , acetone-treated animals. Note the lack of protein
incorporation into the undeveloped ovaries of experimental females.
males (eg, bands 9 and 10 in Fig. 3). Band 6, which typically appeared in low
concentrations in the hemolymph of control females from the second period,
largely accumulated in all precocene-treated animals examined (Fig, 3). It is
worth noting that 8 of the 28 experimental animals examined later than two
weeks after precocene treatment revealed the 17’0,000 dalton female-specific
polypeptide in the hemolymph. Most of those females also showed the
200,000 dalton polypeptide (Fig. 3).
Ovaries of precocene-treated animals were carefully examined under the
microscope, and no maturing oocytes were found. The electrophoretic patterns of crude ovary extracts indirectly confirmed the CA inactivation by
precocene 11, since no substantial protein incorporation was detected in those
ovaries, even in the animals that showed the female-specific bands and a
large protein accumulation in the hemolymph.
JH Effects
To confirm that the effects of precocene I1 on the female-specific hemolymph polypeptides were due to its allatotoxic action, we applied JH I to
precocene-treated females. The hemolymph electrophoretic pattern of chemically allatectomized females posteriorly treated with JH revealed the presence of the two female-specific polypeptides both 5 and 10 days after
precocene treatment. Control animals, treated with acetone after exposure to
precocene 11, did not reveal, as before, any of those bands in the hemolymph
(Fig. 5).
54
Martinez and Carcera
20
PI I
OV
C
PI I
HF
HF
OV
HF
OV
Fig. 3. SDS-PACE of hemolymph (HF) and crude ovary extracts (OV) of 0. fasciatus adult
females 20 days after treatment. Notice the accumulation of general polypeptides in the
hemolymph of precocene-treated females (PII) and the presence of band 3 (a female-specific
polypeptide) in one of them. C, acetone-treatedfemale.
2
200
3
4
.-(
4
116
93
66
45
Fig. 4. SDS-electrophoretic analysis of hemolymph and ovarian proteins of an 0. fasciatus
mature female. 1: Yolk from mature eggs; 2: anterior region of ovarioles including vitellogenic
oocytes (ca 0.5 mm); 3: vitellogenic oocytes (ca 0.5 mm); 4 hemolymph; S: molecular weight
standards. Arrows indicate the female-specific polypeptides.
Precocene Effects on Female-Specific Polypeptides
5
PI I
55
10
+JH
+JH
PI I
Fig. 5. SDS-PACE of hemolymph of 0. fasciatus adult females treated with precocene II
immediately after emergence. Number of days after precocene treatment is indicated at top.
PII, precocene 11-treated animals; + JH, precocene Il-treated animals, later treated with JH I.
Arrows indicate the two female-specific bands present in female hemolymph after JH
application.
DISCUSSION
The major hemolymph polypeptides of 200,000 and 170,000 daltons from
mature females constituted two main bands of crude ovary extracts. These
observations suggested that those female-specific polypeptides could be vitellogenins (since they were both detected in the hernolymph and in the
ovaries of reproductive females). Although they were not present in the yolk
of mature eggs, they were found to be the main bands of early vitellogenic
oocytes (ca 0.5 mm in length). These results indicate that the two femalespecific polypeptides are probably yolk precursors.
The slower-migrating band possibly corresponds to a female-specific vitellogenic protein of 200,000 daltons identified in 0. fusciutus as vitellogenin A
[10,12]. This protein, which is not detected in mature eggs, is thought to be
a precursor form of an antigenically complete vitellogenin, called AB (or B),
with a molecular weight of 68,000 daltons [10,12]. It has been suggested that
vitellogenin A is cleaved in the hemolymph to produce vitellogenin AB and
that this mature protein would be taken up by the oocytes in modified form,
since the electrophoretic mobility and the peptide composition of the major
yolk protein of mature eggs were found to be slightly different to those of
vitellogenin AB [10,12].
The results obtained in the present study are in agreement with the above
model in that the 200,000 dalton polypeptide, the putative vitellogenin A,
may be a precursor of the mature yolk protein. However, results also indicate
that the 200,000 dalton band could be incorporated directly into the oocytes
and that the cleavage or molecular rearrangement might take place within
the ovary during oocyte maturation.
The 170,000 dalton female-specific polypeptide could be a vitellogenic
protein as well and might correspond to a female-specific band that has been
56
Martinez and Garcera
observed to migrate ahead of vitellogenin A [12]. However, the lack of any
further information about that protein precludes the possibility of establishing a relationship between these two bands.
With the present electrophoretic conditions, we visualized a polypeptide
band in female hemolymph with an approximate molecular weight of 68,000
daltons (band 6). However, a band with the same electrophoretic mobility
was also found to be a major component of male hemolymph. No femalespecific polypeptide could be detected in that region of the gel, even after
decreasing the concentration of acrylamide below 8%, as was suggested by
Rankin and Jackle [El. We were unable, therefore, to confirm the presence
of the mature form of vitellogenin (AB).
The present results show some similarities with another heteropteran
species, Dysdercus intemedius [4], in which the hemolymph electrophoretic
patterns from males and vitellogenic females also differ in two major polypeptides (M, = 180,000 and 175,000).Those female-specific bands were found
in yolk from both early vitellogenic follicles and mature eggs. The latter,
however, also contained some polypeptide bands that were absent from
female hemolymph, indicating a possible processing of vitellogenin during
oocyte maturation in this species as well.
Treatment of newly emerged adult females with precocene I1 inhibited the
appearance of the 200,000 and 170,000 dalton female-specific polypeptides in
the hemolymph of young mature females. JH application restored the appearance of both bands in the hemolymph. These results suggest at least a
partial control by JH on the synthesis of major female-specific polypeptides
in 0. fusciatus. It has been observed that vitellogenin synthesis in ethoxyprecocene-treated females of Locusta rnigrutoria could also be restored with the
JH analog methoprene [lq.
Previous studies by Kelly and Telfer [lo] revealed the presence of vitellogenin A in starved and diapausing females of Oncopeltus, animals with a
supposedly inactive CA. These observations suggested that synthesis of
vitellogenin A could be independent of JH. However, starved and diapausing
females were found to have a certain JH titer in the hemolymph [18,19].
In addition, Rankin and Jackle [12] reported no evidence for JH control of
vitellogenin synthesis in 0. fusciatus, since they could find both forms of
vitellogenin in the hemolymph of females treated with precocene 11. However, in those experiments, precocene treatment was initiated 1-3 days after
adult emergence, and it seems likely that this is too late to start treatment.
We have observed that Oncopeltus females could be far along in ovarian
development (ie, presence of advanced vitellogenic oocytes) when treatment
with precocene I1 was initiated at that time of the adult age. This development could be due to the presence of some JH synthesized in those females
after emergence, since vitellogenesis in Oncopeltus is known to be dependent
on JH [8-lo].
In newly emerged 0. fusciutus females treated with precocene 11, degenerative changes of the CA parenchymal cells have been observed at the ultrastructural level; however, the extent of degeneration was found to vary in
different cells 1201. It is possible then that some CA cells of precocene-treated
females are producing JH.
Precocene Effects on Female-Specific Polypeptides
57
Those previous reports showing the presence of vitellogenins in females
with a CA thought to be inactive, together with our observations that the
female-specific bands are present in the hemolymph of some precocenetreated animals examined some time (ie, 15-30 days) after treatment, indicate
that low levels of JH may stimulate synthesis of vitellogenic proteins in 0.
fusciutus. This possibility is also supported by the observation that vitellogenins appear in the hemolymph of 3-4-day-old females [ll], in which the JH
titer is reported to be low [19].
On the other hand, the important accumulation of other hemolymph
polypeptides observed in precocene-treated females suggests that JH is not
required, at least at high concentrations, for general protein synthesis in 0.
fusciutus. A similar accumulation of proteins in hemolymph was reported in
surgically allatectomized females of two orthopteran species [21,22]. However, major female-specific proteins, some of them identified as vitellogenins,
were found to be absent from allatectomized females of different species
[22-251.
The lack of any important protein incorporation into the oocytes of all
precocene-treated females examined is in agreement with previous findings
in starved and diapausing females of 0.fusciutus, in which no yolk deposition
was observed [9]. These observations also indicate that in Oncopeltus, as in
other insect species [26], the presence of proteins in the hemolymph cannot,
by itself, induce yolk deposition. Although a certain JH titer is known to
exist in starved and diapausing females [18,19], and probably in some precocene-treated animals, that titer appears not to be high enough to stimulate
incorporation of proteins into the oocytes. The present results agree with the
hypothesis that high levels of JH may be required for vitellogenin uptake in
Oncopeltus, whereas low JH levels could be adequate for vitellogenin synthesis [2].
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(1979).
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Martinez and Garcera
10. Kelly TJ, Telfer WH: Antigenic and electrophoretic variants of vitellogenin in Oncopeltus
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